Hindawi Publishing CorporationScientificaVolume 2013 Article ID 505714 17 pageshttpdxdoiorg1011552013505714

Review ArticleProtein Machineries Involved in the Attachment of Heme toCytochrome c Protein Structures and Molecular Mechanisms

Carlo Travaglini-Allocatelli

Department of Biochemical Sciences University of Rome ldquoSapienzardquo Ple A Moro 5 00185 Rome Italy

Correspondence should be addressed to Carlo Travaglini-Allocatelli carlotravagliniuniroma1it

Received 21 October 2013 Accepted 24 November 2013

Academic Editors D K Dube H Iwano C Riganti and J D Warren

Copyright copy 2013 Carlo Travaglini-Allocatelli This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Cytochromes c (Cyt c) are ubiquitous heme-containing proteins mainly involved in electron transfer processes whose structureand functions have been and still are intensely studied Surprisingly our understanding of the molecular mechanism whereby theheme group is covalently attached to the apoprotein (apoCyt) in the cell is still largely unknown This posttranslational processknown as Cyt c biogenesis or Cyt c maturation ensures the stereospecific formation of the thioether bonds between the heme vinylgroups and the cysteine thiols of the apoCyt heme binding motif To accomplish this task prokaryotic and eukaryotic cells haveevolved distinctive protein machineries composed of different proteins In this review the structural and functional properties ofthe main maturation apparatuses found in gram-negative and gram-positive bacteria and in the mitochondria of eukaryotic cellswill be presented dissecting theCyt cmaturation process into three functional steps (i) heme translocation and delivery (ii) apoCytthioreductive pathway and (iii) apoCyt chaperoning and heme ligation Moreover current hypotheses and open questions aboutthe molecular mechanisms of each of the three steps will be discussed with special attention to System I the maturation apparatusfound in gram-negative bacteria

1 Introduction

Cytochromes c (Cyts c) are ubiquitous heme-containingproteins involved in a variety of critical processes of cellularmetabolism since their discovery by Keilin in the early1920s they have been the focus of multidisciplinary scientificinterests and nowadays are considered textbook proteinsin biochemistry courses However many aspects of c-typecytochromes are still to be unveiled from the control andfine-tuning of electron transfer reactions and heme reactivity[1ndash3] to the description of Cyt c folding pathways and stability[4ndash6] The presence of the covalently bound heme prostheticgroup dictates the functions of Cyts c which are associ-ated mainly with electron transfer processes in aerobic andanaerobic respiration and in photosynthesis [7 8] howeverit is now clear that Cyts c play important roles also in othercellular processes such as H

2O2scavenging cytochrome

c oxidase assembly [9] lipid signaling [10] or apoptoticprocesses in the eukaryotic cells [11 12] This review dealswith a complex and still largely unknown process whereby

the heme is covalently and stereospecifically attached to theapoprotein (apoCyt) in the cell this posttranslational processis known as Cyt c biogenesis or Cyt c maturation Overand above its scientific relevance a full understanding ofthis posttranslational process may pave the way for futurebiotechnological applications such as the design and theproduction in vivo of novel heme-proteins and biosensorsendowed with innovative redox functions [13]

The heme b (Fe-protoporphyrin IX) is synthesized inprokaryotes and eukaryotes along a conserved pathwaywith highly related enzymes and biosynthetic intermediates[14] heme c is defined as a heme b covalently linked tothe protein by thioether bonds (Figure 1) In bacteria hemebiosynthesis occurs in the cytoplasm and the final step is theinsertion of iron into protoporphyrin IX by ferrochelatase inthe eukaryotic cell the heme biosynthetic pathway is splittedbetween the cytosol and the mitochondrion here at the levelof the mitochondrial inner membrane the ferrochelataseenzyme catalyzes the heme iron insertionAlthough the hemebiosynthetic pathway is well characterized the molecular

2 Scientifica

Figure 1 The heme-binding site typically observed in c-typecytochromes as exemplified by a close-up view of the structure of Paeruginosa Cyt c551 (Pa-Cytc PDB 351c) The heme is shown in redwhile the atoms of the residues from the heme-binding motif of Pa-Cytc (C

12VAC15H) and the distal Met61 are color-coded (C green

O red N blue S yellow) The figure highlights the thioether bondsbetween the Cys12 (on the right) and the vinyl-2 and between Cys15(on the left) and the vinyl-4 The iron atom of the heme (in gray) isaxially coordinated by the distal methionine residue (Met61 shownabove the heme plane) and by the proximal histidine residue (His16shown below the heme plane)

mechanism(s) underlying the process of heme traffickingacross the membranes is still largely obscure (see [15 16] forreviews on heme synthesis and trafficking in eukaryotes) Inall known Cyts c the heme is covalently linked to the apoCytwith the same stereochemistry two thioether bonds arepresent between the vinyls at positions 2 and 4 of the tetrapyr-role ring of heme b and the thiols of the N- and C-terminalcysteines (Cys1 and Cys2 resp) of a conserved heme-bindingmotif (C1XXC2H where X denotes any residues) The ironatom of the Fe-protoporphyrin IX is always axially coor-dinated to the histidine of the heme-binding motif (on theproximal side of the heme cavity) while amethionine residueon the distal side generally represents the second axial ligand(Figure 1) C-type cytochromes may contain more than oneheme c linked to the protein through different C1XXC2Hmotifs From a structural point of view Cyt c proteins definea well-defined 120572-helical fold (see SCOPmdashhttpscopmrc-lmbcamacukscop and CATHmdashhttpwwwcathdbinfoprotein structure databases) characterized by the presenceof three 120572-helices the N- and C-terminal 120572-helices interacteach other in the native structure while an additional 120572-helix(historically known as the 601015840 helix) overlays part of the hemecavity Since the seminal experiment of Anfinsen on horseheart Cyt c [17] it is generally accepted that Cyt c withoutits covalently bound heme (apoCyt) is an unfolded proteindevoid of appreciable secondary and tertiary structure andthat the polypeptide chain is able to fold into its typical Cytc structure only when the thioether bonds with the hemeare formed As it will be discussed below these observations

raise interesting questions as to how an unfolded proteinsuch as apoCyt is specifically recognized by the differentprotein components of the maturation apparatus of the cellRecently however evidence has been presented that at leastin some cases the Cyt c fold may be attained even in theabsence of the heme [18] challenging our current view of theCyt c folding mechanism [4 19]

C-type cytochromes are synthesized in the cytoplasm (n-side of the membrane) but they exert their functions inother subcellular compartments (p-side of the membrane)that is the periplasm of gram-negative bacteria the bacterialextracytoplasmic space of gram-positive bacteria the inter-membrane spacemdashIMS of mitochondria or the chloroplastthylakoid lumen It is in these subcellular compartments thatthe heme b is covalently attached to apoCyt by the appropriatematuration apparatus In prokaryotes the necessary translo-cation of apoCyt across the membrane is carried out by theSec machinery [26] this apparatus composed of the SecAB-DYEFG proteins is able to translocate unfolded proteinscarrying a specific targeting sequence [27] In eukaryotesthe newly synthesized apoCyt is probably translocated intothe mitochondrion via a different mechanism involvingcomponents of the TOM complex on the outer side of themembrane and the cytochrome c heme lyase which probablyacts also as an apoCyt receptor in the mitochondrial IMS[28 29] However the process is not completely clear as westill do not know whether the apoCyt is delivered to themitochondrial matrix and then exported to the IMS [30] or itis translocated directly to the IMS via a different mechanism[31] It should be noticed that in plants the translocation ofc-type cytochromes into the chloroplast lumen is probablyindependent on the heme attachment reaction [32 33]

Despite that in all c-type cytochromes both prokaryoticor eukaryotic the heme is always covalently linked to theconserved CXXCH heme-binding motif different matura-tion apparatuses composed of different proteins have beenidentified ([34 35] see Figures 2 3 and 4 and Table 1) Struc-tural and functional properties of the protein componentsof Systems IndashIII are the focus of the present review othermaturation apparatuses involved in the unusual attachmentof heme b to the protein moiety via a single thioetherbond (Systems IVndashVI) have been described and reviewedelsewhere [36 37]

With the exception of system III which is present ineukaryotic cells the distribution of the other Systems amongBacteria Archaea and plant cells is complicated by theobservation that in many cases the maturation machinery isnot conserved [38] rendering the analysis of their evolution-ary origins and relationships difficult [39ndash41] In 120572- and 120574-proteobacteria in some 120573- and 120575-proteobacteria in Archaeaand in themitochondria of plants and algae Cyt cmaturationis carried out by a set of eight or nine proteins belongingto System I [42] (Figure 2) in gram-positive bacteria incyanobacteria in the chloroplasts of plants and algae in 120576-120573- and some 120575-proteobacteria the Cyt c maturation processis carried out by three or four proteins belonging to System II[43 44] (Figure 3) while System III occurs in mitochondriaof fungi metazoans and some protozoa [16 45] (Figure 4)The observation that in plants three systems are present

Scientifica 3

CcmACcmA

CcmC

CcmE

Ccm

D

Ccm

B

Ccm

B

CcmF

CcmICcmH

CcmG

Periplasm

HemeATP ADP

Cytoplasm

Figure 2 Schematic representation of the protein components ofSystem I Proteins involved in the heme translocation and deliverypathway are shown in light brown proteins involved in the apoCytthioreduction pathway are shown in green proteins involved inapoCyt chaperoning and heme attachment processes are shownin light purple Cyt c (the 3D structure is that of the Cyt c551from P aeruginosa) Protein Data Bank accession number 2EXV[20] and apoCyt (represented as a cartoon) are shown in blueThe translocation process of heme (shown in red) is unknownThe 3D structures of the soluble periplasmic domains of Ec-CcmE Pa-CcmG and Pa-CcmH are shown (Protein Data Bankaccession numbers are 1LIZ [21] 3KH7 [22] and 2HL7 [23] resp)Organisms employing System I 120572- and 120574-proteobacteria some120573-proteobacteria (eg Nitrosomonas) and 120575-proteobacteria (egDesulfovibrio) and Deinococci and Archaea Additionally System Iis observed in plant mitochondria and in the mitochondria of someprotozoa (eg Tetrahymena)

(System I in mitochondria System III in the p-side of thethylakoid membrane and System IV in the n-side of thethylakoidmembrane [16 38])makes the classification and thedistribution of the different maturation systems even moredifficultWith the exception of System III which is apparentlycomposed of a single protein able to carry out the differenttasks of the Cytc maturation process (see below) the variousproteins of Systems I and II carry out different functionsincluding the translocation and delivery of heme b from thecytoplasm where it is synthesized to the relevant subcellularcompartment the chaperoning of apoCyt and the reductionof its disulfide the formation of the covalent bonds betweenthe heme b and the CXXCH heme-binding motif of theapoprotein (Table 1) The complexity of System I comparedto the protein composition of other Cyt cmaturation systemshas long been discussed in particular it has been proposedthat a possible explanation is to be found in the ability evolvedby organisms employing System I to utilize lower levels ofendogenous heme than those necessary for organisms whichevolved Systems II or III [46]

2 System I

The proteins belonging to System I (named CcmABCDE-FGH(I) from Cytochrome c maturation) are membraneproteins exposing their soluble domains (when present) intothe periplasm (Figure 2) All of these proteins are encodedby a single operon in 120572- 120573- and 120574-proteobacteria [4247] The availability of the entire Ccm operon in a single

Ccs

A

Ccs

B

Heme

Cytoplasm

Cyt capoCyt

ResA

Figure 3 Schematic representation of the protein componentsof System II Proteins involved in the heme translocation anddelivery and in the apoCyt chaperoning and heme attachmentprocesses are shown in light brown proteins involved in the apoCytthioreduction pathway are shown in green Cyt c and apoCyt(represented as a cartoon) are shown in blueThe 3D structure of thesoluble periplasmic domain of Bs-ResA is shown in green (ProteinData Bank accession number is 1ST9 [24] System II is foundin plant chloroplasts in gram-positive bacteria cyanobacteria 120576-proteobacteria most 120573-proteobacteria (eg Bordetella Burkholde-ria) and some 120575-proteobacteria (eg Geobacter)

Heme

Intermembrane space

Inner membrane

Outer membrane

Cyt c

apoCytHCCS

Figure 4 Schematic representation of System III A single pro-tein (HCCS) associated to the mitochondrial inner membraneis required for Cyt c maturation The translocation process ofheme (shown in red) is unknown System III is found in themitochondria of fungi (eg S cerevisiae) vertebrates (eg human)and invertebrates (eg C elegans Drosophila)

plasmid (pEC86 [48]) greatly facilitated the heterologousover-expression of many c-type cytochromes in E coli [49]Apparently c-type cytochromes specificity of the Ccm appa-ratus is rather low indeed in an attempt to characterize theminimal sequence requirements of the apoCyt polypeptiderecognized by System I it was shown that this complexmultiprotein apparatus is able to attach the heme even toshort microperoxidase-like peptides carrying the CXXCHmotif [50]

As discussed below different studies mainly carried outby immunoprecipitation experiments suggest that the Ccmproteins may be assembled in the bacterial membrane in a

4 Scientifica

Table1Proteincompo

nentsof

Syste

msIIIand

IIIa

long

with

theirstructural

features

(orPD

Bcodeswhenkn

own)

andfunctio

nalrolesS

ystem

Iproteinsarefoun

din120572-and120574-

proteobacteriasom

e120573-a

nd120575-proteob

acteria

and

Dein

ococciandArchaeaPlantm

itochon

driaM

itochon

driaof

somep

rotozoaSyste

mIIproteins

arefou

ndin

plantchlorop

lasts

Gram-

positiveb

acteria

cyano

bacteria120576-proteob

acteria

most120573

-proteob

acteria

and

some120575

-proteob

acteria

HCC

SofSystemIIIisfou

ndinthem

itochon

driaoffung

ivertebratesand

invertebrates

Syste

mI(SI)

SIstr

ucturalfeatures

Syste

mII(SII)

SIIstructuralfeatures

Syste

mIII(SIII)

SIIIstr

ucturalfeatures

Functio

n(s)

Ccm

AABC

transportermem

brane

n-sid

enu

cleotide-bind

ing

domain

ResB

(CcsB)

5-6TM

helices

1large

perip

lasm

icdo

main

conservedHis

resid

ues

HCC

SMem

brane-associated

protein

conservedHis

resid

ues

Hem

etranslocatio

nand

delivery

Ccm

BABC

transporter6TM

helices

ResC

(CcsA)

6ndash8TM

helices

WWDdo

main

conservedHis

resid

ues

Ccm

C6T

Mhelicesperiplasm

icWWDdo

main

Ccm

DSm

allm

embranep

rotein1

TMhelix

Ccm

E1T

MhelixO

B-fold

1SR3

1LM

01J6

Q2KC

T

Ccm

G1T

MhelixT

RX-like

fold

1Z5Y2B1K

1KNG3KH

73K

H93K

8NRe

sATR

X-lik

efold

2H1B1SU

91ST9

2F9

SapoC

ytthioredu

ction

Ccm

H1T

Mhelix3-helixbu

ndlefold

2HL72KW

0CcdA

6TM

helices

Ccm

F10ndash15TM

helicesperiplasm

icWWDdo

main

conservedHis

resid

ues

ResB

(CcsB)

ResC

(CcsA)

HCC

SapoC

ytchaperon

ingand

hemea

ttachment

Ccm

IPerip

lasm

icTP

Rand120572120573

domains

Scientifica 5

maturase multiprotein complex(es) However the existenceandor stoichiometry of these complexes remains to bedetermined either because of the experimental difficultiesin handling membrane protein complexes or because itis possible that these complexes are unstable and onlytransiently populated Independently from their functionalexistence in the bacterial periplasm as independent units or ascomponents of a multisubunit complex it is clear that each oftheCcmproteins plays a different role from the transport andchaperoning of the heme cofactor to the necessary reductionof the disulfide bond between the sulfur atoms of the twoCys residues of the conserved CXXCH motif of apoCytand finally to the catalysis of covalent heme attachment Anadditional interesting aspect is that over and above theirrole in the biogenesis of Cytc there is also evidence thatinactivation of some ccm genes induces phenotypes thatcannot be explained only in terms of absence of synthesis ofCyt c all of these pleiotropic effects are linked to impairmentof heme andor iron trafficking in the periplasm [47] Inparticular it has been recently shown that in 120572- and 120574-proteobacteria (including the human opportunistic pathogenP aeruginosa) mutations in the ccmC ccmI and ccmF genesinduce phenotypes such as reduced pyoverdine produc-tion reduced bacterial motility or impaired growth in low-iron conditions ([51] and refs therein) These observationssuggesting that Ccm proteins perform additional functionscritical for bacterial physiology growth and virulence pro-vide a rationale to explain why bacteria at variance withthe eukaryotic cell have evolved a metabolically expensiveoperon to accomplish an apparently simple task such asheme ligation to apoCyt Novel hypotheses addressing theseaspects and awaiting experimental investigation include (i)the utilization of Ccm-associated heme for additional cellularprocesses besides attachment to apoCyt (ii) trafficking a non-heme compound through the Ccm system required for ironacquisition such a siderophore (iii) the Ccm inactivation-dependent accumulation of heme b a photoreactivemoleculewhose degradation leads to reactive oxygen species and (iv)the destructive effect on [Fe-S] clusters of ferrisiderophoresreductases

In the following the structural (when known) andfunctional properties of the different components of theSystem I maturation apparatus will be discussed dissectingthe Cytc maturation process into three main functionalsteps heme translocation and delivery apoCyt thioreductivepathway and apoCyt chaperoning and heme ligation (asimilar modular description can also be found elsewhere(see [34 52])) However it should be remembered that inmany cases the proteins involved in the three steps are notuniquely assigned to a specific module as they interact witheach other moreover it has been shown that in some casesmore than one system can be present [41] This modularorganization of Cyt c biogenesis should therefore be intendedonly as a way to simplify the description of an overall highlyintegrated process For each of the three functional stepspresentation of the structural and functional properties of thedifferent protein components is followed by a discussion ofthe proposed molecular mechanism(s)

21 System I Components of the Heme Translocation andDelivery Pathway CcmA and CcmB proteins show the typ-ical sequence features of the ABC (ATP binding Cassette)transporter family and therefore these components of SystemI were initially considered as the proteins responsible forthe translocation of the newly synthesized heme from thecytoplasm to the periplasmic space ABC transporters areubiquitous multidomain integral membrane proteins thattranslocate a large variety of substrates across cellular mem-branes using ATP hydrolysis as a source of energy they aregenerally composed of a transmembrane (TM) domain and aconserved cytosolic nucleotide-binding domain [53]

CcmA is a cytoplasmic soluble protein representing thenucleotide-binding domain of the hypothetical ABC trans-porter according to this hypothesis its sequence containsa nucleotide-binding domain and Walker A and B motifsfor ATP hydrolysis [46] It has also been shown that CcmApossesses ATPase activity in vitro and that the protein isassociated with the membrane fraction only when CcmB isalso present [54]

CcmB and CcmC are both integral membrane proteinspredicted to contain six TM helices CcmC contains a shortWWDdomain in its second periplasmic domain and belongsto the heme handling protein family (HHP) [55] WWDdomains are short tryptophan-rich aminoacid stretcheswith the conserved WGX120601WXWDXRLT sequence (where 120601represents an aromatic amino acid residue and X representsany residue) [40 56] it has been proposed that proteinscontaining WWD domains are involved in heme-bindingand as we will see below a WWD domain is also presentin the CcmF protein another crucial heme-binding proteinof System I CcmC also contains two absolutely conservedhistidine residues in its first (between TM helices 1 and 2)and third (between TMhelices 5 and 6) periplasmic domainsAn attractive hypothesis still requiring experimental proofis that the hydrophobic residues within the tryptophan-richmotif provide a platform for the binding of heme whereasthe two conserved His residues (H60 and H184 in E coliCcmC) act as axial heme ligands [57] this hypothesis isstrengthened by the observation that CcmC indeed interactsdirectly with heme [58 59] Immunoprecipitation experi-ments have shown that in E coli CcmABC proteins forma multiprotein complex with a CcmA

2CcmB

1CcmC

1stoi-

chiometry confirming that these components form an ABC-type transporter complex with unusual functional propertiesassociated to the release of holoCcmE from CcmC [41 46]rather than to heme transport per se CcmC is an interestingprotein worth of future experimental efforts as it is knownthat in some pathogenic bacteria CcmC mutations are asso-ciated to specific phenotypes apparently not related to Cytc maturation such as siderophore production in Paracoccusand Pseudomonas [51] and iron utilization in Legionella [60]

Limited information is available about the structure andfunction of CcmD which appears to be a small membraneprotein (about 70 aminoacid residues) with no conservedsequence features whose topology is currently debatedcontrary to the original proposal [61] additional experimentshave shown that in E coli and R capsulatus CcmD isan integral membrane protein composed of a single TM

6 Scientifica

helix a periplasmic-orientedN-terminus and a cytoplasmic-oriented C-terminus [62] Immunoprecipitation experimentsindicate that CcmD interacts with the CcmA

2CcmB

1CcmC

1

complex even if it is not essential for heme transfer andattachment from CcmC to CcmE CcmD is strictly requiredfor the release of holoCcmE from the ABC transporter [6162]

CcmE is a heme-binding protein discovered as an essen-tial System I component as early as the late 1990rsquos [63]CcmE is a monotopic membrane protein anchored to themembrane via its N-terminal TM segment and exposing itsactive site to the periplasm it is the only Ccm componentof the heme trafficking and delivery module of System I forwhich a three-dimensional structure is available ([21] PDB1SR3 [64] PDB 1LM0) The 3D structure of the apo-state(without bound heme) consists of a six-stranded antiparallel120573-sheet reminiscent of the classical OB-fold [65] with N-and C-terminal extensions CcmE can be considered a ldquohemechaperonerdquo as it protects the cell from a potentially dangerouscompound by sequestering free heme in the periplasm [66]it is thought to act as an intermediate in the heme deliverypathway of Cytc maturation The structure of apoCcmEshowed no recognizable heme-binding cavities and in theabsence of a 3D structure of CcmEwith bound heme (holoC-cmE) the heme-binding region could only be predicted byin silico modeling It is generally believed that the hemein holoCcmE is solvent exposed but recent mutagenesisexperiments challenged this view [67] The unusual covalentbond between the nitrogen atomof a histidine residue presentin the conserved VLAKHDE motif located in a solventexposed environment (H130 in E coli CcmE) and a 120573-carbonof one of the heme vinyl groups has been described in greatdetail by NMR spectroscopy [68] Recently it was shown thatCcmE proteins from the proteobacteria D desulfuricans andD vulgaris contain the unusual CXXXY heme-bindingmotifwhere the Cys residue replaces the canonical His bindingresidue NMR solution structure of D vulgaris CcmE (PDB2KCT) revealed that the proteins adopt the same OB-foldcharacteristic of the CcmE superfamily Contrary to whatreported for theD desulfuricansCcmE [69] the homologousprotein from D vulgaris binds ferric heme noncovalentlythrough the conserved C127 residue [70] An additionalconserved residue in CcmE proteins is Tyr134 which wasshown to provide a coordination bond to the heme ironof holoCcmE [71 72] once it is released from CcmABCDcomplex [36] as discussed below

22 System I Heme Translocation and Delivery PathwayMechanisms We still do not know how the b heme istranslocated from the cytoplasm (where it is synthesized)to the periplasm where Cyt c maturation occurs Differ-ent mechanisms such as translocation through a proteinchannel or free diffusion across the membrane have beenproposed [73] The CcmAB proteins show structural featurestypical of the ABC transporters and for these reasons theywere originally hypothesized to be involved in the heme btranslocation process [57 63 74 75] However it is nowclear that an alternative process must exist since it has

been shown that periplasmic b-type cytochromes can beproduced in the absence of Ccm proteins [76] and thatinactivation of the ATPase activity of CcmA does not abolishheme accumulation in the periplasm [46 54] We have nowevidence that CcmC has the ability to bind heme at its WWDdomain present in the second periplasmic domain but itis still not clear if this membrane protein acts as a proteinchannel for heme translocation or simply collects it in theperiplasm [77]

Another important aspect concerns the oxidation state ofthe heme iron during translocation and delivery processesindeed this property of the heme iron may determine thereaction mechanism by which the unusual CcmE H130nitrogen is covalently linked to the vinyl 120573-carbon of theheme (see [36] for a detailed discussion of this topic) Basedon mutagenesis studies on CcmC [59] a model has beenpresented whereby oxidized heme is bound to CcmC onlyin the presence of CcmE forming a ternary complex BothCcmC and CcmE provide critical residues for heme-bindingthe two conserved His residues (H60 and H180 coordinatingthe heme iron) and theWWDdomain ofCcmCandHis130 ofCcmE forming the unusual covalent bond with heme vinyl-2 [59] The ATPase activity of CcmA is then required torelease holoCcmE from the CcmABCD complex a processthat depends also on the presence of CcmD [46 54] It shouldbe noticed that purified holoCcmE alone or in the CcmCDEcomplex [36 59] contains the heme iron in the oxidized statean observation that is apparently in contrast with the fact thatthe heme must be in its reduced state before attachment toapoCyt can occur Although the oxidation state of the hemeiron is currently debated [36 78] it is possible that CcmFwhich was recently shown to contain a heme b cofactor mayact as specific heme oxidoreductase (see Section 25)

23 System I ApoCyt Thioreduction Pathway ComponentsThe periplasm can be considered a relatively oxidizing envi-ronment due to the presence of an efficient oxidative systemcomposed of the DsbAB proteins [79 80] DsbA is a highlyoxidizing protein (1198641015840

0= 120mV) that is responsible for

the introduction of disulfide bonds into extracytoplasmicproteins [81] On the basis of the results obtained on E colidsbA deletion mutants that are unable to synthesize c-typecytochromes [82 83] it was generally accepted that formationof the intramolecular disulfide bond in apoCyt was a nec-essary step in the Cyt c biogenesis However only reducedapoCyt is clearly competent for heme ligation It is possiblethat this seemingly paradoxical thioreduction process hasevolved in order to protect the apoCyt from proteolyticdegradation aggregation andor formation of intermolec-ular disulfide bonds with thiols from other molecules (seealso Section 31 for a discussion about this aspect in SystemII) Recently however an analysis of c-type cytochromesproduction in several E coli dsb genes deletion strains ledto the hypothesis that DsbA is not necessary for Cyt cmaturation and that heme ligation to apoCyt and apoCytoxidation pathways is alternative competing processes [84]

In gram-negative bacteria a thioreduction pathway hasevolved to specifically reduce the oxidized apoCyt substrate

Scientifica 7

which includes the Ccm proteins CcmG and CcmH Thenecessary reducing power is transferred from the cytoplasmicthioredoxin (TRX) to CcmG via DsbD a large membraneprotein organized in three structural domains an N-terminalperiplasmic domain with a IgG-like fold (nDsbD) a C-terminal periplasmic domain with a thioredoxin-like (TRX-like) fold (cDsbD) and a central domain composed ofeight TM helices [85] Each of these domains contains apair of Cys residues and transfer electrons via a cascadeof disulfide exchange reactions making DsbD a ldquoredox-hubrdquo in the periplasm performing disulfide bond exchangereactions with different oxidized proteins [79] In particulara combination of X-ray crystallography experiments andkinetic analyses showed that electrons are transferred fromthe cytoplasmic TRX to the membrane domain of DsbDfollowed by reduction of cDsbD and finally of nDsbD whichis the direct electron donor to CcmG [85]

CcmG is a membrane-anchored protein linked to themembrane via an N-terminal TM helix and exposing itssoluble TRX-like domain in the periplasm The 3D structureof the TRX-like domain of CcmG from different bacteriahas been solved by X-ray crystallography (E coli PDB 1Z5Y[85] PDB 2B1K [86] B japonicum PDB 1KNG [87] Paeruginosa PDB 3KH7 3KH9 [22]) and is generally wellconserved as proved by the low RMSD (08 A between Pa-CcmG and Ec-CcmG 135 A between Pa-CcmG and Bj-CcmG) Although all these proteins adopt a TRX-like foldand contain the redox-active motif CXXC in the first 120572-helixthey are inactive in the classic insulin reduction assay [7588] CcmG proteins are therefore considered specific thiol-oxidoreductase able to recognize and selectively interactonly with their upstream and downstream binding partnersin the thioreduction process leading to reduced apoCytcLooking at the 3D structure of the periplasmic domain ofthe prototypical Pa-CcmG it is possible to identify the 120573120572120573and 120573120573120572 structural motifs of the TRX fold linked by a short120572-helix and forming a four-stranded 120573-sheet surrounded bythree helices the protein contains an additional N-terminalextension (residues 26ndash62) and a central insert (residues 102ndash123) The redox-active motif of Pa-CcmG (CPSC) is locatedin the first 120572-helix of the TRX fold as usually observed in allTRX-like proteins As for any molecular machinery whereeach component must recognize and interact with more thanone target (ie the substrate and the other components ofthe apparatus) an open question concerns the mechanismwhereby CcmG is able to recognize its different partnersThe availability of the crystal structures of Pa-CcmG bothin the oxidized (22 A resolution) and reduced state (18 Aresolution) [22] allowed highlighting the structural similaritybetween the two redox states (Rmsd of the C120572 atoms inthe two redox forms is 019 A) and therefore to excludestructural rearrangement as the mechanism used by Pa-CcmG to discriminate between reduced (such as the nDsbDdomain) and oxidized partners (Pa-CcmH andor apoCyt)

The standard redox potential of Pa-CcmG (11986410158400= 0213V

at pH 70 [22] as well as that of Ec-CcmG (11986410158400= 0212V

[86]) indicates that these proteins act as mild reductants inthe thioreductive pathway of Cytc biogenesis However the

Figure 5 Three-dimensional structure of Pa-CcmH shown inribbon representation The figure shows the three-helix bundleforming the characteristic fold of Pa-CcmHThe active site disulfidebond between residues Cys25 andCys28 in the long loop connectinghelices 120572-helix1 and 120572-helix 2 is highlighted in yellow

function of thiol-oxidoreductases obviously depends on thepKa values of their activesite Cys residues The pKa of CysX(613 plusmn 005) and CysY (105 plusmn 007) are consistent with thepKa values measured in different TRXs where the active N-terminal Cys residue has a pKa close to pH 70 whereas theC-terminal Cys has a much higher pKa [89 90] Such a largedifference between the two pKa values in the TRX family isfunctionally relevant because it allows the N-terminal Cysto perform the nucleophilic attack on the target disulfidewhile the C-terminal Cys is involved in the resolution of theresulting mixed-disulfide [90]

CcmH is the other component of System I involvedin the reduction of apoCyt Notably CcmH proteins fromdifferent bacterial subgroups may display structural vari-ability indeed while in E coli Ec-CcmH is a bipartiteprotein characterized by two soluble domains exposed to theperiplasm and two TM segments CcmH from P aeruginosa(Pa-CcmH) is a one-domain redox-active protein anchoredto the membrane via a single TM helix and homologous tothe N-terminal redox-active domain of Ec-CcmH Surpris-ingly the 3D structure of the soluble periplasmic domainof Pa-CcmH revealed that it adopts a peculiar three-helixbundle fold strikingly different from that of canonical thiol-oxidoreductases (Figure 5 PDB 2HL7 [23])TheN-terminaldomain of Ec-CcmH was also shown to have the same 3Dstructure although helix-swapping and dimerization havebeen observed in this case (PDB 2KW0 [91 92]) Theconserved redox-active motif (LRCPKC) is located in theloop connecting helices 1 and 2 close to the activesite thecrystal structure reveals the presence of a small pocket on thesurface of Pa-CcmH surrounded by conserved hydrophobicand polar residues which could represent the recognition sitefor the heme-binding motif of apoCyt

Concerning the functional properties of this unusualthiol-oxidoreductase it is interesting to note that its standardredox potential (1198641015840

0= 0215V) [23] is similar to that ob-

tained for Pa-CcmG This observation stands against thelinear redox cascade hypothesis whereby CcmG reducesCcmH While in the canonical redox-active CXXC motif

8 Scientifica

S SSS

SH

SH

Scheme 1 Scheme 2

SH

SH

SHSHS

S

CcmG 7477

CcmG 7477

CcmH 2528

CcmH 2528

apoCytox

apoCytred

S

SHCcmG 74

77

SH

SH

HSHS

CcmG 7477

S

S

SS

3

1

2CcmG 7477

SH

SHCcmH 25

28SH

HSS S

CcmH 2528

SHS

CcmH2528

apoCytox

apoCytred

+

Figure 6 Alternative thioreduction pathways whichmay be operative in System I and hypothesized on the basis of structural and functionalcharacterization of the redox-active Ccm proteins from P aeruginosa [22 23 25] Scheme 1 is a linear redox cascade whereby CcmG is thedirect reductant of CcmH which reduces oxidized apoCyt Scheme 2 envisages a more complex scenario involving the formation of a mixed-disulfide complex between CcmH and apoCyt (Step 1) This complex is the substrate for the attack by reduced CcmG (Step 2) that liberatesreduced apoCyt The resulting disulfide bond between CcmH and CcmG is then resolved by the free Cys thiol of CcmG (probably Cys77 inPa-CcmG) Adapted from [25]

of the TRX family the N-terminal Cys is always solventexposed in CcmH proteins the arrangement of the two Cysresidues is reversed the N-terminal Cys residue is buriedwhereas the C-terminal Cys residue is solvent exposed Onthe basis of this observation it was suggested that differentfrom the canonical TRX redox mechanism CcmH proteinsperform the nucleophilic attack on the apoCyt disulfide viatheir C-terminal Cys residue [23] This mechanism which isin agreement with the mechanism proposed earlier for Ec-CcmH on the basis of mutational-complementation studies[93 94] is substantiated by the peculiar pKa values of theactive site Cys residues of Pa-CcmH which were found tobe similar for both cysteines (84 plusmn 01 and 86 plusmn 01 [23])Again this is different from what is generally observed in thecase of TRX proteins where the pKa value of the Cys residueperforming the initial nucleophilic attack is significantlylower than the pKa value of theCys residue responsible for theresolution of the intermediate mixed-disulfide It is temptingto speculate that the unusual pKa values of the Pa-CcmHactive site thiols may ensure the necessary specificity of thiscomponent of the Ccm apparatus toward the CXXCH motifof the apoCyt substrate

24 System I ApoCyt Thioreduction Pathway MechanismAlthough we know that CcmG and CcmH are the redox-active components of System I involved in the thioreductivepathway of Cyt c biogenesis not only an acceptedmechanismfor the reduction of apoCyt disulfide bond is still lackingbut also the absolute requirement of such a process is nowdebated [38 84] Focusing our attention on the reductionof the apoCyt internal disulfide at least two mechanismscan been hypothesized which involve either a linear redoxcascade of disulfide exchange reactions or a nonlinear redox

process involving transient formation of a mixed-disulfidecomplex as depicted in Figure 6 and Schemes 1 and 2respectively

Both the thiol-disulfide exchange mechanisms depictedin Figure 6 suggest that CcmH is the direct reductant ofthe apoCytc disulfide however even if immunoprecipitationexperiments failed to detect the formation of a mixed-disulfide complex between apoCyt and CcmH proteins [95]some in vitro evidence supporting the formation of sucha complex has been presented In particular it has beenshown that Rhodobacter capsulatus and Arabidopsis thalianaCcmH homologues (Rc-CcmH and At-CcmH) are able toreduce the CXXCH motif of an apoCyt-mimicking peptide[75 96] In the latter case yeast two-hybrid experimentscarried out on At-CcmH indeed revealed an interactionbetween the protein and a peptide mimicking the A thalianaCyt c sequence In the case of Pa-CcmH FRET kineticexperiments employing a Trp-containing fluorescent variantof the protein and a dansylated nonapeptide encompassingthe heme-binding motif of P aeruginosa cytochrome c551(dans-KGCVACHAI) [23] allowed to directly observe theformation of themixed-disulfide complex and tomeasure theoff-rate constant of the bound peptide The results of these invitro binding experiments allowed to calculate an equilibriumdissociation constant which combines an adequate affinity(low 120583M) with the need to release efficiently reduced apoCytto other component(s) of the System I maturase complex[23] More recently the results obtained by FRET bindingexperiments carried out with single Cys-containing mutantsof Pa-CcmH and Pa-CcmG [25] substantiated the hypothesisdepicted in Scheme 2 (Figure 6) Altogether these structuraland functional results suggest that the thioreduction pathwaymechanism leading to reduced apoCyt is better describedby Scheme 2 and that reducing equivalents might not be

Scientifica 9

transferred directly from CcmG to apoCyt as depicted inScheme 1 According to Scheme 2 reduced CcmH (a non-TRX-like thiol-oxidoreductase) specifically recognizes andreduces oxidized apoCyt via the formation of a mixed-disulfide complex which is subsequently resolved by CcmGThe resulting disulfide bond between CcmH and CcmG isthen resolved by the free Cys thiol of CcmG (probably Cys77in Pa-CcmG)

However further in vitro experiments with CcmH andapoCyt single Cys-containing mutants are needed to unveilthe details of the thioreduction of oxidized apoCyt by CcmHIn particular it would be crucial to identify the Cys residueof apoCyt that remains free in the apoCyt-CcmH mixed-disulfide complex intermediate (see Scheme 2 and Section 26below) and available to thioether bond formation with oneof the heme vinyl groups Clearly structure determinationof the trapped mixed-disulfide complexes between CcmHCcmG and apoCyt (or apoCyt peptides) would providekey information for our understanding of this specializedthioreduction pathway mechanism

25 System I ApoCyt Chaperoning and Heme AttachmentComponents The reduced heme-binding motif of apoCyt isnow available to the heme ligation reaction However themolecularmechanismwhereby the Ccmmachinery catalyzesor promotes the formation of the heme-apoCyt covalentbonds is still largely obscure representing themost importantgoal in the field Past observations and recent experimentssuggest that CcmF and CcmI possibly together with CcmHare involved in these final steps [16 34 36]

CcmF is a large integral membrane protein of more than600 residues belonging to the heme handling protein family(HHP [55]) and predicted to contain 10ndash15 TM helices (notethat some discrepancy exists as to the number of TM helicespredicted by computer programs and those predicted onthe basis of phoA and lacZ fusion experiments [40 97])a conserved WWD domain and a larger domain devoidof any recognizable sequence features both exposed to theperiplasm Only recently E coli CcmF (Ec-CcmF) has beenoverexpressed solubilized from the membrane fraction andspectroscopically characterized in vitro [36 41] Surprisinglythe biochemical characterization of recombinant Ec-CcmFallowed to show that the purified protein contains heme b ascofactor in a 1 1 stoichiometry this observation led to thehypothesis that in addition to its heme lyase function Ec-CcmF may act as a heme oxidoreductase In particular it ispossible that the heme b of Ec-CcmF may act as a reductantfor the oxidized iron of the heme bound to CcmE [41]indeed the in vitro reduction of Ec-CcmF by quinones hasbeen experimentally observed strengthening the hypothesisabout the quinolhemeoxidoreductase function of this elusiveproteinThe structuralmodel proposed for Ec-CcmFpredicts13 TMhelices and notably the location of the four completelyconserved His residues according to the model two of them(His173 andHis303) are located in periplasmic exposed loopsnext to the conserved WWD domain which is believedto provide a platform for the heme bound to holoCcmEwhile His261 is located in one of the TM helices and it is

predicted to act as an axial ligand to the heme b of Ec-CcmFthe other conserved His residue (H491) could provide thesecond axial coordination bond to the heme although thishas not been experimentally addressed This model of Ec-CcmF therefore envisages that this large membrane proteinis characterized by two heme-binding sites one of themis embedded in the membrane and coordinates a heme bprosthetic group necessary to reduce the CcmE-bound hemehosted in the second heme-binding site and constituted by itsWWD domain

It is interesting to note that in plants mitochondria theCcmF ortholog appears to be split into three different pro-teins (At-CcmFN1 At-CcmFN2 and At-CcmFC) possiblyinteracting each other [16] Since each of these proteins issimilar to the corresponding domain in the bacterial CcmFortholog this observation may provide useful informationin the design of engineered fragments of bacterial CcmFproteins amenable to structural analyses

The other System I component which is generallybelieved to be involved in the final steps of Cyt c maturationis CcmI As stated above the ccmI gene is present only insome Ccm operons while in others the corresponding ORFis present within the ccmH gene (as in E coli)The functionalrole of CcmI in Cytc biogenesis is revealed by geneticstudies showing that in R capsulatus and B japonicuminactivation of the ccmI gene leads to inability to synthesizefunctional c-type cytochromes [98 99] In R capsulatus andP aeruginosa the CcmI protein (Rc-CcmI and Pa-CcmIresp) can be described as being composed of two domainsstarting from the N-terminus a first domain composed oftwo TM helices connected by a short cytoplasmic regionand a large periplasmic domain Structural variations maybe observed among CcmI members from different bacteriaindeed multiple sequence alignment indicates that the cyto-plasmic region of Rc-CcmI contains a leucine zipper motifwhich is not present in the putative cytoplasmic region of Pa-CcmI [100ndash102] Surprisingly no crystallographic structureis available up to now for the soluble domain of any CcmIprotein with the exception of the ortholog protein NrfGfrom E coli (Ec-NrfG) [103] This protein is necessary toattach the heme to the unusual heme-binding motif CWSCK(where a Lys residue substitutes the conservedHis) present inNrfA a pentaheme c-type cytochrome [103 104] Accordingto secondary structure prediction methods [105] it hasbeen proposed that the periplasmic domain of Pa-CcmI iscomposed of aN-terminal120572-helical region containing at leastthree TPR motifs connected by a disordered linker to a 120572-120573C-terminal regionMultiple sequence analyses and secondarystructure predictionmethods show that the TPR region of Pa-CcmI can be successfully aligned with many TPR-containingproteins including Ec-NrfG [106]

26 System I ApoCyt Chaperoning and Heme AttachmentMechanisms TPR domain-containing proteins are commonto eukaryotes prokaryotes and archaea these proteins aregenerally involved in the assembly of multiprotein complexesand to the chaperoning of unfolded proteins [103 107] Itis therefore plausible that CcmI (or the TPR C-terminal

10 Scientifica

domain of Ec-CcmH) may act to provide a platform for theunfolded apoCyt chaperoning it to the heme attachment sitepresumably located on the WWD domain of CcmF CcmImay thus be considered a component of amembrane-integralmultisubunit heme ligation complex together with CcmFand CcmH as experimentally observed by affinity purifi-cation experiments carried out with Rc-CcmFHI proteins[97 99 108] According to the proposed function of CcmI acritical requirement is represented by its ability to recognizedifferent protein targets over and above apoCyt such asCcmFandCcmHHowever until now direct evidence has been pre-sented only for the interaction of CcmI with apoCyt but thepossibility remains that CcmFHI proteins interact each othervia their TM helices and not via their periplasmic domainsInterestingly both for Pa-CcmI [106] or Rc-CcmI [99] CDspectroscopy experiments carried out on the CcmIapoCytcomplex highlighted major conformational changes at thesecondary structure level It is tempting to speculate on thebasis of these results that in vivo the folding of apoCyt maybe induced by the interaction with CcmI In the case of Paeruginosa System I proteins the binding process betweenPa-CcmI and its target protein apoCyt c551 (Pa-apoCyt) hasbeen studied both at equilibrium and kinetically [106] the119870119863measured for this interaction (in the 120583M range) appeared

to be low enough to ensure apoCyt delivery to the othercomponents of the Ccmmachinery Clearly a major questionconcerns the molecular determinants of such recognitionprocess interestingly both affinity coprecipitation assays[99] and equilibrium and kinetic binding experiments [106]highlighted the role played by the C-terminal 120572-helix of Cytc Similar observations have been made for the interaction ofEc-NrfGwith a peptidemimickingNrfA its apoCyt substrate[103] in this case isothermal titration calorimetry (ITC)experiments indicate that the TPR-domain of NrfG serves asa binding site for the C-terminal motif of NrfA Altogetherthese observations are in agreement with the fact that TPRproteins generally bind to their targets by recognizing theirC-terminal region [107]

The CcmI chaperoning activity has been experimentallysupported for the first time in the case of Pa-CcmI by citratesynthase tests [106] it has been proposed that the observedability to suppress protein aggregation in vitromay reflect thecapacity of CcmI to avoid apoCyt aggregation in vivo Stillanother piece of the Cyt c biogenesis puzzle has been addedrecently by showing that Rc-CcmI is able to interact withapoCcmE either alone or together with its substrate apoCytc2 forming a stable ternary complex in the absence of heme[109] This unexpected observation obtained by reciprocalcopurification experiments provides supporting evidence forthe existence of a large multisubunit complex composed ofCcmFHI andCcmE possibly interactingwith theCcmABCDcomplex It is interesting to note that while in the case ofthe CcmIapoCyt recognition different studies highlightedthe crucial role of the C-terminal helical region of apoCyt(see above) in the case of the apoCcmE apoCyt recognitionthe N-terminal region of apoCyt seems to represent a criticalregion

It is generally accepted that CcmF is the Ccm compo-nent responsible for heme covalent attachment to apoCyt

however as discussed above it is possible that this largemembrane protein plays such a role only together withother Ccm proteins such as CcmH and CcmI Moreover asrecently discovered by Kranz and coworkers [36 41] CcmFmay also act as a quinoleheme oxidoreductase ensuringthe necessary reduction of the oxidized heme b bound toCcmE Why it is necessary that the heme iron be in itsreduced state rather than in its oxidized state is not completelyclear although it is possible that this is a prerequisite to themechanism of thioether bond formation [110] According tocurrent hypotheses it is likely that the periplasmic WWDdomain of CcmF provides a platform for heme b bindingSanders et al and Verissimo et al [34 109] have presenteda mechanistic view of the heme attachment process whichtakes into account all the available experimental observationson the different Ccmproteins According to thismodel stere-ospecific heme ligation to reduced apoCyt occurs becauseonly the vinyl-4 group is available to form the first thioetherbond with a free cysteine at the apoCyt heme-binding motifsince the vinyl-2 group is involved (at least in the Ec-CcmE)in the covalent bond with His130 of CcmE [67] Howeverexperimental proof for this hypothesis requires a detailedinvestigation of the apoCyt thioreduction process catalyzedby CcmH (see Section 24)

It should be noticed that the mechanisms described sofar for the function(s) played by CcmF (see [34 36 109]) donot envisage a clear role for its large C-terminal periplasmicdomain (residues 510 to 611 in Ec-CcmF) It would be inter-esting to see if this domain apparently devoid of recognizablesequence features may mediate intermolecular recognitionprocesses with one (or more) component(s) involved in thehemeapoCyt ligation process

3 System II

System II is typically found in gram-positive bacteria and inin 120576-proteobacteria it is also present in most 120573- and some120575-proteobacteria in Aquificales and cyanobacteria as wellas in algal and plant chloroplasts System II is composedof three or four membrane-bound proteins CcdA ResACcsA (also known as ResC) and CcsB (also known as ResB)(Figure 3) CcdA andResA are redox-active proteins involvedin the reduction of the disulfide bond in the heme-bindingmotif of apoCyt whereas CcsA and CcsB are responsible forthe heme-apoCyt ligation process and are considered Cyt csynthethases (CCS) BothCcsAwhich is evolutionary relatedto the CcmC and CcmF proteins of System I [55] and CcsBare integral membrane proteins In some 120576-proteobacteriasuch asHelicobacter hepaticus andHelicobacter pylori a singlefusion protein composed of CcsA and CcsB polypeptides ispresent [41 111] Although as discussed below evidence hasbeen put forward to support the hypothesis that the CcsBAcomplex acts a heme translocase we still do not know if theheme is transported across the membrane by component(s)of System II itself or by a different unidentified process

31 System II ApoCyt Thioreduction Pathway After the Secmachinery secretes the newly synthesized apoCyt it readily

Scientifica 11

becomes a substrate for the oxidative system present on theouter surface of the cytoplasmic membrane of the gram-positive bacteria In B subtilis the BdbCD system [112113] is functionally but not structurally equivalent to thewell-characterized DsbAB system present in gram-negativebacteria As discussed above for System I the disulfide ofthe apoCyt heme-binding motif must be reduced in orderto allow thioether bonds formation and heme attachmentResA is the extracytoplasmic membrane-anchored TRX-likeprotein involved in the specific reduction of the apoCytdisulfide bond after the disulfide bond exchange reactionhas occurred oxidized ResA is reduced by CcdA whichreceives its reducing equivalents from a cytoplasmic TRX[114] Although ResA displays a classical TRX-like foldthe analysis of the 3D structure of oxidized and reducedResA from B subtilis (Bs-ResA) showed redox-dependentconformational modifications not observed in other TRX-like proteins Interestingly such modifications occur at thelevel of a cavity proposed to represent the binding site foroxidized apoCyt [24 115 116] Another peculiar feature of Bs-ResA is represented by the unusually similar pKa values of itsthe active site Cys residues (88 and 82 resp) as observedalso in the case of the active site Cys residues of the SystemI Pa-CcmH (see Section 23) However at variance with Pa-CcmH in Bs-ResA the large separation between the twocysteine thiols observed in the structure of the reduced formof the protein can be invoked to account for this result

CcdA is a large membrane protein containing six TMhelices [117 118] homologous to the TM domain of E coliDsbD [85 119] Its role in the Cyt c biogenesis is supportedby the observation that inactivation of CcdA blocks theproduction of c-type cytochromes in B subtilis [120 121]Moreover over and above the reduction of its apoCyt sub-strate Bs-CcdA is able to reduce the disulfide bond of othersecreted proteins such as StoA [122] It is interesting to notethat ResA and CcdA are not essential for Cyt c synthesisin the absence of BdbD or BdbC or if a disulfide reductantis present in the growth medium [123 124] As discussedabove (Section 24) a similar observation was made on therole of the thio-reductive pathway of System I in this casepersistent production of c-type cytochromes was observedin Dsb-inactivated bacterial strains [38] Following theseobservations the question has been put forward as to why theCyt c biogenesis process involves this apparently redundantthio-reduction route only to correct the effects of Bdb orDsb activity Referring to the case of B subtilis [111] it hasbeen proposed that the main reason is that BdbD mustefficiently oxidize newly secreted proteins since the activityof most of them depends on the presence of disulfide bondsAlternatively it can be hypothesized that the intramoleculardisulfide bondof apoCyt protects the protein fromproteolyticdegradation or aggregation or from cross-linking to otherthiol-containing proteins

32 System II Heme Translocation and Attachment to ApoCytRecent experimental evidence is accumulating supportingthe involvement of the heterodimeric membrane complexResBC or of the fusion protein CcsBA in the translocation

of heme from the n-side to the p-side of the bacterialmembrane In particular it has been shown that CcsBAfrom H hepaticus (Hh-CcsBA) is able to reconstitute Cyt cbiogenesis in the periplasm of E coli [36] Moreover it wasalso observed that Hh-CcsBA is able to bind reduced hemevia two conserved histidines flanking the WWD domainand required both for the translocation of the heme andfor the synthetase function of Hh-CcsBA [36] A differentstudy carried out on B subtilis System II proteins providedsupport for heme-binding capability by the ResBC complexaccording to the results obtained on recombinant ResBC theResB component of the heterodimeric CCS complex is ableto covalently bind the heme in the cytoplasm (probably by aCys residue) and to deliver it to an extracytoplamic domain ofResC which is responsible for the covalent ligation to apoCyt[117] It should be noticed however that the transfer of theCCS-bound heme to apoCyt still awaits direct experimentalproof Moreover it has also been reported that in B subtilisthe inactivation of CCS does not affect the presence ofother heme-containing proteins in the periplasm such ascytochrome b562 [111] this observation which is in contrastto the heme-transport hypothesis by CCSs parallels similarconcerns about heme translocation mechanism that arecurrently discussed in the context of System I (see Section 22above) In the case of the Hh-CcsBA synthetase site-directedmutagenesis experiments allowed to assign a heme-bindingrole for the two pairs of conserved His residues accordingto the topological model of the protein obtained by alkalinephosphatase (PhoA) assays and GFP fusions the conservedHis77 and His858 residues are located on TM helices whileHis761 and His897 are located on the periplasmic side [36]These observations together with the results of site-directedmutagenesis experiments allowed the authors to suggest thatHis77 andHis858 form a low affinitymdashmembrane embeddedheme-binding site for ferrous heme which is subsequentlytranslocated to an external heme-binding domain of Hh-CcsBA where it is coordinated by His761 and His897 Thetopological model therefore predicts that this last His pairis part of a periplasmic-located WWD domain (homologousto theWWDdomains found in theCcmCandCcmFproteinsof System I described above)

4 System III

Strikingly different from Systems I and II the Cyt c mat-uration apparatus found in fungi and in metazoan cells(System III) is composed of a single protein known as Holo-Cytochrome c Synthetase (HCCS) or Cytochrome c HemeLyase (CCHL) apparently responsible for all the subprocessesdescribed above (heme transport and chaperoning reductionand chaperoning of apoCyt and catalysis of the thioetherbonds formation between heme and apoCyt) (Figure 4)Surprisingly although this protein has been identified in Scerevisiaemitochondria several years ago [125 126] only veryrecently the human HCCS has been expressed as a recombi-nant protein in E coli and spectroscopically characterized invitro [127] It is worth noticing that humanHCCS is attractinginterest since over and above its role in Cyt c biogenesis it

12 Scientifica

is involved in diseases such as microphthalmia with linearskin defects syndrome (MLS) an X-linked genetic disorder[128ndash130] and in other processes such as Cytc-independentapoptosis in injured motor neurons [131] Different fromanimal cells where a single HCCS is sufficient for thematuration of both soluble and membrane-anchored Cytc(Cyt c and Cy c1 resp) in S cerevisiae two homologs HCCSand HCC1S located in the inner mitochondrial membraneand facing to the IMS space [132 133] are responsible for hemeattachment to apoCyt c and apoCyt c1 respectively [125 134]It should also be noticed that in fungi an additional FAD-containing protein (Cyc2p) is required for Cyt c synthesisCyc2p is a mitochondrial membrane-anchored flavoproteinexposing its redox domain to the IMS which is requiredfor the maturation of Cyt c but not for that of Cyt c1 [135]This protein does not contain the conserved Cys residuestypically found in disulfide reductases and indeed it is notable to reduce oxidized apoCyt in vitro However it has beenrecently shown that Cyc2p is able to catalyze the NAD(P)H-dependent reduction of heme in vitro [136] a necessarystep before the thioether bond formation can occur asdiscussed above (see Section 26) This result together withthe observation that Cyc2p interacts with HCCS and withapoCyt c and c1 lends support to the proposal that Cyc2p isinvolved in the reduction of the heme iron in vivo [136 137]

AlthoughHCCS proteins are crucial for Cyt cmaturationwe still do not know how these proteins recognize theirsubstrates (heme and apoCyt) and how they promote orcatalyze the formation of thioether bonds between the hemevinyl groups and the cysteine thiols of the apoCyt CXXCHmotif

Contrary to the broad specificity of System I whichis able to recognize and attach the heme to prokaryoticand eukaryotic c-type cytochromes [49 50] and even tovery short microperoxidase-like sequences [49 50] mito-chondrial HCCS is characterized by a higher specificityas it does not recognize bacterial apoCyt sequences Theseobservations prompted the investigation of the recognitionprocess between apoCyt and HCCS Recently by using arecombinant yeast HCCS and chimerical apoCyt sequencesexpressed in E coli it was possible to conclude that a crucialrecognition determinant is represented by the N-terminalregion of apoCyt containing the heme-binding motif [35]notice that in the context of System I the same N-terminalregion of the Cyt c sequence has been recently identified to beimportant for the recognition by apoCcmE (see Section 26)Over and above the role played by the intervening residuesin the CXXCH heme-binding motif [135] it has been shownthat a conserved Phe residue occurring in the N-terminalregion before the CXXCH motif is important for HCCSrecognition [35 138] These results support the hypothesisthat this residue known to be a key determinant of Cytcfolding and stability [19 20 139] may also be crucial for Cytcmaturation by HCCS

Another relevant question concerns the ability of HCCSto recognize and bind the heme molecule as the region ofthe protein responsible for heme recognition remains to beidentified Initially it was hypothesized that the recognitionof heme could be mediated by the CP motifs present in

the HCCS protein [140] these short sequences are indeedknown to bind heme in a variety of heme-containing proteins[141 142] However it has been recently shown that CPmotifsof the recombinant S cerevisiae HCCS are not necessaryfor Cyt c production in E coli [143] excluding them as keydeterminants of heme recognition

The long-awaited in vitro characterization of the recom-binant human HCCS allowed for the first time to pro-pose a molecular mechanism underlying Cytc maturationin eukaryotes which can be experimentally tested [127]According to the proposed model the human HCCS activitycan be described as a four step mechanism involving (i)heme-binding (ii) apoCyt recognition (iii) thioether bondsformation and (iv) holoCyt c release In particular theheme is proposed to play the role of a scaffolding moleculemediating the contacts between HCCS and apoCyt Muta-genesis experiments carried out on the recombinant HCCSstrongly suggest that heme-binding (Step 1) depends on thepresence of a specific His residue (His154) acting as anaxial ligand to ferrous heme Residues present at the N-terminus of apoCyt mediate the recognition with HCCS(Step 2) as discussed above it is known that this regionforms structurally conserved 120572-helix in the fold of all c-type cytochromes Unfortunately no information is availableup to now concerning the region of HCCS involved in therecognition and binding to the N-terminal region of apoCytCoordination of the heme iron by the His residue of theapoCyt heme-binding motif CXXCH provides the secondaxial ligand to the heme iron and is probably importantfor the correct positioning of the two apoCyt Cys residuesand formation of the thioether bonds (Step 3) The finalrelease of functional Cyt c (Step 4) clearly requires thedisplacement of the His154Fe2+ coordination bond sucha displacement is probably mediated by formation of thecoordination bond with the Cyt c conserved Met residueandor by the simultaneous folding of Cyt c Again it isinteresting to note that this last hypothesis is in accordancewith in vitro folding studies on c-type cytochromes thathighlighted the late formation of the Met-Fe coordinationduring Cyt c folding [144 145]

Acknowledgments

The author thanks A Di Matteo and E Di Silvio for fruitfuldiscussions and for help in preparing this paper This work issupported by grants from the University of Rome ldquoSapienzardquo(C26A11MBXJ and C26A13MWF4)

References

[1] M G Galinato J G Kleingardner S E Bowman et alldquoHeme-protein vibrational couplings in cytochrome c providea dynamic link that connects the heme-iron and the proteinsurfacerdquo Proceedings of the National Academy of Sciences vol109 no 23 pp 8896ndash8900 2012

[2] H B Gray and J R Winkler ldquoElectron flow through metal-loproteinsrdquo Biochimica et Biophysica Acta vol 1797 no 9 pp1563ndash1572 2010

Scientifica 13

[3] P Ascenzi R Santucci M Coletta and F PolticellildquoCytochromes reactivity of the ldquodark siderdquo of the hemerdquoBiophysical Chemistry vol 152 no 1ndash3 pp 21ndash27 2010

[4] S Yamada N D Bouley Ford G E Keller W C Ford HB Gray and J R Winkler ldquoSnapshots of a protein foldingintermediaterdquo Proceedings of the National Academy of Sciencesvol 110 no 5 pp 1606ndash1610 2013

[5] P Weinkam J Zimmermann F E Romesberg and P GWolynes ldquoThe folding energy landscape and free energy excita-tions of cytochrome crdquo Accounts of Chemical Research vol 43no 5 pp 652ndash660 2010

[6] M Yamanaka M Masanari and Y Sambongi ldquoConfermentof folding ability to a naturally unfolded apocytochrome cthrough introduction of hydrophobic amino acid residuesrdquoBiochemistry vol 50 no 12 pp 2313ndash2320 2011

[7] G W Moore and G W Pettigrew Cytochrome C EvolutionaryStructural and Physicochemical Aspects Springer Berlin Ger-many 1990

[8] I Bertini G Cavallaro and A Rosato ldquoCytochrome c occur-rence and functionsrdquo Chemical Reviews vol 106 no 1 pp 90ndash115 2006

[9] D A Pearce and F Sherman ldquoDegradation of cytochromeoxidase subunits in mutants of yeast lacking cytochrome c andsuppression of the degradation by mutation of yme1rdquo Journal ofBiological Chemistry vol 270 no 36 pp 20879ndash20882 1995

[10] G Silkstone S M Kapetanaki I Husu M H Vos and MT Wilson ldquoNitric oxide binding to the cardiolipin complex offerric cytochrome crdquo Biochemistry vol 51 no 34 pp 6760ndash6766 2012

[11] M Giorgio E Migliaccio F Orsini et al ldquoElectron transferbetween cytochrome c and p66Shc generates reactive oxygenspecies that trigger mitochondrial apoptosisrdquo Cell vol 122 no2 pp 221ndash233 2005

[12] S M Kilbride and J H Prehn ldquoCentral roles of apoptoticproteins in mitochondrial functionrdquo Oncogene vol 32 no 22pp 2703ndash2711 2013

[13] T Noll and G Noll ldquoStrategies for ldquowiringrdquo redox-activeproteins to electrodes and applications in biosensors biofuelcells and nanotechnologyrdquo Chemical Society Reviews vol 40no 7 pp 3564ndash3576 2011

[14] G Layer J Reichelt D Jahn and D W Heinz ldquoStructure andfunction of enzymes in heme biosynthesisrdquo Protein Science vol19 no 6 pp 1137ndash1161 2010

[15] S Severance and IHamza ldquoTrafficking of heme and porphyrinsin metazoardquo Chemical Reviews vol 109 no 10 pp 4596ndash46162009

[16] P Hamel V Corvest P Giege and G Bonnard ldquoBiochemi-cal requirements for the maturation of mitochondrial c-typecytochromesrdquo Biochimica et Biophysica Acta vol 1793 no 1 pp125ndash138 2009

[17] W R Fisher H Taniuchi and C B Anfinsen ldquoOn the role ofheme in the formation of the structure of cytochrome crdquo Journalof Biological Chemistry vol 248 no 9 pp 3188ndash3195 1973

[18] M Yamanaka H Mita Y Yamamoto and Y Sambongi ldquoHemeis not required for aquifex aeolicus cytochrome c555 polypep-tide foldingrdquoBioscience Biotechnology andBiochemistry vol 73no 9 pp 2022ndash2025 2009

[19] C Travaglini-Allocatelli S Gianni andMBrunori ldquoA commonfolding mechanism in the cytochrome c familyrdquo Trends inBiochemical Sciences vol 29 no 10 pp 535ndash541 2004

[20] A Borgia D Bonivento C Travaglini-Allocatelli A DiMatteoand M Brunori ldquoUnveiling a hidden folding intermediate in c-type cytochromes by protein engineeringrdquo Journal of BiologicalChemistry vol 281 no 14 pp 9331ndash9336 2006

[21] E Enggist L Thony-Meyer P Guntert and K PervushinldquoNMR structure of the heme chaperone CcmE reveals a novelfunctional motifrdquo Structure vol 10 no 11 pp 1551ndash1557 2002

[22] A di Matteo N Calosci S Gianni P Jemth M Brunori and CTravaglini-Allocatelli ldquoStructural and functional characteriza-tion of CcmG from pseudomonas aeruginosa a key componentof the bacterial cytochrome c maturation apparatusrdquo Proteinsvol 78 no 10 pp 2213ndash2221 2010

[23] A diMatteo S GianniM E Schinina et al ldquoA strategic proteinin cytochrome c maturation three-dimensional structure ofCcmH and binding to apocytochrome crdquo Journal of BiologicalChemistry vol 282 no 37 pp 27012ndash27019 2007

[24] A Crow R M Acheson N E Le Brun and A OubrieldquoStructural basis of redox-coupled protein substrate selectionby the cytochrome c biosynthesis protein ResArdquo Journal ofBiological Chemistry vol 279 no 22 pp 23654ndash23660 2004

[25] E di Silvio A di Matteo and C Travaglini-Allocatelli ldquoTheRedox pathway of Pseudomonas aeruginosa cytochrome cbiogenesisrdquo Journal of Proteins and Proteomics vol 3 no 1 pp1ndash7 2012

[26] LThony-Meyer andP Kunzler ldquoTranslocation to the periplasmand signal sequence cleavage of preapocytochrome c depend onsec and lep but not on the ccmgene productsrdquoEuropean Journalof Biochemistry vol 246 no 3 pp 794ndash799 1997

[27] S J Facey and A Kuhn ldquoBiogenesis of bacterial inner-membrane proteinsrdquo Cellular and Molecular Life Sciences vol67 no 14 pp 2343ndash2362 2010

[28] K Diekert A I de Kroon U Ahting et al ldquoApocytochromec requires the TOM complex for translocation across themitochondrial outer membranerdquo The EMBO Journal vol 20no 20 pp 5626ndash5635 2001

[29] N Wiedemann V Kozjak T Prinz et al ldquoBiogenesis of yeastmitochondrial cytochrome c a unique relationship to the TOMmachineryrdquo Journal ofMolecular Biology vol 327 no 2 pp 465ndash474 2003

[30] F-U Hartl N Pfanner and W Neupert ldquoTranslocation inter-mediates on the import pathway of proteins intomitochondriardquoBiochemical Society Transactions vol 15 no 1 pp 95ndash97 1987

[31] B S Glick A Brandt K Cunningham SMuller R L Hallbergand G Schatz ldquoCytochromes c1 and b2 are sorted to theintermembrane space of yeast mitochondria by a stop-transfermechanismrdquo Cell vol 69 no 5 pp 809ndash822 1992

[32] G Howe and S Merchant ldquoRole of heme in the biosynthesis ofcytochrome c6rdquo Journal of Biological Chemistry vol 269 no 8pp 5824ndash5832 1994

[33] S S Nakamoto P Hamel and S Merchant ldquoAssembly ofchloroplast cytochromes b and crdquo Biochimie vol 82 no 6-7 pp603ndash614 2000

[34] C Sanders S Turkarslan D-W Lee and F DaldalldquoCytochrome c biogenesis the Ccm systemrdquo Trends inMicrobiology vol 18 no 6 pp 266ndash274 2010

[35] J M Stevens D A Mavridou R Hamer P Kritsiligkou A DGoddard and S J Ferguson ldquoCytochrome c biogenesis systemIrdquoThe FEBS Journal vol 278 no 22 pp 4170ndash4178 2011

[36] R G Kranz C Richard-Fogal J-S Taylor and E R FrawleyldquoCytochrome c biogenesis mechanisms for covalent modifica-tions and trafficking of heme and for heme-iron redox controlrdquo

14 Scientifica

Microbiology and Molecular Biology Reviews vol 73 no 3 pp510ndash528 2009

[37] J W A Allen ldquoCytochrome c biogenesis in mitochondriamdashsystems III and VrdquoThe FEBS Journal vol 278 no 22 pp 4198ndash4216 2011

[38] D A Mavridou S J Ferguson and J M Stevens ldquoCytochromec assemblyrdquo IUBMB Life vol 65 no 3 pp 209ndash216 2013

[39] I Bertini G Cavallaro and A Rosato ldquoEvolution ofmitochondrial-type cytochrome c domains and of theprotein machinery for their assemblyrdquo Journal of InorganicBiochemistry vol 101 no 11-12 pp 1798ndash1811 2007

[40] B S Goldman and R G Kranz ldquoEvolution and horizontaltransfer of an entire biosynthetic pathway for cytochromec biogenesis helicobacter deinococcus archae and morerdquoMolecular Microbiology vol 27 no 4 pp 871ndash873 1998

[41] C L Richard-Fogal E R Frawley E R Bonner H Zhu BSan Francisco and R G Kranz ldquoA conserved haem redoxand trafficking pathway for cofactor attachmentrdquo The EMBOJournal vol 28 no 16 pp 2349ndash2359 2009

[42] L Thony-Meyer F Fischer P Kunzler D Ritz and H Hen-necke ldquoEscherichia coli genes required for cytochrome cmaturationrdquo Journal of Bacteriology vol 177 no 15 pp 4321ndash4326 1995

[43] C S Beckett J A Loughman K A Karberg G M DonatoW E Goldman and R G Kranz ldquoFour genes are required forthe system II cytochrome c biogenesis pathway in Bordetellapertussis a unique bacterial modelrdquo Molecular Microbiologyvol 38 no 3 pp 465ndash481 2000

[44] N E Le Brun J Bengtsson and L Hederstedt ldquoGenes requiredfor cytochrome c synthesis in Bacillus subtilisrdquo MolecularMicrobiology vol 36 no 3 pp 638ndash650 2000

[45] P Giege J M Grienenberger and G Bonnard ldquoCytochromec biogenesis in mitochondriardquo Mitochondrion vol 8 no 1 pp61ndash73 2008

[46] R E Feissner C L Richard-Fogal E R Frawley J A Lough-man KW Earley and R G Kranz ldquoRecombinant cytochromesc biogenesis systems I and II and analysis of haem deliverypathways in Escherichia colirdquo Molecular Microbiology vol 60no 3 pp 563ndash577 2006

[47] N P Cianciotto P Cornelis and C Baysse ldquoImpact of thebacterial type I cytochrome c maturation system on differentbiological processesrdquoMolecular Microbiology vol 56 no 6 pp1408ndash1415 2005

[48] E Arslan H Schulz R Zufferey P Kunzler and L Thony-Meyer ldquoOverproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in Escherichiacolirdquo Biochemical and Biophysical Research Communicationsvol 251 no 3 pp 744ndash747 1998

[49] C Sanders and H Lill ldquoExpression of prokaryotic and eukary-otic cytochromes c in Escherichia colirdquoBiochimica et BiophysicaActa vol 1459 no 1 pp 131ndash138 2000

[50] M Braun and L Thony-Meyer ldquoBiosynthesis of artificialmicroperoxidases by exploiting the secretion and cytochromec maturation apparatuses of Escherichia colirdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 35 pp 12830ndash12835 2004

[51] B Baert C Baysse S Matthijs and P Cornelis ldquoMultiplephenotypic alterations caused by a c-type cytochrome mat-uration ccmC gene mutation in Pseudomonas aeruginosardquoMicrobiology vol 154 no 1 pp 127ndash138 2008

[52] S Turkarslan C Sanders and F Daldal ldquoExtracytoplasmicprosthetic group ligation to apoproteins maturation of c-typecytochromesrdquo Molecular Microbiology vol 60 no 3 pp 537ndash541 2006

[53] P M Jones and A M George ldquoThe ABC transporter structureand mechanism perspectives on recent researchrdquo Cellular andMolecular Life Sciences vol 61 no 6 pp 682ndash699 2004

[54] O Christensen E M Harvat L Thony-Meyer S J Fergusonand J M Stevens ldquoLoss of ATP hydrolysis activity by CcmABresults in loss of c-type cytochrome synthesis and incompleteprocessing ofCcmErdquoTheFEBS Journal vol 274 no 9 pp 2322ndash2332 2007

[55] J-H Lee E M Harvat J M Stevens S J Ferguson and M HSaier Jr ldquoEvolutionary origins of members of a superfamily ofintegralmembrane cytochrome c biogenesis proteinsrdquoBiochim-ica et Biophysica Acta vol 1768 no 9 pp 2164ndash2181 2007

[56] D L Beckman D R Trawick and R G Kranz ldquoBacterialcytochromes c biogenesisrdquo Genes and Development vol 6 no2 pp 268ndash283 1992

[57] H Schulz R A Fabianek E C Pellicioli H Hennecke andL Thony-Meyer ldquoHeme transfer to the heme chaperone CcmEduring cytochrome c maturation requires the CcmC proteinwhich may function independently of the ABC-transporterCcmABrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 96 no 11 pp 6462ndash6467 1999

[58] Q Ren and L Thony-Meyer ldquoPhysical Interaction ofCcmC with Heme and the Heme Chaperone CcmE duringCytochrome cMaturationrdquo Journal of Biological Chemistry vol276 no 35 pp 32591ndash32596 2001

[59] C Richard-Fogal and R G Kranz ldquoThe CcmChemeCcmEcomplex in heme trafficking and cytochrome c biosynthesisrdquoJournal of Molecular Biology vol 401 no 3 pp 350ndash362 2010

[60] V K Viswanathan S Kurtz L L Pedersen et al ldquoThecytochrome C maturation locus of Legionella pneumophilapromotes iron assimilation and intracellular infection andcontains a strain-specific insertion sequence elementrdquo Infectionand Immunity vol 70 no 4 pp 1842ndash1852 2002

[61] U Ahuja and L Thony-Meyer ldquoCcmD is involved in complexformation between CcmC and the heme chaperone CcmE dur-ing cytochrome c maturationrdquo Journal of Biological Chemistryvol 280 no 1 pp 236ndash243 2005

[62] C L Richard-Fogal E R Frawley and R G Kranz ldquoTopologyand function of CcmD in cytochrome Cmaturationrdquo Journal ofBacteriology vol 190 no 10 pp 3489ndash3493 2008

[63] H Schulz H Hennecke and L Thony-Meyer ldquoPrototype ofa heme chaperone essential for cytochrome c maturationrdquoScience vol 281 no 5380 pp 1197ndash1200 1998

[64] F Arnesano L Banci P D Barker et al ldquoSolution structure andcharacterization of the heme chaperone CcmErdquo Biochemistryvol 41 no 46 pp 13587ndash13594 2002

[65] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[66] L Thony-Meyer ldquoA heme chaperone for cytochrome c biosyn-thesisrdquo Biochemistry vol 42 no 45 pp 13099ndash13105 2003

[67] E M Harvat C Redfield J M Stevens and S J FergusonldquoProbing the heme-binding site of the cytochrome cmaturationprotein CcmErdquoBiochemistry vol 48 no 8 pp 1820ndash1828 2009

[68] D Lee K Pervushin D Bischof M Braun and L Thony-Meyer ldquoUnusual heme-histidine bond in the active site of a

Scientifica 15

chaperonerdquo Journal of the American Chemical Society vol 127no 11 pp 3716ndash3717 2005

[69] A D Goddard J M Stevens F Rao et al ldquoc-Type cytochromebiogenesis can occur via a natural Ccm system lacking CcmHCcmG and the heme-binding histidine of CcmErdquo Journal ofBiological Chemistry vol 285 no 30 pp 22882ndash22889 2010

[70] J M Aramini K Hamilton P Rossi et al ldquoSolution NMRstructure backbone dynamics and heme-binding propertiesof a novel cytochrome c maturation protein CcmE fromDesulfovibrio vulgarisrdquo Biochemistry vol 51 no 18 pp 3705ndash3707 2012

[71] T Uchida J M Stevens O Daltrop et al ldquoThe interactionof covalently bound heme with the cytochrome c maturationprotein CcmErdquo Journal of Biological Chemistry vol 279 no 50pp 51981ndash51988 2004

[72] I Garcıa-Rubio M Braun I Gromov L Thony-Meyer andA Schweiger ldquoAxial coordination of heme in ferric CcmEchaperone characterized by EPR spectroscopyrdquo BiophysicalJournal vol 92 no 4 pp 1361ndash1373 2007

[73] L Thony-Meyer ldquoHaem-polypeptide interactions duringcytochrome c maturationrdquo Biochimica et Biophysica Acta vol1459 no 2-3 pp 316ndash324 2000

[74] J A Keightley D Sanders T R Todaro A Pastuszyn and J AFee ldquoCloning expression in Escherichia coli of the cytochromec552 gene from Thermus thermophilus HB8 Evidence forgenetic linkage to an ATP- binding cassette protein and initialcharacterization of the cycA gene productsrdquo Journal of Biologi-cal Chemistry vol 273 no 20 pp 12006ndash12016 1998

[75] E M Monika B S Goldman D L Beckman and R GKranz ldquoA thioreduction pathway tethered to the membranefor periplasmic cytochromes c biogenesis In vitro and in vivostudiesrdquo Journal of Molecular Biology vol 271 no 5 pp 679ndash692 1997

[76] M Throne-Holst L Thony-Meyer and L HederstedtldquoEscherichia coli ccm in-frame deletion mutants can produceperiplasmic cytochrome b but not cytochrome crdquo FEBS Lettersvol 410 no 2-3 pp 351ndash355 1997

[77] U Ahuja and L Thony-Meyer ldquoDynamic features of a hemedelivery system for cytochrome c maturationrdquo Journal of Bio-logical Chemistry vol 278 no 52 pp 52061ndash52070 2003

[78] J W A Allen P D Barker O Daltrop et al ldquoWhy isnrsquot ldquostan-dardrdquo heme good enough for c-type and di-type cytochromesrdquoDalton Transactions no 21 pp 3410ndash3418 2005

[79] K Inaba and K Ito ldquoStructure and mechanisms of the DsbB-DsbA disulfide bond generation machinerdquo Biochimica et Bio-physica Acta vol 1783 no 4 pp 520ndash529 2008

[80] H Kadokura F Katzen and J Beckwith ldquoProtein disulfidebond formation in prokaryotesrdquoAnnual Review of Biochemistryvol 72 pp 111ndash135 2003

[81] S R Shouldice B Heras P M Walden M Totsika M ASchembri and J L Martin ldquoStructure and function of DsbA akey bacterial oxidative folding catalystrdquoAntioxidants and RedoxSignaling vol 14 no 9 pp 1729ndash1760 2011

[82] R Metheringham L Griffiths H Crooke S Forsythe and JCole ldquoAn essential role for DsbA in cytochrome c synthesis andformate-dependent nitrite reduction by Escherichia coli K-12rdquoArchives of Microbiology vol 164 no 4 pp 301ndash307 1995

[83] Y Sambongi and S J Ferguson ldquoMutants of Escherichia colilacking disulphide oxidoreductases DsbA and DsbB cannotsynthesise an exogenous monohaem c-type cytochrome exceptin the presence of disulphide compoundsrdquo FEBS Letters vol398 no 2-3 pp 265ndash268 1996

[84] D AMavridou S J Ferguson and JM Stevens ldquoThe interplaybetween the disulfide bond formation pathway and cytochromec maturation in Escherichia colirdquo FEBS Letters vol 586 no 12pp 1702ndash1707 2012

[85] C U Stirnimann A Rozhkova U Grauschopf M G GrutterR Glockshuber and G Capitani ldquoStructural basis and kineticsof DsbD-dependent cytochrome c maturationrdquo Structure vol13 no 7 pp 985ndash993 2005

[86] N Ouyang Y-G Gao H-Y Hu and Z-X Xia ldquoCrystalstructures of E coli CcmGand itsmutants reveal key roles of theN-terminal120573-sheet and the fingerprint regionrdquoProteins vol 65no 4 pp 1021ndash1031 2006

[87] M A Edeling L W Guddat R A Fabianek et al ldquoCrys-tallization and preliminary diffraction studies of native andselenomethionine CcmG (CycY DsbE)rdquoActa CrystallographicaD vol 57 no 9 pp 1293ndash1295 2001

[88] R A Fabianek M Huber-Wunderlich R Glockshuber PKunzler H Hennecke and L Thony-Meyer ldquoCharacterizationof the Bradyrhizobium japonicum CycY protein a membrane-anchored periplasmic thioredoxin that may play a role as areductant in the biogenesis of c-type cytochromesrdquo Journal ofBiological Chemistry vol 272 no 7 pp 4467ndash4473 1997

[89] G B Kallis and A Holmgren ldquoDifferential reactivity of thefunctional sulfhydryl groups of cysteine-32 and cysteine-32present in the reduced form of thioredoxin from Escherichiacolirdquo Journal of Biological Chemistry vol 255 no 21 pp 10261ndash10265 1980

[90] P T Chivers andR T Raines ldquoGeneral acidbase catalysis in theactive site of Escherichia coli thioredoxinrdquoBiochemistry vol 36no 50 pp 15810ndash15816 1997

[91] X M Zheng J Hong H Y Li D H Lin and H Y HuldquoBiochemical properties and catalytic domain structure of theCcmH protein from Escherichia colirdquo Biochim Biophys Actavol 1824 no 12 pp 1394ndash1400 2012

[92] UAhujaA Rozhkova RGlockshuber LThony-Meyer andOEinsle ldquoHelix swapping leads to dimerization of the N-terminaldomain of the c-type cytochrome maturation protein CcmHfrom Escherichia colirdquo FEBS Letters vol 582 no 18 pp 2779ndash2786 2008

[93] R A Fabianek T Hofer and L Thony-Meyer ldquoCharacteriza-tion of the Escherichia coli CcmH protein reveals new insightsinto the redox pathway required for cytochrome c maturationrdquoArchives of Microbiology vol 171 no 2 pp 92ndash100 1999

[94] E Reid J Cole and D J Eaves ldquoThe Escherichia coli CcmGprotein fulfils a specific role in cytochrome c assemblyrdquo Bio-chemical Journal vol 355 no 1 pp 51ndash58 2001

[95] Q Ren U Ahuja and LThony-Meyer ldquoA bacterial cytochromec heme lyase CcmF forms a complex with the heme chaperoneCcmE and CcmH but not with apocytochrome crdquo Journal ofBiological Chemistry vol 277 no 10 pp 7657ndash7663 2002

[96] E H Meyer P Giege E Gelhaye et al ldquoAtCCMH an essentialcomponent of the c-type cytochrome maturation pathway inArabidopsis mitochondria interacts with apocytochrome crdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 44 pp 16113ndash16118 2005

[97] C Rios-Velazquez R Coller and T J Donohue ldquoFeatures ofRhodobacter sphaeroides CcmFHrdquo Journal of Bacteriology vol185 no 2 pp 422ndash431 2003

[98] C Sanders M Deshmukh D Astor R G Kranz and F DaldalldquoOverproduction of CcmG and CcmFHRc fully suppresses thec-type cytochrome biogenesis defect of Rhodobacter capsulatus

16 Scientifica

CcmI-null mutantsrdquo Journal of Bacteriology vol 187 no 12 pp4245ndash4256 2005

[99] A F Verissimo H Yang X Wu C Sanders and F DaldalldquoCcmI subunit of CcmFHI heme ligation complex functionsas an apocytochrome c chaperone during c-type cytochromematurationrdquo Journal of Biological Chemistry vol 286 no 47 pp40452ndash40463 2011

[100] K Bryson L J McGuffin R L Marsden J J Ward J SSodhi and D T Jones ldquoProtein structure prediction servers atUniversity College Londonrdquo Nucleic Acids Research vol 33 no2 pp W36ndashW38 2005

[101] D T Jones ldquoProtein secondary structure prediction basedon position-specific scoring matricesrdquo Journal of MolecularBiology vol 292 no 2 pp 195ndash202 1999

[102] C Sanders C Boulay and F Daldal ldquoMembrane-spanning andperiplasmic segments of CcmI have distinct functions duringcytochrome c biogenesis in Rhodobacter capsulatusrdquo Journal ofBacteriology vol 189 no 3 pp 789ndash800 2007

[103] D Han K Kim J Oh J Park and Y Kim ldquoTPR domainof NrfG mediates complex formation between heme lyaseand formate-dependent nitrite reductase in Escherichia coliO157H7rdquo Proteins vol 70 no 3 pp 900ndash914 2008

[104] D J Eaves J Grove W Staudenmann et al ldquoInvolvement ofproducts of the nrfEFG genes in the covalent attachment ofhaem c to a novel cysteine-lysine motif in the cytochrome c552nitrite reductase from Escherichia colirdquo Molecular Microbiol-ogy vol 28 no 1 pp 205ndash216 1998

[105] J Nilsson B Persson and G Von Heijne ldquoConsensus predic-tions of membrane protein topologyrdquo FEBS Letters vol 486 no3 pp 267ndash269 2000

[106] E di Silvio A di Matteo F Malatesta and C Travaglini-Allocatelli ldquoRecognition and binding of apocytochrome c toP aeruginosa CcmI a component of cytochrome c maturationmachineryrdquo Biochim Biophys Acta vol 1834 no 8 pp 1554ndash1561 2013

[107] N Zeytuni and R Zarivach ldquoStructural and functional dis-cussion of the tetra-trico-peptide repeat a protein interactionmodulerdquo Structure vol 20 no 3 pp 397ndash405 2012

[108] C Sanders S Turkarslan D-W Lee O Onder R G Kranz andF Daldal ldquoThe cytochrome c maturation components CcmFCcmH and CcmI form a membrane-integral multisubunitheme ligation complexrdquo Journal of Biological Chemistry vol283 no 44 pp 29715ndash29722 2008

[109] A F Verissimo M A Mohtar and F Daldal ldquoThe hemechaperone ApoCcmE forms a ternary complex with CcmI andapocytochrome crdquoThe Journal of Biological Chemistry vol 288no 9 pp 6272ndash6283 2013

[110] P D Barker J C Ferrer M Mylrajan et al ldquoTransmutation of aheme proteinrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 90 no 14 pp 6542ndash65461993

[111] J Simon and L Hederstedt ldquoComposition and function ofcytochrome c biogenesis system IIrdquoThe FEBS Journal vol 278no 22 pp 4179ndash4188 2011

[112] T R H M Kouwen and J M van Dijl ldquoInterchangeablemodules in bacterial thiol-disulfide exchange pathwaysrdquo Trendsin Microbiology vol 17 no 1 pp 6ndash12 2009

[113] A Crow A Lewin O Hecht et al ldquoCrystal structure andbiophysical properties of Bacillus subtilis BdbD An oxidizingthioldisulfide oxidoreductase containing a novel metal siterdquoJournal of Biological Chemistry vol 284 no 35 pp 23719ndash23733 2009

[114] M C Moller and L Hederstedt ldquoExtracytoplasmic processesimpaired by inactivation of trxA (thioredoxin gene) in Bacillussubtilisrdquo Journal of Bacteriology vol 190 no 13 pp 4660ndash46652008

[115] A Lewin A Crow A Oubrie and N E Le Brun ldquoMolecularbasis for specificity of the extracytoplasmic thioredoxin ResArdquoJournal of Biological Chemistry vol 281 no 46 pp 35467ndash35477 2006

[116] C L Colbert QWu P J A Erbel K H Gardner and J Deisen-hofer ldquoMechanism of substrate specificity in Bacillus subtilisResA a thioredoxin-like protein involved in cytochrome cmaturationrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 103 no 12 pp 4410ndash44152006

[117] UAhuja P Kjelgaard B L Schulz LThony-Meyer andLHed-erstedt ldquoHaem-delivery proteins in cytochrome c maturationsystem IIrdquoMolecular Microbiology vol 73 no 6 pp 1058ndash10712009

[118] M L D Page P P Hamel S T Gabilly et al ldquoA homologof prokaryotic thiol disulfide transporter CcdA is required forthe assembly of the cytochrome b6f complex in Arabidopsischloroplastsrdquo Journal of Biological Chemistry vol 279 no 31pp 32474ndash32482 2004

[119] A Rozhkova C U Stirnimann P Frei et al ldquoStructural basisand kinetics of inter- and intramolecular disulfide exchange inthe redox catalyst DsbDrdquoThe EMBO Journal vol 23 no 8 pp1709ndash1719 2004

[120] T Schiott M Throne-Holst and L Hederstedt ldquoBacillussubtilis CcdA-defective mutants are blocked in a late step ofcytochrome C biogenesisrdquo Journal of Bacteriology vol 179 no14 pp 4523ndash4529 1997

[121] R E Feissner C S Beckett J A Loughman and R G KranzldquoMutations in cytochrome assembly and periplasmic redoxpathways in Bordetella pertussisrdquo Journal of Bacteriology vol187 no 12 pp 3941ndash3949 2005

[122] L S ErlendssonMMoller and L Hederstedt ldquoBacillus subtilisStoA is a thiol-disulfide oxidoreductase important for sporecortex synthesisrdquo Journal of Bacteriology vol 186 no 18 pp6230ndash6238 2004

[123] L S Erlendsson R M Acheson L Hederstedt and N E LeBrun ldquoBacillus subtilis ResA is a thiol-disulfide oxidoreductaseinvolved in cytochrome c synthesisrdquo Journal of BiologicalChemistry vol 278 no 20 pp 17852ndash17858 2003

[124] L S Erlendsson and L Hederstedt ldquoMutations in the thiol-disulfide oxidoreductases BdbC and BdbD can suppresscytochrome c deficiency of CcdA-defective Bacillus subtiliscellsrdquo Journal of Bacteriology vol 184 no 5 pp 1423ndash1429 2002

[125] M E Dumont J F Ernst D M Hampsey and F ShermanldquoIdentification and sequence of the gene encoding cytochromec heme lyase in the yeast Saccharomyces cerevisiaerdquoThe EMBOJournal vol 6 no 1 pp 235ndash241 1987

[126] M E Dumont J F Ernst and F Sherman ldquoCoupling of hemeattachment to import of cytochrome c into yeast mitochondriaStudies with heme lyase-deficient mitochondria and alteredapocytochromes crdquo Journal of Biological Chemistry vol 263 no31 pp 15928ndash15937 1988

[127] B San Francisco E C Bretsnyder and R G Kranz ldquoHumanmitochondrial holocytochrome c synthasersquos hemebindingmat-uration determinants and complex formationwith cytochromecrdquo Proceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 9 pp E788ndashE797 2012

Scientifica 17

[128] L Schaefer A Ballabio and H Y Zoghbi ldquoCloning andcharacterization of a putative human holocytochrome c-typesynthetase gene (HCCS) isolated from the critical region formicrophthalmia with linear skin defects (MLS)rdquoGenomics vol34 no 2 pp 166ndash172 1996

[129] S K Prakash T A Cormier A E McCall et al ldquoLoss ofholocytochrome c-type synthetase causes the male lethality ofX-linked dominant microphthalmia with linear skin defects(MLS) syndromerdquo Human Molecular Genetics vol 11 no 25pp 3237ndash3248 2002

[130] I Wimplinger M Morleo G Rosenberger et al ldquoMutations ofthe mitochondrial holocytochrome c-type synthase in X-linkeddominant microphthalmia with linear skin defects syndromerdquoTheAmerican Journal of HumanGenetics vol 79 no 5 pp 878ndash889 2006

[131] S Kiryu-Seo KGamo T Tachibana K Tanaka andHKiyamaldquoUnique anti-apoptotic activity of EAAC1 in injured motorneuronsrdquoTheEMBO Journal vol 25 no 14 pp 3411ndash3421 2006

[132] R Lill R A Stuart M E Drygas F E Nargang and W Neu-pert ldquoImport of cytochrome c heme lyase into mitochondriaa novel pathway into the intermembrane spacerdquo The EMBOJournal vol 11 no 2 pp 449ndash456 1992

[133] H Steiner A Zollner A Haid W Neupert and R LillldquoBiogenesis of mitochondrial heme lyases in yeast Importand folding in the intermembrane spacerdquo Journal of BiologicalChemistry vol 270 no 39 pp 22842ndash22849 1995

[134] A Zollner G Rodel and A Haid ldquoMolecular cloning andcharacterization of the Saccharomyces cerevisiae CYT2 geneencoding cytochrome-c1-heme lyaserdquo European Journal of Bio-chemistry vol 207 no 3 pp 1093ndash1100 1992

[135] V Corvest D A Murrey D G Bernard D B Knaff B Guiardand P PHamel ldquoc-Type cytochrome assembly in Saccharomycescerevisiae a key residue for apocytochrome c1lyase interactionrdquoGenetics vol 186 no 2 pp 561ndash571 2010

[136] V Corvest D A Murrey M Hirasawa D B Knaff B Guiardand P P Hamel ldquoThe flavoprotein Cyc2p a mitochondrialcytochrome c assembly factor is a NAD(P)H-dependent haemreductaserdquo Molecular Microbiology vol 83 no 5 pp 968ndash9802012

[137] D G Bernard S Quevillon-Cheruel S Merchant B Guiardand P P Hamel ldquoCyc2p a membrane-bound flavopro-tein involved in the maturation of mitochondrial c-typecytochromesrdquo Journal of Biological Chemistry vol 280 no 48pp 39852ndash39859 2005

[138] J G Kleingardner and K L Bren ldquoComparing substratespecificity between cytochrome cmaturation and cytochrome cheme lyase systems for cytochrome c biogenesisrdquo Metallomicsvol 3 no 4 pp 396ndash403 2011

[139] C Travaglini-Allocatelli S Gianni VMorea A Tramontano TSoulimane and M Brunori ldquoExploring the cytochrome c fold-ing mechanism cytochrome c552 fromThermus thermophilusfolds through an on-pathway intermediaterdquo Journal of BiologicalChemistry vol 278 no 42 pp 41136ndash41140 2003

[140] L Zhang and L Guarente ldquoHeme binds to a short sequencethat serves a regulatory function in diverse proteinsrdquoTheEMBOJournal vol 14 no 2 pp 313ndash320 1995

[141] S Hira T Tomita T Matsui K Igarashi and M Ikeda-SaitoldquoBach1 a heme-dependent transcription factor reveals pres-ence of multiple heme binding sites with distinct coordinationstructurerdquo IUBMB Life vol 59 no 8-9 pp 542ndash551 2007

[142] T K Kuhl A Wiszligbrock N Goradia et al ldquoAnalysis of Fe(III)heme binding to cysteine-containing heme-regulatory motifs

in proteinsrdquo ACS Chemical Biology vol 8 no 8 pp 1785ndash17932013

[143] R L Moore J M Stevens and S J Ferguson ldquoMitochondrialcytochrome c synthase CP motifs are not necessary for hemeattachment to apocytochrome crdquo FEBS Letters vol 585 no 21pp 3415ndash3419 2011

[144] S Gianni M Brunori and C Travaglini-Allocatelli ldquoRefoldingkinetics of cytochrome c551 reveals a mechanistic differencebetween urea and guanidinerdquo Protein Science vol 10 no 8 pp1685ndash1688 2001

[145] S Gianni C Travaglini-Allocatelli F Cutruzzola M BrunoriM C R Shastry and H Roder ldquoParallel pathways incytochrome c551 foldingrdquo Journal of Molecular Biology vol 330no 5 pp 1145ndash1152 2003

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Microbiology

2 Scientifica

Figure 1 The heme-binding site typically observed in c-typecytochromes as exemplified by a close-up view of the structure of Paeruginosa Cyt c551 (Pa-Cytc PDB 351c) The heme is shown in redwhile the atoms of the residues from the heme-binding motif of Pa-Cytc (C

12VAC15H) and the distal Met61 are color-coded (C green

O red N blue S yellow) The figure highlights the thioether bondsbetween the Cys12 (on the right) and the vinyl-2 and between Cys15(on the left) and the vinyl-4 The iron atom of the heme (in gray) isaxially coordinated by the distal methionine residue (Met61 shownabove the heme plane) and by the proximal histidine residue (His16shown below the heme plane)

mechanism(s) underlying the process of heme traffickingacross the membranes is still largely obscure (see [15 16] forreviews on heme synthesis and trafficking in eukaryotes) Inall known Cyts c the heme is covalently linked to the apoCytwith the same stereochemistry two thioether bonds arepresent between the vinyls at positions 2 and 4 of the tetrapyr-role ring of heme b and the thiols of the N- and C-terminalcysteines (Cys1 and Cys2 resp) of a conserved heme-bindingmotif (C1XXC2H where X denotes any residues) The ironatom of the Fe-protoporphyrin IX is always axially coor-dinated to the histidine of the heme-binding motif (on theproximal side of the heme cavity) while amethionine residueon the distal side generally represents the second axial ligand(Figure 1) C-type cytochromes may contain more than oneheme c linked to the protein through different C1XXC2Hmotifs From a structural point of view Cyt c proteins definea well-defined 120572-helical fold (see SCOPmdashhttpscopmrc-lmbcamacukscop and CATHmdashhttpwwwcathdbinfoprotein structure databases) characterized by the presenceof three 120572-helices the N- and C-terminal 120572-helices interacteach other in the native structure while an additional 120572-helix(historically known as the 601015840 helix) overlays part of the hemecavity Since the seminal experiment of Anfinsen on horseheart Cyt c [17] it is generally accepted that Cyt c withoutits covalently bound heme (apoCyt) is an unfolded proteindevoid of appreciable secondary and tertiary structure andthat the polypeptide chain is able to fold into its typical Cytc structure only when the thioether bonds with the hemeare formed As it will be discussed below these observations

raise interesting questions as to how an unfolded proteinsuch as apoCyt is specifically recognized by the differentprotein components of the maturation apparatus of the cellRecently however evidence has been presented that at leastin some cases the Cyt c fold may be attained even in theabsence of the heme [18] challenging our current view of theCyt c folding mechanism [4 19]

C-type cytochromes are synthesized in the cytoplasm (n-side of the membrane) but they exert their functions inother subcellular compartments (p-side of the membrane)that is the periplasm of gram-negative bacteria the bacterialextracytoplasmic space of gram-positive bacteria the inter-membrane spacemdashIMS of mitochondria or the chloroplastthylakoid lumen It is in these subcellular compartments thatthe heme b is covalently attached to apoCyt by the appropriatematuration apparatus In prokaryotes the necessary translo-cation of apoCyt across the membrane is carried out by theSec machinery [26] this apparatus composed of the SecAB-DYEFG proteins is able to translocate unfolded proteinscarrying a specific targeting sequence [27] In eukaryotesthe newly synthesized apoCyt is probably translocated intothe mitochondrion via a different mechanism involvingcomponents of the TOM complex on the outer side of themembrane and the cytochrome c heme lyase which probablyacts also as an apoCyt receptor in the mitochondrial IMS[28 29] However the process is not completely clear as westill do not know whether the apoCyt is delivered to themitochondrial matrix and then exported to the IMS [30] or itis translocated directly to the IMS via a different mechanism[31] It should be noticed that in plants the translocation ofc-type cytochromes into the chloroplast lumen is probablyindependent on the heme attachment reaction [32 33]

Despite that in all c-type cytochromes both prokaryoticor eukaryotic the heme is always covalently linked to theconserved CXXCH heme-binding motif different matura-tion apparatuses composed of different proteins have beenidentified ([34 35] see Figures 2 3 and 4 and Table 1) Struc-tural and functional properties of the protein componentsof Systems IndashIII are the focus of the present review othermaturation apparatuses involved in the unusual attachmentof heme b to the protein moiety via a single thioetherbond (Systems IVndashVI) have been described and reviewedelsewhere [36 37]

With the exception of system III which is present ineukaryotic cells the distribution of the other Systems amongBacteria Archaea and plant cells is complicated by theobservation that in many cases the maturation machinery isnot conserved [38] rendering the analysis of their evolution-ary origins and relationships difficult [39ndash41] In 120572- and 120574-proteobacteria in some 120573- and 120575-proteobacteria in Archaeaand in themitochondria of plants and algae Cyt cmaturationis carried out by a set of eight or nine proteins belongingto System I [42] (Figure 2) in gram-positive bacteria incyanobacteria in the chloroplasts of plants and algae in 120576-120573- and some 120575-proteobacteria the Cyt c maturation processis carried out by three or four proteins belonging to System II[43 44] (Figure 3) while System III occurs in mitochondriaof fungi metazoans and some protozoa [16 45] (Figure 4)The observation that in plants three systems are present

Scientifica 3

CcmACcmA

CcmC

CcmE

Ccm

D

Ccm

B

Ccm

B

CcmF

CcmICcmH

CcmG

Periplasm

HemeATP ADP

Cytoplasm

Figure 2 Schematic representation of the protein components ofSystem I Proteins involved in the heme translocation and deliverypathway are shown in light brown proteins involved in the apoCytthioreduction pathway are shown in green proteins involved inapoCyt chaperoning and heme attachment processes are shownin light purple Cyt c (the 3D structure is that of the Cyt c551from P aeruginosa) Protein Data Bank accession number 2EXV[20] and apoCyt (represented as a cartoon) are shown in blueThe translocation process of heme (shown in red) is unknownThe 3D structures of the soluble periplasmic domains of Ec-CcmE Pa-CcmG and Pa-CcmH are shown (Protein Data Bankaccession numbers are 1LIZ [21] 3KH7 [22] and 2HL7 [23] resp)Organisms employing System I 120572- and 120574-proteobacteria some120573-proteobacteria (eg Nitrosomonas) and 120575-proteobacteria (egDesulfovibrio) and Deinococci and Archaea Additionally System Iis observed in plant mitochondria and in the mitochondria of someprotozoa (eg Tetrahymena)

(System I in mitochondria System III in the p-side of thethylakoid membrane and System IV in the n-side of thethylakoidmembrane [16 38])makes the classification and thedistribution of the different maturation systems even moredifficultWith the exception of System III which is apparentlycomposed of a single protein able to carry out the differenttasks of the Cytc maturation process (see below) the variousproteins of Systems I and II carry out different functionsincluding the translocation and delivery of heme b from thecytoplasm where it is synthesized to the relevant subcellularcompartment the chaperoning of apoCyt and the reductionof its disulfide the formation of the covalent bonds betweenthe heme b and the CXXCH heme-binding motif of theapoprotein (Table 1) The complexity of System I comparedto the protein composition of other Cyt cmaturation systemshas long been discussed in particular it has been proposedthat a possible explanation is to be found in the ability evolvedby organisms employing System I to utilize lower levels ofendogenous heme than those necessary for organisms whichevolved Systems II or III [46]

2 System I

The proteins belonging to System I (named CcmABCDE-FGH(I) from Cytochrome c maturation) are membraneproteins exposing their soluble domains (when present) intothe periplasm (Figure 2) All of these proteins are encodedby a single operon in 120572- 120573- and 120574-proteobacteria [4247] The availability of the entire Ccm operon in a single

Ccs

A

Ccs

B

Heme

Cytoplasm

Cyt capoCyt

ResA

Figure 3 Schematic representation of the protein componentsof System II Proteins involved in the heme translocation anddelivery and in the apoCyt chaperoning and heme attachmentprocesses are shown in light brown proteins involved in the apoCytthioreduction pathway are shown in green Cyt c and apoCyt(represented as a cartoon) are shown in blueThe 3D structure of thesoluble periplasmic domain of Bs-ResA is shown in green (ProteinData Bank accession number is 1ST9 [24] System II is foundin plant chloroplasts in gram-positive bacteria cyanobacteria 120576-proteobacteria most 120573-proteobacteria (eg Bordetella Burkholde-ria) and some 120575-proteobacteria (eg Geobacter)

Heme

Intermembrane space

Inner membrane

Outer membrane

Cyt c

apoCytHCCS

Figure 4 Schematic representation of System III A single pro-tein (HCCS) associated to the mitochondrial inner membraneis required for Cyt c maturation The translocation process ofheme (shown in red) is unknown System III is found in themitochondria of fungi (eg S cerevisiae) vertebrates (eg human)and invertebrates (eg C elegans Drosophila)

plasmid (pEC86 [48]) greatly facilitated the heterologousover-expression of many c-type cytochromes in E coli [49]Apparently c-type cytochromes specificity of the Ccm appa-ratus is rather low indeed in an attempt to characterize theminimal sequence requirements of the apoCyt polypeptiderecognized by System I it was shown that this complexmultiprotein apparatus is able to attach the heme even toshort microperoxidase-like peptides carrying the CXXCHmotif [50]

As discussed below different studies mainly carried outby immunoprecipitation experiments suggest that the Ccmproteins may be assembled in the bacterial membrane in a

4 Scientifica

Table1Proteincompo

nentsof

Syste

msIIIand

IIIa

long

with

theirstructural

features

(orPD

Bcodeswhenkn

own)

andfunctio

nalrolesS

ystem

Iproteinsarefoun

din120572-and120574-

proteobacteriasom

e120573-a

nd120575-proteob

acteria

and

Dein

ococciandArchaeaPlantm

itochon

driaM

itochon

driaof

somep

rotozoaSyste

mIIproteins

arefou

ndin

plantchlorop

lasts

Gram-

positiveb

acteria

cyano

bacteria120576-proteob

acteria

most120573

-proteob

acteria

and

some120575

-proteob

acteria

HCC

SofSystemIIIisfou

ndinthem

itochon

driaoffung

ivertebratesand

invertebrates

Syste

mI(SI)

SIstr

ucturalfeatures

Syste

mII(SII)

SIIstructuralfeatures

Syste

mIII(SIII)

SIIIstr

ucturalfeatures

Functio

n(s)

Ccm

AABC

transportermem

brane

n-sid

enu

cleotide-bind

ing

domain

ResB

(CcsB)

5-6TM

helices

1large

perip

lasm

icdo

main

conservedHis

resid

ues

HCC

SMem

brane-associated

protein

conservedHis

resid

ues

Hem

etranslocatio

nand

delivery

Ccm

BABC

transporter6TM

helices

ResC

(CcsA)

6ndash8TM

helices

WWDdo

main

conservedHis

resid

ues

Ccm

C6T

Mhelicesperiplasm

icWWDdo

main

Ccm

DSm

allm

embranep

rotein1

TMhelix

Ccm

E1T

MhelixO

B-fold

1SR3

1LM

01J6

Q2KC

T

Ccm

G1T

MhelixT

RX-like

fold

1Z5Y2B1K

1KNG3KH

73K

H93K

8NRe

sATR

X-lik

efold

2H1B1SU

91ST9

2F9

SapoC

ytthioredu

ction

Ccm

H1T

Mhelix3-helixbu

ndlefold

2HL72KW

0CcdA

6TM

helices

Ccm

F10ndash15TM

helicesperiplasm

icWWDdo

main

conservedHis

resid

ues

ResB

(CcsB)

ResC

(CcsA)

HCC

SapoC

ytchaperon

ingand

hemea

ttachment

Ccm

IPerip

lasm

icTP

Rand120572120573

domains

Scientifica 5

maturase multiprotein complex(es) However the existenceandor stoichiometry of these complexes remains to bedetermined either because of the experimental difficultiesin handling membrane protein complexes or because itis possible that these complexes are unstable and onlytransiently populated Independently from their functionalexistence in the bacterial periplasm as independent units or ascomponents of a multisubunit complex it is clear that each oftheCcmproteins plays a different role from the transport andchaperoning of the heme cofactor to the necessary reductionof the disulfide bond between the sulfur atoms of the twoCys residues of the conserved CXXCH motif of apoCytand finally to the catalysis of covalent heme attachment Anadditional interesting aspect is that over and above theirrole in the biogenesis of Cytc there is also evidence thatinactivation of some ccm genes induces phenotypes thatcannot be explained only in terms of absence of synthesis ofCyt c all of these pleiotropic effects are linked to impairmentof heme andor iron trafficking in the periplasm [47] Inparticular it has been recently shown that in 120572- and 120574-proteobacteria (including the human opportunistic pathogenP aeruginosa) mutations in the ccmC ccmI and ccmF genesinduce phenotypes such as reduced pyoverdine produc-tion reduced bacterial motility or impaired growth in low-iron conditions ([51] and refs therein) These observationssuggesting that Ccm proteins perform additional functionscritical for bacterial physiology growth and virulence pro-vide a rationale to explain why bacteria at variance withthe eukaryotic cell have evolved a metabolically expensiveoperon to accomplish an apparently simple task such asheme ligation to apoCyt Novel hypotheses addressing theseaspects and awaiting experimental investigation include (i)the utilization of Ccm-associated heme for additional cellularprocesses besides attachment to apoCyt (ii) trafficking a non-heme compound through the Ccm system required for ironacquisition such a siderophore (iii) the Ccm inactivation-dependent accumulation of heme b a photoreactivemoleculewhose degradation leads to reactive oxygen species and (iv)the destructive effect on [Fe-S] clusters of ferrisiderophoresreductases

In the following the structural (when known) andfunctional properties of the different components of theSystem I maturation apparatus will be discussed dissectingthe Cytc maturation process into three main functionalsteps heme translocation and delivery apoCyt thioreductivepathway and apoCyt chaperoning and heme ligation (asimilar modular description can also be found elsewhere(see [34 52])) However it should be remembered that inmany cases the proteins involved in the three steps are notuniquely assigned to a specific module as they interact witheach other moreover it has been shown that in some casesmore than one system can be present [41] This modularorganization of Cyt c biogenesis should therefore be intendedonly as a way to simplify the description of an overall highlyintegrated process For each of the three functional stepspresentation of the structural and functional properties of thedifferent protein components is followed by a discussion ofthe proposed molecular mechanism(s)

21 System I Components of the Heme Translocation andDelivery Pathway CcmA and CcmB proteins show the typ-ical sequence features of the ABC (ATP binding Cassette)transporter family and therefore these components of SystemI were initially considered as the proteins responsible forthe translocation of the newly synthesized heme from thecytoplasm to the periplasmic space ABC transporters areubiquitous multidomain integral membrane proteins thattranslocate a large variety of substrates across cellular mem-branes using ATP hydrolysis as a source of energy they aregenerally composed of a transmembrane (TM) domain and aconserved cytosolic nucleotide-binding domain [53]

CcmA is a cytoplasmic soluble protein representing thenucleotide-binding domain of the hypothetical ABC trans-porter according to this hypothesis its sequence containsa nucleotide-binding domain and Walker A and B motifsfor ATP hydrolysis [46] It has also been shown that CcmApossesses ATPase activity in vitro and that the protein isassociated with the membrane fraction only when CcmB isalso present [54]

CcmB and CcmC are both integral membrane proteinspredicted to contain six TM helices CcmC contains a shortWWDdomain in its second periplasmic domain and belongsto the heme handling protein family (HHP) [55] WWDdomains are short tryptophan-rich aminoacid stretcheswith the conserved WGX120601WXWDXRLT sequence (where 120601represents an aromatic amino acid residue and X representsany residue) [40 56] it has been proposed that proteinscontaining WWD domains are involved in heme-bindingand as we will see below a WWD domain is also presentin the CcmF protein another crucial heme-binding proteinof System I CcmC also contains two absolutely conservedhistidine residues in its first (between TM helices 1 and 2)and third (between TMhelices 5 and 6) periplasmic domainsAn attractive hypothesis still requiring experimental proofis that the hydrophobic residues within the tryptophan-richmotif provide a platform for the binding of heme whereasthe two conserved His residues (H60 and H184 in E coliCcmC) act as axial heme ligands [57] this hypothesis isstrengthened by the observation that CcmC indeed interactsdirectly with heme [58 59] Immunoprecipitation experi-ments have shown that in E coli CcmABC proteins forma multiprotein complex with a CcmA

2CcmB

1CcmC

1stoi-

chiometry confirming that these components form an ABC-type transporter complex with unusual functional propertiesassociated to the release of holoCcmE from CcmC [41 46]rather than to heme transport per se CcmC is an interestingprotein worth of future experimental efforts as it is knownthat in some pathogenic bacteria CcmC mutations are asso-ciated to specific phenotypes apparently not related to Cytc maturation such as siderophore production in Paracoccusand Pseudomonas [51] and iron utilization in Legionella [60]

Limited information is available about the structure andfunction of CcmD which appears to be a small membraneprotein (about 70 aminoacid residues) with no conservedsequence features whose topology is currently debatedcontrary to the original proposal [61] additional experimentshave shown that in E coli and R capsulatus CcmD isan integral membrane protein composed of a single TM

6 Scientifica

helix a periplasmic-orientedN-terminus and a cytoplasmic-oriented C-terminus [62] Immunoprecipitation experimentsindicate that CcmD interacts with the CcmA

2CcmB

1CcmC

1

complex even if it is not essential for heme transfer andattachment from CcmC to CcmE CcmD is strictly requiredfor the release of holoCcmE from the ABC transporter [6162]

CcmE is a heme-binding protein discovered as an essen-tial System I component as early as the late 1990rsquos [63]CcmE is a monotopic membrane protein anchored to themembrane via its N-terminal TM segment and exposing itsactive site to the periplasm it is the only Ccm componentof the heme trafficking and delivery module of System I forwhich a three-dimensional structure is available ([21] PDB1SR3 [64] PDB 1LM0) The 3D structure of the apo-state(without bound heme) consists of a six-stranded antiparallel120573-sheet reminiscent of the classical OB-fold [65] with N-and C-terminal extensions CcmE can be considered a ldquohemechaperonerdquo as it protects the cell from a potentially dangerouscompound by sequestering free heme in the periplasm [66]it is thought to act as an intermediate in the heme deliverypathway of Cytc maturation The structure of apoCcmEshowed no recognizable heme-binding cavities and in theabsence of a 3D structure of CcmEwith bound heme (holoC-cmE) the heme-binding region could only be predicted byin silico modeling It is generally believed that the hemein holoCcmE is solvent exposed but recent mutagenesisexperiments challenged this view [67] The unusual covalentbond between the nitrogen atomof a histidine residue presentin the conserved VLAKHDE motif located in a solventexposed environment (H130 in E coli CcmE) and a 120573-carbonof one of the heme vinyl groups has been described in greatdetail by NMR spectroscopy [68] Recently it was shown thatCcmE proteins from the proteobacteria D desulfuricans andD vulgaris contain the unusual CXXXY heme-bindingmotifwhere the Cys residue replaces the canonical His bindingresidue NMR solution structure of D vulgaris CcmE (PDB2KCT) revealed that the proteins adopt the same OB-foldcharacteristic of the CcmE superfamily Contrary to whatreported for theD desulfuricansCcmE [69] the homologousprotein from D vulgaris binds ferric heme noncovalentlythrough the conserved C127 residue [70] An additionalconserved residue in CcmE proteins is Tyr134 which wasshown to provide a coordination bond to the heme ironof holoCcmE [71 72] once it is released from CcmABCDcomplex [36] as discussed below

22 System I Heme Translocation and Delivery PathwayMechanisms We still do not know how the b heme istranslocated from the cytoplasm (where it is synthesized)to the periplasm where Cyt c maturation occurs Differ-ent mechanisms such as translocation through a proteinchannel or free diffusion across the membrane have beenproposed [73] The CcmAB proteins show structural featurestypical of the ABC transporters and for these reasons theywere originally hypothesized to be involved in the heme btranslocation process [57 63 74 75] However it is nowclear that an alternative process must exist since it has

been shown that periplasmic b-type cytochromes can beproduced in the absence of Ccm proteins [76] and thatinactivation of the ATPase activity of CcmA does not abolishheme accumulation in the periplasm [46 54] We have nowevidence that CcmC has the ability to bind heme at its WWDdomain present in the second periplasmic domain but itis still not clear if this membrane protein acts as a proteinchannel for heme translocation or simply collects it in theperiplasm [77]

Another important aspect concerns the oxidation state ofthe heme iron during translocation and delivery processesindeed this property of the heme iron may determine thereaction mechanism by which the unusual CcmE H130nitrogen is covalently linked to the vinyl 120573-carbon of theheme (see [36] for a detailed discussion of this topic) Basedon mutagenesis studies on CcmC [59] a model has beenpresented whereby oxidized heme is bound to CcmC onlyin the presence of CcmE forming a ternary complex BothCcmC and CcmE provide critical residues for heme-bindingthe two conserved His residues (H60 and H180 coordinatingthe heme iron) and theWWDdomain ofCcmCandHis130 ofCcmE forming the unusual covalent bond with heme vinyl-2 [59] The ATPase activity of CcmA is then required torelease holoCcmE from the CcmABCD complex a processthat depends also on the presence of CcmD [46 54] It shouldbe noticed that purified holoCcmE alone or in the CcmCDEcomplex [36 59] contains the heme iron in the oxidized statean observation that is apparently in contrast with the fact thatthe heme must be in its reduced state before attachment toapoCyt can occur Although the oxidation state of the hemeiron is currently debated [36 78] it is possible that CcmFwhich was recently shown to contain a heme b cofactor mayact as specific heme oxidoreductase (see Section 25)

23 System I ApoCyt Thioreduction Pathway ComponentsThe periplasm can be considered a relatively oxidizing envi-ronment due to the presence of an efficient oxidative systemcomposed of the DsbAB proteins [79 80] DsbA is a highlyoxidizing protein (1198641015840

0= 120mV) that is responsible for

the introduction of disulfide bonds into extracytoplasmicproteins [81] On the basis of the results obtained on E colidsbA deletion mutants that are unable to synthesize c-typecytochromes [82 83] it was generally accepted that formationof the intramolecular disulfide bond in apoCyt was a nec-essary step in the Cyt c biogenesis However only reducedapoCyt is clearly competent for heme ligation It is possiblethat this seemingly paradoxical thioreduction process hasevolved in order to protect the apoCyt from proteolyticdegradation aggregation andor formation of intermolec-ular disulfide bonds with thiols from other molecules (seealso Section 31 for a discussion about this aspect in SystemII) Recently however an analysis of c-type cytochromesproduction in several E coli dsb genes deletion strains ledto the hypothesis that DsbA is not necessary for Cyt cmaturation and that heme ligation to apoCyt and apoCytoxidation pathways is alternative competing processes [84]

In gram-negative bacteria a thioreduction pathway hasevolved to specifically reduce the oxidized apoCyt substrate

Scientifica 7

which includes the Ccm proteins CcmG and CcmH Thenecessary reducing power is transferred from the cytoplasmicthioredoxin (TRX) to CcmG via DsbD a large membraneprotein organized in three structural domains an N-terminalperiplasmic domain with a IgG-like fold (nDsbD) a C-terminal periplasmic domain with a thioredoxin-like (TRX-like) fold (cDsbD) and a central domain composed ofeight TM helices [85] Each of these domains contains apair of Cys residues and transfer electrons via a cascadeof disulfide exchange reactions making DsbD a ldquoredox-hubrdquo in the periplasm performing disulfide bond exchangereactions with different oxidized proteins [79] In particulara combination of X-ray crystallography experiments andkinetic analyses showed that electrons are transferred fromthe cytoplasmic TRX to the membrane domain of DsbDfollowed by reduction of cDsbD and finally of nDsbD whichis the direct electron donor to CcmG [85]

CcmG is a membrane-anchored protein linked to themembrane via an N-terminal TM helix and exposing itssoluble TRX-like domain in the periplasm The 3D structureof the TRX-like domain of CcmG from different bacteriahas been solved by X-ray crystallography (E coli PDB 1Z5Y[85] PDB 2B1K [86] B japonicum PDB 1KNG [87] Paeruginosa PDB 3KH7 3KH9 [22]) and is generally wellconserved as proved by the low RMSD (08 A between Pa-CcmG and Ec-CcmG 135 A between Pa-CcmG and Bj-CcmG) Although all these proteins adopt a TRX-like foldand contain the redox-active motif CXXC in the first 120572-helixthey are inactive in the classic insulin reduction assay [7588] CcmG proteins are therefore considered specific thiol-oxidoreductase able to recognize and selectively interactonly with their upstream and downstream binding partnersin the thioreduction process leading to reduced apoCytcLooking at the 3D structure of the periplasmic domain ofthe prototypical Pa-CcmG it is possible to identify the 120573120572120573and 120573120573120572 structural motifs of the TRX fold linked by a short120572-helix and forming a four-stranded 120573-sheet surrounded bythree helices the protein contains an additional N-terminalextension (residues 26ndash62) and a central insert (residues 102ndash123) The redox-active motif of Pa-CcmG (CPSC) is locatedin the first 120572-helix of the TRX fold as usually observed in allTRX-like proteins As for any molecular machinery whereeach component must recognize and interact with more thanone target (ie the substrate and the other components ofthe apparatus) an open question concerns the mechanismwhereby CcmG is able to recognize its different partnersThe availability of the crystal structures of Pa-CcmG bothin the oxidized (22 A resolution) and reduced state (18 Aresolution) [22] allowed highlighting the structural similaritybetween the two redox states (Rmsd of the C120572 atoms inthe two redox forms is 019 A) and therefore to excludestructural rearrangement as the mechanism used by Pa-CcmG to discriminate between reduced (such as the nDsbDdomain) and oxidized partners (Pa-CcmH andor apoCyt)

The standard redox potential of Pa-CcmG (11986410158400= 0213V

at pH 70 [22] as well as that of Ec-CcmG (11986410158400= 0212V

[86]) indicates that these proteins act as mild reductants inthe thioreductive pathway of Cytc biogenesis However the

Figure 5 Three-dimensional structure of Pa-CcmH shown inribbon representation The figure shows the three-helix bundleforming the characteristic fold of Pa-CcmHThe active site disulfidebond between residues Cys25 andCys28 in the long loop connectinghelices 120572-helix1 and 120572-helix 2 is highlighted in yellow

function of thiol-oxidoreductases obviously depends on thepKa values of their activesite Cys residues The pKa of CysX(613 plusmn 005) and CysY (105 plusmn 007) are consistent with thepKa values measured in different TRXs where the active N-terminal Cys residue has a pKa close to pH 70 whereas theC-terminal Cys has a much higher pKa [89 90] Such a largedifference between the two pKa values in the TRX family isfunctionally relevant because it allows the N-terminal Cysto perform the nucleophilic attack on the target disulfidewhile the C-terminal Cys is involved in the resolution of theresulting mixed-disulfide [90]

CcmH is the other component of System I involvedin the reduction of apoCyt Notably CcmH proteins fromdifferent bacterial subgroups may display structural vari-ability indeed while in E coli Ec-CcmH is a bipartiteprotein characterized by two soluble domains exposed to theperiplasm and two TM segments CcmH from P aeruginosa(Pa-CcmH) is a one-domain redox-active protein anchoredto the membrane via a single TM helix and homologous tothe N-terminal redox-active domain of Ec-CcmH Surpris-ingly the 3D structure of the soluble periplasmic domainof Pa-CcmH revealed that it adopts a peculiar three-helixbundle fold strikingly different from that of canonical thiol-oxidoreductases (Figure 5 PDB 2HL7 [23])TheN-terminaldomain of Ec-CcmH was also shown to have the same 3Dstructure although helix-swapping and dimerization havebeen observed in this case (PDB 2KW0 [91 92]) Theconserved redox-active motif (LRCPKC) is located in theloop connecting helices 1 and 2 close to the activesite thecrystal structure reveals the presence of a small pocket on thesurface of Pa-CcmH surrounded by conserved hydrophobicand polar residues which could represent the recognition sitefor the heme-binding motif of apoCyt

Concerning the functional properties of this unusualthiol-oxidoreductase it is interesting to note that its standardredox potential (1198641015840

0= 0215V) [23] is similar to that ob-

tained for Pa-CcmG This observation stands against thelinear redox cascade hypothesis whereby CcmG reducesCcmH While in the canonical redox-active CXXC motif

8 Scientifica

S SSS

SH

SH

Scheme 1 Scheme 2

SH

SH

SHSHS

S

CcmG 7477

CcmG 7477

CcmH 2528

CcmH 2528

apoCytox

apoCytred

S

SHCcmG 74

77

SH

SH

HSHS

CcmG 7477

S

S

SS

3

1

2CcmG 7477

SH

SHCcmH 25

28SH

HSS S

CcmH 2528

SHS

CcmH2528

apoCytox

apoCytred

+

Figure 6 Alternative thioreduction pathways whichmay be operative in System I and hypothesized on the basis of structural and functionalcharacterization of the redox-active Ccm proteins from P aeruginosa [22 23 25] Scheme 1 is a linear redox cascade whereby CcmG is thedirect reductant of CcmH which reduces oxidized apoCyt Scheme 2 envisages a more complex scenario involving the formation of a mixed-disulfide complex between CcmH and apoCyt (Step 1) This complex is the substrate for the attack by reduced CcmG (Step 2) that liberatesreduced apoCyt The resulting disulfide bond between CcmH and CcmG is then resolved by the free Cys thiol of CcmG (probably Cys77 inPa-CcmG) Adapted from [25]

of the TRX family the N-terminal Cys is always solventexposed in CcmH proteins the arrangement of the two Cysresidues is reversed the N-terminal Cys residue is buriedwhereas the C-terminal Cys residue is solvent exposed Onthe basis of this observation it was suggested that differentfrom the canonical TRX redox mechanism CcmH proteinsperform the nucleophilic attack on the apoCyt disulfide viatheir C-terminal Cys residue [23] This mechanism which isin agreement with the mechanism proposed earlier for Ec-CcmH on the basis of mutational-complementation studies[93 94] is substantiated by the peculiar pKa values of theactive site Cys residues of Pa-CcmH which were found tobe similar for both cysteines (84 plusmn 01 and 86 plusmn 01 [23])Again this is different from what is generally observed in thecase of TRX proteins where the pKa value of the Cys residueperforming the initial nucleophilic attack is significantlylower than the pKa value of theCys residue responsible for theresolution of the intermediate mixed-disulfide It is temptingto speculate that the unusual pKa values of the Pa-CcmHactive site thiols may ensure the necessary specificity of thiscomponent of the Ccm apparatus toward the CXXCH motifof the apoCyt substrate

24 System I ApoCyt Thioreduction Pathway MechanismAlthough we know that CcmG and CcmH are the redox-active components of System I involved in the thioreductivepathway of Cyt c biogenesis not only an acceptedmechanismfor the reduction of apoCyt disulfide bond is still lackingbut also the absolute requirement of such a process is nowdebated [38 84] Focusing our attention on the reductionof the apoCyt internal disulfide at least two mechanismscan been hypothesized which involve either a linear redoxcascade of disulfide exchange reactions or a nonlinear redox

process involving transient formation of a mixed-disulfidecomplex as depicted in Figure 6 and Schemes 1 and 2respectively

Both the thiol-disulfide exchange mechanisms depictedin Figure 6 suggest that CcmH is the direct reductant ofthe apoCytc disulfide however even if immunoprecipitationexperiments failed to detect the formation of a mixed-disulfide complex between apoCyt and CcmH proteins [95]some in vitro evidence supporting the formation of sucha complex has been presented In particular it has beenshown that Rhodobacter capsulatus and Arabidopsis thalianaCcmH homologues (Rc-CcmH and At-CcmH) are able toreduce the CXXCH motif of an apoCyt-mimicking peptide[75 96] In the latter case yeast two-hybrid experimentscarried out on At-CcmH indeed revealed an interactionbetween the protein and a peptide mimicking the A thalianaCyt c sequence In the case of Pa-CcmH FRET kineticexperiments employing a Trp-containing fluorescent variantof the protein and a dansylated nonapeptide encompassingthe heme-binding motif of P aeruginosa cytochrome c551(dans-KGCVACHAI) [23] allowed to directly observe theformation of themixed-disulfide complex and tomeasure theoff-rate constant of the bound peptide The results of these invitro binding experiments allowed to calculate an equilibriumdissociation constant which combines an adequate affinity(low 120583M) with the need to release efficiently reduced apoCytto other component(s) of the System I maturase complex[23] More recently the results obtained by FRET bindingexperiments carried out with single Cys-containing mutantsof Pa-CcmH and Pa-CcmG [25] substantiated the hypothesisdepicted in Scheme 2 (Figure 6) Altogether these structuraland functional results suggest that the thioreduction pathwaymechanism leading to reduced apoCyt is better describedby Scheme 2 and that reducing equivalents might not be

Scientifica 9

transferred directly from CcmG to apoCyt as depicted inScheme 1 According to Scheme 2 reduced CcmH (a non-TRX-like thiol-oxidoreductase) specifically recognizes andreduces oxidized apoCyt via the formation of a mixed-disulfide complex which is subsequently resolved by CcmGThe resulting disulfide bond between CcmH and CcmG isthen resolved by the free Cys thiol of CcmG (probably Cys77in Pa-CcmG)

However further in vitro experiments with CcmH andapoCyt single Cys-containing mutants are needed to unveilthe details of the thioreduction of oxidized apoCyt by CcmHIn particular it would be crucial to identify the Cys residueof apoCyt that remains free in the apoCyt-CcmH mixed-disulfide complex intermediate (see Scheme 2 and Section 26below) and available to thioether bond formation with oneof the heme vinyl groups Clearly structure determinationof the trapped mixed-disulfide complexes between CcmHCcmG and apoCyt (or apoCyt peptides) would providekey information for our understanding of this specializedthioreduction pathway mechanism

25 System I ApoCyt Chaperoning and Heme AttachmentComponents The reduced heme-binding motif of apoCyt isnow available to the heme ligation reaction However themolecularmechanismwhereby the Ccmmachinery catalyzesor promotes the formation of the heme-apoCyt covalentbonds is still largely obscure representing themost importantgoal in the field Past observations and recent experimentssuggest that CcmF and CcmI possibly together with CcmHare involved in these final steps [16 34 36]

CcmF is a large integral membrane protein of more than600 residues belonging to the heme handling protein family(HHP [55]) and predicted to contain 10ndash15 TM helices (notethat some discrepancy exists as to the number of TM helicespredicted by computer programs and those predicted onthe basis of phoA and lacZ fusion experiments [40 97])a conserved WWD domain and a larger domain devoidof any recognizable sequence features both exposed to theperiplasm Only recently E coli CcmF (Ec-CcmF) has beenoverexpressed solubilized from the membrane fraction andspectroscopically characterized in vitro [36 41] Surprisinglythe biochemical characterization of recombinant Ec-CcmFallowed to show that the purified protein contains heme b ascofactor in a 1 1 stoichiometry this observation led to thehypothesis that in addition to its heme lyase function Ec-CcmF may act as a heme oxidoreductase In particular it ispossible that the heme b of Ec-CcmF may act as a reductantfor the oxidized iron of the heme bound to CcmE [41]indeed the in vitro reduction of Ec-CcmF by quinones hasbeen experimentally observed strengthening the hypothesisabout the quinolhemeoxidoreductase function of this elusiveproteinThe structuralmodel proposed for Ec-CcmFpredicts13 TMhelices and notably the location of the four completelyconserved His residues according to the model two of them(His173 andHis303) are located in periplasmic exposed loopsnext to the conserved WWD domain which is believedto provide a platform for the heme bound to holoCcmEwhile His261 is located in one of the TM helices and it is

predicted to act as an axial ligand to the heme b of Ec-CcmFthe other conserved His residue (H491) could provide thesecond axial coordination bond to the heme although thishas not been experimentally addressed This model of Ec-CcmF therefore envisages that this large membrane proteinis characterized by two heme-binding sites one of themis embedded in the membrane and coordinates a heme bprosthetic group necessary to reduce the CcmE-bound hemehosted in the second heme-binding site and constituted by itsWWD domain

It is interesting to note that in plants mitochondria theCcmF ortholog appears to be split into three different pro-teins (At-CcmFN1 At-CcmFN2 and At-CcmFC) possiblyinteracting each other [16] Since each of these proteins issimilar to the corresponding domain in the bacterial CcmFortholog this observation may provide useful informationin the design of engineered fragments of bacterial CcmFproteins amenable to structural analyses

The other System I component which is generallybelieved to be involved in the final steps of Cyt c maturationis CcmI As stated above the ccmI gene is present only insome Ccm operons while in others the corresponding ORFis present within the ccmH gene (as in E coli)The functionalrole of CcmI in Cytc biogenesis is revealed by geneticstudies showing that in R capsulatus and B japonicuminactivation of the ccmI gene leads to inability to synthesizefunctional c-type cytochromes [98 99] In R capsulatus andP aeruginosa the CcmI protein (Rc-CcmI and Pa-CcmIresp) can be described as being composed of two domainsstarting from the N-terminus a first domain composed oftwo TM helices connected by a short cytoplasmic regionand a large periplasmic domain Structural variations maybe observed among CcmI members from different bacteriaindeed multiple sequence alignment indicates that the cyto-plasmic region of Rc-CcmI contains a leucine zipper motifwhich is not present in the putative cytoplasmic region of Pa-CcmI [100ndash102] Surprisingly no crystallographic structureis available up to now for the soluble domain of any CcmIprotein with the exception of the ortholog protein NrfGfrom E coli (Ec-NrfG) [103] This protein is necessary toattach the heme to the unusual heme-binding motif CWSCK(where a Lys residue substitutes the conservedHis) present inNrfA a pentaheme c-type cytochrome [103 104] Accordingto secondary structure prediction methods [105] it hasbeen proposed that the periplasmic domain of Pa-CcmI iscomposed of aN-terminal120572-helical region containing at leastthree TPR motifs connected by a disordered linker to a 120572-120573C-terminal regionMultiple sequence analyses and secondarystructure predictionmethods show that the TPR region of Pa-CcmI can be successfully aligned with many TPR-containingproteins including Ec-NrfG [106]

26 System I ApoCyt Chaperoning and Heme AttachmentMechanisms TPR domain-containing proteins are commonto eukaryotes prokaryotes and archaea these proteins aregenerally involved in the assembly of multiprotein complexesand to the chaperoning of unfolded proteins [103 107] Itis therefore plausible that CcmI (or the TPR C-terminal

10 Scientifica

domain of Ec-CcmH) may act to provide a platform for theunfolded apoCyt chaperoning it to the heme attachment sitepresumably located on the WWD domain of CcmF CcmImay thus be considered a component of amembrane-integralmultisubunit heme ligation complex together with CcmFand CcmH as experimentally observed by affinity purifi-cation experiments carried out with Rc-CcmFHI proteins[97 99 108] According to the proposed function of CcmI acritical requirement is represented by its ability to recognizedifferent protein targets over and above apoCyt such asCcmFandCcmHHowever until now direct evidence has been pre-sented only for the interaction of CcmI with apoCyt but thepossibility remains that CcmFHI proteins interact each othervia their TM helices and not via their periplasmic domainsInterestingly both for Pa-CcmI [106] or Rc-CcmI [99] CDspectroscopy experiments carried out on the CcmIapoCytcomplex highlighted major conformational changes at thesecondary structure level It is tempting to speculate on thebasis of these results that in vivo the folding of apoCyt maybe induced by the interaction with CcmI In the case of Paeruginosa System I proteins the binding process betweenPa-CcmI and its target protein apoCyt c551 (Pa-apoCyt) hasbeen studied both at equilibrium and kinetically [106] the119870119863measured for this interaction (in the 120583M range) appeared

to be low enough to ensure apoCyt delivery to the othercomponents of the Ccmmachinery Clearly a major questionconcerns the molecular determinants of such recognitionprocess interestingly both affinity coprecipitation assays[99] and equilibrium and kinetic binding experiments [106]highlighted the role played by the C-terminal 120572-helix of Cytc Similar observations have been made for the interaction ofEc-NrfGwith a peptidemimickingNrfA its apoCyt substrate[103] in this case isothermal titration calorimetry (ITC)experiments indicate that the TPR-domain of NrfG serves asa binding site for the C-terminal motif of NrfA Altogetherthese observations are in agreement with the fact that TPRproteins generally bind to their targets by recognizing theirC-terminal region [107]

The CcmI chaperoning activity has been experimentallysupported for the first time in the case of Pa-CcmI by citratesynthase tests [106] it has been proposed that the observedability to suppress protein aggregation in vitromay reflect thecapacity of CcmI to avoid apoCyt aggregation in vivo Stillanother piece of the Cyt c biogenesis puzzle has been addedrecently by showing that Rc-CcmI is able to interact withapoCcmE either alone or together with its substrate apoCytc2 forming a stable ternary complex in the absence of heme[109] This unexpected observation obtained by reciprocalcopurification experiments provides supporting evidence forthe existence of a large multisubunit complex composed ofCcmFHI andCcmE possibly interactingwith theCcmABCDcomplex It is interesting to note that while in the case ofthe CcmIapoCyt recognition different studies highlightedthe crucial role of the C-terminal helical region of apoCyt(see above) in the case of the apoCcmE apoCyt recognitionthe N-terminal region of apoCyt seems to represent a criticalregion

It is generally accepted that CcmF is the Ccm compo-nent responsible for heme covalent attachment to apoCyt

however as discussed above it is possible that this largemembrane protein plays such a role only together withother Ccm proteins such as CcmH and CcmI Moreover asrecently discovered by Kranz and coworkers [36 41] CcmFmay also act as a quinoleheme oxidoreductase ensuringthe necessary reduction of the oxidized heme b bound toCcmE Why it is necessary that the heme iron be in itsreduced state rather than in its oxidized state is not completelyclear although it is possible that this is a prerequisite to themechanism of thioether bond formation [110] According tocurrent hypotheses it is likely that the periplasmic WWDdomain of CcmF provides a platform for heme b bindingSanders et al and Verissimo et al [34 109] have presenteda mechanistic view of the heme attachment process whichtakes into account all the available experimental observationson the different Ccmproteins According to thismodel stere-ospecific heme ligation to reduced apoCyt occurs becauseonly the vinyl-4 group is available to form the first thioetherbond with a free cysteine at the apoCyt heme-binding motifsince the vinyl-2 group is involved (at least in the Ec-CcmE)in the covalent bond with His130 of CcmE [67] Howeverexperimental proof for this hypothesis requires a detailedinvestigation of the apoCyt thioreduction process catalyzedby CcmH (see Section 24)

It should be noticed that the mechanisms described sofar for the function(s) played by CcmF (see [34 36 109]) donot envisage a clear role for its large C-terminal periplasmicdomain (residues 510 to 611 in Ec-CcmF) It would be inter-esting to see if this domain apparently devoid of recognizablesequence features may mediate intermolecular recognitionprocesses with one (or more) component(s) involved in thehemeapoCyt ligation process

3 System II

System II is typically found in gram-positive bacteria and inin 120576-proteobacteria it is also present in most 120573- and some120575-proteobacteria in Aquificales and cyanobacteria as wellas in algal and plant chloroplasts System II is composedof three or four membrane-bound proteins CcdA ResACcsA (also known as ResC) and CcsB (also known as ResB)(Figure 3) CcdA andResA are redox-active proteins involvedin the reduction of the disulfide bond in the heme-bindingmotif of apoCyt whereas CcsA and CcsB are responsible forthe heme-apoCyt ligation process and are considered Cyt csynthethases (CCS) BothCcsAwhich is evolutionary relatedto the CcmC and CcmF proteins of System I [55] and CcsBare integral membrane proteins In some 120576-proteobacteriasuch asHelicobacter hepaticus andHelicobacter pylori a singlefusion protein composed of CcsA and CcsB polypeptides ispresent [41 111] Although as discussed below evidence hasbeen put forward to support the hypothesis that the CcsBAcomplex acts a heme translocase we still do not know if theheme is transported across the membrane by component(s)of System II itself or by a different unidentified process

31 System II ApoCyt Thioreduction Pathway After the Secmachinery secretes the newly synthesized apoCyt it readily

Scientifica 11

becomes a substrate for the oxidative system present on theouter surface of the cytoplasmic membrane of the gram-positive bacteria In B subtilis the BdbCD system [112113] is functionally but not structurally equivalent to thewell-characterized DsbAB system present in gram-negativebacteria As discussed above for System I the disulfide ofthe apoCyt heme-binding motif must be reduced in orderto allow thioether bonds formation and heme attachmentResA is the extracytoplasmic membrane-anchored TRX-likeprotein involved in the specific reduction of the apoCytdisulfide bond after the disulfide bond exchange reactionhas occurred oxidized ResA is reduced by CcdA whichreceives its reducing equivalents from a cytoplasmic TRX[114] Although ResA displays a classical TRX-like foldthe analysis of the 3D structure of oxidized and reducedResA from B subtilis (Bs-ResA) showed redox-dependentconformational modifications not observed in other TRX-like proteins Interestingly such modifications occur at thelevel of a cavity proposed to represent the binding site foroxidized apoCyt [24 115 116] Another peculiar feature of Bs-ResA is represented by the unusually similar pKa values of itsthe active site Cys residues (88 and 82 resp) as observedalso in the case of the active site Cys residues of the SystemI Pa-CcmH (see Section 23) However at variance with Pa-CcmH in Bs-ResA the large separation between the twocysteine thiols observed in the structure of the reduced formof the protein can be invoked to account for this result

CcdA is a large membrane protein containing six TMhelices [117 118] homologous to the TM domain of E coliDsbD [85 119] Its role in the Cyt c biogenesis is supportedby the observation that inactivation of CcdA blocks theproduction of c-type cytochromes in B subtilis [120 121]Moreover over and above the reduction of its apoCyt sub-strate Bs-CcdA is able to reduce the disulfide bond of othersecreted proteins such as StoA [122] It is interesting to notethat ResA and CcdA are not essential for Cyt c synthesisin the absence of BdbD or BdbC or if a disulfide reductantis present in the growth medium [123 124] As discussedabove (Section 24) a similar observation was made on therole of the thio-reductive pathway of System I in this casepersistent production of c-type cytochromes was observedin Dsb-inactivated bacterial strains [38] Following theseobservations the question has been put forward as to why theCyt c biogenesis process involves this apparently redundantthio-reduction route only to correct the effects of Bdb orDsb activity Referring to the case of B subtilis [111] it hasbeen proposed that the main reason is that BdbD mustefficiently oxidize newly secreted proteins since the activityof most of them depends on the presence of disulfide bondsAlternatively it can be hypothesized that the intramoleculardisulfide bondof apoCyt protects the protein fromproteolyticdegradation or aggregation or from cross-linking to otherthiol-containing proteins

32 System II Heme Translocation and Attachment to ApoCytRecent experimental evidence is accumulating supportingthe involvement of the heterodimeric membrane complexResBC or of the fusion protein CcsBA in the translocation

of heme from the n-side to the p-side of the bacterialmembrane In particular it has been shown that CcsBAfrom H hepaticus (Hh-CcsBA) is able to reconstitute Cyt cbiogenesis in the periplasm of E coli [36] Moreover it wasalso observed that Hh-CcsBA is able to bind reduced hemevia two conserved histidines flanking the WWD domainand required both for the translocation of the heme andfor the synthetase function of Hh-CcsBA [36] A differentstudy carried out on B subtilis System II proteins providedsupport for heme-binding capability by the ResBC complexaccording to the results obtained on recombinant ResBC theResB component of the heterodimeric CCS complex is ableto covalently bind the heme in the cytoplasm (probably by aCys residue) and to deliver it to an extracytoplamic domain ofResC which is responsible for the covalent ligation to apoCyt[117] It should be noticed however that the transfer of theCCS-bound heme to apoCyt still awaits direct experimentalproof Moreover it has also been reported that in B subtilisthe inactivation of CCS does not affect the presence ofother heme-containing proteins in the periplasm such ascytochrome b562 [111] this observation which is in contrastto the heme-transport hypothesis by CCSs parallels similarconcerns about heme translocation mechanism that arecurrently discussed in the context of System I (see Section 22above) In the case of the Hh-CcsBA synthetase site-directedmutagenesis experiments allowed to assign a heme-bindingrole for the two pairs of conserved His residues accordingto the topological model of the protein obtained by alkalinephosphatase (PhoA) assays and GFP fusions the conservedHis77 and His858 residues are located on TM helices whileHis761 and His897 are located on the periplasmic side [36]These observations together with the results of site-directedmutagenesis experiments allowed the authors to suggest thatHis77 andHis858 form a low affinitymdashmembrane embeddedheme-binding site for ferrous heme which is subsequentlytranslocated to an external heme-binding domain of Hh-CcsBA where it is coordinated by His761 and His897 Thetopological model therefore predicts that this last His pairis part of a periplasmic-located WWD domain (homologousto theWWDdomains found in theCcmCandCcmFproteinsof System I described above)

4 System III

Strikingly different from Systems I and II the Cyt c mat-uration apparatus found in fungi and in metazoan cells(System III) is composed of a single protein known as Holo-Cytochrome c Synthetase (HCCS) or Cytochrome c HemeLyase (CCHL) apparently responsible for all the subprocessesdescribed above (heme transport and chaperoning reductionand chaperoning of apoCyt and catalysis of the thioetherbonds formation between heme and apoCyt) (Figure 4)Surprisingly although this protein has been identified in Scerevisiaemitochondria several years ago [125 126] only veryrecently the human HCCS has been expressed as a recombi-nant protein in E coli and spectroscopically characterized invitro [127] It is worth noticing that humanHCCS is attractinginterest since over and above its role in Cyt c biogenesis it

12 Scientifica

is involved in diseases such as microphthalmia with linearskin defects syndrome (MLS) an X-linked genetic disorder[128ndash130] and in other processes such as Cytc-independentapoptosis in injured motor neurons [131] Different fromanimal cells where a single HCCS is sufficient for thematuration of both soluble and membrane-anchored Cytc(Cyt c and Cy c1 resp) in S cerevisiae two homologs HCCSand HCC1S located in the inner mitochondrial membraneand facing to the IMS space [132 133] are responsible for hemeattachment to apoCyt c and apoCyt c1 respectively [125 134]It should also be noticed that in fungi an additional FAD-containing protein (Cyc2p) is required for Cyt c synthesisCyc2p is a mitochondrial membrane-anchored flavoproteinexposing its redox domain to the IMS which is requiredfor the maturation of Cyt c but not for that of Cyt c1 [135]This protein does not contain the conserved Cys residuestypically found in disulfide reductases and indeed it is notable to reduce oxidized apoCyt in vitro However it has beenrecently shown that Cyc2p is able to catalyze the NAD(P)H-dependent reduction of heme in vitro [136] a necessarystep before the thioether bond formation can occur asdiscussed above (see Section 26) This result together withthe observation that Cyc2p interacts with HCCS and withapoCyt c and c1 lends support to the proposal that Cyc2p isinvolved in the reduction of the heme iron in vivo [136 137]

AlthoughHCCS proteins are crucial for Cyt cmaturationwe still do not know how these proteins recognize theirsubstrates (heme and apoCyt) and how they promote orcatalyze the formation of thioether bonds between the hemevinyl groups and the cysteine thiols of the apoCyt CXXCHmotif

Contrary to the broad specificity of System I whichis able to recognize and attach the heme to prokaryoticand eukaryotic c-type cytochromes [49 50] and even tovery short microperoxidase-like sequences [49 50] mito-chondrial HCCS is characterized by a higher specificityas it does not recognize bacterial apoCyt sequences Theseobservations prompted the investigation of the recognitionprocess between apoCyt and HCCS Recently by using arecombinant yeast HCCS and chimerical apoCyt sequencesexpressed in E coli it was possible to conclude that a crucialrecognition determinant is represented by the N-terminalregion of apoCyt containing the heme-binding motif [35]notice that in the context of System I the same N-terminalregion of the Cyt c sequence has been recently identified to beimportant for the recognition by apoCcmE (see Section 26)Over and above the role played by the intervening residuesin the CXXCH heme-binding motif [135] it has been shownthat a conserved Phe residue occurring in the N-terminalregion before the CXXCH motif is important for HCCSrecognition [35 138] These results support the hypothesisthat this residue known to be a key determinant of Cytcfolding and stability [19 20 139] may also be crucial for Cytcmaturation by HCCS

Another relevant question concerns the ability of HCCSto recognize and bind the heme molecule as the region ofthe protein responsible for heme recognition remains to beidentified Initially it was hypothesized that the recognitionof heme could be mediated by the CP motifs present in

the HCCS protein [140] these short sequences are indeedknown to bind heme in a variety of heme-containing proteins[141 142] However it has been recently shown that CPmotifsof the recombinant S cerevisiae HCCS are not necessaryfor Cyt c production in E coli [143] excluding them as keydeterminants of heme recognition

The long-awaited in vitro characterization of the recom-binant human HCCS allowed for the first time to pro-pose a molecular mechanism underlying Cytc maturationin eukaryotes which can be experimentally tested [127]According to the proposed model the human HCCS activitycan be described as a four step mechanism involving (i)heme-binding (ii) apoCyt recognition (iii) thioether bondsformation and (iv) holoCyt c release In particular theheme is proposed to play the role of a scaffolding moleculemediating the contacts between HCCS and apoCyt Muta-genesis experiments carried out on the recombinant HCCSstrongly suggest that heme-binding (Step 1) depends on thepresence of a specific His residue (His154) acting as anaxial ligand to ferrous heme Residues present at the N-terminus of apoCyt mediate the recognition with HCCS(Step 2) as discussed above it is known that this regionforms structurally conserved 120572-helix in the fold of all c-type cytochromes Unfortunately no information is availableup to now concerning the region of HCCS involved in therecognition and binding to the N-terminal region of apoCytCoordination of the heme iron by the His residue of theapoCyt heme-binding motif CXXCH provides the secondaxial ligand to the heme iron and is probably importantfor the correct positioning of the two apoCyt Cys residuesand formation of the thioether bonds (Step 3) The finalrelease of functional Cyt c (Step 4) clearly requires thedisplacement of the His154Fe2+ coordination bond sucha displacement is probably mediated by formation of thecoordination bond with the Cyt c conserved Met residueandor by the simultaneous folding of Cyt c Again it isinteresting to note that this last hypothesis is in accordancewith in vitro folding studies on c-type cytochromes thathighlighted the late formation of the Met-Fe coordinationduring Cyt c folding [144 145]

Acknowledgments

The author thanks A Di Matteo and E Di Silvio for fruitfuldiscussions and for help in preparing this paper This work issupported by grants from the University of Rome ldquoSapienzardquo(C26A11MBXJ and C26A13MWF4)

References

[1] M G Galinato J G Kleingardner S E Bowman et alldquoHeme-protein vibrational couplings in cytochrome c providea dynamic link that connects the heme-iron and the proteinsurfacerdquo Proceedings of the National Academy of Sciences vol109 no 23 pp 8896ndash8900 2012

[2] H B Gray and J R Winkler ldquoElectron flow through metal-loproteinsrdquo Biochimica et Biophysica Acta vol 1797 no 9 pp1563ndash1572 2010

Scientifica 13

[3] P Ascenzi R Santucci M Coletta and F PolticellildquoCytochromes reactivity of the ldquodark siderdquo of the hemerdquoBiophysical Chemistry vol 152 no 1ndash3 pp 21ndash27 2010

[4] S Yamada N D Bouley Ford G E Keller W C Ford HB Gray and J R Winkler ldquoSnapshots of a protein foldingintermediaterdquo Proceedings of the National Academy of Sciencesvol 110 no 5 pp 1606ndash1610 2013

[5] P Weinkam J Zimmermann F E Romesberg and P GWolynes ldquoThe folding energy landscape and free energy excita-tions of cytochrome crdquo Accounts of Chemical Research vol 43no 5 pp 652ndash660 2010

[6] M Yamanaka M Masanari and Y Sambongi ldquoConfermentof folding ability to a naturally unfolded apocytochrome cthrough introduction of hydrophobic amino acid residuesrdquoBiochemistry vol 50 no 12 pp 2313ndash2320 2011

[7] G W Moore and G W Pettigrew Cytochrome C EvolutionaryStructural and Physicochemical Aspects Springer Berlin Ger-many 1990

[8] I Bertini G Cavallaro and A Rosato ldquoCytochrome c occur-rence and functionsrdquo Chemical Reviews vol 106 no 1 pp 90ndash115 2006

[9] D A Pearce and F Sherman ldquoDegradation of cytochromeoxidase subunits in mutants of yeast lacking cytochrome c andsuppression of the degradation by mutation of yme1rdquo Journal ofBiological Chemistry vol 270 no 36 pp 20879ndash20882 1995

[10] G Silkstone S M Kapetanaki I Husu M H Vos and MT Wilson ldquoNitric oxide binding to the cardiolipin complex offerric cytochrome crdquo Biochemistry vol 51 no 34 pp 6760ndash6766 2012

[11] M Giorgio E Migliaccio F Orsini et al ldquoElectron transferbetween cytochrome c and p66Shc generates reactive oxygenspecies that trigger mitochondrial apoptosisrdquo Cell vol 122 no2 pp 221ndash233 2005

[12] S M Kilbride and J H Prehn ldquoCentral roles of apoptoticproteins in mitochondrial functionrdquo Oncogene vol 32 no 22pp 2703ndash2711 2013

[13] T Noll and G Noll ldquoStrategies for ldquowiringrdquo redox-activeproteins to electrodes and applications in biosensors biofuelcells and nanotechnologyrdquo Chemical Society Reviews vol 40no 7 pp 3564ndash3576 2011

[14] G Layer J Reichelt D Jahn and D W Heinz ldquoStructure andfunction of enzymes in heme biosynthesisrdquo Protein Science vol19 no 6 pp 1137ndash1161 2010

[15] S Severance and IHamza ldquoTrafficking of heme and porphyrinsin metazoardquo Chemical Reviews vol 109 no 10 pp 4596ndash46162009

[16] P Hamel V Corvest P Giege and G Bonnard ldquoBiochemi-cal requirements for the maturation of mitochondrial c-typecytochromesrdquo Biochimica et Biophysica Acta vol 1793 no 1 pp125ndash138 2009

[17] W R Fisher H Taniuchi and C B Anfinsen ldquoOn the role ofheme in the formation of the structure of cytochrome crdquo Journalof Biological Chemistry vol 248 no 9 pp 3188ndash3195 1973

[18] M Yamanaka H Mita Y Yamamoto and Y Sambongi ldquoHemeis not required for aquifex aeolicus cytochrome c555 polypep-tide foldingrdquoBioscience Biotechnology andBiochemistry vol 73no 9 pp 2022ndash2025 2009

[19] C Travaglini-Allocatelli S Gianni andMBrunori ldquoA commonfolding mechanism in the cytochrome c familyrdquo Trends inBiochemical Sciences vol 29 no 10 pp 535ndash541 2004

[20] A Borgia D Bonivento C Travaglini-Allocatelli A DiMatteoand M Brunori ldquoUnveiling a hidden folding intermediate in c-type cytochromes by protein engineeringrdquo Journal of BiologicalChemistry vol 281 no 14 pp 9331ndash9336 2006

[21] E Enggist L Thony-Meyer P Guntert and K PervushinldquoNMR structure of the heme chaperone CcmE reveals a novelfunctional motifrdquo Structure vol 10 no 11 pp 1551ndash1557 2002

[22] A di Matteo N Calosci S Gianni P Jemth M Brunori and CTravaglini-Allocatelli ldquoStructural and functional characteriza-tion of CcmG from pseudomonas aeruginosa a key componentof the bacterial cytochrome c maturation apparatusrdquo Proteinsvol 78 no 10 pp 2213ndash2221 2010

[23] A diMatteo S GianniM E Schinina et al ldquoA strategic proteinin cytochrome c maturation three-dimensional structure ofCcmH and binding to apocytochrome crdquo Journal of BiologicalChemistry vol 282 no 37 pp 27012ndash27019 2007

[24] A Crow R M Acheson N E Le Brun and A OubrieldquoStructural basis of redox-coupled protein substrate selectionby the cytochrome c biosynthesis protein ResArdquo Journal ofBiological Chemistry vol 279 no 22 pp 23654ndash23660 2004

[25] E di Silvio A di Matteo and C Travaglini-Allocatelli ldquoTheRedox pathway of Pseudomonas aeruginosa cytochrome cbiogenesisrdquo Journal of Proteins and Proteomics vol 3 no 1 pp1ndash7 2012

[26] LThony-Meyer andP Kunzler ldquoTranslocation to the periplasmand signal sequence cleavage of preapocytochrome c depend onsec and lep but not on the ccmgene productsrdquoEuropean Journalof Biochemistry vol 246 no 3 pp 794ndash799 1997

[27] S J Facey and A Kuhn ldquoBiogenesis of bacterial inner-membrane proteinsrdquo Cellular and Molecular Life Sciences vol67 no 14 pp 2343ndash2362 2010

[28] K Diekert A I de Kroon U Ahting et al ldquoApocytochromec requires the TOM complex for translocation across themitochondrial outer membranerdquo The EMBO Journal vol 20no 20 pp 5626ndash5635 2001

[29] N Wiedemann V Kozjak T Prinz et al ldquoBiogenesis of yeastmitochondrial cytochrome c a unique relationship to the TOMmachineryrdquo Journal ofMolecular Biology vol 327 no 2 pp 465ndash474 2003

[30] F-U Hartl N Pfanner and W Neupert ldquoTranslocation inter-mediates on the import pathway of proteins intomitochondriardquoBiochemical Society Transactions vol 15 no 1 pp 95ndash97 1987

[31] B S Glick A Brandt K Cunningham SMuller R L Hallbergand G Schatz ldquoCytochromes c1 and b2 are sorted to theintermembrane space of yeast mitochondria by a stop-transfermechanismrdquo Cell vol 69 no 5 pp 809ndash822 1992

[32] G Howe and S Merchant ldquoRole of heme in the biosynthesis ofcytochrome c6rdquo Journal of Biological Chemistry vol 269 no 8pp 5824ndash5832 1994

[33] S S Nakamoto P Hamel and S Merchant ldquoAssembly ofchloroplast cytochromes b and crdquo Biochimie vol 82 no 6-7 pp603ndash614 2000

[34] C Sanders S Turkarslan D-W Lee and F DaldalldquoCytochrome c biogenesis the Ccm systemrdquo Trends inMicrobiology vol 18 no 6 pp 266ndash274 2010

[35] J M Stevens D A Mavridou R Hamer P Kritsiligkou A DGoddard and S J Ferguson ldquoCytochrome c biogenesis systemIrdquoThe FEBS Journal vol 278 no 22 pp 4170ndash4178 2011

[36] R G Kranz C Richard-Fogal J-S Taylor and E R FrawleyldquoCytochrome c biogenesis mechanisms for covalent modifica-tions and trafficking of heme and for heme-iron redox controlrdquo

14 Scientifica

Microbiology and Molecular Biology Reviews vol 73 no 3 pp510ndash528 2009

[37] J W A Allen ldquoCytochrome c biogenesis in mitochondriamdashsystems III and VrdquoThe FEBS Journal vol 278 no 22 pp 4198ndash4216 2011

[38] D A Mavridou S J Ferguson and J M Stevens ldquoCytochromec assemblyrdquo IUBMB Life vol 65 no 3 pp 209ndash216 2013

[39] I Bertini G Cavallaro and A Rosato ldquoEvolution ofmitochondrial-type cytochrome c domains and of theprotein machinery for their assemblyrdquo Journal of InorganicBiochemistry vol 101 no 11-12 pp 1798ndash1811 2007

[40] B S Goldman and R G Kranz ldquoEvolution and horizontaltransfer of an entire biosynthetic pathway for cytochromec biogenesis helicobacter deinococcus archae and morerdquoMolecular Microbiology vol 27 no 4 pp 871ndash873 1998

[41] C L Richard-Fogal E R Frawley E R Bonner H Zhu BSan Francisco and R G Kranz ldquoA conserved haem redoxand trafficking pathway for cofactor attachmentrdquo The EMBOJournal vol 28 no 16 pp 2349ndash2359 2009

[42] L Thony-Meyer F Fischer P Kunzler D Ritz and H Hen-necke ldquoEscherichia coli genes required for cytochrome cmaturationrdquo Journal of Bacteriology vol 177 no 15 pp 4321ndash4326 1995

[43] C S Beckett J A Loughman K A Karberg G M DonatoW E Goldman and R G Kranz ldquoFour genes are required forthe system II cytochrome c biogenesis pathway in Bordetellapertussis a unique bacterial modelrdquo Molecular Microbiologyvol 38 no 3 pp 465ndash481 2000

[44] N E Le Brun J Bengtsson and L Hederstedt ldquoGenes requiredfor cytochrome c synthesis in Bacillus subtilisrdquo MolecularMicrobiology vol 36 no 3 pp 638ndash650 2000

[45] P Giege J M Grienenberger and G Bonnard ldquoCytochromec biogenesis in mitochondriardquo Mitochondrion vol 8 no 1 pp61ndash73 2008

[46] R E Feissner C L Richard-Fogal E R Frawley J A Lough-man KW Earley and R G Kranz ldquoRecombinant cytochromesc biogenesis systems I and II and analysis of haem deliverypathways in Escherichia colirdquo Molecular Microbiology vol 60no 3 pp 563ndash577 2006

[47] N P Cianciotto P Cornelis and C Baysse ldquoImpact of thebacterial type I cytochrome c maturation system on differentbiological processesrdquoMolecular Microbiology vol 56 no 6 pp1408ndash1415 2005

[48] E Arslan H Schulz R Zufferey P Kunzler and L Thony-Meyer ldquoOverproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in Escherichiacolirdquo Biochemical and Biophysical Research Communicationsvol 251 no 3 pp 744ndash747 1998

[49] C Sanders and H Lill ldquoExpression of prokaryotic and eukary-otic cytochromes c in Escherichia colirdquoBiochimica et BiophysicaActa vol 1459 no 1 pp 131ndash138 2000

[50] M Braun and L Thony-Meyer ldquoBiosynthesis of artificialmicroperoxidases by exploiting the secretion and cytochromec maturation apparatuses of Escherichia colirdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 35 pp 12830ndash12835 2004

[51] B Baert C Baysse S Matthijs and P Cornelis ldquoMultiplephenotypic alterations caused by a c-type cytochrome mat-uration ccmC gene mutation in Pseudomonas aeruginosardquoMicrobiology vol 154 no 1 pp 127ndash138 2008

[52] S Turkarslan C Sanders and F Daldal ldquoExtracytoplasmicprosthetic group ligation to apoproteins maturation of c-typecytochromesrdquo Molecular Microbiology vol 60 no 3 pp 537ndash541 2006

[53] P M Jones and A M George ldquoThe ABC transporter structureand mechanism perspectives on recent researchrdquo Cellular andMolecular Life Sciences vol 61 no 6 pp 682ndash699 2004

[54] O Christensen E M Harvat L Thony-Meyer S J Fergusonand J M Stevens ldquoLoss of ATP hydrolysis activity by CcmABresults in loss of c-type cytochrome synthesis and incompleteprocessing ofCcmErdquoTheFEBS Journal vol 274 no 9 pp 2322ndash2332 2007

[55] J-H Lee E M Harvat J M Stevens S J Ferguson and M HSaier Jr ldquoEvolutionary origins of members of a superfamily ofintegralmembrane cytochrome c biogenesis proteinsrdquoBiochim-ica et Biophysica Acta vol 1768 no 9 pp 2164ndash2181 2007

[56] D L Beckman D R Trawick and R G Kranz ldquoBacterialcytochromes c biogenesisrdquo Genes and Development vol 6 no2 pp 268ndash283 1992

[57] H Schulz R A Fabianek E C Pellicioli H Hennecke andL Thony-Meyer ldquoHeme transfer to the heme chaperone CcmEduring cytochrome c maturation requires the CcmC proteinwhich may function independently of the ABC-transporterCcmABrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 96 no 11 pp 6462ndash6467 1999

[58] Q Ren and L Thony-Meyer ldquoPhysical Interaction ofCcmC with Heme and the Heme Chaperone CcmE duringCytochrome cMaturationrdquo Journal of Biological Chemistry vol276 no 35 pp 32591ndash32596 2001

[59] C Richard-Fogal and R G Kranz ldquoThe CcmChemeCcmEcomplex in heme trafficking and cytochrome c biosynthesisrdquoJournal of Molecular Biology vol 401 no 3 pp 350ndash362 2010

[60] V K Viswanathan S Kurtz L L Pedersen et al ldquoThecytochrome C maturation locus of Legionella pneumophilapromotes iron assimilation and intracellular infection andcontains a strain-specific insertion sequence elementrdquo Infectionand Immunity vol 70 no 4 pp 1842ndash1852 2002

[61] U Ahuja and L Thony-Meyer ldquoCcmD is involved in complexformation between CcmC and the heme chaperone CcmE dur-ing cytochrome c maturationrdquo Journal of Biological Chemistryvol 280 no 1 pp 236ndash243 2005

[62] C L Richard-Fogal E R Frawley and R G Kranz ldquoTopologyand function of CcmD in cytochrome Cmaturationrdquo Journal ofBacteriology vol 190 no 10 pp 3489ndash3493 2008

[63] H Schulz H Hennecke and L Thony-Meyer ldquoPrototype ofa heme chaperone essential for cytochrome c maturationrdquoScience vol 281 no 5380 pp 1197ndash1200 1998

[64] F Arnesano L Banci P D Barker et al ldquoSolution structure andcharacterization of the heme chaperone CcmErdquo Biochemistryvol 41 no 46 pp 13587ndash13594 2002

[65] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[66] L Thony-Meyer ldquoA heme chaperone for cytochrome c biosyn-thesisrdquo Biochemistry vol 42 no 45 pp 13099ndash13105 2003

[67] E M Harvat C Redfield J M Stevens and S J FergusonldquoProbing the heme-binding site of the cytochrome cmaturationprotein CcmErdquoBiochemistry vol 48 no 8 pp 1820ndash1828 2009

[68] D Lee K Pervushin D Bischof M Braun and L Thony-Meyer ldquoUnusual heme-histidine bond in the active site of a

Scientifica 15

chaperonerdquo Journal of the American Chemical Society vol 127no 11 pp 3716ndash3717 2005

[69] A D Goddard J M Stevens F Rao et al ldquoc-Type cytochromebiogenesis can occur via a natural Ccm system lacking CcmHCcmG and the heme-binding histidine of CcmErdquo Journal ofBiological Chemistry vol 285 no 30 pp 22882ndash22889 2010

[70] J M Aramini K Hamilton P Rossi et al ldquoSolution NMRstructure backbone dynamics and heme-binding propertiesof a novel cytochrome c maturation protein CcmE fromDesulfovibrio vulgarisrdquo Biochemistry vol 51 no 18 pp 3705ndash3707 2012

[71] T Uchida J M Stevens O Daltrop et al ldquoThe interactionof covalently bound heme with the cytochrome c maturationprotein CcmErdquo Journal of Biological Chemistry vol 279 no 50pp 51981ndash51988 2004

[72] I Garcıa-Rubio M Braun I Gromov L Thony-Meyer andA Schweiger ldquoAxial coordination of heme in ferric CcmEchaperone characterized by EPR spectroscopyrdquo BiophysicalJournal vol 92 no 4 pp 1361ndash1373 2007

[73] L Thony-Meyer ldquoHaem-polypeptide interactions duringcytochrome c maturationrdquo Biochimica et Biophysica Acta vol1459 no 2-3 pp 316ndash324 2000

[74] J A Keightley D Sanders T R Todaro A Pastuszyn and J AFee ldquoCloning expression in Escherichia coli of the cytochromec552 gene from Thermus thermophilus HB8 Evidence forgenetic linkage to an ATP- binding cassette protein and initialcharacterization of the cycA gene productsrdquo Journal of Biologi-cal Chemistry vol 273 no 20 pp 12006ndash12016 1998

[75] E M Monika B S Goldman D L Beckman and R GKranz ldquoA thioreduction pathway tethered to the membranefor periplasmic cytochromes c biogenesis In vitro and in vivostudiesrdquo Journal of Molecular Biology vol 271 no 5 pp 679ndash692 1997

[76] M Throne-Holst L Thony-Meyer and L HederstedtldquoEscherichia coli ccm in-frame deletion mutants can produceperiplasmic cytochrome b but not cytochrome crdquo FEBS Lettersvol 410 no 2-3 pp 351ndash355 1997

[77] U Ahuja and L Thony-Meyer ldquoDynamic features of a hemedelivery system for cytochrome c maturationrdquo Journal of Bio-logical Chemistry vol 278 no 52 pp 52061ndash52070 2003

[78] J W A Allen P D Barker O Daltrop et al ldquoWhy isnrsquot ldquostan-dardrdquo heme good enough for c-type and di-type cytochromesrdquoDalton Transactions no 21 pp 3410ndash3418 2005

[79] K Inaba and K Ito ldquoStructure and mechanisms of the DsbB-DsbA disulfide bond generation machinerdquo Biochimica et Bio-physica Acta vol 1783 no 4 pp 520ndash529 2008

[80] H Kadokura F Katzen and J Beckwith ldquoProtein disulfidebond formation in prokaryotesrdquoAnnual Review of Biochemistryvol 72 pp 111ndash135 2003

[81] S R Shouldice B Heras P M Walden M Totsika M ASchembri and J L Martin ldquoStructure and function of DsbA akey bacterial oxidative folding catalystrdquoAntioxidants and RedoxSignaling vol 14 no 9 pp 1729ndash1760 2011

[82] R Metheringham L Griffiths H Crooke S Forsythe and JCole ldquoAn essential role for DsbA in cytochrome c synthesis andformate-dependent nitrite reduction by Escherichia coli K-12rdquoArchives of Microbiology vol 164 no 4 pp 301ndash307 1995

[83] Y Sambongi and S J Ferguson ldquoMutants of Escherichia colilacking disulphide oxidoreductases DsbA and DsbB cannotsynthesise an exogenous monohaem c-type cytochrome exceptin the presence of disulphide compoundsrdquo FEBS Letters vol398 no 2-3 pp 265ndash268 1996

[84] D AMavridou S J Ferguson and JM Stevens ldquoThe interplaybetween the disulfide bond formation pathway and cytochromec maturation in Escherichia colirdquo FEBS Letters vol 586 no 12pp 1702ndash1707 2012

[85] C U Stirnimann A Rozhkova U Grauschopf M G GrutterR Glockshuber and G Capitani ldquoStructural basis and kineticsof DsbD-dependent cytochrome c maturationrdquo Structure vol13 no 7 pp 985ndash993 2005

[86] N Ouyang Y-G Gao H-Y Hu and Z-X Xia ldquoCrystalstructures of E coli CcmGand itsmutants reveal key roles of theN-terminal120573-sheet and the fingerprint regionrdquoProteins vol 65no 4 pp 1021ndash1031 2006

[87] M A Edeling L W Guddat R A Fabianek et al ldquoCrys-tallization and preliminary diffraction studies of native andselenomethionine CcmG (CycY DsbE)rdquoActa CrystallographicaD vol 57 no 9 pp 1293ndash1295 2001

[88] R A Fabianek M Huber-Wunderlich R Glockshuber PKunzler H Hennecke and L Thony-Meyer ldquoCharacterizationof the Bradyrhizobium japonicum CycY protein a membrane-anchored periplasmic thioredoxin that may play a role as areductant in the biogenesis of c-type cytochromesrdquo Journal ofBiological Chemistry vol 272 no 7 pp 4467ndash4473 1997

[89] G B Kallis and A Holmgren ldquoDifferential reactivity of thefunctional sulfhydryl groups of cysteine-32 and cysteine-32present in the reduced form of thioredoxin from Escherichiacolirdquo Journal of Biological Chemistry vol 255 no 21 pp 10261ndash10265 1980

[90] P T Chivers andR T Raines ldquoGeneral acidbase catalysis in theactive site of Escherichia coli thioredoxinrdquoBiochemistry vol 36no 50 pp 15810ndash15816 1997

[91] X M Zheng J Hong H Y Li D H Lin and H Y HuldquoBiochemical properties and catalytic domain structure of theCcmH protein from Escherichia colirdquo Biochim Biophys Actavol 1824 no 12 pp 1394ndash1400 2012

[92] UAhujaA Rozhkova RGlockshuber LThony-Meyer andOEinsle ldquoHelix swapping leads to dimerization of the N-terminaldomain of the c-type cytochrome maturation protein CcmHfrom Escherichia colirdquo FEBS Letters vol 582 no 18 pp 2779ndash2786 2008

[93] R A Fabianek T Hofer and L Thony-Meyer ldquoCharacteriza-tion of the Escherichia coli CcmH protein reveals new insightsinto the redox pathway required for cytochrome c maturationrdquoArchives of Microbiology vol 171 no 2 pp 92ndash100 1999

[94] E Reid J Cole and D J Eaves ldquoThe Escherichia coli CcmGprotein fulfils a specific role in cytochrome c assemblyrdquo Bio-chemical Journal vol 355 no 1 pp 51ndash58 2001

[95] Q Ren U Ahuja and LThony-Meyer ldquoA bacterial cytochromec heme lyase CcmF forms a complex with the heme chaperoneCcmE and CcmH but not with apocytochrome crdquo Journal ofBiological Chemistry vol 277 no 10 pp 7657ndash7663 2002

[96] E H Meyer P Giege E Gelhaye et al ldquoAtCCMH an essentialcomponent of the c-type cytochrome maturation pathway inArabidopsis mitochondria interacts with apocytochrome crdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 44 pp 16113ndash16118 2005

[97] C Rios-Velazquez R Coller and T J Donohue ldquoFeatures ofRhodobacter sphaeroides CcmFHrdquo Journal of Bacteriology vol185 no 2 pp 422ndash431 2003

[98] C Sanders M Deshmukh D Astor R G Kranz and F DaldalldquoOverproduction of CcmG and CcmFHRc fully suppresses thec-type cytochrome biogenesis defect of Rhodobacter capsulatus

16 Scientifica

CcmI-null mutantsrdquo Journal of Bacteriology vol 187 no 12 pp4245ndash4256 2005

[99] A F Verissimo H Yang X Wu C Sanders and F DaldalldquoCcmI subunit of CcmFHI heme ligation complex functionsas an apocytochrome c chaperone during c-type cytochromematurationrdquo Journal of Biological Chemistry vol 286 no 47 pp40452ndash40463 2011

[100] K Bryson L J McGuffin R L Marsden J J Ward J SSodhi and D T Jones ldquoProtein structure prediction servers atUniversity College Londonrdquo Nucleic Acids Research vol 33 no2 pp W36ndashW38 2005

[101] D T Jones ldquoProtein secondary structure prediction basedon position-specific scoring matricesrdquo Journal of MolecularBiology vol 292 no 2 pp 195ndash202 1999

[102] C Sanders C Boulay and F Daldal ldquoMembrane-spanning andperiplasmic segments of CcmI have distinct functions duringcytochrome c biogenesis in Rhodobacter capsulatusrdquo Journal ofBacteriology vol 189 no 3 pp 789ndash800 2007

[103] D Han K Kim J Oh J Park and Y Kim ldquoTPR domainof NrfG mediates complex formation between heme lyaseand formate-dependent nitrite reductase in Escherichia coliO157H7rdquo Proteins vol 70 no 3 pp 900ndash914 2008

[104] D J Eaves J Grove W Staudenmann et al ldquoInvolvement ofproducts of the nrfEFG genes in the covalent attachment ofhaem c to a novel cysteine-lysine motif in the cytochrome c552nitrite reductase from Escherichia colirdquo Molecular Microbiol-ogy vol 28 no 1 pp 205ndash216 1998

[105] J Nilsson B Persson and G Von Heijne ldquoConsensus predic-tions of membrane protein topologyrdquo FEBS Letters vol 486 no3 pp 267ndash269 2000

[106] E di Silvio A di Matteo F Malatesta and C Travaglini-Allocatelli ldquoRecognition and binding of apocytochrome c toP aeruginosa CcmI a component of cytochrome c maturationmachineryrdquo Biochim Biophys Acta vol 1834 no 8 pp 1554ndash1561 2013

[107] N Zeytuni and R Zarivach ldquoStructural and functional dis-cussion of the tetra-trico-peptide repeat a protein interactionmodulerdquo Structure vol 20 no 3 pp 397ndash405 2012

[108] C Sanders S Turkarslan D-W Lee O Onder R G Kranz andF Daldal ldquoThe cytochrome c maturation components CcmFCcmH and CcmI form a membrane-integral multisubunitheme ligation complexrdquo Journal of Biological Chemistry vol283 no 44 pp 29715ndash29722 2008

[109] A F Verissimo M A Mohtar and F Daldal ldquoThe hemechaperone ApoCcmE forms a ternary complex with CcmI andapocytochrome crdquoThe Journal of Biological Chemistry vol 288no 9 pp 6272ndash6283 2013

[110] P D Barker J C Ferrer M Mylrajan et al ldquoTransmutation of aheme proteinrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 90 no 14 pp 6542ndash65461993

[111] J Simon and L Hederstedt ldquoComposition and function ofcytochrome c biogenesis system IIrdquoThe FEBS Journal vol 278no 22 pp 4179ndash4188 2011

[112] T R H M Kouwen and J M van Dijl ldquoInterchangeablemodules in bacterial thiol-disulfide exchange pathwaysrdquo Trendsin Microbiology vol 17 no 1 pp 6ndash12 2009

[113] A Crow A Lewin O Hecht et al ldquoCrystal structure andbiophysical properties of Bacillus subtilis BdbD An oxidizingthioldisulfide oxidoreductase containing a novel metal siterdquoJournal of Biological Chemistry vol 284 no 35 pp 23719ndash23733 2009

[114] M C Moller and L Hederstedt ldquoExtracytoplasmic processesimpaired by inactivation of trxA (thioredoxin gene) in Bacillussubtilisrdquo Journal of Bacteriology vol 190 no 13 pp 4660ndash46652008

[115] A Lewin A Crow A Oubrie and N E Le Brun ldquoMolecularbasis for specificity of the extracytoplasmic thioredoxin ResArdquoJournal of Biological Chemistry vol 281 no 46 pp 35467ndash35477 2006

[116] C L Colbert QWu P J A Erbel K H Gardner and J Deisen-hofer ldquoMechanism of substrate specificity in Bacillus subtilisResA a thioredoxin-like protein involved in cytochrome cmaturationrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 103 no 12 pp 4410ndash44152006

[117] UAhuja P Kjelgaard B L Schulz LThony-Meyer andLHed-erstedt ldquoHaem-delivery proteins in cytochrome c maturationsystem IIrdquoMolecular Microbiology vol 73 no 6 pp 1058ndash10712009

[118] M L D Page P P Hamel S T Gabilly et al ldquoA homologof prokaryotic thiol disulfide transporter CcdA is required forthe assembly of the cytochrome b6f complex in Arabidopsischloroplastsrdquo Journal of Biological Chemistry vol 279 no 31pp 32474ndash32482 2004

[119] A Rozhkova C U Stirnimann P Frei et al ldquoStructural basisand kinetics of inter- and intramolecular disulfide exchange inthe redox catalyst DsbDrdquoThe EMBO Journal vol 23 no 8 pp1709ndash1719 2004

[120] T Schiott M Throne-Holst and L Hederstedt ldquoBacillussubtilis CcdA-defective mutants are blocked in a late step ofcytochrome C biogenesisrdquo Journal of Bacteriology vol 179 no14 pp 4523ndash4529 1997

[121] R E Feissner C S Beckett J A Loughman and R G KranzldquoMutations in cytochrome assembly and periplasmic redoxpathways in Bordetella pertussisrdquo Journal of Bacteriology vol187 no 12 pp 3941ndash3949 2005

[122] L S ErlendssonMMoller and L Hederstedt ldquoBacillus subtilisStoA is a thiol-disulfide oxidoreductase important for sporecortex synthesisrdquo Journal of Bacteriology vol 186 no 18 pp6230ndash6238 2004

[123] L S Erlendsson R M Acheson L Hederstedt and N E LeBrun ldquoBacillus subtilis ResA is a thiol-disulfide oxidoreductaseinvolved in cytochrome c synthesisrdquo Journal of BiologicalChemistry vol 278 no 20 pp 17852ndash17858 2003

[124] L S Erlendsson and L Hederstedt ldquoMutations in the thiol-disulfide oxidoreductases BdbC and BdbD can suppresscytochrome c deficiency of CcdA-defective Bacillus subtiliscellsrdquo Journal of Bacteriology vol 184 no 5 pp 1423ndash1429 2002

[125] M E Dumont J F Ernst D M Hampsey and F ShermanldquoIdentification and sequence of the gene encoding cytochromec heme lyase in the yeast Saccharomyces cerevisiaerdquoThe EMBOJournal vol 6 no 1 pp 235ndash241 1987

[126] M E Dumont J F Ernst and F Sherman ldquoCoupling of hemeattachment to import of cytochrome c into yeast mitochondriaStudies with heme lyase-deficient mitochondria and alteredapocytochromes crdquo Journal of Biological Chemistry vol 263 no31 pp 15928ndash15937 1988

[127] B San Francisco E C Bretsnyder and R G Kranz ldquoHumanmitochondrial holocytochrome c synthasersquos hemebindingmat-uration determinants and complex formationwith cytochromecrdquo Proceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 9 pp E788ndashE797 2012

Scientifica 17

[128] L Schaefer A Ballabio and H Y Zoghbi ldquoCloning andcharacterization of a putative human holocytochrome c-typesynthetase gene (HCCS) isolated from the critical region formicrophthalmia with linear skin defects (MLS)rdquoGenomics vol34 no 2 pp 166ndash172 1996

[129] S K Prakash T A Cormier A E McCall et al ldquoLoss ofholocytochrome c-type synthetase causes the male lethality ofX-linked dominant microphthalmia with linear skin defects(MLS) syndromerdquo Human Molecular Genetics vol 11 no 25pp 3237ndash3248 2002

[130] I Wimplinger M Morleo G Rosenberger et al ldquoMutations ofthe mitochondrial holocytochrome c-type synthase in X-linkeddominant microphthalmia with linear skin defects syndromerdquoTheAmerican Journal of HumanGenetics vol 79 no 5 pp 878ndash889 2006

[131] S Kiryu-Seo KGamo T Tachibana K Tanaka andHKiyamaldquoUnique anti-apoptotic activity of EAAC1 in injured motorneuronsrdquoTheEMBO Journal vol 25 no 14 pp 3411ndash3421 2006

[132] R Lill R A Stuart M E Drygas F E Nargang and W Neu-pert ldquoImport of cytochrome c heme lyase into mitochondriaa novel pathway into the intermembrane spacerdquo The EMBOJournal vol 11 no 2 pp 449ndash456 1992

[133] H Steiner A Zollner A Haid W Neupert and R LillldquoBiogenesis of mitochondrial heme lyases in yeast Importand folding in the intermembrane spacerdquo Journal of BiologicalChemistry vol 270 no 39 pp 22842ndash22849 1995

[134] A Zollner G Rodel and A Haid ldquoMolecular cloning andcharacterization of the Saccharomyces cerevisiae CYT2 geneencoding cytochrome-c1-heme lyaserdquo European Journal of Bio-chemistry vol 207 no 3 pp 1093ndash1100 1992

[135] V Corvest D A Murrey D G Bernard D B Knaff B Guiardand P PHamel ldquoc-Type cytochrome assembly in Saccharomycescerevisiae a key residue for apocytochrome c1lyase interactionrdquoGenetics vol 186 no 2 pp 561ndash571 2010

[136] V Corvest D A Murrey M Hirasawa D B Knaff B Guiardand P P Hamel ldquoThe flavoprotein Cyc2p a mitochondrialcytochrome c assembly factor is a NAD(P)H-dependent haemreductaserdquo Molecular Microbiology vol 83 no 5 pp 968ndash9802012

[137] D G Bernard S Quevillon-Cheruel S Merchant B Guiardand P P Hamel ldquoCyc2p a membrane-bound flavopro-tein involved in the maturation of mitochondrial c-typecytochromesrdquo Journal of Biological Chemistry vol 280 no 48pp 39852ndash39859 2005

[138] J G Kleingardner and K L Bren ldquoComparing substratespecificity between cytochrome cmaturation and cytochrome cheme lyase systems for cytochrome c biogenesisrdquo Metallomicsvol 3 no 4 pp 396ndash403 2011

[139] C Travaglini-Allocatelli S Gianni VMorea A Tramontano TSoulimane and M Brunori ldquoExploring the cytochrome c fold-ing mechanism cytochrome c552 fromThermus thermophilusfolds through an on-pathway intermediaterdquo Journal of BiologicalChemistry vol 278 no 42 pp 41136ndash41140 2003

[140] L Zhang and L Guarente ldquoHeme binds to a short sequencethat serves a regulatory function in diverse proteinsrdquoTheEMBOJournal vol 14 no 2 pp 313ndash320 1995

[141] S Hira T Tomita T Matsui K Igarashi and M Ikeda-SaitoldquoBach1 a heme-dependent transcription factor reveals pres-ence of multiple heme binding sites with distinct coordinationstructurerdquo IUBMB Life vol 59 no 8-9 pp 542ndash551 2007

[142] T K Kuhl A Wiszligbrock N Goradia et al ldquoAnalysis of Fe(III)heme binding to cysteine-containing heme-regulatory motifs

in proteinsrdquo ACS Chemical Biology vol 8 no 8 pp 1785ndash17932013

[143] R L Moore J M Stevens and S J Ferguson ldquoMitochondrialcytochrome c synthase CP motifs are not necessary for hemeattachment to apocytochrome crdquo FEBS Letters vol 585 no 21pp 3415ndash3419 2011

[144] S Gianni M Brunori and C Travaglini-Allocatelli ldquoRefoldingkinetics of cytochrome c551 reveals a mechanistic differencebetween urea and guanidinerdquo Protein Science vol 10 no 8 pp1685ndash1688 2001

[145] S Gianni C Travaglini-Allocatelli F Cutruzzola M BrunoriM C R Shastry and H Roder ldquoParallel pathways incytochrome c551 foldingrdquo Journal of Molecular Biology vol 330no 5 pp 1145ndash1152 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Microbiology

Scientifica 3

CcmACcmA

CcmC

CcmE

Ccm

D

Ccm

B

Ccm

B

CcmF

CcmICcmH

CcmG

Periplasm

HemeATP ADP

Cytoplasm

Figure 2 Schematic representation of the protein components ofSystem I Proteins involved in the heme translocation and deliverypathway are shown in light brown proteins involved in the apoCytthioreduction pathway are shown in green proteins involved inapoCyt chaperoning and heme attachment processes are shownin light purple Cyt c (the 3D structure is that of the Cyt c551from P aeruginosa) Protein Data Bank accession number 2EXV[20] and apoCyt (represented as a cartoon) are shown in blueThe translocation process of heme (shown in red) is unknownThe 3D structures of the soluble periplasmic domains of Ec-CcmE Pa-CcmG and Pa-CcmH are shown (Protein Data Bankaccession numbers are 1LIZ [21] 3KH7 [22] and 2HL7 [23] resp)Organisms employing System I 120572- and 120574-proteobacteria some120573-proteobacteria (eg Nitrosomonas) and 120575-proteobacteria (egDesulfovibrio) and Deinococci and Archaea Additionally System Iis observed in plant mitochondria and in the mitochondria of someprotozoa (eg Tetrahymena)

(System I in mitochondria System III in the p-side of thethylakoid membrane and System IV in the n-side of thethylakoidmembrane [16 38])makes the classification and thedistribution of the different maturation systems even moredifficultWith the exception of System III which is apparentlycomposed of a single protein able to carry out the differenttasks of the Cytc maturation process (see below) the variousproteins of Systems I and II carry out different functionsincluding the translocation and delivery of heme b from thecytoplasm where it is synthesized to the relevant subcellularcompartment the chaperoning of apoCyt and the reductionof its disulfide the formation of the covalent bonds betweenthe heme b and the CXXCH heme-binding motif of theapoprotein (Table 1) The complexity of System I comparedto the protein composition of other Cyt cmaturation systemshas long been discussed in particular it has been proposedthat a possible explanation is to be found in the ability evolvedby organisms employing System I to utilize lower levels ofendogenous heme than those necessary for organisms whichevolved Systems II or III [46]

2 System I

The proteins belonging to System I (named CcmABCDE-FGH(I) from Cytochrome c maturation) are membraneproteins exposing their soluble domains (when present) intothe periplasm (Figure 2) All of these proteins are encodedby a single operon in 120572- 120573- and 120574-proteobacteria [4247] The availability of the entire Ccm operon in a single

Ccs

A

Ccs

B

Heme

Cytoplasm

Cyt capoCyt

ResA

Figure 3 Schematic representation of the protein componentsof System II Proteins involved in the heme translocation anddelivery and in the apoCyt chaperoning and heme attachmentprocesses are shown in light brown proteins involved in the apoCytthioreduction pathway are shown in green Cyt c and apoCyt(represented as a cartoon) are shown in blueThe 3D structure of thesoluble periplasmic domain of Bs-ResA is shown in green (ProteinData Bank accession number is 1ST9 [24] System II is foundin plant chloroplasts in gram-positive bacteria cyanobacteria 120576-proteobacteria most 120573-proteobacteria (eg Bordetella Burkholde-ria) and some 120575-proteobacteria (eg Geobacter)

Heme

Intermembrane space

Inner membrane

Outer membrane

Cyt c

apoCytHCCS

Figure 4 Schematic representation of System III A single pro-tein (HCCS) associated to the mitochondrial inner membraneis required for Cyt c maturation The translocation process ofheme (shown in red) is unknown System III is found in themitochondria of fungi (eg S cerevisiae) vertebrates (eg human)and invertebrates (eg C elegans Drosophila)

plasmid (pEC86 [48]) greatly facilitated the heterologousover-expression of many c-type cytochromes in E coli [49]Apparently c-type cytochromes specificity of the Ccm appa-ratus is rather low indeed in an attempt to characterize theminimal sequence requirements of the apoCyt polypeptiderecognized by System I it was shown that this complexmultiprotein apparatus is able to attach the heme even toshort microperoxidase-like peptides carrying the CXXCHmotif [50]

As discussed below different studies mainly carried outby immunoprecipitation experiments suggest that the Ccmproteins may be assembled in the bacterial membrane in a

4 Scientifica

Table1Proteincompo

nentsof

Syste

msIIIand

IIIa

long

with

theirstructural

features

(orPD

Bcodeswhenkn

own)

andfunctio

nalrolesS

ystem

Iproteinsarefoun

din120572-and120574-

proteobacteriasom

e120573-a

nd120575-proteob

acteria

and

Dein

ococciandArchaeaPlantm

itochon

driaM

itochon

driaof

somep

rotozoaSyste

mIIproteins

arefou

ndin

plantchlorop

lasts

Gram-

positiveb

acteria

cyano

bacteria120576-proteob

acteria

most120573

-proteob

acteria

and

some120575

-proteob

acteria

HCC

SofSystemIIIisfou

ndinthem

itochon

driaoffung

ivertebratesand

invertebrates

Syste

mI(SI)

SIstr

ucturalfeatures

Syste

mII(SII)

SIIstructuralfeatures

Syste

mIII(SIII)

SIIIstr

ucturalfeatures

Functio

n(s)

Ccm

AABC

transportermem

brane

n-sid

enu

cleotide-bind

ing

domain

ResB

(CcsB)

5-6TM

helices

1large

perip

lasm

icdo

main

conservedHis

resid

ues

HCC

SMem

brane-associated

protein

conservedHis

resid

ues

Hem

etranslocatio

nand

delivery

Ccm

BABC

transporter6TM

helices

ResC

(CcsA)

6ndash8TM

helices

WWDdo

main

conservedHis

resid

ues

Ccm

C6T

Mhelicesperiplasm

icWWDdo

main

Ccm

DSm

allm

embranep

rotein1

TMhelix

Ccm

E1T

MhelixO

B-fold

1SR3

1LM

01J6

Q2KC

T

Ccm

G1T

MhelixT

RX-like

fold

1Z5Y2B1K

1KNG3KH

73K

H93K

8NRe

sATR

X-lik

efold

2H1B1SU

91ST9

2F9

SapoC

ytthioredu

ction

Ccm

H1T

Mhelix3-helixbu

ndlefold

2HL72KW

0CcdA

6TM

helices

Ccm

F10ndash15TM

helicesperiplasm

icWWDdo

main

conservedHis

resid

ues

ResB

(CcsB)

ResC

(CcsA)

HCC

SapoC

ytchaperon

ingand

hemea

ttachment

Ccm

IPerip

lasm

icTP

Rand120572120573

domains

Scientifica 5

maturase multiprotein complex(es) However the existenceandor stoichiometry of these complexes remains to bedetermined either because of the experimental difficultiesin handling membrane protein complexes or because itis possible that these complexes are unstable and onlytransiently populated Independently from their functionalexistence in the bacterial periplasm as independent units or ascomponents of a multisubunit complex it is clear that each oftheCcmproteins plays a different role from the transport andchaperoning of the heme cofactor to the necessary reductionof the disulfide bond between the sulfur atoms of the twoCys residues of the conserved CXXCH motif of apoCytand finally to the catalysis of covalent heme attachment Anadditional interesting aspect is that over and above theirrole in the biogenesis of Cytc there is also evidence thatinactivation of some ccm genes induces phenotypes thatcannot be explained only in terms of absence of synthesis ofCyt c all of these pleiotropic effects are linked to impairmentof heme andor iron trafficking in the periplasm [47] Inparticular it has been recently shown that in 120572- and 120574-proteobacteria (including the human opportunistic pathogenP aeruginosa) mutations in the ccmC ccmI and ccmF genesinduce phenotypes such as reduced pyoverdine produc-tion reduced bacterial motility or impaired growth in low-iron conditions ([51] and refs therein) These observationssuggesting that Ccm proteins perform additional functionscritical for bacterial physiology growth and virulence pro-vide a rationale to explain why bacteria at variance withthe eukaryotic cell have evolved a metabolically expensiveoperon to accomplish an apparently simple task such asheme ligation to apoCyt Novel hypotheses addressing theseaspects and awaiting experimental investigation include (i)the utilization of Ccm-associated heme for additional cellularprocesses besides attachment to apoCyt (ii) trafficking a non-heme compound through the Ccm system required for ironacquisition such a siderophore (iii) the Ccm inactivation-dependent accumulation of heme b a photoreactivemoleculewhose degradation leads to reactive oxygen species and (iv)the destructive effect on [Fe-S] clusters of ferrisiderophoresreductases

In the following the structural (when known) andfunctional properties of the different components of theSystem I maturation apparatus will be discussed dissectingthe Cytc maturation process into three main functionalsteps heme translocation and delivery apoCyt thioreductivepathway and apoCyt chaperoning and heme ligation (asimilar modular description can also be found elsewhere(see [34 52])) However it should be remembered that inmany cases the proteins involved in the three steps are notuniquely assigned to a specific module as they interact witheach other moreover it has been shown that in some casesmore than one system can be present [41] This modularorganization of Cyt c biogenesis should therefore be intendedonly as a way to simplify the description of an overall highlyintegrated process For each of the three functional stepspresentation of the structural and functional properties of thedifferent protein components is followed by a discussion ofthe proposed molecular mechanism(s)

21 System I Components of the Heme Translocation andDelivery Pathway CcmA and CcmB proteins show the typ-ical sequence features of the ABC (ATP binding Cassette)transporter family and therefore these components of SystemI were initially considered as the proteins responsible forthe translocation of the newly synthesized heme from thecytoplasm to the periplasmic space ABC transporters areubiquitous multidomain integral membrane proteins thattranslocate a large variety of substrates across cellular mem-branes using ATP hydrolysis as a source of energy they aregenerally composed of a transmembrane (TM) domain and aconserved cytosolic nucleotide-binding domain [53]

CcmA is a cytoplasmic soluble protein representing thenucleotide-binding domain of the hypothetical ABC trans-porter according to this hypothesis its sequence containsa nucleotide-binding domain and Walker A and B motifsfor ATP hydrolysis [46] It has also been shown that CcmApossesses ATPase activity in vitro and that the protein isassociated with the membrane fraction only when CcmB isalso present [54]

CcmB and CcmC are both integral membrane proteinspredicted to contain six TM helices CcmC contains a shortWWDdomain in its second periplasmic domain and belongsto the heme handling protein family (HHP) [55] WWDdomains are short tryptophan-rich aminoacid stretcheswith the conserved WGX120601WXWDXRLT sequence (where 120601represents an aromatic amino acid residue and X representsany residue) [40 56] it has been proposed that proteinscontaining WWD domains are involved in heme-bindingand as we will see below a WWD domain is also presentin the CcmF protein another crucial heme-binding proteinof System I CcmC also contains two absolutely conservedhistidine residues in its first (between TM helices 1 and 2)and third (between TMhelices 5 and 6) periplasmic domainsAn attractive hypothesis still requiring experimental proofis that the hydrophobic residues within the tryptophan-richmotif provide a platform for the binding of heme whereasthe two conserved His residues (H60 and H184 in E coliCcmC) act as axial heme ligands [57] this hypothesis isstrengthened by the observation that CcmC indeed interactsdirectly with heme [58 59] Immunoprecipitation experi-ments have shown that in E coli CcmABC proteins forma multiprotein complex with a CcmA

2CcmB

1CcmC

1stoi-

chiometry confirming that these components form an ABC-type transporter complex with unusual functional propertiesassociated to the release of holoCcmE from CcmC [41 46]rather than to heme transport per se CcmC is an interestingprotein worth of future experimental efforts as it is knownthat in some pathogenic bacteria CcmC mutations are asso-ciated to specific phenotypes apparently not related to Cytc maturation such as siderophore production in Paracoccusand Pseudomonas [51] and iron utilization in Legionella [60]

Limited information is available about the structure andfunction of CcmD which appears to be a small membraneprotein (about 70 aminoacid residues) with no conservedsequence features whose topology is currently debatedcontrary to the original proposal [61] additional experimentshave shown that in E coli and R capsulatus CcmD isan integral membrane protein composed of a single TM

6 Scientifica

helix a periplasmic-orientedN-terminus and a cytoplasmic-oriented C-terminus [62] Immunoprecipitation experimentsindicate that CcmD interacts with the CcmA

2CcmB

1CcmC

1

complex even if it is not essential for heme transfer andattachment from CcmC to CcmE CcmD is strictly requiredfor the release of holoCcmE from the ABC transporter [6162]

CcmE is a heme-binding protein discovered as an essen-tial System I component as early as the late 1990rsquos [63]CcmE is a monotopic membrane protein anchored to themembrane via its N-terminal TM segment and exposing itsactive site to the periplasm it is the only Ccm componentof the heme trafficking and delivery module of System I forwhich a three-dimensional structure is available ([21] PDB1SR3 [64] PDB 1LM0) The 3D structure of the apo-state(without bound heme) consists of a six-stranded antiparallel120573-sheet reminiscent of the classical OB-fold [65] with N-and C-terminal extensions CcmE can be considered a ldquohemechaperonerdquo as it protects the cell from a potentially dangerouscompound by sequestering free heme in the periplasm [66]it is thought to act as an intermediate in the heme deliverypathway of Cytc maturation The structure of apoCcmEshowed no recognizable heme-binding cavities and in theabsence of a 3D structure of CcmEwith bound heme (holoC-cmE) the heme-binding region could only be predicted byin silico modeling It is generally believed that the hemein holoCcmE is solvent exposed but recent mutagenesisexperiments challenged this view [67] The unusual covalentbond between the nitrogen atomof a histidine residue presentin the conserved VLAKHDE motif located in a solventexposed environment (H130 in E coli CcmE) and a 120573-carbonof one of the heme vinyl groups has been described in greatdetail by NMR spectroscopy [68] Recently it was shown thatCcmE proteins from the proteobacteria D desulfuricans andD vulgaris contain the unusual CXXXY heme-bindingmotifwhere the Cys residue replaces the canonical His bindingresidue NMR solution structure of D vulgaris CcmE (PDB2KCT) revealed that the proteins adopt the same OB-foldcharacteristic of the CcmE superfamily Contrary to whatreported for theD desulfuricansCcmE [69] the homologousprotein from D vulgaris binds ferric heme noncovalentlythrough the conserved C127 residue [70] An additionalconserved residue in CcmE proteins is Tyr134 which wasshown to provide a coordination bond to the heme ironof holoCcmE [71 72] once it is released from CcmABCDcomplex [36] as discussed below

22 System I Heme Translocation and Delivery PathwayMechanisms We still do not know how the b heme istranslocated from the cytoplasm (where it is synthesized)to the periplasm where Cyt c maturation occurs Differ-ent mechanisms such as translocation through a proteinchannel or free diffusion across the membrane have beenproposed [73] The CcmAB proteins show structural featurestypical of the ABC transporters and for these reasons theywere originally hypothesized to be involved in the heme btranslocation process [57 63 74 75] However it is nowclear that an alternative process must exist since it has

been shown that periplasmic b-type cytochromes can beproduced in the absence of Ccm proteins [76] and thatinactivation of the ATPase activity of CcmA does not abolishheme accumulation in the periplasm [46 54] We have nowevidence that CcmC has the ability to bind heme at its WWDdomain present in the second periplasmic domain but itis still not clear if this membrane protein acts as a proteinchannel for heme translocation or simply collects it in theperiplasm [77]

Another important aspect concerns the oxidation state ofthe heme iron during translocation and delivery processesindeed this property of the heme iron may determine thereaction mechanism by which the unusual CcmE H130nitrogen is covalently linked to the vinyl 120573-carbon of theheme (see [36] for a detailed discussion of this topic) Basedon mutagenesis studies on CcmC [59] a model has beenpresented whereby oxidized heme is bound to CcmC onlyin the presence of CcmE forming a ternary complex BothCcmC and CcmE provide critical residues for heme-bindingthe two conserved His residues (H60 and H180 coordinatingthe heme iron) and theWWDdomain ofCcmCandHis130 ofCcmE forming the unusual covalent bond with heme vinyl-2 [59] The ATPase activity of CcmA is then required torelease holoCcmE from the CcmABCD complex a processthat depends also on the presence of CcmD [46 54] It shouldbe noticed that purified holoCcmE alone or in the CcmCDEcomplex [36 59] contains the heme iron in the oxidized statean observation that is apparently in contrast with the fact thatthe heme must be in its reduced state before attachment toapoCyt can occur Although the oxidation state of the hemeiron is currently debated [36 78] it is possible that CcmFwhich was recently shown to contain a heme b cofactor mayact as specific heme oxidoreductase (see Section 25)

23 System I ApoCyt Thioreduction Pathway ComponentsThe periplasm can be considered a relatively oxidizing envi-ronment due to the presence of an efficient oxidative systemcomposed of the DsbAB proteins [79 80] DsbA is a highlyoxidizing protein (1198641015840

0= 120mV) that is responsible for

the introduction of disulfide bonds into extracytoplasmicproteins [81] On the basis of the results obtained on E colidsbA deletion mutants that are unable to synthesize c-typecytochromes [82 83] it was generally accepted that formationof the intramolecular disulfide bond in apoCyt was a nec-essary step in the Cyt c biogenesis However only reducedapoCyt is clearly competent for heme ligation It is possiblethat this seemingly paradoxical thioreduction process hasevolved in order to protect the apoCyt from proteolyticdegradation aggregation andor formation of intermolec-ular disulfide bonds with thiols from other molecules (seealso Section 31 for a discussion about this aspect in SystemII) Recently however an analysis of c-type cytochromesproduction in several E coli dsb genes deletion strains ledto the hypothesis that DsbA is not necessary for Cyt cmaturation and that heme ligation to apoCyt and apoCytoxidation pathways is alternative competing processes [84]

In gram-negative bacteria a thioreduction pathway hasevolved to specifically reduce the oxidized apoCyt substrate

Scientifica 7

which includes the Ccm proteins CcmG and CcmH Thenecessary reducing power is transferred from the cytoplasmicthioredoxin (TRX) to CcmG via DsbD a large membraneprotein organized in three structural domains an N-terminalperiplasmic domain with a IgG-like fold (nDsbD) a C-terminal periplasmic domain with a thioredoxin-like (TRX-like) fold (cDsbD) and a central domain composed ofeight TM helices [85] Each of these domains contains apair of Cys residues and transfer electrons via a cascadeof disulfide exchange reactions making DsbD a ldquoredox-hubrdquo in the periplasm performing disulfide bond exchangereactions with different oxidized proteins [79] In particulara combination of X-ray crystallography experiments andkinetic analyses showed that electrons are transferred fromthe cytoplasmic TRX to the membrane domain of DsbDfollowed by reduction of cDsbD and finally of nDsbD whichis the direct electron donor to CcmG [85]

CcmG is a membrane-anchored protein linked to themembrane via an N-terminal TM helix and exposing itssoluble TRX-like domain in the periplasm The 3D structureof the TRX-like domain of CcmG from different bacteriahas been solved by X-ray crystallography (E coli PDB 1Z5Y[85] PDB 2B1K [86] B japonicum PDB 1KNG [87] Paeruginosa PDB 3KH7 3KH9 [22]) and is generally wellconserved as proved by the low RMSD (08 A between Pa-CcmG and Ec-CcmG 135 A between Pa-CcmG and Bj-CcmG) Although all these proteins adopt a TRX-like foldand contain the redox-active motif CXXC in the first 120572-helixthey are inactive in the classic insulin reduction assay [7588] CcmG proteins are therefore considered specific thiol-oxidoreductase able to recognize and selectively interactonly with their upstream and downstream binding partnersin the thioreduction process leading to reduced apoCytcLooking at the 3D structure of the periplasmic domain ofthe prototypical Pa-CcmG it is possible to identify the 120573120572120573and 120573120573120572 structural motifs of the TRX fold linked by a short120572-helix and forming a four-stranded 120573-sheet surrounded bythree helices the protein contains an additional N-terminalextension (residues 26ndash62) and a central insert (residues 102ndash123) The redox-active motif of Pa-CcmG (CPSC) is locatedin the first 120572-helix of the TRX fold as usually observed in allTRX-like proteins As for any molecular machinery whereeach component must recognize and interact with more thanone target (ie the substrate and the other components ofthe apparatus) an open question concerns the mechanismwhereby CcmG is able to recognize its different partnersThe availability of the crystal structures of Pa-CcmG bothin the oxidized (22 A resolution) and reduced state (18 Aresolution) [22] allowed highlighting the structural similaritybetween the two redox states (Rmsd of the C120572 atoms inthe two redox forms is 019 A) and therefore to excludestructural rearrangement as the mechanism used by Pa-CcmG to discriminate between reduced (such as the nDsbDdomain) and oxidized partners (Pa-CcmH andor apoCyt)

The standard redox potential of Pa-CcmG (11986410158400= 0213V

at pH 70 [22] as well as that of Ec-CcmG (11986410158400= 0212V

[86]) indicates that these proteins act as mild reductants inthe thioreductive pathway of Cytc biogenesis However the

Figure 5 Three-dimensional structure of Pa-CcmH shown inribbon representation The figure shows the three-helix bundleforming the characteristic fold of Pa-CcmHThe active site disulfidebond between residues Cys25 andCys28 in the long loop connectinghelices 120572-helix1 and 120572-helix 2 is highlighted in yellow

function of thiol-oxidoreductases obviously depends on thepKa values of their activesite Cys residues The pKa of CysX(613 plusmn 005) and CysY (105 plusmn 007) are consistent with thepKa values measured in different TRXs where the active N-terminal Cys residue has a pKa close to pH 70 whereas theC-terminal Cys has a much higher pKa [89 90] Such a largedifference between the two pKa values in the TRX family isfunctionally relevant because it allows the N-terminal Cysto perform the nucleophilic attack on the target disulfidewhile the C-terminal Cys is involved in the resolution of theresulting mixed-disulfide [90]

CcmH is the other component of System I involvedin the reduction of apoCyt Notably CcmH proteins fromdifferent bacterial subgroups may display structural vari-ability indeed while in E coli Ec-CcmH is a bipartiteprotein characterized by two soluble domains exposed to theperiplasm and two TM segments CcmH from P aeruginosa(Pa-CcmH) is a one-domain redox-active protein anchoredto the membrane via a single TM helix and homologous tothe N-terminal redox-active domain of Ec-CcmH Surpris-ingly the 3D structure of the soluble periplasmic domainof Pa-CcmH revealed that it adopts a peculiar three-helixbundle fold strikingly different from that of canonical thiol-oxidoreductases (Figure 5 PDB 2HL7 [23])TheN-terminaldomain of Ec-CcmH was also shown to have the same 3Dstructure although helix-swapping and dimerization havebeen observed in this case (PDB 2KW0 [91 92]) Theconserved redox-active motif (LRCPKC) is located in theloop connecting helices 1 and 2 close to the activesite thecrystal structure reveals the presence of a small pocket on thesurface of Pa-CcmH surrounded by conserved hydrophobicand polar residues which could represent the recognition sitefor the heme-binding motif of apoCyt

Concerning the functional properties of this unusualthiol-oxidoreductase it is interesting to note that its standardredox potential (1198641015840

0= 0215V) [23] is similar to that ob-

tained for Pa-CcmG This observation stands against thelinear redox cascade hypothesis whereby CcmG reducesCcmH While in the canonical redox-active CXXC motif

8 Scientifica

S SSS

SH

SH

Scheme 1 Scheme 2

SH

SH

SHSHS

S

CcmG 7477

CcmG 7477

CcmH 2528

CcmH 2528

apoCytox

apoCytred

S

SHCcmG 74

77

SH

SH

HSHS

CcmG 7477

S

S

SS

3

1

2CcmG 7477

SH

SHCcmH 25

28SH

HSS S

CcmH 2528

SHS

CcmH2528

apoCytox

apoCytred

+

Figure 6 Alternative thioreduction pathways whichmay be operative in System I and hypothesized on the basis of structural and functionalcharacterization of the redox-active Ccm proteins from P aeruginosa [22 23 25] Scheme 1 is a linear redox cascade whereby CcmG is thedirect reductant of CcmH which reduces oxidized apoCyt Scheme 2 envisages a more complex scenario involving the formation of a mixed-disulfide complex between CcmH and apoCyt (Step 1) This complex is the substrate for the attack by reduced CcmG (Step 2) that liberatesreduced apoCyt The resulting disulfide bond between CcmH and CcmG is then resolved by the free Cys thiol of CcmG (probably Cys77 inPa-CcmG) Adapted from [25]

of the TRX family the N-terminal Cys is always solventexposed in CcmH proteins the arrangement of the two Cysresidues is reversed the N-terminal Cys residue is buriedwhereas the C-terminal Cys residue is solvent exposed Onthe basis of this observation it was suggested that differentfrom the canonical TRX redox mechanism CcmH proteinsperform the nucleophilic attack on the apoCyt disulfide viatheir C-terminal Cys residue [23] This mechanism which isin agreement with the mechanism proposed earlier for Ec-CcmH on the basis of mutational-complementation studies[93 94] is substantiated by the peculiar pKa values of theactive site Cys residues of Pa-CcmH which were found tobe similar for both cysteines (84 plusmn 01 and 86 plusmn 01 [23])Again this is different from what is generally observed in thecase of TRX proteins where the pKa value of the Cys residueperforming the initial nucleophilic attack is significantlylower than the pKa value of theCys residue responsible for theresolution of the intermediate mixed-disulfide It is temptingto speculate that the unusual pKa values of the Pa-CcmHactive site thiols may ensure the necessary specificity of thiscomponent of the Ccm apparatus toward the CXXCH motifof the apoCyt substrate

24 System I ApoCyt Thioreduction Pathway MechanismAlthough we know that CcmG and CcmH are the redox-active components of System I involved in the thioreductivepathway of Cyt c biogenesis not only an acceptedmechanismfor the reduction of apoCyt disulfide bond is still lackingbut also the absolute requirement of such a process is nowdebated [38 84] Focusing our attention on the reductionof the apoCyt internal disulfide at least two mechanismscan been hypothesized which involve either a linear redoxcascade of disulfide exchange reactions or a nonlinear redox

process involving transient formation of a mixed-disulfidecomplex as depicted in Figure 6 and Schemes 1 and 2respectively

Both the thiol-disulfide exchange mechanisms depictedin Figure 6 suggest that CcmH is the direct reductant ofthe apoCytc disulfide however even if immunoprecipitationexperiments failed to detect the formation of a mixed-disulfide complex between apoCyt and CcmH proteins [95]some in vitro evidence supporting the formation of sucha complex has been presented In particular it has beenshown that Rhodobacter capsulatus and Arabidopsis thalianaCcmH homologues (Rc-CcmH and At-CcmH) are able toreduce the CXXCH motif of an apoCyt-mimicking peptide[75 96] In the latter case yeast two-hybrid experimentscarried out on At-CcmH indeed revealed an interactionbetween the protein and a peptide mimicking the A thalianaCyt c sequence In the case of Pa-CcmH FRET kineticexperiments employing a Trp-containing fluorescent variantof the protein and a dansylated nonapeptide encompassingthe heme-binding motif of P aeruginosa cytochrome c551(dans-KGCVACHAI) [23] allowed to directly observe theformation of themixed-disulfide complex and tomeasure theoff-rate constant of the bound peptide The results of these invitro binding experiments allowed to calculate an equilibriumdissociation constant which combines an adequate affinity(low 120583M) with the need to release efficiently reduced apoCytto other component(s) of the System I maturase complex[23] More recently the results obtained by FRET bindingexperiments carried out with single Cys-containing mutantsof Pa-CcmH and Pa-CcmG [25] substantiated the hypothesisdepicted in Scheme 2 (Figure 6) Altogether these structuraland functional results suggest that the thioreduction pathwaymechanism leading to reduced apoCyt is better describedby Scheme 2 and that reducing equivalents might not be

Scientifica 9

transferred directly from CcmG to apoCyt as depicted inScheme 1 According to Scheme 2 reduced CcmH (a non-TRX-like thiol-oxidoreductase) specifically recognizes andreduces oxidized apoCyt via the formation of a mixed-disulfide complex which is subsequently resolved by CcmGThe resulting disulfide bond between CcmH and CcmG isthen resolved by the free Cys thiol of CcmG (probably Cys77in Pa-CcmG)

However further in vitro experiments with CcmH andapoCyt single Cys-containing mutants are needed to unveilthe details of the thioreduction of oxidized apoCyt by CcmHIn particular it would be crucial to identify the Cys residueof apoCyt that remains free in the apoCyt-CcmH mixed-disulfide complex intermediate (see Scheme 2 and Section 26below) and available to thioether bond formation with oneof the heme vinyl groups Clearly structure determinationof the trapped mixed-disulfide complexes between CcmHCcmG and apoCyt (or apoCyt peptides) would providekey information for our understanding of this specializedthioreduction pathway mechanism

25 System I ApoCyt Chaperoning and Heme AttachmentComponents The reduced heme-binding motif of apoCyt isnow available to the heme ligation reaction However themolecularmechanismwhereby the Ccmmachinery catalyzesor promotes the formation of the heme-apoCyt covalentbonds is still largely obscure representing themost importantgoal in the field Past observations and recent experimentssuggest that CcmF and CcmI possibly together with CcmHare involved in these final steps [16 34 36]

CcmF is a large integral membrane protein of more than600 residues belonging to the heme handling protein family(HHP [55]) and predicted to contain 10ndash15 TM helices (notethat some discrepancy exists as to the number of TM helicespredicted by computer programs and those predicted onthe basis of phoA and lacZ fusion experiments [40 97])a conserved WWD domain and a larger domain devoidof any recognizable sequence features both exposed to theperiplasm Only recently E coli CcmF (Ec-CcmF) has beenoverexpressed solubilized from the membrane fraction andspectroscopically characterized in vitro [36 41] Surprisinglythe biochemical characterization of recombinant Ec-CcmFallowed to show that the purified protein contains heme b ascofactor in a 1 1 stoichiometry this observation led to thehypothesis that in addition to its heme lyase function Ec-CcmF may act as a heme oxidoreductase In particular it ispossible that the heme b of Ec-CcmF may act as a reductantfor the oxidized iron of the heme bound to CcmE [41]indeed the in vitro reduction of Ec-CcmF by quinones hasbeen experimentally observed strengthening the hypothesisabout the quinolhemeoxidoreductase function of this elusiveproteinThe structuralmodel proposed for Ec-CcmFpredicts13 TMhelices and notably the location of the four completelyconserved His residues according to the model two of them(His173 andHis303) are located in periplasmic exposed loopsnext to the conserved WWD domain which is believedto provide a platform for the heme bound to holoCcmEwhile His261 is located in one of the TM helices and it is

predicted to act as an axial ligand to the heme b of Ec-CcmFthe other conserved His residue (H491) could provide thesecond axial coordination bond to the heme although thishas not been experimentally addressed This model of Ec-CcmF therefore envisages that this large membrane proteinis characterized by two heme-binding sites one of themis embedded in the membrane and coordinates a heme bprosthetic group necessary to reduce the CcmE-bound hemehosted in the second heme-binding site and constituted by itsWWD domain

It is interesting to note that in plants mitochondria theCcmF ortholog appears to be split into three different pro-teins (At-CcmFN1 At-CcmFN2 and At-CcmFC) possiblyinteracting each other [16] Since each of these proteins issimilar to the corresponding domain in the bacterial CcmFortholog this observation may provide useful informationin the design of engineered fragments of bacterial CcmFproteins amenable to structural analyses

The other System I component which is generallybelieved to be involved in the final steps of Cyt c maturationis CcmI As stated above the ccmI gene is present only insome Ccm operons while in others the corresponding ORFis present within the ccmH gene (as in E coli)The functionalrole of CcmI in Cytc biogenesis is revealed by geneticstudies showing that in R capsulatus and B japonicuminactivation of the ccmI gene leads to inability to synthesizefunctional c-type cytochromes [98 99] In R capsulatus andP aeruginosa the CcmI protein (Rc-CcmI and Pa-CcmIresp) can be described as being composed of two domainsstarting from the N-terminus a first domain composed oftwo TM helices connected by a short cytoplasmic regionand a large periplasmic domain Structural variations maybe observed among CcmI members from different bacteriaindeed multiple sequence alignment indicates that the cyto-plasmic region of Rc-CcmI contains a leucine zipper motifwhich is not present in the putative cytoplasmic region of Pa-CcmI [100ndash102] Surprisingly no crystallographic structureis available up to now for the soluble domain of any CcmIprotein with the exception of the ortholog protein NrfGfrom E coli (Ec-NrfG) [103] This protein is necessary toattach the heme to the unusual heme-binding motif CWSCK(where a Lys residue substitutes the conservedHis) present inNrfA a pentaheme c-type cytochrome [103 104] Accordingto secondary structure prediction methods [105] it hasbeen proposed that the periplasmic domain of Pa-CcmI iscomposed of aN-terminal120572-helical region containing at leastthree TPR motifs connected by a disordered linker to a 120572-120573C-terminal regionMultiple sequence analyses and secondarystructure predictionmethods show that the TPR region of Pa-CcmI can be successfully aligned with many TPR-containingproteins including Ec-NrfG [106]

26 System I ApoCyt Chaperoning and Heme AttachmentMechanisms TPR domain-containing proteins are commonto eukaryotes prokaryotes and archaea these proteins aregenerally involved in the assembly of multiprotein complexesand to the chaperoning of unfolded proteins [103 107] Itis therefore plausible that CcmI (or the TPR C-terminal

10 Scientifica

domain of Ec-CcmH) may act to provide a platform for theunfolded apoCyt chaperoning it to the heme attachment sitepresumably located on the WWD domain of CcmF CcmImay thus be considered a component of amembrane-integralmultisubunit heme ligation complex together with CcmFand CcmH as experimentally observed by affinity purifi-cation experiments carried out with Rc-CcmFHI proteins[97 99 108] According to the proposed function of CcmI acritical requirement is represented by its ability to recognizedifferent protein targets over and above apoCyt such asCcmFandCcmHHowever until now direct evidence has been pre-sented only for the interaction of CcmI with apoCyt but thepossibility remains that CcmFHI proteins interact each othervia their TM helices and not via their periplasmic domainsInterestingly both for Pa-CcmI [106] or Rc-CcmI [99] CDspectroscopy experiments carried out on the CcmIapoCytcomplex highlighted major conformational changes at thesecondary structure level It is tempting to speculate on thebasis of these results that in vivo the folding of apoCyt maybe induced by the interaction with CcmI In the case of Paeruginosa System I proteins the binding process betweenPa-CcmI and its target protein apoCyt c551 (Pa-apoCyt) hasbeen studied both at equilibrium and kinetically [106] the119870119863measured for this interaction (in the 120583M range) appeared

to be low enough to ensure apoCyt delivery to the othercomponents of the Ccmmachinery Clearly a major questionconcerns the molecular determinants of such recognitionprocess interestingly both affinity coprecipitation assays[99] and equilibrium and kinetic binding experiments [106]highlighted the role played by the C-terminal 120572-helix of Cytc Similar observations have been made for the interaction ofEc-NrfGwith a peptidemimickingNrfA its apoCyt substrate[103] in this case isothermal titration calorimetry (ITC)experiments indicate that the TPR-domain of NrfG serves asa binding site for the C-terminal motif of NrfA Altogetherthese observations are in agreement with the fact that TPRproteins generally bind to their targets by recognizing theirC-terminal region [107]

The CcmI chaperoning activity has been experimentallysupported for the first time in the case of Pa-CcmI by citratesynthase tests [106] it has been proposed that the observedability to suppress protein aggregation in vitromay reflect thecapacity of CcmI to avoid apoCyt aggregation in vivo Stillanother piece of the Cyt c biogenesis puzzle has been addedrecently by showing that Rc-CcmI is able to interact withapoCcmE either alone or together with its substrate apoCytc2 forming a stable ternary complex in the absence of heme[109] This unexpected observation obtained by reciprocalcopurification experiments provides supporting evidence forthe existence of a large multisubunit complex composed ofCcmFHI andCcmE possibly interactingwith theCcmABCDcomplex It is interesting to note that while in the case ofthe CcmIapoCyt recognition different studies highlightedthe crucial role of the C-terminal helical region of apoCyt(see above) in the case of the apoCcmE apoCyt recognitionthe N-terminal region of apoCyt seems to represent a criticalregion

It is generally accepted that CcmF is the Ccm compo-nent responsible for heme covalent attachment to apoCyt

however as discussed above it is possible that this largemembrane protein plays such a role only together withother Ccm proteins such as CcmH and CcmI Moreover asrecently discovered by Kranz and coworkers [36 41] CcmFmay also act as a quinoleheme oxidoreductase ensuringthe necessary reduction of the oxidized heme b bound toCcmE Why it is necessary that the heme iron be in itsreduced state rather than in its oxidized state is not completelyclear although it is possible that this is a prerequisite to themechanism of thioether bond formation [110] According tocurrent hypotheses it is likely that the periplasmic WWDdomain of CcmF provides a platform for heme b bindingSanders et al and Verissimo et al [34 109] have presenteda mechanistic view of the heme attachment process whichtakes into account all the available experimental observationson the different Ccmproteins According to thismodel stere-ospecific heme ligation to reduced apoCyt occurs becauseonly the vinyl-4 group is available to form the first thioetherbond with a free cysteine at the apoCyt heme-binding motifsince the vinyl-2 group is involved (at least in the Ec-CcmE)in the covalent bond with His130 of CcmE [67] Howeverexperimental proof for this hypothesis requires a detailedinvestigation of the apoCyt thioreduction process catalyzedby CcmH (see Section 24)

It should be noticed that the mechanisms described sofar for the function(s) played by CcmF (see [34 36 109]) donot envisage a clear role for its large C-terminal periplasmicdomain (residues 510 to 611 in Ec-CcmF) It would be inter-esting to see if this domain apparently devoid of recognizablesequence features may mediate intermolecular recognitionprocesses with one (or more) component(s) involved in thehemeapoCyt ligation process

3 System II

System II is typically found in gram-positive bacteria and inin 120576-proteobacteria it is also present in most 120573- and some120575-proteobacteria in Aquificales and cyanobacteria as wellas in algal and plant chloroplasts System II is composedof three or four membrane-bound proteins CcdA ResACcsA (also known as ResC) and CcsB (also known as ResB)(Figure 3) CcdA andResA are redox-active proteins involvedin the reduction of the disulfide bond in the heme-bindingmotif of apoCyt whereas CcsA and CcsB are responsible forthe heme-apoCyt ligation process and are considered Cyt csynthethases (CCS) BothCcsAwhich is evolutionary relatedto the CcmC and CcmF proteins of System I [55] and CcsBare integral membrane proteins In some 120576-proteobacteriasuch asHelicobacter hepaticus andHelicobacter pylori a singlefusion protein composed of CcsA and CcsB polypeptides ispresent [41 111] Although as discussed below evidence hasbeen put forward to support the hypothesis that the CcsBAcomplex acts a heme translocase we still do not know if theheme is transported across the membrane by component(s)of System II itself or by a different unidentified process

31 System II ApoCyt Thioreduction Pathway After the Secmachinery secretes the newly synthesized apoCyt it readily

Scientifica 11

becomes a substrate for the oxidative system present on theouter surface of the cytoplasmic membrane of the gram-positive bacteria In B subtilis the BdbCD system [112113] is functionally but not structurally equivalent to thewell-characterized DsbAB system present in gram-negativebacteria As discussed above for System I the disulfide ofthe apoCyt heme-binding motif must be reduced in orderto allow thioether bonds formation and heme attachmentResA is the extracytoplasmic membrane-anchored TRX-likeprotein involved in the specific reduction of the apoCytdisulfide bond after the disulfide bond exchange reactionhas occurred oxidized ResA is reduced by CcdA whichreceives its reducing equivalents from a cytoplasmic TRX[114] Although ResA displays a classical TRX-like foldthe analysis of the 3D structure of oxidized and reducedResA from B subtilis (Bs-ResA) showed redox-dependentconformational modifications not observed in other TRX-like proteins Interestingly such modifications occur at thelevel of a cavity proposed to represent the binding site foroxidized apoCyt [24 115 116] Another peculiar feature of Bs-ResA is represented by the unusually similar pKa values of itsthe active site Cys residues (88 and 82 resp) as observedalso in the case of the active site Cys residues of the SystemI Pa-CcmH (see Section 23) However at variance with Pa-CcmH in Bs-ResA the large separation between the twocysteine thiols observed in the structure of the reduced formof the protein can be invoked to account for this result

CcdA is a large membrane protein containing six TMhelices [117 118] homologous to the TM domain of E coliDsbD [85 119] Its role in the Cyt c biogenesis is supportedby the observation that inactivation of CcdA blocks theproduction of c-type cytochromes in B subtilis [120 121]Moreover over and above the reduction of its apoCyt sub-strate Bs-CcdA is able to reduce the disulfide bond of othersecreted proteins such as StoA [122] It is interesting to notethat ResA and CcdA are not essential for Cyt c synthesisin the absence of BdbD or BdbC or if a disulfide reductantis present in the growth medium [123 124] As discussedabove (Section 24) a similar observation was made on therole of the thio-reductive pathway of System I in this casepersistent production of c-type cytochromes was observedin Dsb-inactivated bacterial strains [38] Following theseobservations the question has been put forward as to why theCyt c biogenesis process involves this apparently redundantthio-reduction route only to correct the effects of Bdb orDsb activity Referring to the case of B subtilis [111] it hasbeen proposed that the main reason is that BdbD mustefficiently oxidize newly secreted proteins since the activityof most of them depends on the presence of disulfide bondsAlternatively it can be hypothesized that the intramoleculardisulfide bondof apoCyt protects the protein fromproteolyticdegradation or aggregation or from cross-linking to otherthiol-containing proteins

32 System II Heme Translocation and Attachment to ApoCytRecent experimental evidence is accumulating supportingthe involvement of the heterodimeric membrane complexResBC or of the fusion protein CcsBA in the translocation

of heme from the n-side to the p-side of the bacterialmembrane In particular it has been shown that CcsBAfrom H hepaticus (Hh-CcsBA) is able to reconstitute Cyt cbiogenesis in the periplasm of E coli [36] Moreover it wasalso observed that Hh-CcsBA is able to bind reduced hemevia two conserved histidines flanking the WWD domainand required both for the translocation of the heme andfor the synthetase function of Hh-CcsBA [36] A differentstudy carried out on B subtilis System II proteins providedsupport for heme-binding capability by the ResBC complexaccording to the results obtained on recombinant ResBC theResB component of the heterodimeric CCS complex is ableto covalently bind the heme in the cytoplasm (probably by aCys residue) and to deliver it to an extracytoplamic domain ofResC which is responsible for the covalent ligation to apoCyt[117] It should be noticed however that the transfer of theCCS-bound heme to apoCyt still awaits direct experimentalproof Moreover it has also been reported that in B subtilisthe inactivation of CCS does not affect the presence ofother heme-containing proteins in the periplasm such ascytochrome b562 [111] this observation which is in contrastto the heme-transport hypothesis by CCSs parallels similarconcerns about heme translocation mechanism that arecurrently discussed in the context of System I (see Section 22above) In the case of the Hh-CcsBA synthetase site-directedmutagenesis experiments allowed to assign a heme-bindingrole for the two pairs of conserved His residues accordingto the topological model of the protein obtained by alkalinephosphatase (PhoA) assays and GFP fusions the conservedHis77 and His858 residues are located on TM helices whileHis761 and His897 are located on the periplasmic side [36]These observations together with the results of site-directedmutagenesis experiments allowed the authors to suggest thatHis77 andHis858 form a low affinitymdashmembrane embeddedheme-binding site for ferrous heme which is subsequentlytranslocated to an external heme-binding domain of Hh-CcsBA where it is coordinated by His761 and His897 Thetopological model therefore predicts that this last His pairis part of a periplasmic-located WWD domain (homologousto theWWDdomains found in theCcmCandCcmFproteinsof System I described above)

4 System III

Strikingly different from Systems I and II the Cyt c mat-uration apparatus found in fungi and in metazoan cells(System III) is composed of a single protein known as Holo-Cytochrome c Synthetase (HCCS) or Cytochrome c HemeLyase (CCHL) apparently responsible for all the subprocessesdescribed above (heme transport and chaperoning reductionand chaperoning of apoCyt and catalysis of the thioetherbonds formation between heme and apoCyt) (Figure 4)Surprisingly although this protein has been identified in Scerevisiaemitochondria several years ago [125 126] only veryrecently the human HCCS has been expressed as a recombi-nant protein in E coli and spectroscopically characterized invitro [127] It is worth noticing that humanHCCS is attractinginterest since over and above its role in Cyt c biogenesis it

12 Scientifica

is involved in diseases such as microphthalmia with linearskin defects syndrome (MLS) an X-linked genetic disorder[128ndash130] and in other processes such as Cytc-independentapoptosis in injured motor neurons [131] Different fromanimal cells where a single HCCS is sufficient for thematuration of both soluble and membrane-anchored Cytc(Cyt c and Cy c1 resp) in S cerevisiae two homologs HCCSand HCC1S located in the inner mitochondrial membraneand facing to the IMS space [132 133] are responsible for hemeattachment to apoCyt c and apoCyt c1 respectively [125 134]It should also be noticed that in fungi an additional FAD-containing protein (Cyc2p) is required for Cyt c synthesisCyc2p is a mitochondrial membrane-anchored flavoproteinexposing its redox domain to the IMS which is requiredfor the maturation of Cyt c but not for that of Cyt c1 [135]This protein does not contain the conserved Cys residuestypically found in disulfide reductases and indeed it is notable to reduce oxidized apoCyt in vitro However it has beenrecently shown that Cyc2p is able to catalyze the NAD(P)H-dependent reduction of heme in vitro [136] a necessarystep before the thioether bond formation can occur asdiscussed above (see Section 26) This result together withthe observation that Cyc2p interacts with HCCS and withapoCyt c and c1 lends support to the proposal that Cyc2p isinvolved in the reduction of the heme iron in vivo [136 137]

AlthoughHCCS proteins are crucial for Cyt cmaturationwe still do not know how these proteins recognize theirsubstrates (heme and apoCyt) and how they promote orcatalyze the formation of thioether bonds between the hemevinyl groups and the cysteine thiols of the apoCyt CXXCHmotif

Contrary to the broad specificity of System I whichis able to recognize and attach the heme to prokaryoticand eukaryotic c-type cytochromes [49 50] and even tovery short microperoxidase-like sequences [49 50] mito-chondrial HCCS is characterized by a higher specificityas it does not recognize bacterial apoCyt sequences Theseobservations prompted the investigation of the recognitionprocess between apoCyt and HCCS Recently by using arecombinant yeast HCCS and chimerical apoCyt sequencesexpressed in E coli it was possible to conclude that a crucialrecognition determinant is represented by the N-terminalregion of apoCyt containing the heme-binding motif [35]notice that in the context of System I the same N-terminalregion of the Cyt c sequence has been recently identified to beimportant for the recognition by apoCcmE (see Section 26)Over and above the role played by the intervening residuesin the CXXCH heme-binding motif [135] it has been shownthat a conserved Phe residue occurring in the N-terminalregion before the CXXCH motif is important for HCCSrecognition [35 138] These results support the hypothesisthat this residue known to be a key determinant of Cytcfolding and stability [19 20 139] may also be crucial for Cytcmaturation by HCCS

Another relevant question concerns the ability of HCCSto recognize and bind the heme molecule as the region ofthe protein responsible for heme recognition remains to beidentified Initially it was hypothesized that the recognitionof heme could be mediated by the CP motifs present in

the HCCS protein [140] these short sequences are indeedknown to bind heme in a variety of heme-containing proteins[141 142] However it has been recently shown that CPmotifsof the recombinant S cerevisiae HCCS are not necessaryfor Cyt c production in E coli [143] excluding them as keydeterminants of heme recognition

The long-awaited in vitro characterization of the recom-binant human HCCS allowed for the first time to pro-pose a molecular mechanism underlying Cytc maturationin eukaryotes which can be experimentally tested [127]According to the proposed model the human HCCS activitycan be described as a four step mechanism involving (i)heme-binding (ii) apoCyt recognition (iii) thioether bondsformation and (iv) holoCyt c release In particular theheme is proposed to play the role of a scaffolding moleculemediating the contacts between HCCS and apoCyt Muta-genesis experiments carried out on the recombinant HCCSstrongly suggest that heme-binding (Step 1) depends on thepresence of a specific His residue (His154) acting as anaxial ligand to ferrous heme Residues present at the N-terminus of apoCyt mediate the recognition with HCCS(Step 2) as discussed above it is known that this regionforms structurally conserved 120572-helix in the fold of all c-type cytochromes Unfortunately no information is availableup to now concerning the region of HCCS involved in therecognition and binding to the N-terminal region of apoCytCoordination of the heme iron by the His residue of theapoCyt heme-binding motif CXXCH provides the secondaxial ligand to the heme iron and is probably importantfor the correct positioning of the two apoCyt Cys residuesand formation of the thioether bonds (Step 3) The finalrelease of functional Cyt c (Step 4) clearly requires thedisplacement of the His154Fe2+ coordination bond sucha displacement is probably mediated by formation of thecoordination bond with the Cyt c conserved Met residueandor by the simultaneous folding of Cyt c Again it isinteresting to note that this last hypothesis is in accordancewith in vitro folding studies on c-type cytochromes thathighlighted the late formation of the Met-Fe coordinationduring Cyt c folding [144 145]

Acknowledgments

The author thanks A Di Matteo and E Di Silvio for fruitfuldiscussions and for help in preparing this paper This work issupported by grants from the University of Rome ldquoSapienzardquo(C26A11MBXJ and C26A13MWF4)

References

[1] M G Galinato J G Kleingardner S E Bowman et alldquoHeme-protein vibrational couplings in cytochrome c providea dynamic link that connects the heme-iron and the proteinsurfacerdquo Proceedings of the National Academy of Sciences vol109 no 23 pp 8896ndash8900 2012

[2] H B Gray and J R Winkler ldquoElectron flow through metal-loproteinsrdquo Biochimica et Biophysica Acta vol 1797 no 9 pp1563ndash1572 2010

Scientifica 13

[3] P Ascenzi R Santucci M Coletta and F PolticellildquoCytochromes reactivity of the ldquodark siderdquo of the hemerdquoBiophysical Chemistry vol 152 no 1ndash3 pp 21ndash27 2010

[4] S Yamada N D Bouley Ford G E Keller W C Ford HB Gray and J R Winkler ldquoSnapshots of a protein foldingintermediaterdquo Proceedings of the National Academy of Sciencesvol 110 no 5 pp 1606ndash1610 2013

[5] P Weinkam J Zimmermann F E Romesberg and P GWolynes ldquoThe folding energy landscape and free energy excita-tions of cytochrome crdquo Accounts of Chemical Research vol 43no 5 pp 652ndash660 2010

[6] M Yamanaka M Masanari and Y Sambongi ldquoConfermentof folding ability to a naturally unfolded apocytochrome cthrough introduction of hydrophobic amino acid residuesrdquoBiochemistry vol 50 no 12 pp 2313ndash2320 2011

[7] G W Moore and G W Pettigrew Cytochrome C EvolutionaryStructural and Physicochemical Aspects Springer Berlin Ger-many 1990

[8] I Bertini G Cavallaro and A Rosato ldquoCytochrome c occur-rence and functionsrdquo Chemical Reviews vol 106 no 1 pp 90ndash115 2006

[9] D A Pearce and F Sherman ldquoDegradation of cytochromeoxidase subunits in mutants of yeast lacking cytochrome c andsuppression of the degradation by mutation of yme1rdquo Journal ofBiological Chemistry vol 270 no 36 pp 20879ndash20882 1995

[10] G Silkstone S M Kapetanaki I Husu M H Vos and MT Wilson ldquoNitric oxide binding to the cardiolipin complex offerric cytochrome crdquo Biochemistry vol 51 no 34 pp 6760ndash6766 2012

[11] M Giorgio E Migliaccio F Orsini et al ldquoElectron transferbetween cytochrome c and p66Shc generates reactive oxygenspecies that trigger mitochondrial apoptosisrdquo Cell vol 122 no2 pp 221ndash233 2005

[12] S M Kilbride and J H Prehn ldquoCentral roles of apoptoticproteins in mitochondrial functionrdquo Oncogene vol 32 no 22pp 2703ndash2711 2013

[13] T Noll and G Noll ldquoStrategies for ldquowiringrdquo redox-activeproteins to electrodes and applications in biosensors biofuelcells and nanotechnologyrdquo Chemical Society Reviews vol 40no 7 pp 3564ndash3576 2011

[14] G Layer J Reichelt D Jahn and D W Heinz ldquoStructure andfunction of enzymes in heme biosynthesisrdquo Protein Science vol19 no 6 pp 1137ndash1161 2010

[15] S Severance and IHamza ldquoTrafficking of heme and porphyrinsin metazoardquo Chemical Reviews vol 109 no 10 pp 4596ndash46162009

[16] P Hamel V Corvest P Giege and G Bonnard ldquoBiochemi-cal requirements for the maturation of mitochondrial c-typecytochromesrdquo Biochimica et Biophysica Acta vol 1793 no 1 pp125ndash138 2009

[17] W R Fisher H Taniuchi and C B Anfinsen ldquoOn the role ofheme in the formation of the structure of cytochrome crdquo Journalof Biological Chemistry vol 248 no 9 pp 3188ndash3195 1973

[18] M Yamanaka H Mita Y Yamamoto and Y Sambongi ldquoHemeis not required for aquifex aeolicus cytochrome c555 polypep-tide foldingrdquoBioscience Biotechnology andBiochemistry vol 73no 9 pp 2022ndash2025 2009

[19] C Travaglini-Allocatelli S Gianni andMBrunori ldquoA commonfolding mechanism in the cytochrome c familyrdquo Trends inBiochemical Sciences vol 29 no 10 pp 535ndash541 2004

[20] A Borgia D Bonivento C Travaglini-Allocatelli A DiMatteoand M Brunori ldquoUnveiling a hidden folding intermediate in c-type cytochromes by protein engineeringrdquo Journal of BiologicalChemistry vol 281 no 14 pp 9331ndash9336 2006

[21] E Enggist L Thony-Meyer P Guntert and K PervushinldquoNMR structure of the heme chaperone CcmE reveals a novelfunctional motifrdquo Structure vol 10 no 11 pp 1551ndash1557 2002

[22] A di Matteo N Calosci S Gianni P Jemth M Brunori and CTravaglini-Allocatelli ldquoStructural and functional characteriza-tion of CcmG from pseudomonas aeruginosa a key componentof the bacterial cytochrome c maturation apparatusrdquo Proteinsvol 78 no 10 pp 2213ndash2221 2010

[23] A diMatteo S GianniM E Schinina et al ldquoA strategic proteinin cytochrome c maturation three-dimensional structure ofCcmH and binding to apocytochrome crdquo Journal of BiologicalChemistry vol 282 no 37 pp 27012ndash27019 2007

[24] A Crow R M Acheson N E Le Brun and A OubrieldquoStructural basis of redox-coupled protein substrate selectionby the cytochrome c biosynthesis protein ResArdquo Journal ofBiological Chemistry vol 279 no 22 pp 23654ndash23660 2004

[25] E di Silvio A di Matteo and C Travaglini-Allocatelli ldquoTheRedox pathway of Pseudomonas aeruginosa cytochrome cbiogenesisrdquo Journal of Proteins and Proteomics vol 3 no 1 pp1ndash7 2012

[26] LThony-Meyer andP Kunzler ldquoTranslocation to the periplasmand signal sequence cleavage of preapocytochrome c depend onsec and lep but not on the ccmgene productsrdquoEuropean Journalof Biochemistry vol 246 no 3 pp 794ndash799 1997

[27] S J Facey and A Kuhn ldquoBiogenesis of bacterial inner-membrane proteinsrdquo Cellular and Molecular Life Sciences vol67 no 14 pp 2343ndash2362 2010

[28] K Diekert A I de Kroon U Ahting et al ldquoApocytochromec requires the TOM complex for translocation across themitochondrial outer membranerdquo The EMBO Journal vol 20no 20 pp 5626ndash5635 2001

[29] N Wiedemann V Kozjak T Prinz et al ldquoBiogenesis of yeastmitochondrial cytochrome c a unique relationship to the TOMmachineryrdquo Journal ofMolecular Biology vol 327 no 2 pp 465ndash474 2003

[30] F-U Hartl N Pfanner and W Neupert ldquoTranslocation inter-mediates on the import pathway of proteins intomitochondriardquoBiochemical Society Transactions vol 15 no 1 pp 95ndash97 1987

[31] B S Glick A Brandt K Cunningham SMuller R L Hallbergand G Schatz ldquoCytochromes c1 and b2 are sorted to theintermembrane space of yeast mitochondria by a stop-transfermechanismrdquo Cell vol 69 no 5 pp 809ndash822 1992

[32] G Howe and S Merchant ldquoRole of heme in the biosynthesis ofcytochrome c6rdquo Journal of Biological Chemistry vol 269 no 8pp 5824ndash5832 1994

[33] S S Nakamoto P Hamel and S Merchant ldquoAssembly ofchloroplast cytochromes b and crdquo Biochimie vol 82 no 6-7 pp603ndash614 2000

[34] C Sanders S Turkarslan D-W Lee and F DaldalldquoCytochrome c biogenesis the Ccm systemrdquo Trends inMicrobiology vol 18 no 6 pp 266ndash274 2010

[35] J M Stevens D A Mavridou R Hamer P Kritsiligkou A DGoddard and S J Ferguson ldquoCytochrome c biogenesis systemIrdquoThe FEBS Journal vol 278 no 22 pp 4170ndash4178 2011

[36] R G Kranz C Richard-Fogal J-S Taylor and E R FrawleyldquoCytochrome c biogenesis mechanisms for covalent modifica-tions and trafficking of heme and for heme-iron redox controlrdquo

14 Scientifica

Microbiology and Molecular Biology Reviews vol 73 no 3 pp510ndash528 2009

[37] J W A Allen ldquoCytochrome c biogenesis in mitochondriamdashsystems III and VrdquoThe FEBS Journal vol 278 no 22 pp 4198ndash4216 2011

[38] D A Mavridou S J Ferguson and J M Stevens ldquoCytochromec assemblyrdquo IUBMB Life vol 65 no 3 pp 209ndash216 2013

[39] I Bertini G Cavallaro and A Rosato ldquoEvolution ofmitochondrial-type cytochrome c domains and of theprotein machinery for their assemblyrdquo Journal of InorganicBiochemistry vol 101 no 11-12 pp 1798ndash1811 2007

[40] B S Goldman and R G Kranz ldquoEvolution and horizontaltransfer of an entire biosynthetic pathway for cytochromec biogenesis helicobacter deinococcus archae and morerdquoMolecular Microbiology vol 27 no 4 pp 871ndash873 1998

[41] C L Richard-Fogal E R Frawley E R Bonner H Zhu BSan Francisco and R G Kranz ldquoA conserved haem redoxand trafficking pathway for cofactor attachmentrdquo The EMBOJournal vol 28 no 16 pp 2349ndash2359 2009

[42] L Thony-Meyer F Fischer P Kunzler D Ritz and H Hen-necke ldquoEscherichia coli genes required for cytochrome cmaturationrdquo Journal of Bacteriology vol 177 no 15 pp 4321ndash4326 1995

[43] C S Beckett J A Loughman K A Karberg G M DonatoW E Goldman and R G Kranz ldquoFour genes are required forthe system II cytochrome c biogenesis pathway in Bordetellapertussis a unique bacterial modelrdquo Molecular Microbiologyvol 38 no 3 pp 465ndash481 2000

[44] N E Le Brun J Bengtsson and L Hederstedt ldquoGenes requiredfor cytochrome c synthesis in Bacillus subtilisrdquo MolecularMicrobiology vol 36 no 3 pp 638ndash650 2000

[45] P Giege J M Grienenberger and G Bonnard ldquoCytochromec biogenesis in mitochondriardquo Mitochondrion vol 8 no 1 pp61ndash73 2008

[46] R E Feissner C L Richard-Fogal E R Frawley J A Lough-man KW Earley and R G Kranz ldquoRecombinant cytochromesc biogenesis systems I and II and analysis of haem deliverypathways in Escherichia colirdquo Molecular Microbiology vol 60no 3 pp 563ndash577 2006

[47] N P Cianciotto P Cornelis and C Baysse ldquoImpact of thebacterial type I cytochrome c maturation system on differentbiological processesrdquoMolecular Microbiology vol 56 no 6 pp1408ndash1415 2005

[48] E Arslan H Schulz R Zufferey P Kunzler and L Thony-Meyer ldquoOverproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in Escherichiacolirdquo Biochemical and Biophysical Research Communicationsvol 251 no 3 pp 744ndash747 1998

[49] C Sanders and H Lill ldquoExpression of prokaryotic and eukary-otic cytochromes c in Escherichia colirdquoBiochimica et BiophysicaActa vol 1459 no 1 pp 131ndash138 2000

[50] M Braun and L Thony-Meyer ldquoBiosynthesis of artificialmicroperoxidases by exploiting the secretion and cytochromec maturation apparatuses of Escherichia colirdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 35 pp 12830ndash12835 2004

[51] B Baert C Baysse S Matthijs and P Cornelis ldquoMultiplephenotypic alterations caused by a c-type cytochrome mat-uration ccmC gene mutation in Pseudomonas aeruginosardquoMicrobiology vol 154 no 1 pp 127ndash138 2008

[52] S Turkarslan C Sanders and F Daldal ldquoExtracytoplasmicprosthetic group ligation to apoproteins maturation of c-typecytochromesrdquo Molecular Microbiology vol 60 no 3 pp 537ndash541 2006

[53] P M Jones and A M George ldquoThe ABC transporter structureand mechanism perspectives on recent researchrdquo Cellular andMolecular Life Sciences vol 61 no 6 pp 682ndash699 2004

[54] O Christensen E M Harvat L Thony-Meyer S J Fergusonand J M Stevens ldquoLoss of ATP hydrolysis activity by CcmABresults in loss of c-type cytochrome synthesis and incompleteprocessing ofCcmErdquoTheFEBS Journal vol 274 no 9 pp 2322ndash2332 2007

[55] J-H Lee E M Harvat J M Stevens S J Ferguson and M HSaier Jr ldquoEvolutionary origins of members of a superfamily ofintegralmembrane cytochrome c biogenesis proteinsrdquoBiochim-ica et Biophysica Acta vol 1768 no 9 pp 2164ndash2181 2007

[56] D L Beckman D R Trawick and R G Kranz ldquoBacterialcytochromes c biogenesisrdquo Genes and Development vol 6 no2 pp 268ndash283 1992

[57] H Schulz R A Fabianek E C Pellicioli H Hennecke andL Thony-Meyer ldquoHeme transfer to the heme chaperone CcmEduring cytochrome c maturation requires the CcmC proteinwhich may function independently of the ABC-transporterCcmABrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 96 no 11 pp 6462ndash6467 1999

[58] Q Ren and L Thony-Meyer ldquoPhysical Interaction ofCcmC with Heme and the Heme Chaperone CcmE duringCytochrome cMaturationrdquo Journal of Biological Chemistry vol276 no 35 pp 32591ndash32596 2001

[59] C Richard-Fogal and R G Kranz ldquoThe CcmChemeCcmEcomplex in heme trafficking and cytochrome c biosynthesisrdquoJournal of Molecular Biology vol 401 no 3 pp 350ndash362 2010

[60] V K Viswanathan S Kurtz L L Pedersen et al ldquoThecytochrome C maturation locus of Legionella pneumophilapromotes iron assimilation and intracellular infection andcontains a strain-specific insertion sequence elementrdquo Infectionand Immunity vol 70 no 4 pp 1842ndash1852 2002

[61] U Ahuja and L Thony-Meyer ldquoCcmD is involved in complexformation between CcmC and the heme chaperone CcmE dur-ing cytochrome c maturationrdquo Journal of Biological Chemistryvol 280 no 1 pp 236ndash243 2005

[62] C L Richard-Fogal E R Frawley and R G Kranz ldquoTopologyand function of CcmD in cytochrome Cmaturationrdquo Journal ofBacteriology vol 190 no 10 pp 3489ndash3493 2008

[63] H Schulz H Hennecke and L Thony-Meyer ldquoPrototype ofa heme chaperone essential for cytochrome c maturationrdquoScience vol 281 no 5380 pp 1197ndash1200 1998

[64] F Arnesano L Banci P D Barker et al ldquoSolution structure andcharacterization of the heme chaperone CcmErdquo Biochemistryvol 41 no 46 pp 13587ndash13594 2002

[65] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[66] L Thony-Meyer ldquoA heme chaperone for cytochrome c biosyn-thesisrdquo Biochemistry vol 42 no 45 pp 13099ndash13105 2003

[67] E M Harvat C Redfield J M Stevens and S J FergusonldquoProbing the heme-binding site of the cytochrome cmaturationprotein CcmErdquoBiochemistry vol 48 no 8 pp 1820ndash1828 2009

[68] D Lee K Pervushin D Bischof M Braun and L Thony-Meyer ldquoUnusual heme-histidine bond in the active site of a

Scientifica 15

chaperonerdquo Journal of the American Chemical Society vol 127no 11 pp 3716ndash3717 2005

[69] A D Goddard J M Stevens F Rao et al ldquoc-Type cytochromebiogenesis can occur via a natural Ccm system lacking CcmHCcmG and the heme-binding histidine of CcmErdquo Journal ofBiological Chemistry vol 285 no 30 pp 22882ndash22889 2010

[70] J M Aramini K Hamilton P Rossi et al ldquoSolution NMRstructure backbone dynamics and heme-binding propertiesof a novel cytochrome c maturation protein CcmE fromDesulfovibrio vulgarisrdquo Biochemistry vol 51 no 18 pp 3705ndash3707 2012

[71] T Uchida J M Stevens O Daltrop et al ldquoThe interactionof covalently bound heme with the cytochrome c maturationprotein CcmErdquo Journal of Biological Chemistry vol 279 no 50pp 51981ndash51988 2004

[72] I Garcıa-Rubio M Braun I Gromov L Thony-Meyer andA Schweiger ldquoAxial coordination of heme in ferric CcmEchaperone characterized by EPR spectroscopyrdquo BiophysicalJournal vol 92 no 4 pp 1361ndash1373 2007

[73] L Thony-Meyer ldquoHaem-polypeptide interactions duringcytochrome c maturationrdquo Biochimica et Biophysica Acta vol1459 no 2-3 pp 316ndash324 2000

[74] J A Keightley D Sanders T R Todaro A Pastuszyn and J AFee ldquoCloning expression in Escherichia coli of the cytochromec552 gene from Thermus thermophilus HB8 Evidence forgenetic linkage to an ATP- binding cassette protein and initialcharacterization of the cycA gene productsrdquo Journal of Biologi-cal Chemistry vol 273 no 20 pp 12006ndash12016 1998

[75] E M Monika B S Goldman D L Beckman and R GKranz ldquoA thioreduction pathway tethered to the membranefor periplasmic cytochromes c biogenesis In vitro and in vivostudiesrdquo Journal of Molecular Biology vol 271 no 5 pp 679ndash692 1997

[76] M Throne-Holst L Thony-Meyer and L HederstedtldquoEscherichia coli ccm in-frame deletion mutants can produceperiplasmic cytochrome b but not cytochrome crdquo FEBS Lettersvol 410 no 2-3 pp 351ndash355 1997

[77] U Ahuja and L Thony-Meyer ldquoDynamic features of a hemedelivery system for cytochrome c maturationrdquo Journal of Bio-logical Chemistry vol 278 no 52 pp 52061ndash52070 2003

[78] J W A Allen P D Barker O Daltrop et al ldquoWhy isnrsquot ldquostan-dardrdquo heme good enough for c-type and di-type cytochromesrdquoDalton Transactions no 21 pp 3410ndash3418 2005

[79] K Inaba and K Ito ldquoStructure and mechanisms of the DsbB-DsbA disulfide bond generation machinerdquo Biochimica et Bio-physica Acta vol 1783 no 4 pp 520ndash529 2008

[80] H Kadokura F Katzen and J Beckwith ldquoProtein disulfidebond formation in prokaryotesrdquoAnnual Review of Biochemistryvol 72 pp 111ndash135 2003

[81] S R Shouldice B Heras P M Walden M Totsika M ASchembri and J L Martin ldquoStructure and function of DsbA akey bacterial oxidative folding catalystrdquoAntioxidants and RedoxSignaling vol 14 no 9 pp 1729ndash1760 2011

[82] R Metheringham L Griffiths H Crooke S Forsythe and JCole ldquoAn essential role for DsbA in cytochrome c synthesis andformate-dependent nitrite reduction by Escherichia coli K-12rdquoArchives of Microbiology vol 164 no 4 pp 301ndash307 1995

[83] Y Sambongi and S J Ferguson ldquoMutants of Escherichia colilacking disulphide oxidoreductases DsbA and DsbB cannotsynthesise an exogenous monohaem c-type cytochrome exceptin the presence of disulphide compoundsrdquo FEBS Letters vol398 no 2-3 pp 265ndash268 1996

[84] D AMavridou S J Ferguson and JM Stevens ldquoThe interplaybetween the disulfide bond formation pathway and cytochromec maturation in Escherichia colirdquo FEBS Letters vol 586 no 12pp 1702ndash1707 2012

[85] C U Stirnimann A Rozhkova U Grauschopf M G GrutterR Glockshuber and G Capitani ldquoStructural basis and kineticsof DsbD-dependent cytochrome c maturationrdquo Structure vol13 no 7 pp 985ndash993 2005

[86] N Ouyang Y-G Gao H-Y Hu and Z-X Xia ldquoCrystalstructures of E coli CcmGand itsmutants reveal key roles of theN-terminal120573-sheet and the fingerprint regionrdquoProteins vol 65no 4 pp 1021ndash1031 2006

[87] M A Edeling L W Guddat R A Fabianek et al ldquoCrys-tallization and preliminary diffraction studies of native andselenomethionine CcmG (CycY DsbE)rdquoActa CrystallographicaD vol 57 no 9 pp 1293ndash1295 2001

[88] R A Fabianek M Huber-Wunderlich R Glockshuber PKunzler H Hennecke and L Thony-Meyer ldquoCharacterizationof the Bradyrhizobium japonicum CycY protein a membrane-anchored periplasmic thioredoxin that may play a role as areductant in the biogenesis of c-type cytochromesrdquo Journal ofBiological Chemistry vol 272 no 7 pp 4467ndash4473 1997

[89] G B Kallis and A Holmgren ldquoDifferential reactivity of thefunctional sulfhydryl groups of cysteine-32 and cysteine-32present in the reduced form of thioredoxin from Escherichiacolirdquo Journal of Biological Chemistry vol 255 no 21 pp 10261ndash10265 1980

[90] P T Chivers andR T Raines ldquoGeneral acidbase catalysis in theactive site of Escherichia coli thioredoxinrdquoBiochemistry vol 36no 50 pp 15810ndash15816 1997

[91] X M Zheng J Hong H Y Li D H Lin and H Y HuldquoBiochemical properties and catalytic domain structure of theCcmH protein from Escherichia colirdquo Biochim Biophys Actavol 1824 no 12 pp 1394ndash1400 2012

[92] UAhujaA Rozhkova RGlockshuber LThony-Meyer andOEinsle ldquoHelix swapping leads to dimerization of the N-terminaldomain of the c-type cytochrome maturation protein CcmHfrom Escherichia colirdquo FEBS Letters vol 582 no 18 pp 2779ndash2786 2008

[93] R A Fabianek T Hofer and L Thony-Meyer ldquoCharacteriza-tion of the Escherichia coli CcmH protein reveals new insightsinto the redox pathway required for cytochrome c maturationrdquoArchives of Microbiology vol 171 no 2 pp 92ndash100 1999

[94] E Reid J Cole and D J Eaves ldquoThe Escherichia coli CcmGprotein fulfils a specific role in cytochrome c assemblyrdquo Bio-chemical Journal vol 355 no 1 pp 51ndash58 2001

[95] Q Ren U Ahuja and LThony-Meyer ldquoA bacterial cytochromec heme lyase CcmF forms a complex with the heme chaperoneCcmE and CcmH but not with apocytochrome crdquo Journal ofBiological Chemistry vol 277 no 10 pp 7657ndash7663 2002

[96] E H Meyer P Giege E Gelhaye et al ldquoAtCCMH an essentialcomponent of the c-type cytochrome maturation pathway inArabidopsis mitochondria interacts with apocytochrome crdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 44 pp 16113ndash16118 2005

[97] C Rios-Velazquez R Coller and T J Donohue ldquoFeatures ofRhodobacter sphaeroides CcmFHrdquo Journal of Bacteriology vol185 no 2 pp 422ndash431 2003

[98] C Sanders M Deshmukh D Astor R G Kranz and F DaldalldquoOverproduction of CcmG and CcmFHRc fully suppresses thec-type cytochrome biogenesis defect of Rhodobacter capsulatus

16 Scientifica

CcmI-null mutantsrdquo Journal of Bacteriology vol 187 no 12 pp4245ndash4256 2005

[99] A F Verissimo H Yang X Wu C Sanders and F DaldalldquoCcmI subunit of CcmFHI heme ligation complex functionsas an apocytochrome c chaperone during c-type cytochromematurationrdquo Journal of Biological Chemistry vol 286 no 47 pp40452ndash40463 2011

[100] K Bryson L J McGuffin R L Marsden J J Ward J SSodhi and D T Jones ldquoProtein structure prediction servers atUniversity College Londonrdquo Nucleic Acids Research vol 33 no2 pp W36ndashW38 2005

[101] D T Jones ldquoProtein secondary structure prediction basedon position-specific scoring matricesrdquo Journal of MolecularBiology vol 292 no 2 pp 195ndash202 1999

[102] C Sanders C Boulay and F Daldal ldquoMembrane-spanning andperiplasmic segments of CcmI have distinct functions duringcytochrome c biogenesis in Rhodobacter capsulatusrdquo Journal ofBacteriology vol 189 no 3 pp 789ndash800 2007

[103] D Han K Kim J Oh J Park and Y Kim ldquoTPR domainof NrfG mediates complex formation between heme lyaseand formate-dependent nitrite reductase in Escherichia coliO157H7rdquo Proteins vol 70 no 3 pp 900ndash914 2008

[104] D J Eaves J Grove W Staudenmann et al ldquoInvolvement ofproducts of the nrfEFG genes in the covalent attachment ofhaem c to a novel cysteine-lysine motif in the cytochrome c552nitrite reductase from Escherichia colirdquo Molecular Microbiol-ogy vol 28 no 1 pp 205ndash216 1998

[105] J Nilsson B Persson and G Von Heijne ldquoConsensus predic-tions of membrane protein topologyrdquo FEBS Letters vol 486 no3 pp 267ndash269 2000

[106] E di Silvio A di Matteo F Malatesta and C Travaglini-Allocatelli ldquoRecognition and binding of apocytochrome c toP aeruginosa CcmI a component of cytochrome c maturationmachineryrdquo Biochim Biophys Acta vol 1834 no 8 pp 1554ndash1561 2013

[107] N Zeytuni and R Zarivach ldquoStructural and functional dis-cussion of the tetra-trico-peptide repeat a protein interactionmodulerdquo Structure vol 20 no 3 pp 397ndash405 2012

[108] C Sanders S Turkarslan D-W Lee O Onder R G Kranz andF Daldal ldquoThe cytochrome c maturation components CcmFCcmH and CcmI form a membrane-integral multisubunitheme ligation complexrdquo Journal of Biological Chemistry vol283 no 44 pp 29715ndash29722 2008

[109] A F Verissimo M A Mohtar and F Daldal ldquoThe hemechaperone ApoCcmE forms a ternary complex with CcmI andapocytochrome crdquoThe Journal of Biological Chemistry vol 288no 9 pp 6272ndash6283 2013

[110] P D Barker J C Ferrer M Mylrajan et al ldquoTransmutation of aheme proteinrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 90 no 14 pp 6542ndash65461993

[111] J Simon and L Hederstedt ldquoComposition and function ofcytochrome c biogenesis system IIrdquoThe FEBS Journal vol 278no 22 pp 4179ndash4188 2011

[112] T R H M Kouwen and J M van Dijl ldquoInterchangeablemodules in bacterial thiol-disulfide exchange pathwaysrdquo Trendsin Microbiology vol 17 no 1 pp 6ndash12 2009

[113] A Crow A Lewin O Hecht et al ldquoCrystal structure andbiophysical properties of Bacillus subtilis BdbD An oxidizingthioldisulfide oxidoreductase containing a novel metal siterdquoJournal of Biological Chemistry vol 284 no 35 pp 23719ndash23733 2009

[114] M C Moller and L Hederstedt ldquoExtracytoplasmic processesimpaired by inactivation of trxA (thioredoxin gene) in Bacillussubtilisrdquo Journal of Bacteriology vol 190 no 13 pp 4660ndash46652008

[115] A Lewin A Crow A Oubrie and N E Le Brun ldquoMolecularbasis for specificity of the extracytoplasmic thioredoxin ResArdquoJournal of Biological Chemistry vol 281 no 46 pp 35467ndash35477 2006

[116] C L Colbert QWu P J A Erbel K H Gardner and J Deisen-hofer ldquoMechanism of substrate specificity in Bacillus subtilisResA a thioredoxin-like protein involved in cytochrome cmaturationrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 103 no 12 pp 4410ndash44152006

[117] UAhuja P Kjelgaard B L Schulz LThony-Meyer andLHed-erstedt ldquoHaem-delivery proteins in cytochrome c maturationsystem IIrdquoMolecular Microbiology vol 73 no 6 pp 1058ndash10712009

[118] M L D Page P P Hamel S T Gabilly et al ldquoA homologof prokaryotic thiol disulfide transporter CcdA is required forthe assembly of the cytochrome b6f complex in Arabidopsischloroplastsrdquo Journal of Biological Chemistry vol 279 no 31pp 32474ndash32482 2004

[119] A Rozhkova C U Stirnimann P Frei et al ldquoStructural basisand kinetics of inter- and intramolecular disulfide exchange inthe redox catalyst DsbDrdquoThe EMBO Journal vol 23 no 8 pp1709ndash1719 2004

[120] T Schiott M Throne-Holst and L Hederstedt ldquoBacillussubtilis CcdA-defective mutants are blocked in a late step ofcytochrome C biogenesisrdquo Journal of Bacteriology vol 179 no14 pp 4523ndash4529 1997

[121] R E Feissner C S Beckett J A Loughman and R G KranzldquoMutations in cytochrome assembly and periplasmic redoxpathways in Bordetella pertussisrdquo Journal of Bacteriology vol187 no 12 pp 3941ndash3949 2005

[122] L S ErlendssonMMoller and L Hederstedt ldquoBacillus subtilisStoA is a thiol-disulfide oxidoreductase important for sporecortex synthesisrdquo Journal of Bacteriology vol 186 no 18 pp6230ndash6238 2004

[123] L S Erlendsson R M Acheson L Hederstedt and N E LeBrun ldquoBacillus subtilis ResA is a thiol-disulfide oxidoreductaseinvolved in cytochrome c synthesisrdquo Journal of BiologicalChemistry vol 278 no 20 pp 17852ndash17858 2003

[124] L S Erlendsson and L Hederstedt ldquoMutations in the thiol-disulfide oxidoreductases BdbC and BdbD can suppresscytochrome c deficiency of CcdA-defective Bacillus subtiliscellsrdquo Journal of Bacteriology vol 184 no 5 pp 1423ndash1429 2002

[125] M E Dumont J F Ernst D M Hampsey and F ShermanldquoIdentification and sequence of the gene encoding cytochromec heme lyase in the yeast Saccharomyces cerevisiaerdquoThe EMBOJournal vol 6 no 1 pp 235ndash241 1987

[126] M E Dumont J F Ernst and F Sherman ldquoCoupling of hemeattachment to import of cytochrome c into yeast mitochondriaStudies with heme lyase-deficient mitochondria and alteredapocytochromes crdquo Journal of Biological Chemistry vol 263 no31 pp 15928ndash15937 1988

[127] B San Francisco E C Bretsnyder and R G Kranz ldquoHumanmitochondrial holocytochrome c synthasersquos hemebindingmat-uration determinants and complex formationwith cytochromecrdquo Proceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 9 pp E788ndashE797 2012

Scientifica 17

[128] L Schaefer A Ballabio and H Y Zoghbi ldquoCloning andcharacterization of a putative human holocytochrome c-typesynthetase gene (HCCS) isolated from the critical region formicrophthalmia with linear skin defects (MLS)rdquoGenomics vol34 no 2 pp 166ndash172 1996

[129] S K Prakash T A Cormier A E McCall et al ldquoLoss ofholocytochrome c-type synthetase causes the male lethality ofX-linked dominant microphthalmia with linear skin defects(MLS) syndromerdquo Human Molecular Genetics vol 11 no 25pp 3237ndash3248 2002

[130] I Wimplinger M Morleo G Rosenberger et al ldquoMutations ofthe mitochondrial holocytochrome c-type synthase in X-linkeddominant microphthalmia with linear skin defects syndromerdquoTheAmerican Journal of HumanGenetics vol 79 no 5 pp 878ndash889 2006

[131] S Kiryu-Seo KGamo T Tachibana K Tanaka andHKiyamaldquoUnique anti-apoptotic activity of EAAC1 in injured motorneuronsrdquoTheEMBO Journal vol 25 no 14 pp 3411ndash3421 2006

[132] R Lill R A Stuart M E Drygas F E Nargang and W Neu-pert ldquoImport of cytochrome c heme lyase into mitochondriaa novel pathway into the intermembrane spacerdquo The EMBOJournal vol 11 no 2 pp 449ndash456 1992

[133] H Steiner A Zollner A Haid W Neupert and R LillldquoBiogenesis of mitochondrial heme lyases in yeast Importand folding in the intermembrane spacerdquo Journal of BiologicalChemistry vol 270 no 39 pp 22842ndash22849 1995

[134] A Zollner G Rodel and A Haid ldquoMolecular cloning andcharacterization of the Saccharomyces cerevisiae CYT2 geneencoding cytochrome-c1-heme lyaserdquo European Journal of Bio-chemistry vol 207 no 3 pp 1093ndash1100 1992

[135] V Corvest D A Murrey D G Bernard D B Knaff B Guiardand P PHamel ldquoc-Type cytochrome assembly in Saccharomycescerevisiae a key residue for apocytochrome c1lyase interactionrdquoGenetics vol 186 no 2 pp 561ndash571 2010

[136] V Corvest D A Murrey M Hirasawa D B Knaff B Guiardand P P Hamel ldquoThe flavoprotein Cyc2p a mitochondrialcytochrome c assembly factor is a NAD(P)H-dependent haemreductaserdquo Molecular Microbiology vol 83 no 5 pp 968ndash9802012

[137] D G Bernard S Quevillon-Cheruel S Merchant B Guiardand P P Hamel ldquoCyc2p a membrane-bound flavopro-tein involved in the maturation of mitochondrial c-typecytochromesrdquo Journal of Biological Chemistry vol 280 no 48pp 39852ndash39859 2005

[138] J G Kleingardner and K L Bren ldquoComparing substratespecificity between cytochrome cmaturation and cytochrome cheme lyase systems for cytochrome c biogenesisrdquo Metallomicsvol 3 no 4 pp 396ndash403 2011

[139] C Travaglini-Allocatelli S Gianni VMorea A Tramontano TSoulimane and M Brunori ldquoExploring the cytochrome c fold-ing mechanism cytochrome c552 fromThermus thermophilusfolds through an on-pathway intermediaterdquo Journal of BiologicalChemistry vol 278 no 42 pp 41136ndash41140 2003

[140] L Zhang and L Guarente ldquoHeme binds to a short sequencethat serves a regulatory function in diverse proteinsrdquoTheEMBOJournal vol 14 no 2 pp 313ndash320 1995

[141] S Hira T Tomita T Matsui K Igarashi and M Ikeda-SaitoldquoBach1 a heme-dependent transcription factor reveals pres-ence of multiple heme binding sites with distinct coordinationstructurerdquo IUBMB Life vol 59 no 8-9 pp 542ndash551 2007

[142] T K Kuhl A Wiszligbrock N Goradia et al ldquoAnalysis of Fe(III)heme binding to cysteine-containing heme-regulatory motifs

in proteinsrdquo ACS Chemical Biology vol 8 no 8 pp 1785ndash17932013

[143] R L Moore J M Stevens and S J Ferguson ldquoMitochondrialcytochrome c synthase CP motifs are not necessary for hemeattachment to apocytochrome crdquo FEBS Letters vol 585 no 21pp 3415ndash3419 2011

[144] S Gianni M Brunori and C Travaglini-Allocatelli ldquoRefoldingkinetics of cytochrome c551 reveals a mechanistic differencebetween urea and guanidinerdquo Protein Science vol 10 no 8 pp1685ndash1688 2001

[145] S Gianni C Travaglini-Allocatelli F Cutruzzola M BrunoriM C R Shastry and H Roder ldquoParallel pathways incytochrome c551 foldingrdquo Journal of Molecular Biology vol 330no 5 pp 1145ndash1152 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 Scientifica

Table1Proteincompo

nentsof

Syste

msIIIand

IIIa

long

with

theirstructural

features

(orPD

Bcodeswhenkn

own)

andfunctio

nalrolesS

ystem

Iproteinsarefoun

din120572-and120574-

proteobacteriasom

e120573-a

nd120575-proteob

acteria

and

Dein

ococciandArchaeaPlantm

itochon

driaM

itochon

driaof

somep

rotozoaSyste

mIIproteins

arefou

ndin

plantchlorop

lasts

Gram-

positiveb

acteria

cyano

bacteria120576-proteob

acteria

most120573

-proteob

acteria

and

some120575

-proteob

acteria

HCC

SofSystemIIIisfou

ndinthem

itochon

driaoffung

ivertebratesand

invertebrates

Syste

mI(SI)

SIstr

ucturalfeatures

Syste

mII(SII)

SIIstructuralfeatures

Syste

mIII(SIII)

SIIIstr

ucturalfeatures

Functio

n(s)

Ccm

AABC

transportermem

brane

n-sid

enu

cleotide-bind

ing

domain

ResB

(CcsB)

5-6TM

helices

1large

perip

lasm

icdo

main

conservedHis

resid

ues

HCC

SMem

brane-associated

protein

conservedHis

resid

ues

Hem

etranslocatio

nand

delivery

Ccm

BABC

transporter6TM

helices

ResC

(CcsA)

6ndash8TM

helices

WWDdo

main

conservedHis

resid

ues

Ccm

C6T

Mhelicesperiplasm

icWWDdo

main

Ccm

DSm

allm

embranep

rotein1

TMhelix

Ccm

E1T

MhelixO

B-fold

1SR3

1LM

01J6

Q2KC

T

Ccm

G1T

MhelixT

RX-like

fold

1Z5Y2B1K

1KNG3KH

73K

H93K

8NRe

sATR

X-lik

efold

2H1B1SU

91ST9

2F9

SapoC

ytthioredu

ction

Ccm

H1T

Mhelix3-helixbu

ndlefold

2HL72KW

0CcdA

6TM

helices

Ccm

F10ndash15TM

helicesperiplasm

icWWDdo

main

conservedHis

resid

ues

ResB

(CcsB)

ResC

(CcsA)

HCC

SapoC

ytchaperon

ingand

hemea

ttachment

Ccm

IPerip

lasm

icTP

Rand120572120573

domains

Scientifica 5

maturase multiprotein complex(es) However the existenceandor stoichiometry of these complexes remains to bedetermined either because of the experimental difficultiesin handling membrane protein complexes or because itis possible that these complexes are unstable and onlytransiently populated Independently from their functionalexistence in the bacterial periplasm as independent units or ascomponents of a multisubunit complex it is clear that each oftheCcmproteins plays a different role from the transport andchaperoning of the heme cofactor to the necessary reductionof the disulfide bond between the sulfur atoms of the twoCys residues of the conserved CXXCH motif of apoCytand finally to the catalysis of covalent heme attachment Anadditional interesting aspect is that over and above theirrole in the biogenesis of Cytc there is also evidence thatinactivation of some ccm genes induces phenotypes thatcannot be explained only in terms of absence of synthesis ofCyt c all of these pleiotropic effects are linked to impairmentof heme andor iron trafficking in the periplasm [47] Inparticular it has been recently shown that in 120572- and 120574-proteobacteria (including the human opportunistic pathogenP aeruginosa) mutations in the ccmC ccmI and ccmF genesinduce phenotypes such as reduced pyoverdine produc-tion reduced bacterial motility or impaired growth in low-iron conditions ([51] and refs therein) These observationssuggesting that Ccm proteins perform additional functionscritical for bacterial physiology growth and virulence pro-vide a rationale to explain why bacteria at variance withthe eukaryotic cell have evolved a metabolically expensiveoperon to accomplish an apparently simple task such asheme ligation to apoCyt Novel hypotheses addressing theseaspects and awaiting experimental investigation include (i)the utilization of Ccm-associated heme for additional cellularprocesses besides attachment to apoCyt (ii) trafficking a non-heme compound through the Ccm system required for ironacquisition such a siderophore (iii) the Ccm inactivation-dependent accumulation of heme b a photoreactivemoleculewhose degradation leads to reactive oxygen species and (iv)the destructive effect on [Fe-S] clusters of ferrisiderophoresreductases

In the following the structural (when known) andfunctional properties of the different components of theSystem I maturation apparatus will be discussed dissectingthe Cytc maturation process into three main functionalsteps heme translocation and delivery apoCyt thioreductivepathway and apoCyt chaperoning and heme ligation (asimilar modular description can also be found elsewhere(see [34 52])) However it should be remembered that inmany cases the proteins involved in the three steps are notuniquely assigned to a specific module as they interact witheach other moreover it has been shown that in some casesmore than one system can be present [41] This modularorganization of Cyt c biogenesis should therefore be intendedonly as a way to simplify the description of an overall highlyintegrated process For each of the three functional stepspresentation of the structural and functional properties of thedifferent protein components is followed by a discussion ofthe proposed molecular mechanism(s)

21 System I Components of the Heme Translocation andDelivery Pathway CcmA and CcmB proteins show the typ-ical sequence features of the ABC (ATP binding Cassette)transporter family and therefore these components of SystemI were initially considered as the proteins responsible forthe translocation of the newly synthesized heme from thecytoplasm to the periplasmic space ABC transporters areubiquitous multidomain integral membrane proteins thattranslocate a large variety of substrates across cellular mem-branes using ATP hydrolysis as a source of energy they aregenerally composed of a transmembrane (TM) domain and aconserved cytosolic nucleotide-binding domain [53]

CcmA is a cytoplasmic soluble protein representing thenucleotide-binding domain of the hypothetical ABC trans-porter according to this hypothesis its sequence containsa nucleotide-binding domain and Walker A and B motifsfor ATP hydrolysis [46] It has also been shown that CcmApossesses ATPase activity in vitro and that the protein isassociated with the membrane fraction only when CcmB isalso present [54]

CcmB and CcmC are both integral membrane proteinspredicted to contain six TM helices CcmC contains a shortWWDdomain in its second periplasmic domain and belongsto the heme handling protein family (HHP) [55] WWDdomains are short tryptophan-rich aminoacid stretcheswith the conserved WGX120601WXWDXRLT sequence (where 120601represents an aromatic amino acid residue and X representsany residue) [40 56] it has been proposed that proteinscontaining WWD domains are involved in heme-bindingand as we will see below a WWD domain is also presentin the CcmF protein another crucial heme-binding proteinof System I CcmC also contains two absolutely conservedhistidine residues in its first (between TM helices 1 and 2)and third (between TMhelices 5 and 6) periplasmic domainsAn attractive hypothesis still requiring experimental proofis that the hydrophobic residues within the tryptophan-richmotif provide a platform for the binding of heme whereasthe two conserved His residues (H60 and H184 in E coliCcmC) act as axial heme ligands [57] this hypothesis isstrengthened by the observation that CcmC indeed interactsdirectly with heme [58 59] Immunoprecipitation experi-ments have shown that in E coli CcmABC proteins forma multiprotein complex with a CcmA

2CcmB

1CcmC

1stoi-

chiometry confirming that these components form an ABC-type transporter complex with unusual functional propertiesassociated to the release of holoCcmE from CcmC [41 46]rather than to heme transport per se CcmC is an interestingprotein worth of future experimental efforts as it is knownthat in some pathogenic bacteria CcmC mutations are asso-ciated to specific phenotypes apparently not related to Cytc maturation such as siderophore production in Paracoccusand Pseudomonas [51] and iron utilization in Legionella [60]

Limited information is available about the structure andfunction of CcmD which appears to be a small membraneprotein (about 70 aminoacid residues) with no conservedsequence features whose topology is currently debatedcontrary to the original proposal [61] additional experimentshave shown that in E coli and R capsulatus CcmD isan integral membrane protein composed of a single TM

6 Scientifica

helix a periplasmic-orientedN-terminus and a cytoplasmic-oriented C-terminus [62] Immunoprecipitation experimentsindicate that CcmD interacts with the CcmA

2CcmB

1CcmC

1

complex even if it is not essential for heme transfer andattachment from CcmC to CcmE CcmD is strictly requiredfor the release of holoCcmE from the ABC transporter [6162]

CcmE is a heme-binding protein discovered as an essen-tial System I component as early as the late 1990rsquos [63]CcmE is a monotopic membrane protein anchored to themembrane via its N-terminal TM segment and exposing itsactive site to the periplasm it is the only Ccm componentof the heme trafficking and delivery module of System I forwhich a three-dimensional structure is available ([21] PDB1SR3 [64] PDB 1LM0) The 3D structure of the apo-state(without bound heme) consists of a six-stranded antiparallel120573-sheet reminiscent of the classical OB-fold [65] with N-and C-terminal extensions CcmE can be considered a ldquohemechaperonerdquo as it protects the cell from a potentially dangerouscompound by sequestering free heme in the periplasm [66]it is thought to act as an intermediate in the heme deliverypathway of Cytc maturation The structure of apoCcmEshowed no recognizable heme-binding cavities and in theabsence of a 3D structure of CcmEwith bound heme (holoC-cmE) the heme-binding region could only be predicted byin silico modeling It is generally believed that the hemein holoCcmE is solvent exposed but recent mutagenesisexperiments challenged this view [67] The unusual covalentbond between the nitrogen atomof a histidine residue presentin the conserved VLAKHDE motif located in a solventexposed environment (H130 in E coli CcmE) and a 120573-carbonof one of the heme vinyl groups has been described in greatdetail by NMR spectroscopy [68] Recently it was shown thatCcmE proteins from the proteobacteria D desulfuricans andD vulgaris contain the unusual CXXXY heme-bindingmotifwhere the Cys residue replaces the canonical His bindingresidue NMR solution structure of D vulgaris CcmE (PDB2KCT) revealed that the proteins adopt the same OB-foldcharacteristic of the CcmE superfamily Contrary to whatreported for theD desulfuricansCcmE [69] the homologousprotein from D vulgaris binds ferric heme noncovalentlythrough the conserved C127 residue [70] An additionalconserved residue in CcmE proteins is Tyr134 which wasshown to provide a coordination bond to the heme ironof holoCcmE [71 72] once it is released from CcmABCDcomplex [36] as discussed below

22 System I Heme Translocation and Delivery PathwayMechanisms We still do not know how the b heme istranslocated from the cytoplasm (where it is synthesized)to the periplasm where Cyt c maturation occurs Differ-ent mechanisms such as translocation through a proteinchannel or free diffusion across the membrane have beenproposed [73] The CcmAB proteins show structural featurestypical of the ABC transporters and for these reasons theywere originally hypothesized to be involved in the heme btranslocation process [57 63 74 75] However it is nowclear that an alternative process must exist since it has

been shown that periplasmic b-type cytochromes can beproduced in the absence of Ccm proteins [76] and thatinactivation of the ATPase activity of CcmA does not abolishheme accumulation in the periplasm [46 54] We have nowevidence that CcmC has the ability to bind heme at its WWDdomain present in the second periplasmic domain but itis still not clear if this membrane protein acts as a proteinchannel for heme translocation or simply collects it in theperiplasm [77]

Another important aspect concerns the oxidation state ofthe heme iron during translocation and delivery processesindeed this property of the heme iron may determine thereaction mechanism by which the unusual CcmE H130nitrogen is covalently linked to the vinyl 120573-carbon of theheme (see [36] for a detailed discussion of this topic) Basedon mutagenesis studies on CcmC [59] a model has beenpresented whereby oxidized heme is bound to CcmC onlyin the presence of CcmE forming a ternary complex BothCcmC and CcmE provide critical residues for heme-bindingthe two conserved His residues (H60 and H180 coordinatingthe heme iron) and theWWDdomain ofCcmCandHis130 ofCcmE forming the unusual covalent bond with heme vinyl-2 [59] The ATPase activity of CcmA is then required torelease holoCcmE from the CcmABCD complex a processthat depends also on the presence of CcmD [46 54] It shouldbe noticed that purified holoCcmE alone or in the CcmCDEcomplex [36 59] contains the heme iron in the oxidized statean observation that is apparently in contrast with the fact thatthe heme must be in its reduced state before attachment toapoCyt can occur Although the oxidation state of the hemeiron is currently debated [36 78] it is possible that CcmFwhich was recently shown to contain a heme b cofactor mayact as specific heme oxidoreductase (see Section 25)

23 System I ApoCyt Thioreduction Pathway ComponentsThe periplasm can be considered a relatively oxidizing envi-ronment due to the presence of an efficient oxidative systemcomposed of the DsbAB proteins [79 80] DsbA is a highlyoxidizing protein (1198641015840

0= 120mV) that is responsible for

the introduction of disulfide bonds into extracytoplasmicproteins [81] On the basis of the results obtained on E colidsbA deletion mutants that are unable to synthesize c-typecytochromes [82 83] it was generally accepted that formationof the intramolecular disulfide bond in apoCyt was a nec-essary step in the Cyt c biogenesis However only reducedapoCyt is clearly competent for heme ligation It is possiblethat this seemingly paradoxical thioreduction process hasevolved in order to protect the apoCyt from proteolyticdegradation aggregation andor formation of intermolec-ular disulfide bonds with thiols from other molecules (seealso Section 31 for a discussion about this aspect in SystemII) Recently however an analysis of c-type cytochromesproduction in several E coli dsb genes deletion strains ledto the hypothesis that DsbA is not necessary for Cyt cmaturation and that heme ligation to apoCyt and apoCytoxidation pathways is alternative competing processes [84]

In gram-negative bacteria a thioreduction pathway hasevolved to specifically reduce the oxidized apoCyt substrate

Scientifica 7

which includes the Ccm proteins CcmG and CcmH Thenecessary reducing power is transferred from the cytoplasmicthioredoxin (TRX) to CcmG via DsbD a large membraneprotein organized in three structural domains an N-terminalperiplasmic domain with a IgG-like fold (nDsbD) a C-terminal periplasmic domain with a thioredoxin-like (TRX-like) fold (cDsbD) and a central domain composed ofeight TM helices [85] Each of these domains contains apair of Cys residues and transfer electrons via a cascadeof disulfide exchange reactions making DsbD a ldquoredox-hubrdquo in the periplasm performing disulfide bond exchangereactions with different oxidized proteins [79] In particulara combination of X-ray crystallography experiments andkinetic analyses showed that electrons are transferred fromthe cytoplasmic TRX to the membrane domain of DsbDfollowed by reduction of cDsbD and finally of nDsbD whichis the direct electron donor to CcmG [85]

CcmG is a membrane-anchored protein linked to themembrane via an N-terminal TM helix and exposing itssoluble TRX-like domain in the periplasm The 3D structureof the TRX-like domain of CcmG from different bacteriahas been solved by X-ray crystallography (E coli PDB 1Z5Y[85] PDB 2B1K [86] B japonicum PDB 1KNG [87] Paeruginosa PDB 3KH7 3KH9 [22]) and is generally wellconserved as proved by the low RMSD (08 A between Pa-CcmG and Ec-CcmG 135 A between Pa-CcmG and Bj-CcmG) Although all these proteins adopt a TRX-like foldand contain the redox-active motif CXXC in the first 120572-helixthey are inactive in the classic insulin reduction assay [7588] CcmG proteins are therefore considered specific thiol-oxidoreductase able to recognize and selectively interactonly with their upstream and downstream binding partnersin the thioreduction process leading to reduced apoCytcLooking at the 3D structure of the periplasmic domain ofthe prototypical Pa-CcmG it is possible to identify the 120573120572120573and 120573120573120572 structural motifs of the TRX fold linked by a short120572-helix and forming a four-stranded 120573-sheet surrounded bythree helices the protein contains an additional N-terminalextension (residues 26ndash62) and a central insert (residues 102ndash123) The redox-active motif of Pa-CcmG (CPSC) is locatedin the first 120572-helix of the TRX fold as usually observed in allTRX-like proteins As for any molecular machinery whereeach component must recognize and interact with more thanone target (ie the substrate and the other components ofthe apparatus) an open question concerns the mechanismwhereby CcmG is able to recognize its different partnersThe availability of the crystal structures of Pa-CcmG bothin the oxidized (22 A resolution) and reduced state (18 Aresolution) [22] allowed highlighting the structural similaritybetween the two redox states (Rmsd of the C120572 atoms inthe two redox forms is 019 A) and therefore to excludestructural rearrangement as the mechanism used by Pa-CcmG to discriminate between reduced (such as the nDsbDdomain) and oxidized partners (Pa-CcmH andor apoCyt)

The standard redox potential of Pa-CcmG (11986410158400= 0213V

at pH 70 [22] as well as that of Ec-CcmG (11986410158400= 0212V

[86]) indicates that these proteins act as mild reductants inthe thioreductive pathway of Cytc biogenesis However the

Figure 5 Three-dimensional structure of Pa-CcmH shown inribbon representation The figure shows the three-helix bundleforming the characteristic fold of Pa-CcmHThe active site disulfidebond between residues Cys25 andCys28 in the long loop connectinghelices 120572-helix1 and 120572-helix 2 is highlighted in yellow

function of thiol-oxidoreductases obviously depends on thepKa values of their activesite Cys residues The pKa of CysX(613 plusmn 005) and CysY (105 plusmn 007) are consistent with thepKa values measured in different TRXs where the active N-terminal Cys residue has a pKa close to pH 70 whereas theC-terminal Cys has a much higher pKa [89 90] Such a largedifference between the two pKa values in the TRX family isfunctionally relevant because it allows the N-terminal Cysto perform the nucleophilic attack on the target disulfidewhile the C-terminal Cys is involved in the resolution of theresulting mixed-disulfide [90]

CcmH is the other component of System I involvedin the reduction of apoCyt Notably CcmH proteins fromdifferent bacterial subgroups may display structural vari-ability indeed while in E coli Ec-CcmH is a bipartiteprotein characterized by two soluble domains exposed to theperiplasm and two TM segments CcmH from P aeruginosa(Pa-CcmH) is a one-domain redox-active protein anchoredto the membrane via a single TM helix and homologous tothe N-terminal redox-active domain of Ec-CcmH Surpris-ingly the 3D structure of the soluble periplasmic domainof Pa-CcmH revealed that it adopts a peculiar three-helixbundle fold strikingly different from that of canonical thiol-oxidoreductases (Figure 5 PDB 2HL7 [23])TheN-terminaldomain of Ec-CcmH was also shown to have the same 3Dstructure although helix-swapping and dimerization havebeen observed in this case (PDB 2KW0 [91 92]) Theconserved redox-active motif (LRCPKC) is located in theloop connecting helices 1 and 2 close to the activesite thecrystal structure reveals the presence of a small pocket on thesurface of Pa-CcmH surrounded by conserved hydrophobicand polar residues which could represent the recognition sitefor the heme-binding motif of apoCyt

Concerning the functional properties of this unusualthiol-oxidoreductase it is interesting to note that its standardredox potential (1198641015840

0= 0215V) [23] is similar to that ob-

tained for Pa-CcmG This observation stands against thelinear redox cascade hypothesis whereby CcmG reducesCcmH While in the canonical redox-active CXXC motif

8 Scientifica

S SSS

SH

SH

Scheme 1 Scheme 2

SH

SH

SHSHS

S

CcmG 7477

CcmG 7477

CcmH 2528

CcmH 2528

apoCytox

apoCytred

S

SHCcmG 74

77

SH

SH

HSHS

CcmG 7477

S

S

SS

3

1

2CcmG 7477

SH

SHCcmH 25

28SH

HSS S

CcmH 2528

SHS

CcmH2528

apoCytox

apoCytred

+

Figure 6 Alternative thioreduction pathways whichmay be operative in System I and hypothesized on the basis of structural and functionalcharacterization of the redox-active Ccm proteins from P aeruginosa [22 23 25] Scheme 1 is a linear redox cascade whereby CcmG is thedirect reductant of CcmH which reduces oxidized apoCyt Scheme 2 envisages a more complex scenario involving the formation of a mixed-disulfide complex between CcmH and apoCyt (Step 1) This complex is the substrate for the attack by reduced CcmG (Step 2) that liberatesreduced apoCyt The resulting disulfide bond between CcmH and CcmG is then resolved by the free Cys thiol of CcmG (probably Cys77 inPa-CcmG) Adapted from [25]

of the TRX family the N-terminal Cys is always solventexposed in CcmH proteins the arrangement of the two Cysresidues is reversed the N-terminal Cys residue is buriedwhereas the C-terminal Cys residue is solvent exposed Onthe basis of this observation it was suggested that differentfrom the canonical TRX redox mechanism CcmH proteinsperform the nucleophilic attack on the apoCyt disulfide viatheir C-terminal Cys residue [23] This mechanism which isin agreement with the mechanism proposed earlier for Ec-CcmH on the basis of mutational-complementation studies[93 94] is substantiated by the peculiar pKa values of theactive site Cys residues of Pa-CcmH which were found tobe similar for both cysteines (84 plusmn 01 and 86 plusmn 01 [23])Again this is different from what is generally observed in thecase of TRX proteins where the pKa value of the Cys residueperforming the initial nucleophilic attack is significantlylower than the pKa value of theCys residue responsible for theresolution of the intermediate mixed-disulfide It is temptingto speculate that the unusual pKa values of the Pa-CcmHactive site thiols may ensure the necessary specificity of thiscomponent of the Ccm apparatus toward the CXXCH motifof the apoCyt substrate

24 System I ApoCyt Thioreduction Pathway MechanismAlthough we know that CcmG and CcmH are the redox-active components of System I involved in the thioreductivepathway of Cyt c biogenesis not only an acceptedmechanismfor the reduction of apoCyt disulfide bond is still lackingbut also the absolute requirement of such a process is nowdebated [38 84] Focusing our attention on the reductionof the apoCyt internal disulfide at least two mechanismscan been hypothesized which involve either a linear redoxcascade of disulfide exchange reactions or a nonlinear redox

process involving transient formation of a mixed-disulfidecomplex as depicted in Figure 6 and Schemes 1 and 2respectively

Both the thiol-disulfide exchange mechanisms depictedin Figure 6 suggest that CcmH is the direct reductant ofthe apoCytc disulfide however even if immunoprecipitationexperiments failed to detect the formation of a mixed-disulfide complex between apoCyt and CcmH proteins [95]some in vitro evidence supporting the formation of sucha complex has been presented In particular it has beenshown that Rhodobacter capsulatus and Arabidopsis thalianaCcmH homologues (Rc-CcmH and At-CcmH) are able toreduce the CXXCH motif of an apoCyt-mimicking peptide[75 96] In the latter case yeast two-hybrid experimentscarried out on At-CcmH indeed revealed an interactionbetween the protein and a peptide mimicking the A thalianaCyt c sequence In the case of Pa-CcmH FRET kineticexperiments employing a Trp-containing fluorescent variantof the protein and a dansylated nonapeptide encompassingthe heme-binding motif of P aeruginosa cytochrome c551(dans-KGCVACHAI) [23] allowed to directly observe theformation of themixed-disulfide complex and tomeasure theoff-rate constant of the bound peptide The results of these invitro binding experiments allowed to calculate an equilibriumdissociation constant which combines an adequate affinity(low 120583M) with the need to release efficiently reduced apoCytto other component(s) of the System I maturase complex[23] More recently the results obtained by FRET bindingexperiments carried out with single Cys-containing mutantsof Pa-CcmH and Pa-CcmG [25] substantiated the hypothesisdepicted in Scheme 2 (Figure 6) Altogether these structuraland functional results suggest that the thioreduction pathwaymechanism leading to reduced apoCyt is better describedby Scheme 2 and that reducing equivalents might not be

Scientifica 9

transferred directly from CcmG to apoCyt as depicted inScheme 1 According to Scheme 2 reduced CcmH (a non-TRX-like thiol-oxidoreductase) specifically recognizes andreduces oxidized apoCyt via the formation of a mixed-disulfide complex which is subsequently resolved by CcmGThe resulting disulfide bond between CcmH and CcmG isthen resolved by the free Cys thiol of CcmG (probably Cys77in Pa-CcmG)

However further in vitro experiments with CcmH andapoCyt single Cys-containing mutants are needed to unveilthe details of the thioreduction of oxidized apoCyt by CcmHIn particular it would be crucial to identify the Cys residueof apoCyt that remains free in the apoCyt-CcmH mixed-disulfide complex intermediate (see Scheme 2 and Section 26below) and available to thioether bond formation with oneof the heme vinyl groups Clearly structure determinationof the trapped mixed-disulfide complexes between CcmHCcmG and apoCyt (or apoCyt peptides) would providekey information for our understanding of this specializedthioreduction pathway mechanism

25 System I ApoCyt Chaperoning and Heme AttachmentComponents The reduced heme-binding motif of apoCyt isnow available to the heme ligation reaction However themolecularmechanismwhereby the Ccmmachinery catalyzesor promotes the formation of the heme-apoCyt covalentbonds is still largely obscure representing themost importantgoal in the field Past observations and recent experimentssuggest that CcmF and CcmI possibly together with CcmHare involved in these final steps [16 34 36]

CcmF is a large integral membrane protein of more than600 residues belonging to the heme handling protein family(HHP [55]) and predicted to contain 10ndash15 TM helices (notethat some discrepancy exists as to the number of TM helicespredicted by computer programs and those predicted onthe basis of phoA and lacZ fusion experiments [40 97])a conserved WWD domain and a larger domain devoidof any recognizable sequence features both exposed to theperiplasm Only recently E coli CcmF (Ec-CcmF) has beenoverexpressed solubilized from the membrane fraction andspectroscopically characterized in vitro [36 41] Surprisinglythe biochemical characterization of recombinant Ec-CcmFallowed to show that the purified protein contains heme b ascofactor in a 1 1 stoichiometry this observation led to thehypothesis that in addition to its heme lyase function Ec-CcmF may act as a heme oxidoreductase In particular it ispossible that the heme b of Ec-CcmF may act as a reductantfor the oxidized iron of the heme bound to CcmE [41]indeed the in vitro reduction of Ec-CcmF by quinones hasbeen experimentally observed strengthening the hypothesisabout the quinolhemeoxidoreductase function of this elusiveproteinThe structuralmodel proposed for Ec-CcmFpredicts13 TMhelices and notably the location of the four completelyconserved His residues according to the model two of them(His173 andHis303) are located in periplasmic exposed loopsnext to the conserved WWD domain which is believedto provide a platform for the heme bound to holoCcmEwhile His261 is located in one of the TM helices and it is

predicted to act as an axial ligand to the heme b of Ec-CcmFthe other conserved His residue (H491) could provide thesecond axial coordination bond to the heme although thishas not been experimentally addressed This model of Ec-CcmF therefore envisages that this large membrane proteinis characterized by two heme-binding sites one of themis embedded in the membrane and coordinates a heme bprosthetic group necessary to reduce the CcmE-bound hemehosted in the second heme-binding site and constituted by itsWWD domain

It is interesting to note that in plants mitochondria theCcmF ortholog appears to be split into three different pro-teins (At-CcmFN1 At-CcmFN2 and At-CcmFC) possiblyinteracting each other [16] Since each of these proteins issimilar to the corresponding domain in the bacterial CcmFortholog this observation may provide useful informationin the design of engineered fragments of bacterial CcmFproteins amenable to structural analyses

The other System I component which is generallybelieved to be involved in the final steps of Cyt c maturationis CcmI As stated above the ccmI gene is present only insome Ccm operons while in others the corresponding ORFis present within the ccmH gene (as in E coli)The functionalrole of CcmI in Cytc biogenesis is revealed by geneticstudies showing that in R capsulatus and B japonicuminactivation of the ccmI gene leads to inability to synthesizefunctional c-type cytochromes [98 99] In R capsulatus andP aeruginosa the CcmI protein (Rc-CcmI and Pa-CcmIresp) can be described as being composed of two domainsstarting from the N-terminus a first domain composed oftwo TM helices connected by a short cytoplasmic regionand a large periplasmic domain Structural variations maybe observed among CcmI members from different bacteriaindeed multiple sequence alignment indicates that the cyto-plasmic region of Rc-CcmI contains a leucine zipper motifwhich is not present in the putative cytoplasmic region of Pa-CcmI [100ndash102] Surprisingly no crystallographic structureis available up to now for the soluble domain of any CcmIprotein with the exception of the ortholog protein NrfGfrom E coli (Ec-NrfG) [103] This protein is necessary toattach the heme to the unusual heme-binding motif CWSCK(where a Lys residue substitutes the conservedHis) present inNrfA a pentaheme c-type cytochrome [103 104] Accordingto secondary structure prediction methods [105] it hasbeen proposed that the periplasmic domain of Pa-CcmI iscomposed of aN-terminal120572-helical region containing at leastthree TPR motifs connected by a disordered linker to a 120572-120573C-terminal regionMultiple sequence analyses and secondarystructure predictionmethods show that the TPR region of Pa-CcmI can be successfully aligned with many TPR-containingproteins including Ec-NrfG [106]

26 System I ApoCyt Chaperoning and Heme AttachmentMechanisms TPR domain-containing proteins are commonto eukaryotes prokaryotes and archaea these proteins aregenerally involved in the assembly of multiprotein complexesand to the chaperoning of unfolded proteins [103 107] Itis therefore plausible that CcmI (or the TPR C-terminal

10 Scientifica

domain of Ec-CcmH) may act to provide a platform for theunfolded apoCyt chaperoning it to the heme attachment sitepresumably located on the WWD domain of CcmF CcmImay thus be considered a component of amembrane-integralmultisubunit heme ligation complex together with CcmFand CcmH as experimentally observed by affinity purifi-cation experiments carried out with Rc-CcmFHI proteins[97 99 108] According to the proposed function of CcmI acritical requirement is represented by its ability to recognizedifferent protein targets over and above apoCyt such asCcmFandCcmHHowever until now direct evidence has been pre-sented only for the interaction of CcmI with apoCyt but thepossibility remains that CcmFHI proteins interact each othervia their TM helices and not via their periplasmic domainsInterestingly both for Pa-CcmI [106] or Rc-CcmI [99] CDspectroscopy experiments carried out on the CcmIapoCytcomplex highlighted major conformational changes at thesecondary structure level It is tempting to speculate on thebasis of these results that in vivo the folding of apoCyt maybe induced by the interaction with CcmI In the case of Paeruginosa System I proteins the binding process betweenPa-CcmI and its target protein apoCyt c551 (Pa-apoCyt) hasbeen studied both at equilibrium and kinetically [106] the119870119863measured for this interaction (in the 120583M range) appeared

to be low enough to ensure apoCyt delivery to the othercomponents of the Ccmmachinery Clearly a major questionconcerns the molecular determinants of such recognitionprocess interestingly both affinity coprecipitation assays[99] and equilibrium and kinetic binding experiments [106]highlighted the role played by the C-terminal 120572-helix of Cytc Similar observations have been made for the interaction ofEc-NrfGwith a peptidemimickingNrfA its apoCyt substrate[103] in this case isothermal titration calorimetry (ITC)experiments indicate that the TPR-domain of NrfG serves asa binding site for the C-terminal motif of NrfA Altogetherthese observations are in agreement with the fact that TPRproteins generally bind to their targets by recognizing theirC-terminal region [107]

The CcmI chaperoning activity has been experimentallysupported for the first time in the case of Pa-CcmI by citratesynthase tests [106] it has been proposed that the observedability to suppress protein aggregation in vitromay reflect thecapacity of CcmI to avoid apoCyt aggregation in vivo Stillanother piece of the Cyt c biogenesis puzzle has been addedrecently by showing that Rc-CcmI is able to interact withapoCcmE either alone or together with its substrate apoCytc2 forming a stable ternary complex in the absence of heme[109] This unexpected observation obtained by reciprocalcopurification experiments provides supporting evidence forthe existence of a large multisubunit complex composed ofCcmFHI andCcmE possibly interactingwith theCcmABCDcomplex It is interesting to note that while in the case ofthe CcmIapoCyt recognition different studies highlightedthe crucial role of the C-terminal helical region of apoCyt(see above) in the case of the apoCcmE apoCyt recognitionthe N-terminal region of apoCyt seems to represent a criticalregion

It is generally accepted that CcmF is the Ccm compo-nent responsible for heme covalent attachment to apoCyt

however as discussed above it is possible that this largemembrane protein plays such a role only together withother Ccm proteins such as CcmH and CcmI Moreover asrecently discovered by Kranz and coworkers [36 41] CcmFmay also act as a quinoleheme oxidoreductase ensuringthe necessary reduction of the oxidized heme b bound toCcmE Why it is necessary that the heme iron be in itsreduced state rather than in its oxidized state is not completelyclear although it is possible that this is a prerequisite to themechanism of thioether bond formation [110] According tocurrent hypotheses it is likely that the periplasmic WWDdomain of CcmF provides a platform for heme b bindingSanders et al and Verissimo et al [34 109] have presenteda mechanistic view of the heme attachment process whichtakes into account all the available experimental observationson the different Ccmproteins According to thismodel stere-ospecific heme ligation to reduced apoCyt occurs becauseonly the vinyl-4 group is available to form the first thioetherbond with a free cysteine at the apoCyt heme-binding motifsince the vinyl-2 group is involved (at least in the Ec-CcmE)in the covalent bond with His130 of CcmE [67] Howeverexperimental proof for this hypothesis requires a detailedinvestigation of the apoCyt thioreduction process catalyzedby CcmH (see Section 24)

It should be noticed that the mechanisms described sofar for the function(s) played by CcmF (see [34 36 109]) donot envisage a clear role for its large C-terminal periplasmicdomain (residues 510 to 611 in Ec-CcmF) It would be inter-esting to see if this domain apparently devoid of recognizablesequence features may mediate intermolecular recognitionprocesses with one (or more) component(s) involved in thehemeapoCyt ligation process

3 System II

System II is typically found in gram-positive bacteria and inin 120576-proteobacteria it is also present in most 120573- and some120575-proteobacteria in Aquificales and cyanobacteria as wellas in algal and plant chloroplasts System II is composedof three or four membrane-bound proteins CcdA ResACcsA (also known as ResC) and CcsB (also known as ResB)(Figure 3) CcdA andResA are redox-active proteins involvedin the reduction of the disulfide bond in the heme-bindingmotif of apoCyt whereas CcsA and CcsB are responsible forthe heme-apoCyt ligation process and are considered Cyt csynthethases (CCS) BothCcsAwhich is evolutionary relatedto the CcmC and CcmF proteins of System I [55] and CcsBare integral membrane proteins In some 120576-proteobacteriasuch asHelicobacter hepaticus andHelicobacter pylori a singlefusion protein composed of CcsA and CcsB polypeptides ispresent [41 111] Although as discussed below evidence hasbeen put forward to support the hypothesis that the CcsBAcomplex acts a heme translocase we still do not know if theheme is transported across the membrane by component(s)of System II itself or by a different unidentified process

31 System II ApoCyt Thioreduction Pathway After the Secmachinery secretes the newly synthesized apoCyt it readily

Scientifica 11

becomes a substrate for the oxidative system present on theouter surface of the cytoplasmic membrane of the gram-positive bacteria In B subtilis the BdbCD system [112113] is functionally but not structurally equivalent to thewell-characterized DsbAB system present in gram-negativebacteria As discussed above for System I the disulfide ofthe apoCyt heme-binding motif must be reduced in orderto allow thioether bonds formation and heme attachmentResA is the extracytoplasmic membrane-anchored TRX-likeprotein involved in the specific reduction of the apoCytdisulfide bond after the disulfide bond exchange reactionhas occurred oxidized ResA is reduced by CcdA whichreceives its reducing equivalents from a cytoplasmic TRX[114] Although ResA displays a classical TRX-like foldthe analysis of the 3D structure of oxidized and reducedResA from B subtilis (Bs-ResA) showed redox-dependentconformational modifications not observed in other TRX-like proteins Interestingly such modifications occur at thelevel of a cavity proposed to represent the binding site foroxidized apoCyt [24 115 116] Another peculiar feature of Bs-ResA is represented by the unusually similar pKa values of itsthe active site Cys residues (88 and 82 resp) as observedalso in the case of the active site Cys residues of the SystemI Pa-CcmH (see Section 23) However at variance with Pa-CcmH in Bs-ResA the large separation between the twocysteine thiols observed in the structure of the reduced formof the protein can be invoked to account for this result

CcdA is a large membrane protein containing six TMhelices [117 118] homologous to the TM domain of E coliDsbD [85 119] Its role in the Cyt c biogenesis is supportedby the observation that inactivation of CcdA blocks theproduction of c-type cytochromes in B subtilis [120 121]Moreover over and above the reduction of its apoCyt sub-strate Bs-CcdA is able to reduce the disulfide bond of othersecreted proteins such as StoA [122] It is interesting to notethat ResA and CcdA are not essential for Cyt c synthesisin the absence of BdbD or BdbC or if a disulfide reductantis present in the growth medium [123 124] As discussedabove (Section 24) a similar observation was made on therole of the thio-reductive pathway of System I in this casepersistent production of c-type cytochromes was observedin Dsb-inactivated bacterial strains [38] Following theseobservations the question has been put forward as to why theCyt c biogenesis process involves this apparently redundantthio-reduction route only to correct the effects of Bdb orDsb activity Referring to the case of B subtilis [111] it hasbeen proposed that the main reason is that BdbD mustefficiently oxidize newly secreted proteins since the activityof most of them depends on the presence of disulfide bondsAlternatively it can be hypothesized that the intramoleculardisulfide bondof apoCyt protects the protein fromproteolyticdegradation or aggregation or from cross-linking to otherthiol-containing proteins

32 System II Heme Translocation and Attachment to ApoCytRecent experimental evidence is accumulating supportingthe involvement of the heterodimeric membrane complexResBC or of the fusion protein CcsBA in the translocation

of heme from the n-side to the p-side of the bacterialmembrane In particular it has been shown that CcsBAfrom H hepaticus (Hh-CcsBA) is able to reconstitute Cyt cbiogenesis in the periplasm of E coli [36] Moreover it wasalso observed that Hh-CcsBA is able to bind reduced hemevia two conserved histidines flanking the WWD domainand required both for the translocation of the heme andfor the synthetase function of Hh-CcsBA [36] A differentstudy carried out on B subtilis System II proteins providedsupport for heme-binding capability by the ResBC complexaccording to the results obtained on recombinant ResBC theResB component of the heterodimeric CCS complex is ableto covalently bind the heme in the cytoplasm (probably by aCys residue) and to deliver it to an extracytoplamic domain ofResC which is responsible for the covalent ligation to apoCyt[117] It should be noticed however that the transfer of theCCS-bound heme to apoCyt still awaits direct experimentalproof Moreover it has also been reported that in B subtilisthe inactivation of CCS does not affect the presence ofother heme-containing proteins in the periplasm such ascytochrome b562 [111] this observation which is in contrastto the heme-transport hypothesis by CCSs parallels similarconcerns about heme translocation mechanism that arecurrently discussed in the context of System I (see Section 22above) In the case of the Hh-CcsBA synthetase site-directedmutagenesis experiments allowed to assign a heme-bindingrole for the two pairs of conserved His residues accordingto the topological model of the protein obtained by alkalinephosphatase (PhoA) assays and GFP fusions the conservedHis77 and His858 residues are located on TM helices whileHis761 and His897 are located on the periplasmic side [36]These observations together with the results of site-directedmutagenesis experiments allowed the authors to suggest thatHis77 andHis858 form a low affinitymdashmembrane embeddedheme-binding site for ferrous heme which is subsequentlytranslocated to an external heme-binding domain of Hh-CcsBA where it is coordinated by His761 and His897 Thetopological model therefore predicts that this last His pairis part of a periplasmic-located WWD domain (homologousto theWWDdomains found in theCcmCandCcmFproteinsof System I described above)

4 System III

Strikingly different from Systems I and II the Cyt c mat-uration apparatus found in fungi and in metazoan cells(System III) is composed of a single protein known as Holo-Cytochrome c Synthetase (HCCS) or Cytochrome c HemeLyase (CCHL) apparently responsible for all the subprocessesdescribed above (heme transport and chaperoning reductionand chaperoning of apoCyt and catalysis of the thioetherbonds formation between heme and apoCyt) (Figure 4)Surprisingly although this protein has been identified in Scerevisiaemitochondria several years ago [125 126] only veryrecently the human HCCS has been expressed as a recombi-nant protein in E coli and spectroscopically characterized invitro [127] It is worth noticing that humanHCCS is attractinginterest since over and above its role in Cyt c biogenesis it

12 Scientifica

is involved in diseases such as microphthalmia with linearskin defects syndrome (MLS) an X-linked genetic disorder[128ndash130] and in other processes such as Cytc-independentapoptosis in injured motor neurons [131] Different fromanimal cells where a single HCCS is sufficient for thematuration of both soluble and membrane-anchored Cytc(Cyt c and Cy c1 resp) in S cerevisiae two homologs HCCSand HCC1S located in the inner mitochondrial membraneand facing to the IMS space [132 133] are responsible for hemeattachment to apoCyt c and apoCyt c1 respectively [125 134]It should also be noticed that in fungi an additional FAD-containing protein (Cyc2p) is required for Cyt c synthesisCyc2p is a mitochondrial membrane-anchored flavoproteinexposing its redox domain to the IMS which is requiredfor the maturation of Cyt c but not for that of Cyt c1 [135]This protein does not contain the conserved Cys residuestypically found in disulfide reductases and indeed it is notable to reduce oxidized apoCyt in vitro However it has beenrecently shown that Cyc2p is able to catalyze the NAD(P)H-dependent reduction of heme in vitro [136] a necessarystep before the thioether bond formation can occur asdiscussed above (see Section 26) This result together withthe observation that Cyc2p interacts with HCCS and withapoCyt c and c1 lends support to the proposal that Cyc2p isinvolved in the reduction of the heme iron in vivo [136 137]

AlthoughHCCS proteins are crucial for Cyt cmaturationwe still do not know how these proteins recognize theirsubstrates (heme and apoCyt) and how they promote orcatalyze the formation of thioether bonds between the hemevinyl groups and the cysteine thiols of the apoCyt CXXCHmotif

Contrary to the broad specificity of System I whichis able to recognize and attach the heme to prokaryoticand eukaryotic c-type cytochromes [49 50] and even tovery short microperoxidase-like sequences [49 50] mito-chondrial HCCS is characterized by a higher specificityas it does not recognize bacterial apoCyt sequences Theseobservations prompted the investigation of the recognitionprocess between apoCyt and HCCS Recently by using arecombinant yeast HCCS and chimerical apoCyt sequencesexpressed in E coli it was possible to conclude that a crucialrecognition determinant is represented by the N-terminalregion of apoCyt containing the heme-binding motif [35]notice that in the context of System I the same N-terminalregion of the Cyt c sequence has been recently identified to beimportant for the recognition by apoCcmE (see Section 26)Over and above the role played by the intervening residuesin the CXXCH heme-binding motif [135] it has been shownthat a conserved Phe residue occurring in the N-terminalregion before the CXXCH motif is important for HCCSrecognition [35 138] These results support the hypothesisthat this residue known to be a key determinant of Cytcfolding and stability [19 20 139] may also be crucial for Cytcmaturation by HCCS

Another relevant question concerns the ability of HCCSto recognize and bind the heme molecule as the region ofthe protein responsible for heme recognition remains to beidentified Initially it was hypothesized that the recognitionof heme could be mediated by the CP motifs present in

the HCCS protein [140] these short sequences are indeedknown to bind heme in a variety of heme-containing proteins[141 142] However it has been recently shown that CPmotifsof the recombinant S cerevisiae HCCS are not necessaryfor Cyt c production in E coli [143] excluding them as keydeterminants of heme recognition

The long-awaited in vitro characterization of the recom-binant human HCCS allowed for the first time to pro-pose a molecular mechanism underlying Cytc maturationin eukaryotes which can be experimentally tested [127]According to the proposed model the human HCCS activitycan be described as a four step mechanism involving (i)heme-binding (ii) apoCyt recognition (iii) thioether bondsformation and (iv) holoCyt c release In particular theheme is proposed to play the role of a scaffolding moleculemediating the contacts between HCCS and apoCyt Muta-genesis experiments carried out on the recombinant HCCSstrongly suggest that heme-binding (Step 1) depends on thepresence of a specific His residue (His154) acting as anaxial ligand to ferrous heme Residues present at the N-terminus of apoCyt mediate the recognition with HCCS(Step 2) as discussed above it is known that this regionforms structurally conserved 120572-helix in the fold of all c-type cytochromes Unfortunately no information is availableup to now concerning the region of HCCS involved in therecognition and binding to the N-terminal region of apoCytCoordination of the heme iron by the His residue of theapoCyt heme-binding motif CXXCH provides the secondaxial ligand to the heme iron and is probably importantfor the correct positioning of the two apoCyt Cys residuesand formation of the thioether bonds (Step 3) The finalrelease of functional Cyt c (Step 4) clearly requires thedisplacement of the His154Fe2+ coordination bond sucha displacement is probably mediated by formation of thecoordination bond with the Cyt c conserved Met residueandor by the simultaneous folding of Cyt c Again it isinteresting to note that this last hypothesis is in accordancewith in vitro folding studies on c-type cytochromes thathighlighted the late formation of the Met-Fe coordinationduring Cyt c folding [144 145]

Acknowledgments

The author thanks A Di Matteo and E Di Silvio for fruitfuldiscussions and for help in preparing this paper This work issupported by grants from the University of Rome ldquoSapienzardquo(C26A11MBXJ and C26A13MWF4)

References

[1] M G Galinato J G Kleingardner S E Bowman et alldquoHeme-protein vibrational couplings in cytochrome c providea dynamic link that connects the heme-iron and the proteinsurfacerdquo Proceedings of the National Academy of Sciences vol109 no 23 pp 8896ndash8900 2012

[2] H B Gray and J R Winkler ldquoElectron flow through metal-loproteinsrdquo Biochimica et Biophysica Acta vol 1797 no 9 pp1563ndash1572 2010

Scientifica 13

[3] P Ascenzi R Santucci M Coletta and F PolticellildquoCytochromes reactivity of the ldquodark siderdquo of the hemerdquoBiophysical Chemistry vol 152 no 1ndash3 pp 21ndash27 2010

[4] S Yamada N D Bouley Ford G E Keller W C Ford HB Gray and J R Winkler ldquoSnapshots of a protein foldingintermediaterdquo Proceedings of the National Academy of Sciencesvol 110 no 5 pp 1606ndash1610 2013

[5] P Weinkam J Zimmermann F E Romesberg and P GWolynes ldquoThe folding energy landscape and free energy excita-tions of cytochrome crdquo Accounts of Chemical Research vol 43no 5 pp 652ndash660 2010

[6] M Yamanaka M Masanari and Y Sambongi ldquoConfermentof folding ability to a naturally unfolded apocytochrome cthrough introduction of hydrophobic amino acid residuesrdquoBiochemistry vol 50 no 12 pp 2313ndash2320 2011

[7] G W Moore and G W Pettigrew Cytochrome C EvolutionaryStructural and Physicochemical Aspects Springer Berlin Ger-many 1990

[8] I Bertini G Cavallaro and A Rosato ldquoCytochrome c occur-rence and functionsrdquo Chemical Reviews vol 106 no 1 pp 90ndash115 2006

[9] D A Pearce and F Sherman ldquoDegradation of cytochromeoxidase subunits in mutants of yeast lacking cytochrome c andsuppression of the degradation by mutation of yme1rdquo Journal ofBiological Chemistry vol 270 no 36 pp 20879ndash20882 1995

[10] G Silkstone S M Kapetanaki I Husu M H Vos and MT Wilson ldquoNitric oxide binding to the cardiolipin complex offerric cytochrome crdquo Biochemistry vol 51 no 34 pp 6760ndash6766 2012

[11] M Giorgio E Migliaccio F Orsini et al ldquoElectron transferbetween cytochrome c and p66Shc generates reactive oxygenspecies that trigger mitochondrial apoptosisrdquo Cell vol 122 no2 pp 221ndash233 2005

[12] S M Kilbride and J H Prehn ldquoCentral roles of apoptoticproteins in mitochondrial functionrdquo Oncogene vol 32 no 22pp 2703ndash2711 2013

[13] T Noll and G Noll ldquoStrategies for ldquowiringrdquo redox-activeproteins to electrodes and applications in biosensors biofuelcells and nanotechnologyrdquo Chemical Society Reviews vol 40no 7 pp 3564ndash3576 2011

[14] G Layer J Reichelt D Jahn and D W Heinz ldquoStructure andfunction of enzymes in heme biosynthesisrdquo Protein Science vol19 no 6 pp 1137ndash1161 2010

[15] S Severance and IHamza ldquoTrafficking of heme and porphyrinsin metazoardquo Chemical Reviews vol 109 no 10 pp 4596ndash46162009

[16] P Hamel V Corvest P Giege and G Bonnard ldquoBiochemi-cal requirements for the maturation of mitochondrial c-typecytochromesrdquo Biochimica et Biophysica Acta vol 1793 no 1 pp125ndash138 2009

[17] W R Fisher H Taniuchi and C B Anfinsen ldquoOn the role ofheme in the formation of the structure of cytochrome crdquo Journalof Biological Chemistry vol 248 no 9 pp 3188ndash3195 1973

[18] M Yamanaka H Mita Y Yamamoto and Y Sambongi ldquoHemeis not required for aquifex aeolicus cytochrome c555 polypep-tide foldingrdquoBioscience Biotechnology andBiochemistry vol 73no 9 pp 2022ndash2025 2009

[19] C Travaglini-Allocatelli S Gianni andMBrunori ldquoA commonfolding mechanism in the cytochrome c familyrdquo Trends inBiochemical Sciences vol 29 no 10 pp 535ndash541 2004

[20] A Borgia D Bonivento C Travaglini-Allocatelli A DiMatteoand M Brunori ldquoUnveiling a hidden folding intermediate in c-type cytochromes by protein engineeringrdquo Journal of BiologicalChemistry vol 281 no 14 pp 9331ndash9336 2006

[21] E Enggist L Thony-Meyer P Guntert and K PervushinldquoNMR structure of the heme chaperone CcmE reveals a novelfunctional motifrdquo Structure vol 10 no 11 pp 1551ndash1557 2002

[22] A di Matteo N Calosci S Gianni P Jemth M Brunori and CTravaglini-Allocatelli ldquoStructural and functional characteriza-tion of CcmG from pseudomonas aeruginosa a key componentof the bacterial cytochrome c maturation apparatusrdquo Proteinsvol 78 no 10 pp 2213ndash2221 2010

[23] A diMatteo S GianniM E Schinina et al ldquoA strategic proteinin cytochrome c maturation three-dimensional structure ofCcmH and binding to apocytochrome crdquo Journal of BiologicalChemistry vol 282 no 37 pp 27012ndash27019 2007

[24] A Crow R M Acheson N E Le Brun and A OubrieldquoStructural basis of redox-coupled protein substrate selectionby the cytochrome c biosynthesis protein ResArdquo Journal ofBiological Chemistry vol 279 no 22 pp 23654ndash23660 2004

[25] E di Silvio A di Matteo and C Travaglini-Allocatelli ldquoTheRedox pathway of Pseudomonas aeruginosa cytochrome cbiogenesisrdquo Journal of Proteins and Proteomics vol 3 no 1 pp1ndash7 2012

[26] LThony-Meyer andP Kunzler ldquoTranslocation to the periplasmand signal sequence cleavage of preapocytochrome c depend onsec and lep but not on the ccmgene productsrdquoEuropean Journalof Biochemistry vol 246 no 3 pp 794ndash799 1997

[27] S J Facey and A Kuhn ldquoBiogenesis of bacterial inner-membrane proteinsrdquo Cellular and Molecular Life Sciences vol67 no 14 pp 2343ndash2362 2010

[28] K Diekert A I de Kroon U Ahting et al ldquoApocytochromec requires the TOM complex for translocation across themitochondrial outer membranerdquo The EMBO Journal vol 20no 20 pp 5626ndash5635 2001

[29] N Wiedemann V Kozjak T Prinz et al ldquoBiogenesis of yeastmitochondrial cytochrome c a unique relationship to the TOMmachineryrdquo Journal ofMolecular Biology vol 327 no 2 pp 465ndash474 2003

[30] F-U Hartl N Pfanner and W Neupert ldquoTranslocation inter-mediates on the import pathway of proteins intomitochondriardquoBiochemical Society Transactions vol 15 no 1 pp 95ndash97 1987

[31] B S Glick A Brandt K Cunningham SMuller R L Hallbergand G Schatz ldquoCytochromes c1 and b2 are sorted to theintermembrane space of yeast mitochondria by a stop-transfermechanismrdquo Cell vol 69 no 5 pp 809ndash822 1992

[32] G Howe and S Merchant ldquoRole of heme in the biosynthesis ofcytochrome c6rdquo Journal of Biological Chemistry vol 269 no 8pp 5824ndash5832 1994

[33] S S Nakamoto P Hamel and S Merchant ldquoAssembly ofchloroplast cytochromes b and crdquo Biochimie vol 82 no 6-7 pp603ndash614 2000

[34] C Sanders S Turkarslan D-W Lee and F DaldalldquoCytochrome c biogenesis the Ccm systemrdquo Trends inMicrobiology vol 18 no 6 pp 266ndash274 2010

[35] J M Stevens D A Mavridou R Hamer P Kritsiligkou A DGoddard and S J Ferguson ldquoCytochrome c biogenesis systemIrdquoThe FEBS Journal vol 278 no 22 pp 4170ndash4178 2011

[36] R G Kranz C Richard-Fogal J-S Taylor and E R FrawleyldquoCytochrome c biogenesis mechanisms for covalent modifica-tions and trafficking of heme and for heme-iron redox controlrdquo

14 Scientifica

Microbiology and Molecular Biology Reviews vol 73 no 3 pp510ndash528 2009

[37] J W A Allen ldquoCytochrome c biogenesis in mitochondriamdashsystems III and VrdquoThe FEBS Journal vol 278 no 22 pp 4198ndash4216 2011

[38] D A Mavridou S J Ferguson and J M Stevens ldquoCytochromec assemblyrdquo IUBMB Life vol 65 no 3 pp 209ndash216 2013

[39] I Bertini G Cavallaro and A Rosato ldquoEvolution ofmitochondrial-type cytochrome c domains and of theprotein machinery for their assemblyrdquo Journal of InorganicBiochemistry vol 101 no 11-12 pp 1798ndash1811 2007

[40] B S Goldman and R G Kranz ldquoEvolution and horizontaltransfer of an entire biosynthetic pathway for cytochromec biogenesis helicobacter deinococcus archae and morerdquoMolecular Microbiology vol 27 no 4 pp 871ndash873 1998

[41] C L Richard-Fogal E R Frawley E R Bonner H Zhu BSan Francisco and R G Kranz ldquoA conserved haem redoxand trafficking pathway for cofactor attachmentrdquo The EMBOJournal vol 28 no 16 pp 2349ndash2359 2009

[42] L Thony-Meyer F Fischer P Kunzler D Ritz and H Hen-necke ldquoEscherichia coli genes required for cytochrome cmaturationrdquo Journal of Bacteriology vol 177 no 15 pp 4321ndash4326 1995

[43] C S Beckett J A Loughman K A Karberg G M DonatoW E Goldman and R G Kranz ldquoFour genes are required forthe system II cytochrome c biogenesis pathway in Bordetellapertussis a unique bacterial modelrdquo Molecular Microbiologyvol 38 no 3 pp 465ndash481 2000

[44] N E Le Brun J Bengtsson and L Hederstedt ldquoGenes requiredfor cytochrome c synthesis in Bacillus subtilisrdquo MolecularMicrobiology vol 36 no 3 pp 638ndash650 2000

[45] P Giege J M Grienenberger and G Bonnard ldquoCytochromec biogenesis in mitochondriardquo Mitochondrion vol 8 no 1 pp61ndash73 2008

[46] R E Feissner C L Richard-Fogal E R Frawley J A Lough-man KW Earley and R G Kranz ldquoRecombinant cytochromesc biogenesis systems I and II and analysis of haem deliverypathways in Escherichia colirdquo Molecular Microbiology vol 60no 3 pp 563ndash577 2006

[47] N P Cianciotto P Cornelis and C Baysse ldquoImpact of thebacterial type I cytochrome c maturation system on differentbiological processesrdquoMolecular Microbiology vol 56 no 6 pp1408ndash1415 2005

[48] E Arslan H Schulz R Zufferey P Kunzler and L Thony-Meyer ldquoOverproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in Escherichiacolirdquo Biochemical and Biophysical Research Communicationsvol 251 no 3 pp 744ndash747 1998

[49] C Sanders and H Lill ldquoExpression of prokaryotic and eukary-otic cytochromes c in Escherichia colirdquoBiochimica et BiophysicaActa vol 1459 no 1 pp 131ndash138 2000

[50] M Braun and L Thony-Meyer ldquoBiosynthesis of artificialmicroperoxidases by exploiting the secretion and cytochromec maturation apparatuses of Escherichia colirdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 35 pp 12830ndash12835 2004

[51] B Baert C Baysse S Matthijs and P Cornelis ldquoMultiplephenotypic alterations caused by a c-type cytochrome mat-uration ccmC gene mutation in Pseudomonas aeruginosardquoMicrobiology vol 154 no 1 pp 127ndash138 2008

[52] S Turkarslan C Sanders and F Daldal ldquoExtracytoplasmicprosthetic group ligation to apoproteins maturation of c-typecytochromesrdquo Molecular Microbiology vol 60 no 3 pp 537ndash541 2006

[53] P M Jones and A M George ldquoThe ABC transporter structureand mechanism perspectives on recent researchrdquo Cellular andMolecular Life Sciences vol 61 no 6 pp 682ndash699 2004

[54] O Christensen E M Harvat L Thony-Meyer S J Fergusonand J M Stevens ldquoLoss of ATP hydrolysis activity by CcmABresults in loss of c-type cytochrome synthesis and incompleteprocessing ofCcmErdquoTheFEBS Journal vol 274 no 9 pp 2322ndash2332 2007

[55] J-H Lee E M Harvat J M Stevens S J Ferguson and M HSaier Jr ldquoEvolutionary origins of members of a superfamily ofintegralmembrane cytochrome c biogenesis proteinsrdquoBiochim-ica et Biophysica Acta vol 1768 no 9 pp 2164ndash2181 2007

[56] D L Beckman D R Trawick and R G Kranz ldquoBacterialcytochromes c biogenesisrdquo Genes and Development vol 6 no2 pp 268ndash283 1992

[57] H Schulz R A Fabianek E C Pellicioli H Hennecke andL Thony-Meyer ldquoHeme transfer to the heme chaperone CcmEduring cytochrome c maturation requires the CcmC proteinwhich may function independently of the ABC-transporterCcmABrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 96 no 11 pp 6462ndash6467 1999

[58] Q Ren and L Thony-Meyer ldquoPhysical Interaction ofCcmC with Heme and the Heme Chaperone CcmE duringCytochrome cMaturationrdquo Journal of Biological Chemistry vol276 no 35 pp 32591ndash32596 2001

[59] C Richard-Fogal and R G Kranz ldquoThe CcmChemeCcmEcomplex in heme trafficking and cytochrome c biosynthesisrdquoJournal of Molecular Biology vol 401 no 3 pp 350ndash362 2010

[60] V K Viswanathan S Kurtz L L Pedersen et al ldquoThecytochrome C maturation locus of Legionella pneumophilapromotes iron assimilation and intracellular infection andcontains a strain-specific insertion sequence elementrdquo Infectionand Immunity vol 70 no 4 pp 1842ndash1852 2002

[61] U Ahuja and L Thony-Meyer ldquoCcmD is involved in complexformation between CcmC and the heme chaperone CcmE dur-ing cytochrome c maturationrdquo Journal of Biological Chemistryvol 280 no 1 pp 236ndash243 2005

[62] C L Richard-Fogal E R Frawley and R G Kranz ldquoTopologyand function of CcmD in cytochrome Cmaturationrdquo Journal ofBacteriology vol 190 no 10 pp 3489ndash3493 2008

[63] H Schulz H Hennecke and L Thony-Meyer ldquoPrototype ofa heme chaperone essential for cytochrome c maturationrdquoScience vol 281 no 5380 pp 1197ndash1200 1998

[64] F Arnesano L Banci P D Barker et al ldquoSolution structure andcharacterization of the heme chaperone CcmErdquo Biochemistryvol 41 no 46 pp 13587ndash13594 2002

[65] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[66] L Thony-Meyer ldquoA heme chaperone for cytochrome c biosyn-thesisrdquo Biochemistry vol 42 no 45 pp 13099ndash13105 2003

[67] E M Harvat C Redfield J M Stevens and S J FergusonldquoProbing the heme-binding site of the cytochrome cmaturationprotein CcmErdquoBiochemistry vol 48 no 8 pp 1820ndash1828 2009

[68] D Lee K Pervushin D Bischof M Braun and L Thony-Meyer ldquoUnusual heme-histidine bond in the active site of a

Scientifica 15

chaperonerdquo Journal of the American Chemical Society vol 127no 11 pp 3716ndash3717 2005

[69] A D Goddard J M Stevens F Rao et al ldquoc-Type cytochromebiogenesis can occur via a natural Ccm system lacking CcmHCcmG and the heme-binding histidine of CcmErdquo Journal ofBiological Chemistry vol 285 no 30 pp 22882ndash22889 2010

[70] J M Aramini K Hamilton P Rossi et al ldquoSolution NMRstructure backbone dynamics and heme-binding propertiesof a novel cytochrome c maturation protein CcmE fromDesulfovibrio vulgarisrdquo Biochemistry vol 51 no 18 pp 3705ndash3707 2012

[71] T Uchida J M Stevens O Daltrop et al ldquoThe interactionof covalently bound heme with the cytochrome c maturationprotein CcmErdquo Journal of Biological Chemistry vol 279 no 50pp 51981ndash51988 2004

[72] I Garcıa-Rubio M Braun I Gromov L Thony-Meyer andA Schweiger ldquoAxial coordination of heme in ferric CcmEchaperone characterized by EPR spectroscopyrdquo BiophysicalJournal vol 92 no 4 pp 1361ndash1373 2007

[73] L Thony-Meyer ldquoHaem-polypeptide interactions duringcytochrome c maturationrdquo Biochimica et Biophysica Acta vol1459 no 2-3 pp 316ndash324 2000

[74] J A Keightley D Sanders T R Todaro A Pastuszyn and J AFee ldquoCloning expression in Escherichia coli of the cytochromec552 gene from Thermus thermophilus HB8 Evidence forgenetic linkage to an ATP- binding cassette protein and initialcharacterization of the cycA gene productsrdquo Journal of Biologi-cal Chemistry vol 273 no 20 pp 12006ndash12016 1998

[75] E M Monika B S Goldman D L Beckman and R GKranz ldquoA thioreduction pathway tethered to the membranefor periplasmic cytochromes c biogenesis In vitro and in vivostudiesrdquo Journal of Molecular Biology vol 271 no 5 pp 679ndash692 1997

[76] M Throne-Holst L Thony-Meyer and L HederstedtldquoEscherichia coli ccm in-frame deletion mutants can produceperiplasmic cytochrome b but not cytochrome crdquo FEBS Lettersvol 410 no 2-3 pp 351ndash355 1997

[77] U Ahuja and L Thony-Meyer ldquoDynamic features of a hemedelivery system for cytochrome c maturationrdquo Journal of Bio-logical Chemistry vol 278 no 52 pp 52061ndash52070 2003

[78] J W A Allen P D Barker O Daltrop et al ldquoWhy isnrsquot ldquostan-dardrdquo heme good enough for c-type and di-type cytochromesrdquoDalton Transactions no 21 pp 3410ndash3418 2005

[79] K Inaba and K Ito ldquoStructure and mechanisms of the DsbB-DsbA disulfide bond generation machinerdquo Biochimica et Bio-physica Acta vol 1783 no 4 pp 520ndash529 2008

[80] H Kadokura F Katzen and J Beckwith ldquoProtein disulfidebond formation in prokaryotesrdquoAnnual Review of Biochemistryvol 72 pp 111ndash135 2003

[81] S R Shouldice B Heras P M Walden M Totsika M ASchembri and J L Martin ldquoStructure and function of DsbA akey bacterial oxidative folding catalystrdquoAntioxidants and RedoxSignaling vol 14 no 9 pp 1729ndash1760 2011

[82] R Metheringham L Griffiths H Crooke S Forsythe and JCole ldquoAn essential role for DsbA in cytochrome c synthesis andformate-dependent nitrite reduction by Escherichia coli K-12rdquoArchives of Microbiology vol 164 no 4 pp 301ndash307 1995

[83] Y Sambongi and S J Ferguson ldquoMutants of Escherichia colilacking disulphide oxidoreductases DsbA and DsbB cannotsynthesise an exogenous monohaem c-type cytochrome exceptin the presence of disulphide compoundsrdquo FEBS Letters vol398 no 2-3 pp 265ndash268 1996

[84] D AMavridou S J Ferguson and JM Stevens ldquoThe interplaybetween the disulfide bond formation pathway and cytochromec maturation in Escherichia colirdquo FEBS Letters vol 586 no 12pp 1702ndash1707 2012

[85] C U Stirnimann A Rozhkova U Grauschopf M G GrutterR Glockshuber and G Capitani ldquoStructural basis and kineticsof DsbD-dependent cytochrome c maturationrdquo Structure vol13 no 7 pp 985ndash993 2005

[86] N Ouyang Y-G Gao H-Y Hu and Z-X Xia ldquoCrystalstructures of E coli CcmGand itsmutants reveal key roles of theN-terminal120573-sheet and the fingerprint regionrdquoProteins vol 65no 4 pp 1021ndash1031 2006

[87] M A Edeling L W Guddat R A Fabianek et al ldquoCrys-tallization and preliminary diffraction studies of native andselenomethionine CcmG (CycY DsbE)rdquoActa CrystallographicaD vol 57 no 9 pp 1293ndash1295 2001

[88] R A Fabianek M Huber-Wunderlich R Glockshuber PKunzler H Hennecke and L Thony-Meyer ldquoCharacterizationof the Bradyrhizobium japonicum CycY protein a membrane-anchored periplasmic thioredoxin that may play a role as areductant in the biogenesis of c-type cytochromesrdquo Journal ofBiological Chemistry vol 272 no 7 pp 4467ndash4473 1997

[89] G B Kallis and A Holmgren ldquoDifferential reactivity of thefunctional sulfhydryl groups of cysteine-32 and cysteine-32present in the reduced form of thioredoxin from Escherichiacolirdquo Journal of Biological Chemistry vol 255 no 21 pp 10261ndash10265 1980

[90] P T Chivers andR T Raines ldquoGeneral acidbase catalysis in theactive site of Escherichia coli thioredoxinrdquoBiochemistry vol 36no 50 pp 15810ndash15816 1997

[91] X M Zheng J Hong H Y Li D H Lin and H Y HuldquoBiochemical properties and catalytic domain structure of theCcmH protein from Escherichia colirdquo Biochim Biophys Actavol 1824 no 12 pp 1394ndash1400 2012

[92] UAhujaA Rozhkova RGlockshuber LThony-Meyer andOEinsle ldquoHelix swapping leads to dimerization of the N-terminaldomain of the c-type cytochrome maturation protein CcmHfrom Escherichia colirdquo FEBS Letters vol 582 no 18 pp 2779ndash2786 2008

[93] R A Fabianek T Hofer and L Thony-Meyer ldquoCharacteriza-tion of the Escherichia coli CcmH protein reveals new insightsinto the redox pathway required for cytochrome c maturationrdquoArchives of Microbiology vol 171 no 2 pp 92ndash100 1999

[94] E Reid J Cole and D J Eaves ldquoThe Escherichia coli CcmGprotein fulfils a specific role in cytochrome c assemblyrdquo Bio-chemical Journal vol 355 no 1 pp 51ndash58 2001

[95] Q Ren U Ahuja and LThony-Meyer ldquoA bacterial cytochromec heme lyase CcmF forms a complex with the heme chaperoneCcmE and CcmH but not with apocytochrome crdquo Journal ofBiological Chemistry vol 277 no 10 pp 7657ndash7663 2002

[96] E H Meyer P Giege E Gelhaye et al ldquoAtCCMH an essentialcomponent of the c-type cytochrome maturation pathway inArabidopsis mitochondria interacts with apocytochrome crdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 44 pp 16113ndash16118 2005

[97] C Rios-Velazquez R Coller and T J Donohue ldquoFeatures ofRhodobacter sphaeroides CcmFHrdquo Journal of Bacteriology vol185 no 2 pp 422ndash431 2003

[98] C Sanders M Deshmukh D Astor R G Kranz and F DaldalldquoOverproduction of CcmG and CcmFHRc fully suppresses thec-type cytochrome biogenesis defect of Rhodobacter capsulatus

16 Scientifica

CcmI-null mutantsrdquo Journal of Bacteriology vol 187 no 12 pp4245ndash4256 2005

[99] A F Verissimo H Yang X Wu C Sanders and F DaldalldquoCcmI subunit of CcmFHI heme ligation complex functionsas an apocytochrome c chaperone during c-type cytochromematurationrdquo Journal of Biological Chemistry vol 286 no 47 pp40452ndash40463 2011

[100] K Bryson L J McGuffin R L Marsden J J Ward J SSodhi and D T Jones ldquoProtein structure prediction servers atUniversity College Londonrdquo Nucleic Acids Research vol 33 no2 pp W36ndashW38 2005

[101] D T Jones ldquoProtein secondary structure prediction basedon position-specific scoring matricesrdquo Journal of MolecularBiology vol 292 no 2 pp 195ndash202 1999

[102] C Sanders C Boulay and F Daldal ldquoMembrane-spanning andperiplasmic segments of CcmI have distinct functions duringcytochrome c biogenesis in Rhodobacter capsulatusrdquo Journal ofBacteriology vol 189 no 3 pp 789ndash800 2007

[103] D Han K Kim J Oh J Park and Y Kim ldquoTPR domainof NrfG mediates complex formation between heme lyaseand formate-dependent nitrite reductase in Escherichia coliO157H7rdquo Proteins vol 70 no 3 pp 900ndash914 2008

[104] D J Eaves J Grove W Staudenmann et al ldquoInvolvement ofproducts of the nrfEFG genes in the covalent attachment ofhaem c to a novel cysteine-lysine motif in the cytochrome c552nitrite reductase from Escherichia colirdquo Molecular Microbiol-ogy vol 28 no 1 pp 205ndash216 1998

[105] J Nilsson B Persson and G Von Heijne ldquoConsensus predic-tions of membrane protein topologyrdquo FEBS Letters vol 486 no3 pp 267ndash269 2000

[106] E di Silvio A di Matteo F Malatesta and C Travaglini-Allocatelli ldquoRecognition and binding of apocytochrome c toP aeruginosa CcmI a component of cytochrome c maturationmachineryrdquo Biochim Biophys Acta vol 1834 no 8 pp 1554ndash1561 2013

[107] N Zeytuni and R Zarivach ldquoStructural and functional dis-cussion of the tetra-trico-peptide repeat a protein interactionmodulerdquo Structure vol 20 no 3 pp 397ndash405 2012

[108] C Sanders S Turkarslan D-W Lee O Onder R G Kranz andF Daldal ldquoThe cytochrome c maturation components CcmFCcmH and CcmI form a membrane-integral multisubunitheme ligation complexrdquo Journal of Biological Chemistry vol283 no 44 pp 29715ndash29722 2008

[109] A F Verissimo M A Mohtar and F Daldal ldquoThe hemechaperone ApoCcmE forms a ternary complex with CcmI andapocytochrome crdquoThe Journal of Biological Chemistry vol 288no 9 pp 6272ndash6283 2013

[110] P D Barker J C Ferrer M Mylrajan et al ldquoTransmutation of aheme proteinrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 90 no 14 pp 6542ndash65461993

[111] J Simon and L Hederstedt ldquoComposition and function ofcytochrome c biogenesis system IIrdquoThe FEBS Journal vol 278no 22 pp 4179ndash4188 2011

[112] T R H M Kouwen and J M van Dijl ldquoInterchangeablemodules in bacterial thiol-disulfide exchange pathwaysrdquo Trendsin Microbiology vol 17 no 1 pp 6ndash12 2009

[113] A Crow A Lewin O Hecht et al ldquoCrystal structure andbiophysical properties of Bacillus subtilis BdbD An oxidizingthioldisulfide oxidoreductase containing a novel metal siterdquoJournal of Biological Chemistry vol 284 no 35 pp 23719ndash23733 2009

[114] M C Moller and L Hederstedt ldquoExtracytoplasmic processesimpaired by inactivation of trxA (thioredoxin gene) in Bacillussubtilisrdquo Journal of Bacteriology vol 190 no 13 pp 4660ndash46652008

[115] A Lewin A Crow A Oubrie and N E Le Brun ldquoMolecularbasis for specificity of the extracytoplasmic thioredoxin ResArdquoJournal of Biological Chemistry vol 281 no 46 pp 35467ndash35477 2006

[116] C L Colbert QWu P J A Erbel K H Gardner and J Deisen-hofer ldquoMechanism of substrate specificity in Bacillus subtilisResA a thioredoxin-like protein involved in cytochrome cmaturationrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 103 no 12 pp 4410ndash44152006

[117] UAhuja P Kjelgaard B L Schulz LThony-Meyer andLHed-erstedt ldquoHaem-delivery proteins in cytochrome c maturationsystem IIrdquoMolecular Microbiology vol 73 no 6 pp 1058ndash10712009

[118] M L D Page P P Hamel S T Gabilly et al ldquoA homologof prokaryotic thiol disulfide transporter CcdA is required forthe assembly of the cytochrome b6f complex in Arabidopsischloroplastsrdquo Journal of Biological Chemistry vol 279 no 31pp 32474ndash32482 2004

[119] A Rozhkova C U Stirnimann P Frei et al ldquoStructural basisand kinetics of inter- and intramolecular disulfide exchange inthe redox catalyst DsbDrdquoThe EMBO Journal vol 23 no 8 pp1709ndash1719 2004

[120] T Schiott M Throne-Holst and L Hederstedt ldquoBacillussubtilis CcdA-defective mutants are blocked in a late step ofcytochrome C biogenesisrdquo Journal of Bacteriology vol 179 no14 pp 4523ndash4529 1997

[121] R E Feissner C S Beckett J A Loughman and R G KranzldquoMutations in cytochrome assembly and periplasmic redoxpathways in Bordetella pertussisrdquo Journal of Bacteriology vol187 no 12 pp 3941ndash3949 2005

[122] L S ErlendssonMMoller and L Hederstedt ldquoBacillus subtilisStoA is a thiol-disulfide oxidoreductase important for sporecortex synthesisrdquo Journal of Bacteriology vol 186 no 18 pp6230ndash6238 2004

[123] L S Erlendsson R M Acheson L Hederstedt and N E LeBrun ldquoBacillus subtilis ResA is a thiol-disulfide oxidoreductaseinvolved in cytochrome c synthesisrdquo Journal of BiologicalChemistry vol 278 no 20 pp 17852ndash17858 2003

[124] L S Erlendsson and L Hederstedt ldquoMutations in the thiol-disulfide oxidoreductases BdbC and BdbD can suppresscytochrome c deficiency of CcdA-defective Bacillus subtiliscellsrdquo Journal of Bacteriology vol 184 no 5 pp 1423ndash1429 2002

[125] M E Dumont J F Ernst D M Hampsey and F ShermanldquoIdentification and sequence of the gene encoding cytochromec heme lyase in the yeast Saccharomyces cerevisiaerdquoThe EMBOJournal vol 6 no 1 pp 235ndash241 1987

[126] M E Dumont J F Ernst and F Sherman ldquoCoupling of hemeattachment to import of cytochrome c into yeast mitochondriaStudies with heme lyase-deficient mitochondria and alteredapocytochromes crdquo Journal of Biological Chemistry vol 263 no31 pp 15928ndash15937 1988

[127] B San Francisco E C Bretsnyder and R G Kranz ldquoHumanmitochondrial holocytochrome c synthasersquos hemebindingmat-uration determinants and complex formationwith cytochromecrdquo Proceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 9 pp E788ndashE797 2012

Scientifica 17

[128] L Schaefer A Ballabio and H Y Zoghbi ldquoCloning andcharacterization of a putative human holocytochrome c-typesynthetase gene (HCCS) isolated from the critical region formicrophthalmia with linear skin defects (MLS)rdquoGenomics vol34 no 2 pp 166ndash172 1996

[129] S K Prakash T A Cormier A E McCall et al ldquoLoss ofholocytochrome c-type synthetase causes the male lethality ofX-linked dominant microphthalmia with linear skin defects(MLS) syndromerdquo Human Molecular Genetics vol 11 no 25pp 3237ndash3248 2002

[130] I Wimplinger M Morleo G Rosenberger et al ldquoMutations ofthe mitochondrial holocytochrome c-type synthase in X-linkeddominant microphthalmia with linear skin defects syndromerdquoTheAmerican Journal of HumanGenetics vol 79 no 5 pp 878ndash889 2006

[131] S Kiryu-Seo KGamo T Tachibana K Tanaka andHKiyamaldquoUnique anti-apoptotic activity of EAAC1 in injured motorneuronsrdquoTheEMBO Journal vol 25 no 14 pp 3411ndash3421 2006

[132] R Lill R A Stuart M E Drygas F E Nargang and W Neu-pert ldquoImport of cytochrome c heme lyase into mitochondriaa novel pathway into the intermembrane spacerdquo The EMBOJournal vol 11 no 2 pp 449ndash456 1992

[133] H Steiner A Zollner A Haid W Neupert and R LillldquoBiogenesis of mitochondrial heme lyases in yeast Importand folding in the intermembrane spacerdquo Journal of BiologicalChemistry vol 270 no 39 pp 22842ndash22849 1995

[134] A Zollner G Rodel and A Haid ldquoMolecular cloning andcharacterization of the Saccharomyces cerevisiae CYT2 geneencoding cytochrome-c1-heme lyaserdquo European Journal of Bio-chemistry vol 207 no 3 pp 1093ndash1100 1992

[135] V Corvest D A Murrey D G Bernard D B Knaff B Guiardand P PHamel ldquoc-Type cytochrome assembly in Saccharomycescerevisiae a key residue for apocytochrome c1lyase interactionrdquoGenetics vol 186 no 2 pp 561ndash571 2010

[136] V Corvest D A Murrey M Hirasawa D B Knaff B Guiardand P P Hamel ldquoThe flavoprotein Cyc2p a mitochondrialcytochrome c assembly factor is a NAD(P)H-dependent haemreductaserdquo Molecular Microbiology vol 83 no 5 pp 968ndash9802012

[137] D G Bernard S Quevillon-Cheruel S Merchant B Guiardand P P Hamel ldquoCyc2p a membrane-bound flavopro-tein involved in the maturation of mitochondrial c-typecytochromesrdquo Journal of Biological Chemistry vol 280 no 48pp 39852ndash39859 2005

[138] J G Kleingardner and K L Bren ldquoComparing substratespecificity between cytochrome cmaturation and cytochrome cheme lyase systems for cytochrome c biogenesisrdquo Metallomicsvol 3 no 4 pp 396ndash403 2011

[139] C Travaglini-Allocatelli S Gianni VMorea A Tramontano TSoulimane and M Brunori ldquoExploring the cytochrome c fold-ing mechanism cytochrome c552 fromThermus thermophilusfolds through an on-pathway intermediaterdquo Journal of BiologicalChemistry vol 278 no 42 pp 41136ndash41140 2003

[140] L Zhang and L Guarente ldquoHeme binds to a short sequencethat serves a regulatory function in diverse proteinsrdquoTheEMBOJournal vol 14 no 2 pp 313ndash320 1995

[141] S Hira T Tomita T Matsui K Igarashi and M Ikeda-SaitoldquoBach1 a heme-dependent transcription factor reveals pres-ence of multiple heme binding sites with distinct coordinationstructurerdquo IUBMB Life vol 59 no 8-9 pp 542ndash551 2007

[142] T K Kuhl A Wiszligbrock N Goradia et al ldquoAnalysis of Fe(III)heme binding to cysteine-containing heme-regulatory motifs

in proteinsrdquo ACS Chemical Biology vol 8 no 8 pp 1785ndash17932013

[143] R L Moore J M Stevens and S J Ferguson ldquoMitochondrialcytochrome c synthase CP motifs are not necessary for hemeattachment to apocytochrome crdquo FEBS Letters vol 585 no 21pp 3415ndash3419 2011

[144] S Gianni M Brunori and C Travaglini-Allocatelli ldquoRefoldingkinetics of cytochrome c551 reveals a mechanistic differencebetween urea and guanidinerdquo Protein Science vol 10 no 8 pp1685ndash1688 2001

[145] S Gianni C Travaglini-Allocatelli F Cutruzzola M BrunoriM C R Shastry and H Roder ldquoParallel pathways incytochrome c551 foldingrdquo Journal of Molecular Biology vol 330no 5 pp 1145ndash1152 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Scientifica 5

maturase multiprotein complex(es) However the existenceandor stoichiometry of these complexes remains to bedetermined either because of the experimental difficultiesin handling membrane protein complexes or because itis possible that these complexes are unstable and onlytransiently populated Independently from their functionalexistence in the bacterial periplasm as independent units or ascomponents of a multisubunit complex it is clear that each oftheCcmproteins plays a different role from the transport andchaperoning of the heme cofactor to the necessary reductionof the disulfide bond between the sulfur atoms of the twoCys residues of the conserved CXXCH motif of apoCytand finally to the catalysis of covalent heme attachment Anadditional interesting aspect is that over and above theirrole in the biogenesis of Cytc there is also evidence thatinactivation of some ccm genes induces phenotypes thatcannot be explained only in terms of absence of synthesis ofCyt c all of these pleiotropic effects are linked to impairmentof heme andor iron trafficking in the periplasm [47] Inparticular it has been recently shown that in 120572- and 120574-proteobacteria (including the human opportunistic pathogenP aeruginosa) mutations in the ccmC ccmI and ccmF genesinduce phenotypes such as reduced pyoverdine produc-tion reduced bacterial motility or impaired growth in low-iron conditions ([51] and refs therein) These observationssuggesting that Ccm proteins perform additional functionscritical for bacterial physiology growth and virulence pro-vide a rationale to explain why bacteria at variance withthe eukaryotic cell have evolved a metabolically expensiveoperon to accomplish an apparently simple task such asheme ligation to apoCyt Novel hypotheses addressing theseaspects and awaiting experimental investigation include (i)the utilization of Ccm-associated heme for additional cellularprocesses besides attachment to apoCyt (ii) trafficking a non-heme compound through the Ccm system required for ironacquisition such a siderophore (iii) the Ccm inactivation-dependent accumulation of heme b a photoreactivemoleculewhose degradation leads to reactive oxygen species and (iv)the destructive effect on [Fe-S] clusters of ferrisiderophoresreductases

In the following the structural (when known) andfunctional properties of the different components of theSystem I maturation apparatus will be discussed dissectingthe Cytc maturation process into three main functionalsteps heme translocation and delivery apoCyt thioreductivepathway and apoCyt chaperoning and heme ligation (asimilar modular description can also be found elsewhere(see [34 52])) However it should be remembered that inmany cases the proteins involved in the three steps are notuniquely assigned to a specific module as they interact witheach other moreover it has been shown that in some casesmore than one system can be present [41] This modularorganization of Cyt c biogenesis should therefore be intendedonly as a way to simplify the description of an overall highlyintegrated process For each of the three functional stepspresentation of the structural and functional properties of thedifferent protein components is followed by a discussion ofthe proposed molecular mechanism(s)

21 System I Components of the Heme Translocation andDelivery Pathway CcmA and CcmB proteins show the typ-ical sequence features of the ABC (ATP binding Cassette)transporter family and therefore these components of SystemI were initially considered as the proteins responsible forthe translocation of the newly synthesized heme from thecytoplasm to the periplasmic space ABC transporters areubiquitous multidomain integral membrane proteins thattranslocate a large variety of substrates across cellular mem-branes using ATP hydrolysis as a source of energy they aregenerally composed of a transmembrane (TM) domain and aconserved cytosolic nucleotide-binding domain [53]

CcmA is a cytoplasmic soluble protein representing thenucleotide-binding domain of the hypothetical ABC trans-porter according to this hypothesis its sequence containsa nucleotide-binding domain and Walker A and B motifsfor ATP hydrolysis [46] It has also been shown that CcmApossesses ATPase activity in vitro and that the protein isassociated with the membrane fraction only when CcmB isalso present [54]

CcmB and CcmC are both integral membrane proteinspredicted to contain six TM helices CcmC contains a shortWWDdomain in its second periplasmic domain and belongsto the heme handling protein family (HHP) [55] WWDdomains are short tryptophan-rich aminoacid stretcheswith the conserved WGX120601WXWDXRLT sequence (where 120601represents an aromatic amino acid residue and X representsany residue) [40 56] it has been proposed that proteinscontaining WWD domains are involved in heme-bindingand as we will see below a WWD domain is also presentin the CcmF protein another crucial heme-binding proteinof System I CcmC also contains two absolutely conservedhistidine residues in its first (between TM helices 1 and 2)and third (between TMhelices 5 and 6) periplasmic domainsAn attractive hypothesis still requiring experimental proofis that the hydrophobic residues within the tryptophan-richmotif provide a platform for the binding of heme whereasthe two conserved His residues (H60 and H184 in E coliCcmC) act as axial heme ligands [57] this hypothesis isstrengthened by the observation that CcmC indeed interactsdirectly with heme [58 59] Immunoprecipitation experi-ments have shown that in E coli CcmABC proteins forma multiprotein complex with a CcmA

2CcmB

1CcmC

1stoi-

chiometry confirming that these components form an ABC-type transporter complex with unusual functional propertiesassociated to the release of holoCcmE from CcmC [41 46]rather than to heme transport per se CcmC is an interestingprotein worth of future experimental efforts as it is knownthat in some pathogenic bacteria CcmC mutations are asso-ciated to specific phenotypes apparently not related to Cytc maturation such as siderophore production in Paracoccusand Pseudomonas [51] and iron utilization in Legionella [60]

Limited information is available about the structure andfunction of CcmD which appears to be a small membraneprotein (about 70 aminoacid residues) with no conservedsequence features whose topology is currently debatedcontrary to the original proposal [61] additional experimentshave shown that in E coli and R capsulatus CcmD isan integral membrane protein composed of a single TM

6 Scientifica

helix a periplasmic-orientedN-terminus and a cytoplasmic-oriented C-terminus [62] Immunoprecipitation experimentsindicate that CcmD interacts with the CcmA

2CcmB

1CcmC

1

complex even if it is not essential for heme transfer andattachment from CcmC to CcmE CcmD is strictly requiredfor the release of holoCcmE from the ABC transporter [6162]

CcmE is a heme-binding protein discovered as an essen-tial System I component as early as the late 1990rsquos [63]CcmE is a monotopic membrane protein anchored to themembrane via its N-terminal TM segment and exposing itsactive site to the periplasm it is the only Ccm componentof the heme trafficking and delivery module of System I forwhich a three-dimensional structure is available ([21] PDB1SR3 [64] PDB 1LM0) The 3D structure of the apo-state(without bound heme) consists of a six-stranded antiparallel120573-sheet reminiscent of the classical OB-fold [65] with N-and C-terminal extensions CcmE can be considered a ldquohemechaperonerdquo as it protects the cell from a potentially dangerouscompound by sequestering free heme in the periplasm [66]it is thought to act as an intermediate in the heme deliverypathway of Cytc maturation The structure of apoCcmEshowed no recognizable heme-binding cavities and in theabsence of a 3D structure of CcmEwith bound heme (holoC-cmE) the heme-binding region could only be predicted byin silico modeling It is generally believed that the hemein holoCcmE is solvent exposed but recent mutagenesisexperiments challenged this view [67] The unusual covalentbond between the nitrogen atomof a histidine residue presentin the conserved VLAKHDE motif located in a solventexposed environment (H130 in E coli CcmE) and a 120573-carbonof one of the heme vinyl groups has been described in greatdetail by NMR spectroscopy [68] Recently it was shown thatCcmE proteins from the proteobacteria D desulfuricans andD vulgaris contain the unusual CXXXY heme-bindingmotifwhere the Cys residue replaces the canonical His bindingresidue NMR solution structure of D vulgaris CcmE (PDB2KCT) revealed that the proteins adopt the same OB-foldcharacteristic of the CcmE superfamily Contrary to whatreported for theD desulfuricansCcmE [69] the homologousprotein from D vulgaris binds ferric heme noncovalentlythrough the conserved C127 residue [70] An additionalconserved residue in CcmE proteins is Tyr134 which wasshown to provide a coordination bond to the heme ironof holoCcmE [71 72] once it is released from CcmABCDcomplex [36] as discussed below

22 System I Heme Translocation and Delivery PathwayMechanisms We still do not know how the b heme istranslocated from the cytoplasm (where it is synthesized)to the periplasm where Cyt c maturation occurs Differ-ent mechanisms such as translocation through a proteinchannel or free diffusion across the membrane have beenproposed [73] The CcmAB proteins show structural featurestypical of the ABC transporters and for these reasons theywere originally hypothesized to be involved in the heme btranslocation process [57 63 74 75] However it is nowclear that an alternative process must exist since it has

been shown that periplasmic b-type cytochromes can beproduced in the absence of Ccm proteins [76] and thatinactivation of the ATPase activity of CcmA does not abolishheme accumulation in the periplasm [46 54] We have nowevidence that CcmC has the ability to bind heme at its WWDdomain present in the second periplasmic domain but itis still not clear if this membrane protein acts as a proteinchannel for heme translocation or simply collects it in theperiplasm [77]

Another important aspect concerns the oxidation state ofthe heme iron during translocation and delivery processesindeed this property of the heme iron may determine thereaction mechanism by which the unusual CcmE H130nitrogen is covalently linked to the vinyl 120573-carbon of theheme (see [36] for a detailed discussion of this topic) Basedon mutagenesis studies on CcmC [59] a model has beenpresented whereby oxidized heme is bound to CcmC onlyin the presence of CcmE forming a ternary complex BothCcmC and CcmE provide critical residues for heme-bindingthe two conserved His residues (H60 and H180 coordinatingthe heme iron) and theWWDdomain ofCcmCandHis130 ofCcmE forming the unusual covalent bond with heme vinyl-2 [59] The ATPase activity of CcmA is then required torelease holoCcmE from the CcmABCD complex a processthat depends also on the presence of CcmD [46 54] It shouldbe noticed that purified holoCcmE alone or in the CcmCDEcomplex [36 59] contains the heme iron in the oxidized statean observation that is apparently in contrast with the fact thatthe heme must be in its reduced state before attachment toapoCyt can occur Although the oxidation state of the hemeiron is currently debated [36 78] it is possible that CcmFwhich was recently shown to contain a heme b cofactor mayact as specific heme oxidoreductase (see Section 25)

23 System I ApoCyt Thioreduction Pathway ComponentsThe periplasm can be considered a relatively oxidizing envi-ronment due to the presence of an efficient oxidative systemcomposed of the DsbAB proteins [79 80] DsbA is a highlyoxidizing protein (1198641015840

0= 120mV) that is responsible for

the introduction of disulfide bonds into extracytoplasmicproteins [81] On the basis of the results obtained on E colidsbA deletion mutants that are unable to synthesize c-typecytochromes [82 83] it was generally accepted that formationof the intramolecular disulfide bond in apoCyt was a nec-essary step in the Cyt c biogenesis However only reducedapoCyt is clearly competent for heme ligation It is possiblethat this seemingly paradoxical thioreduction process hasevolved in order to protect the apoCyt from proteolyticdegradation aggregation andor formation of intermolec-ular disulfide bonds with thiols from other molecules (seealso Section 31 for a discussion about this aspect in SystemII) Recently however an analysis of c-type cytochromesproduction in several E coli dsb genes deletion strains ledto the hypothesis that DsbA is not necessary for Cyt cmaturation and that heme ligation to apoCyt and apoCytoxidation pathways is alternative competing processes [84]

In gram-negative bacteria a thioreduction pathway hasevolved to specifically reduce the oxidized apoCyt substrate

Scientifica 7

which includes the Ccm proteins CcmG and CcmH Thenecessary reducing power is transferred from the cytoplasmicthioredoxin (TRX) to CcmG via DsbD a large membraneprotein organized in three structural domains an N-terminalperiplasmic domain with a IgG-like fold (nDsbD) a C-terminal periplasmic domain with a thioredoxin-like (TRX-like) fold (cDsbD) and a central domain composed ofeight TM helices [85] Each of these domains contains apair of Cys residues and transfer electrons via a cascadeof disulfide exchange reactions making DsbD a ldquoredox-hubrdquo in the periplasm performing disulfide bond exchangereactions with different oxidized proteins [79] In particulara combination of X-ray crystallography experiments andkinetic analyses showed that electrons are transferred fromthe cytoplasmic TRX to the membrane domain of DsbDfollowed by reduction of cDsbD and finally of nDsbD whichis the direct electron donor to CcmG [85]

CcmG is a membrane-anchored protein linked to themembrane via an N-terminal TM helix and exposing itssoluble TRX-like domain in the periplasm The 3D structureof the TRX-like domain of CcmG from different bacteriahas been solved by X-ray crystallography (E coli PDB 1Z5Y[85] PDB 2B1K [86] B japonicum PDB 1KNG [87] Paeruginosa PDB 3KH7 3KH9 [22]) and is generally wellconserved as proved by the low RMSD (08 A between Pa-CcmG and Ec-CcmG 135 A between Pa-CcmG and Bj-CcmG) Although all these proteins adopt a TRX-like foldand contain the redox-active motif CXXC in the first 120572-helixthey are inactive in the classic insulin reduction assay [7588] CcmG proteins are therefore considered specific thiol-oxidoreductase able to recognize and selectively interactonly with their upstream and downstream binding partnersin the thioreduction process leading to reduced apoCytcLooking at the 3D structure of the periplasmic domain ofthe prototypical Pa-CcmG it is possible to identify the 120573120572120573and 120573120573120572 structural motifs of the TRX fold linked by a short120572-helix and forming a four-stranded 120573-sheet surrounded bythree helices the protein contains an additional N-terminalextension (residues 26ndash62) and a central insert (residues 102ndash123) The redox-active motif of Pa-CcmG (CPSC) is locatedin the first 120572-helix of the TRX fold as usually observed in allTRX-like proteins As for any molecular machinery whereeach component must recognize and interact with more thanone target (ie the substrate and the other components ofthe apparatus) an open question concerns the mechanismwhereby CcmG is able to recognize its different partnersThe availability of the crystal structures of Pa-CcmG bothin the oxidized (22 A resolution) and reduced state (18 Aresolution) [22] allowed highlighting the structural similaritybetween the two redox states (Rmsd of the C120572 atoms inthe two redox forms is 019 A) and therefore to excludestructural rearrangement as the mechanism used by Pa-CcmG to discriminate between reduced (such as the nDsbDdomain) and oxidized partners (Pa-CcmH andor apoCyt)

The standard redox potential of Pa-CcmG (11986410158400= 0213V

at pH 70 [22] as well as that of Ec-CcmG (11986410158400= 0212V

[86]) indicates that these proteins act as mild reductants inthe thioreductive pathway of Cytc biogenesis However the

Figure 5 Three-dimensional structure of Pa-CcmH shown inribbon representation The figure shows the three-helix bundleforming the characteristic fold of Pa-CcmHThe active site disulfidebond between residues Cys25 andCys28 in the long loop connectinghelices 120572-helix1 and 120572-helix 2 is highlighted in yellow

function of thiol-oxidoreductases obviously depends on thepKa values of their activesite Cys residues The pKa of CysX(613 plusmn 005) and CysY (105 plusmn 007) are consistent with thepKa values measured in different TRXs where the active N-terminal Cys residue has a pKa close to pH 70 whereas theC-terminal Cys has a much higher pKa [89 90] Such a largedifference between the two pKa values in the TRX family isfunctionally relevant because it allows the N-terminal Cysto perform the nucleophilic attack on the target disulfidewhile the C-terminal Cys is involved in the resolution of theresulting mixed-disulfide [90]

CcmH is the other component of System I involvedin the reduction of apoCyt Notably CcmH proteins fromdifferent bacterial subgroups may display structural vari-ability indeed while in E coli Ec-CcmH is a bipartiteprotein characterized by two soluble domains exposed to theperiplasm and two TM segments CcmH from P aeruginosa(Pa-CcmH) is a one-domain redox-active protein anchoredto the membrane via a single TM helix and homologous tothe N-terminal redox-active domain of Ec-CcmH Surpris-ingly the 3D structure of the soluble periplasmic domainof Pa-CcmH revealed that it adopts a peculiar three-helixbundle fold strikingly different from that of canonical thiol-oxidoreductases (Figure 5 PDB 2HL7 [23])TheN-terminaldomain of Ec-CcmH was also shown to have the same 3Dstructure although helix-swapping and dimerization havebeen observed in this case (PDB 2KW0 [91 92]) Theconserved redox-active motif (LRCPKC) is located in theloop connecting helices 1 and 2 close to the activesite thecrystal structure reveals the presence of a small pocket on thesurface of Pa-CcmH surrounded by conserved hydrophobicand polar residues which could represent the recognition sitefor the heme-binding motif of apoCyt

Concerning the functional properties of this unusualthiol-oxidoreductase it is interesting to note that its standardredox potential (1198641015840

0= 0215V) [23] is similar to that ob-

tained for Pa-CcmG This observation stands against thelinear redox cascade hypothesis whereby CcmG reducesCcmH While in the canonical redox-active CXXC motif

8 Scientifica

S SSS

SH

SH

Scheme 1 Scheme 2

SH

SH

SHSHS

S

CcmG 7477

CcmG 7477

CcmH 2528

CcmH 2528

apoCytox

apoCytred

S

SHCcmG 74

77

SH

SH

HSHS

CcmG 7477

S

S

SS

3

1

2CcmG 7477

SH

SHCcmH 25

28SH

HSS S

CcmH 2528

SHS

CcmH2528

apoCytox

apoCytred

+

Figure 6 Alternative thioreduction pathways whichmay be operative in System I and hypothesized on the basis of structural and functionalcharacterization of the redox-active Ccm proteins from P aeruginosa [22 23 25] Scheme 1 is a linear redox cascade whereby CcmG is thedirect reductant of CcmH which reduces oxidized apoCyt Scheme 2 envisages a more complex scenario involving the formation of a mixed-disulfide complex between CcmH and apoCyt (Step 1) This complex is the substrate for the attack by reduced CcmG (Step 2) that liberatesreduced apoCyt The resulting disulfide bond between CcmH and CcmG is then resolved by the free Cys thiol of CcmG (probably Cys77 inPa-CcmG) Adapted from [25]

of the TRX family the N-terminal Cys is always solventexposed in CcmH proteins the arrangement of the two Cysresidues is reversed the N-terminal Cys residue is buriedwhereas the C-terminal Cys residue is solvent exposed Onthe basis of this observation it was suggested that differentfrom the canonical TRX redox mechanism CcmH proteinsperform the nucleophilic attack on the apoCyt disulfide viatheir C-terminal Cys residue [23] This mechanism which isin agreement with the mechanism proposed earlier for Ec-CcmH on the basis of mutational-complementation studies[93 94] is substantiated by the peculiar pKa values of theactive site Cys residues of Pa-CcmH which were found tobe similar for both cysteines (84 plusmn 01 and 86 plusmn 01 [23])Again this is different from what is generally observed in thecase of TRX proteins where the pKa value of the Cys residueperforming the initial nucleophilic attack is significantlylower than the pKa value of theCys residue responsible for theresolution of the intermediate mixed-disulfide It is temptingto speculate that the unusual pKa values of the Pa-CcmHactive site thiols may ensure the necessary specificity of thiscomponent of the Ccm apparatus toward the CXXCH motifof the apoCyt substrate

24 System I ApoCyt Thioreduction Pathway MechanismAlthough we know that CcmG and CcmH are the redox-active components of System I involved in the thioreductivepathway of Cyt c biogenesis not only an acceptedmechanismfor the reduction of apoCyt disulfide bond is still lackingbut also the absolute requirement of such a process is nowdebated [38 84] Focusing our attention on the reductionof the apoCyt internal disulfide at least two mechanismscan been hypothesized which involve either a linear redoxcascade of disulfide exchange reactions or a nonlinear redox

process involving transient formation of a mixed-disulfidecomplex as depicted in Figure 6 and Schemes 1 and 2respectively

Both the thiol-disulfide exchange mechanisms depictedin Figure 6 suggest that CcmH is the direct reductant ofthe apoCytc disulfide however even if immunoprecipitationexperiments failed to detect the formation of a mixed-disulfide complex between apoCyt and CcmH proteins [95]some in vitro evidence supporting the formation of sucha complex has been presented In particular it has beenshown that Rhodobacter capsulatus and Arabidopsis thalianaCcmH homologues (Rc-CcmH and At-CcmH) are able toreduce the CXXCH motif of an apoCyt-mimicking peptide[75 96] In the latter case yeast two-hybrid experimentscarried out on At-CcmH indeed revealed an interactionbetween the protein and a peptide mimicking the A thalianaCyt c sequence In the case of Pa-CcmH FRET kineticexperiments employing a Trp-containing fluorescent variantof the protein and a dansylated nonapeptide encompassingthe heme-binding motif of P aeruginosa cytochrome c551(dans-KGCVACHAI) [23] allowed to directly observe theformation of themixed-disulfide complex and tomeasure theoff-rate constant of the bound peptide The results of these invitro binding experiments allowed to calculate an equilibriumdissociation constant which combines an adequate affinity(low 120583M) with the need to release efficiently reduced apoCytto other component(s) of the System I maturase complex[23] More recently the results obtained by FRET bindingexperiments carried out with single Cys-containing mutantsof Pa-CcmH and Pa-CcmG [25] substantiated the hypothesisdepicted in Scheme 2 (Figure 6) Altogether these structuraland functional results suggest that the thioreduction pathwaymechanism leading to reduced apoCyt is better describedby Scheme 2 and that reducing equivalents might not be

Scientifica 9

transferred directly from CcmG to apoCyt as depicted inScheme 1 According to Scheme 2 reduced CcmH (a non-TRX-like thiol-oxidoreductase) specifically recognizes andreduces oxidized apoCyt via the formation of a mixed-disulfide complex which is subsequently resolved by CcmGThe resulting disulfide bond between CcmH and CcmG isthen resolved by the free Cys thiol of CcmG (probably Cys77in Pa-CcmG)

However further in vitro experiments with CcmH andapoCyt single Cys-containing mutants are needed to unveilthe details of the thioreduction of oxidized apoCyt by CcmHIn particular it would be crucial to identify the Cys residueof apoCyt that remains free in the apoCyt-CcmH mixed-disulfide complex intermediate (see Scheme 2 and Section 26below) and available to thioether bond formation with oneof the heme vinyl groups Clearly structure determinationof the trapped mixed-disulfide complexes between CcmHCcmG and apoCyt (or apoCyt peptides) would providekey information for our understanding of this specializedthioreduction pathway mechanism

25 System I ApoCyt Chaperoning and Heme AttachmentComponents The reduced heme-binding motif of apoCyt isnow available to the heme ligation reaction However themolecularmechanismwhereby the Ccmmachinery catalyzesor promotes the formation of the heme-apoCyt covalentbonds is still largely obscure representing themost importantgoal in the field Past observations and recent experimentssuggest that CcmF and CcmI possibly together with CcmHare involved in these final steps [16 34 36]

CcmF is a large integral membrane protein of more than600 residues belonging to the heme handling protein family(HHP [55]) and predicted to contain 10ndash15 TM helices (notethat some discrepancy exists as to the number of TM helicespredicted by computer programs and those predicted onthe basis of phoA and lacZ fusion experiments [40 97])a conserved WWD domain and a larger domain devoidof any recognizable sequence features both exposed to theperiplasm Only recently E coli CcmF (Ec-CcmF) has beenoverexpressed solubilized from the membrane fraction andspectroscopically characterized in vitro [36 41] Surprisinglythe biochemical characterization of recombinant Ec-CcmFallowed to show that the purified protein contains heme b ascofactor in a 1 1 stoichiometry this observation led to thehypothesis that in addition to its heme lyase function Ec-CcmF may act as a heme oxidoreductase In particular it ispossible that the heme b of Ec-CcmF may act as a reductantfor the oxidized iron of the heme bound to CcmE [41]indeed the in vitro reduction of Ec-CcmF by quinones hasbeen experimentally observed strengthening the hypothesisabout the quinolhemeoxidoreductase function of this elusiveproteinThe structuralmodel proposed for Ec-CcmFpredicts13 TMhelices and notably the location of the four completelyconserved His residues according to the model two of them(His173 andHis303) are located in periplasmic exposed loopsnext to the conserved WWD domain which is believedto provide a platform for the heme bound to holoCcmEwhile His261 is located in one of the TM helices and it is

predicted to act as an axial ligand to the heme b of Ec-CcmFthe other conserved His residue (H491) could provide thesecond axial coordination bond to the heme although thishas not been experimentally addressed This model of Ec-CcmF therefore envisages that this large membrane proteinis characterized by two heme-binding sites one of themis embedded in the membrane and coordinates a heme bprosthetic group necessary to reduce the CcmE-bound hemehosted in the second heme-binding site and constituted by itsWWD domain

It is interesting to note that in plants mitochondria theCcmF ortholog appears to be split into three different pro-teins (At-CcmFN1 At-CcmFN2 and At-CcmFC) possiblyinteracting each other [16] Since each of these proteins issimilar to the corresponding domain in the bacterial CcmFortholog this observation may provide useful informationin the design of engineered fragments of bacterial CcmFproteins amenable to structural analyses

The other System I component which is generallybelieved to be involved in the final steps of Cyt c maturationis CcmI As stated above the ccmI gene is present only insome Ccm operons while in others the corresponding ORFis present within the ccmH gene (as in E coli)The functionalrole of CcmI in Cytc biogenesis is revealed by geneticstudies showing that in R capsulatus and B japonicuminactivation of the ccmI gene leads to inability to synthesizefunctional c-type cytochromes [98 99] In R capsulatus andP aeruginosa the CcmI protein (Rc-CcmI and Pa-CcmIresp) can be described as being composed of two domainsstarting from the N-terminus a first domain composed oftwo TM helices connected by a short cytoplasmic regionand a large periplasmic domain Structural variations maybe observed among CcmI members from different bacteriaindeed multiple sequence alignment indicates that the cyto-plasmic region of Rc-CcmI contains a leucine zipper motifwhich is not present in the putative cytoplasmic region of Pa-CcmI [100ndash102] Surprisingly no crystallographic structureis available up to now for the soluble domain of any CcmIprotein with the exception of the ortholog protein NrfGfrom E coli (Ec-NrfG) [103] This protein is necessary toattach the heme to the unusual heme-binding motif CWSCK(where a Lys residue substitutes the conservedHis) present inNrfA a pentaheme c-type cytochrome [103 104] Accordingto secondary structure prediction methods [105] it hasbeen proposed that the periplasmic domain of Pa-CcmI iscomposed of aN-terminal120572-helical region containing at leastthree TPR motifs connected by a disordered linker to a 120572-120573C-terminal regionMultiple sequence analyses and secondarystructure predictionmethods show that the TPR region of Pa-CcmI can be successfully aligned with many TPR-containingproteins including Ec-NrfG [106]

26 System I ApoCyt Chaperoning and Heme AttachmentMechanisms TPR domain-containing proteins are commonto eukaryotes prokaryotes and archaea these proteins aregenerally involved in the assembly of multiprotein complexesand to the chaperoning of unfolded proteins [103 107] Itis therefore plausible that CcmI (or the TPR C-terminal

10 Scientifica

domain of Ec-CcmH) may act to provide a platform for theunfolded apoCyt chaperoning it to the heme attachment sitepresumably located on the WWD domain of CcmF CcmImay thus be considered a component of amembrane-integralmultisubunit heme ligation complex together with CcmFand CcmH as experimentally observed by affinity purifi-cation experiments carried out with Rc-CcmFHI proteins[97 99 108] According to the proposed function of CcmI acritical requirement is represented by its ability to recognizedifferent protein targets over and above apoCyt such asCcmFandCcmHHowever until now direct evidence has been pre-sented only for the interaction of CcmI with apoCyt but thepossibility remains that CcmFHI proteins interact each othervia their TM helices and not via their periplasmic domainsInterestingly both for Pa-CcmI [106] or Rc-CcmI [99] CDspectroscopy experiments carried out on the CcmIapoCytcomplex highlighted major conformational changes at thesecondary structure level It is tempting to speculate on thebasis of these results that in vivo the folding of apoCyt maybe induced by the interaction with CcmI In the case of Paeruginosa System I proteins the binding process betweenPa-CcmI and its target protein apoCyt c551 (Pa-apoCyt) hasbeen studied both at equilibrium and kinetically [106] the119870119863measured for this interaction (in the 120583M range) appeared

to be low enough to ensure apoCyt delivery to the othercomponents of the Ccmmachinery Clearly a major questionconcerns the molecular determinants of such recognitionprocess interestingly both affinity coprecipitation assays[99] and equilibrium and kinetic binding experiments [106]highlighted the role played by the C-terminal 120572-helix of Cytc Similar observations have been made for the interaction ofEc-NrfGwith a peptidemimickingNrfA its apoCyt substrate[103] in this case isothermal titration calorimetry (ITC)experiments indicate that the TPR-domain of NrfG serves asa binding site for the C-terminal motif of NrfA Altogetherthese observations are in agreement with the fact that TPRproteins generally bind to their targets by recognizing theirC-terminal region [107]

The CcmI chaperoning activity has been experimentallysupported for the first time in the case of Pa-CcmI by citratesynthase tests [106] it has been proposed that the observedability to suppress protein aggregation in vitromay reflect thecapacity of CcmI to avoid apoCyt aggregation in vivo Stillanother piece of the Cyt c biogenesis puzzle has been addedrecently by showing that Rc-CcmI is able to interact withapoCcmE either alone or together with its substrate apoCytc2 forming a stable ternary complex in the absence of heme[109] This unexpected observation obtained by reciprocalcopurification experiments provides supporting evidence forthe existence of a large multisubunit complex composed ofCcmFHI andCcmE possibly interactingwith theCcmABCDcomplex It is interesting to note that while in the case ofthe CcmIapoCyt recognition different studies highlightedthe crucial role of the C-terminal helical region of apoCyt(see above) in the case of the apoCcmE apoCyt recognitionthe N-terminal region of apoCyt seems to represent a criticalregion

It is generally accepted that CcmF is the Ccm compo-nent responsible for heme covalent attachment to apoCyt

however as discussed above it is possible that this largemembrane protein plays such a role only together withother Ccm proteins such as CcmH and CcmI Moreover asrecently discovered by Kranz and coworkers [36 41] CcmFmay also act as a quinoleheme oxidoreductase ensuringthe necessary reduction of the oxidized heme b bound toCcmE Why it is necessary that the heme iron be in itsreduced state rather than in its oxidized state is not completelyclear although it is possible that this is a prerequisite to themechanism of thioether bond formation [110] According tocurrent hypotheses it is likely that the periplasmic WWDdomain of CcmF provides a platform for heme b bindingSanders et al and Verissimo et al [34 109] have presenteda mechanistic view of the heme attachment process whichtakes into account all the available experimental observationson the different Ccmproteins According to thismodel stere-ospecific heme ligation to reduced apoCyt occurs becauseonly the vinyl-4 group is available to form the first thioetherbond with a free cysteine at the apoCyt heme-binding motifsince the vinyl-2 group is involved (at least in the Ec-CcmE)in the covalent bond with His130 of CcmE [67] Howeverexperimental proof for this hypothesis requires a detailedinvestigation of the apoCyt thioreduction process catalyzedby CcmH (see Section 24)

It should be noticed that the mechanisms described sofar for the function(s) played by CcmF (see [34 36 109]) donot envisage a clear role for its large C-terminal periplasmicdomain (residues 510 to 611 in Ec-CcmF) It would be inter-esting to see if this domain apparently devoid of recognizablesequence features may mediate intermolecular recognitionprocesses with one (or more) component(s) involved in thehemeapoCyt ligation process

3 System II

System II is typically found in gram-positive bacteria and inin 120576-proteobacteria it is also present in most 120573- and some120575-proteobacteria in Aquificales and cyanobacteria as wellas in algal and plant chloroplasts System II is composedof three or four membrane-bound proteins CcdA ResACcsA (also known as ResC) and CcsB (also known as ResB)(Figure 3) CcdA andResA are redox-active proteins involvedin the reduction of the disulfide bond in the heme-bindingmotif of apoCyt whereas CcsA and CcsB are responsible forthe heme-apoCyt ligation process and are considered Cyt csynthethases (CCS) BothCcsAwhich is evolutionary relatedto the CcmC and CcmF proteins of System I [55] and CcsBare integral membrane proteins In some 120576-proteobacteriasuch asHelicobacter hepaticus andHelicobacter pylori a singlefusion protein composed of CcsA and CcsB polypeptides ispresent [41 111] Although as discussed below evidence hasbeen put forward to support the hypothesis that the CcsBAcomplex acts a heme translocase we still do not know if theheme is transported across the membrane by component(s)of System II itself or by a different unidentified process

31 System II ApoCyt Thioreduction Pathway After the Secmachinery secretes the newly synthesized apoCyt it readily

Scientifica 11

becomes a substrate for the oxidative system present on theouter surface of the cytoplasmic membrane of the gram-positive bacteria In B subtilis the BdbCD system [112113] is functionally but not structurally equivalent to thewell-characterized DsbAB system present in gram-negativebacteria As discussed above for System I the disulfide ofthe apoCyt heme-binding motif must be reduced in orderto allow thioether bonds formation and heme attachmentResA is the extracytoplasmic membrane-anchored TRX-likeprotein involved in the specific reduction of the apoCytdisulfide bond after the disulfide bond exchange reactionhas occurred oxidized ResA is reduced by CcdA whichreceives its reducing equivalents from a cytoplasmic TRX[114] Although ResA displays a classical TRX-like foldthe analysis of the 3D structure of oxidized and reducedResA from B subtilis (Bs-ResA) showed redox-dependentconformational modifications not observed in other TRX-like proteins Interestingly such modifications occur at thelevel of a cavity proposed to represent the binding site foroxidized apoCyt [24 115 116] Another peculiar feature of Bs-ResA is represented by the unusually similar pKa values of itsthe active site Cys residues (88 and 82 resp) as observedalso in the case of the active site Cys residues of the SystemI Pa-CcmH (see Section 23) However at variance with Pa-CcmH in Bs-ResA the large separation between the twocysteine thiols observed in the structure of the reduced formof the protein can be invoked to account for this result

CcdA is a large membrane protein containing six TMhelices [117 118] homologous to the TM domain of E coliDsbD [85 119] Its role in the Cyt c biogenesis is supportedby the observation that inactivation of CcdA blocks theproduction of c-type cytochromes in B subtilis [120 121]Moreover over and above the reduction of its apoCyt sub-strate Bs-CcdA is able to reduce the disulfide bond of othersecreted proteins such as StoA [122] It is interesting to notethat ResA and CcdA are not essential for Cyt c synthesisin the absence of BdbD or BdbC or if a disulfide reductantis present in the growth medium [123 124] As discussedabove (Section 24) a similar observation was made on therole of the thio-reductive pathway of System I in this casepersistent production of c-type cytochromes was observedin Dsb-inactivated bacterial strains [38] Following theseobservations the question has been put forward as to why theCyt c biogenesis process involves this apparently redundantthio-reduction route only to correct the effects of Bdb orDsb activity Referring to the case of B subtilis [111] it hasbeen proposed that the main reason is that BdbD mustefficiently oxidize newly secreted proteins since the activityof most of them depends on the presence of disulfide bondsAlternatively it can be hypothesized that the intramoleculardisulfide bondof apoCyt protects the protein fromproteolyticdegradation or aggregation or from cross-linking to otherthiol-containing proteins

32 System II Heme Translocation and Attachment to ApoCytRecent experimental evidence is accumulating supportingthe involvement of the heterodimeric membrane complexResBC or of the fusion protein CcsBA in the translocation

of heme from the n-side to the p-side of the bacterialmembrane In particular it has been shown that CcsBAfrom H hepaticus (Hh-CcsBA) is able to reconstitute Cyt cbiogenesis in the periplasm of E coli [36] Moreover it wasalso observed that Hh-CcsBA is able to bind reduced hemevia two conserved histidines flanking the WWD domainand required both for the translocation of the heme andfor the synthetase function of Hh-CcsBA [36] A differentstudy carried out on B subtilis System II proteins providedsupport for heme-binding capability by the ResBC complexaccording to the results obtained on recombinant ResBC theResB component of the heterodimeric CCS complex is ableto covalently bind the heme in the cytoplasm (probably by aCys residue) and to deliver it to an extracytoplamic domain ofResC which is responsible for the covalent ligation to apoCyt[117] It should be noticed however that the transfer of theCCS-bound heme to apoCyt still awaits direct experimentalproof Moreover it has also been reported that in B subtilisthe inactivation of CCS does not affect the presence ofother heme-containing proteins in the periplasm such ascytochrome b562 [111] this observation which is in contrastto the heme-transport hypothesis by CCSs parallels similarconcerns about heme translocation mechanism that arecurrently discussed in the context of System I (see Section 22above) In the case of the Hh-CcsBA synthetase site-directedmutagenesis experiments allowed to assign a heme-bindingrole for the two pairs of conserved His residues accordingto the topological model of the protein obtained by alkalinephosphatase (PhoA) assays and GFP fusions the conservedHis77 and His858 residues are located on TM helices whileHis761 and His897 are located on the periplasmic side [36]These observations together with the results of site-directedmutagenesis experiments allowed the authors to suggest thatHis77 andHis858 form a low affinitymdashmembrane embeddedheme-binding site for ferrous heme which is subsequentlytranslocated to an external heme-binding domain of Hh-CcsBA where it is coordinated by His761 and His897 Thetopological model therefore predicts that this last His pairis part of a periplasmic-located WWD domain (homologousto theWWDdomains found in theCcmCandCcmFproteinsof System I described above)

4 System III

Strikingly different from Systems I and II the Cyt c mat-uration apparatus found in fungi and in metazoan cells(System III) is composed of a single protein known as Holo-Cytochrome c Synthetase (HCCS) or Cytochrome c HemeLyase (CCHL) apparently responsible for all the subprocessesdescribed above (heme transport and chaperoning reductionand chaperoning of apoCyt and catalysis of the thioetherbonds formation between heme and apoCyt) (Figure 4)Surprisingly although this protein has been identified in Scerevisiaemitochondria several years ago [125 126] only veryrecently the human HCCS has been expressed as a recombi-nant protein in E coli and spectroscopically characterized invitro [127] It is worth noticing that humanHCCS is attractinginterest since over and above its role in Cyt c biogenesis it

12 Scientifica

is involved in diseases such as microphthalmia with linearskin defects syndrome (MLS) an X-linked genetic disorder[128ndash130] and in other processes such as Cytc-independentapoptosis in injured motor neurons [131] Different fromanimal cells where a single HCCS is sufficient for thematuration of both soluble and membrane-anchored Cytc(Cyt c and Cy c1 resp) in S cerevisiae two homologs HCCSand HCC1S located in the inner mitochondrial membraneand facing to the IMS space [132 133] are responsible for hemeattachment to apoCyt c and apoCyt c1 respectively [125 134]It should also be noticed that in fungi an additional FAD-containing protein (Cyc2p) is required for Cyt c synthesisCyc2p is a mitochondrial membrane-anchored flavoproteinexposing its redox domain to the IMS which is requiredfor the maturation of Cyt c but not for that of Cyt c1 [135]This protein does not contain the conserved Cys residuestypically found in disulfide reductases and indeed it is notable to reduce oxidized apoCyt in vitro However it has beenrecently shown that Cyc2p is able to catalyze the NAD(P)H-dependent reduction of heme in vitro [136] a necessarystep before the thioether bond formation can occur asdiscussed above (see Section 26) This result together withthe observation that Cyc2p interacts with HCCS and withapoCyt c and c1 lends support to the proposal that Cyc2p isinvolved in the reduction of the heme iron in vivo [136 137]

AlthoughHCCS proteins are crucial for Cyt cmaturationwe still do not know how these proteins recognize theirsubstrates (heme and apoCyt) and how they promote orcatalyze the formation of thioether bonds between the hemevinyl groups and the cysteine thiols of the apoCyt CXXCHmotif

Contrary to the broad specificity of System I whichis able to recognize and attach the heme to prokaryoticand eukaryotic c-type cytochromes [49 50] and even tovery short microperoxidase-like sequences [49 50] mito-chondrial HCCS is characterized by a higher specificityas it does not recognize bacterial apoCyt sequences Theseobservations prompted the investigation of the recognitionprocess between apoCyt and HCCS Recently by using arecombinant yeast HCCS and chimerical apoCyt sequencesexpressed in E coli it was possible to conclude that a crucialrecognition determinant is represented by the N-terminalregion of apoCyt containing the heme-binding motif [35]notice that in the context of System I the same N-terminalregion of the Cyt c sequence has been recently identified to beimportant for the recognition by apoCcmE (see Section 26)Over and above the role played by the intervening residuesin the CXXCH heme-binding motif [135] it has been shownthat a conserved Phe residue occurring in the N-terminalregion before the CXXCH motif is important for HCCSrecognition [35 138] These results support the hypothesisthat this residue known to be a key determinant of Cytcfolding and stability [19 20 139] may also be crucial for Cytcmaturation by HCCS

Another relevant question concerns the ability of HCCSto recognize and bind the heme molecule as the region ofthe protein responsible for heme recognition remains to beidentified Initially it was hypothesized that the recognitionof heme could be mediated by the CP motifs present in

the HCCS protein [140] these short sequences are indeedknown to bind heme in a variety of heme-containing proteins[141 142] However it has been recently shown that CPmotifsof the recombinant S cerevisiae HCCS are not necessaryfor Cyt c production in E coli [143] excluding them as keydeterminants of heme recognition

The long-awaited in vitro characterization of the recom-binant human HCCS allowed for the first time to pro-pose a molecular mechanism underlying Cytc maturationin eukaryotes which can be experimentally tested [127]According to the proposed model the human HCCS activitycan be described as a four step mechanism involving (i)heme-binding (ii) apoCyt recognition (iii) thioether bondsformation and (iv) holoCyt c release In particular theheme is proposed to play the role of a scaffolding moleculemediating the contacts between HCCS and apoCyt Muta-genesis experiments carried out on the recombinant HCCSstrongly suggest that heme-binding (Step 1) depends on thepresence of a specific His residue (His154) acting as anaxial ligand to ferrous heme Residues present at the N-terminus of apoCyt mediate the recognition with HCCS(Step 2) as discussed above it is known that this regionforms structurally conserved 120572-helix in the fold of all c-type cytochromes Unfortunately no information is availableup to now concerning the region of HCCS involved in therecognition and binding to the N-terminal region of apoCytCoordination of the heme iron by the His residue of theapoCyt heme-binding motif CXXCH provides the secondaxial ligand to the heme iron and is probably importantfor the correct positioning of the two apoCyt Cys residuesand formation of the thioether bonds (Step 3) The finalrelease of functional Cyt c (Step 4) clearly requires thedisplacement of the His154Fe2+ coordination bond sucha displacement is probably mediated by formation of thecoordination bond with the Cyt c conserved Met residueandor by the simultaneous folding of Cyt c Again it isinteresting to note that this last hypothesis is in accordancewith in vitro folding studies on c-type cytochromes thathighlighted the late formation of the Met-Fe coordinationduring Cyt c folding [144 145]

Acknowledgments

The author thanks A Di Matteo and E Di Silvio for fruitfuldiscussions and for help in preparing this paper This work issupported by grants from the University of Rome ldquoSapienzardquo(C26A11MBXJ and C26A13MWF4)

References

[1] M G Galinato J G Kleingardner S E Bowman et alldquoHeme-protein vibrational couplings in cytochrome c providea dynamic link that connects the heme-iron and the proteinsurfacerdquo Proceedings of the National Academy of Sciences vol109 no 23 pp 8896ndash8900 2012

[2] H B Gray and J R Winkler ldquoElectron flow through metal-loproteinsrdquo Biochimica et Biophysica Acta vol 1797 no 9 pp1563ndash1572 2010

Scientifica 13

[3] P Ascenzi R Santucci M Coletta and F PolticellildquoCytochromes reactivity of the ldquodark siderdquo of the hemerdquoBiophysical Chemistry vol 152 no 1ndash3 pp 21ndash27 2010

[4] S Yamada N D Bouley Ford G E Keller W C Ford HB Gray and J R Winkler ldquoSnapshots of a protein foldingintermediaterdquo Proceedings of the National Academy of Sciencesvol 110 no 5 pp 1606ndash1610 2013

[5] P Weinkam J Zimmermann F E Romesberg and P GWolynes ldquoThe folding energy landscape and free energy excita-tions of cytochrome crdquo Accounts of Chemical Research vol 43no 5 pp 652ndash660 2010

[6] M Yamanaka M Masanari and Y Sambongi ldquoConfermentof folding ability to a naturally unfolded apocytochrome cthrough introduction of hydrophobic amino acid residuesrdquoBiochemistry vol 50 no 12 pp 2313ndash2320 2011

[7] G W Moore and G W Pettigrew Cytochrome C EvolutionaryStructural and Physicochemical Aspects Springer Berlin Ger-many 1990

[8] I Bertini G Cavallaro and A Rosato ldquoCytochrome c occur-rence and functionsrdquo Chemical Reviews vol 106 no 1 pp 90ndash115 2006

[9] D A Pearce and F Sherman ldquoDegradation of cytochromeoxidase subunits in mutants of yeast lacking cytochrome c andsuppression of the degradation by mutation of yme1rdquo Journal ofBiological Chemistry vol 270 no 36 pp 20879ndash20882 1995

[10] G Silkstone S M Kapetanaki I Husu M H Vos and MT Wilson ldquoNitric oxide binding to the cardiolipin complex offerric cytochrome crdquo Biochemistry vol 51 no 34 pp 6760ndash6766 2012

[11] M Giorgio E Migliaccio F Orsini et al ldquoElectron transferbetween cytochrome c and p66Shc generates reactive oxygenspecies that trigger mitochondrial apoptosisrdquo Cell vol 122 no2 pp 221ndash233 2005

[12] S M Kilbride and J H Prehn ldquoCentral roles of apoptoticproteins in mitochondrial functionrdquo Oncogene vol 32 no 22pp 2703ndash2711 2013

[13] T Noll and G Noll ldquoStrategies for ldquowiringrdquo redox-activeproteins to electrodes and applications in biosensors biofuelcells and nanotechnologyrdquo Chemical Society Reviews vol 40no 7 pp 3564ndash3576 2011

[14] G Layer J Reichelt D Jahn and D W Heinz ldquoStructure andfunction of enzymes in heme biosynthesisrdquo Protein Science vol19 no 6 pp 1137ndash1161 2010

[15] S Severance and IHamza ldquoTrafficking of heme and porphyrinsin metazoardquo Chemical Reviews vol 109 no 10 pp 4596ndash46162009

[16] P Hamel V Corvest P Giege and G Bonnard ldquoBiochemi-cal requirements for the maturation of mitochondrial c-typecytochromesrdquo Biochimica et Biophysica Acta vol 1793 no 1 pp125ndash138 2009

[17] W R Fisher H Taniuchi and C B Anfinsen ldquoOn the role ofheme in the formation of the structure of cytochrome crdquo Journalof Biological Chemistry vol 248 no 9 pp 3188ndash3195 1973

[18] M Yamanaka H Mita Y Yamamoto and Y Sambongi ldquoHemeis not required for aquifex aeolicus cytochrome c555 polypep-tide foldingrdquoBioscience Biotechnology andBiochemistry vol 73no 9 pp 2022ndash2025 2009

[19] C Travaglini-Allocatelli S Gianni andMBrunori ldquoA commonfolding mechanism in the cytochrome c familyrdquo Trends inBiochemical Sciences vol 29 no 10 pp 535ndash541 2004

[20] A Borgia D Bonivento C Travaglini-Allocatelli A DiMatteoand M Brunori ldquoUnveiling a hidden folding intermediate in c-type cytochromes by protein engineeringrdquo Journal of BiologicalChemistry vol 281 no 14 pp 9331ndash9336 2006

[21] E Enggist L Thony-Meyer P Guntert and K PervushinldquoNMR structure of the heme chaperone CcmE reveals a novelfunctional motifrdquo Structure vol 10 no 11 pp 1551ndash1557 2002

[22] A di Matteo N Calosci S Gianni P Jemth M Brunori and CTravaglini-Allocatelli ldquoStructural and functional characteriza-tion of CcmG from pseudomonas aeruginosa a key componentof the bacterial cytochrome c maturation apparatusrdquo Proteinsvol 78 no 10 pp 2213ndash2221 2010

[23] A diMatteo S GianniM E Schinina et al ldquoA strategic proteinin cytochrome c maturation three-dimensional structure ofCcmH and binding to apocytochrome crdquo Journal of BiologicalChemistry vol 282 no 37 pp 27012ndash27019 2007

[24] A Crow R M Acheson N E Le Brun and A OubrieldquoStructural basis of redox-coupled protein substrate selectionby the cytochrome c biosynthesis protein ResArdquo Journal ofBiological Chemistry vol 279 no 22 pp 23654ndash23660 2004

[25] E di Silvio A di Matteo and C Travaglini-Allocatelli ldquoTheRedox pathway of Pseudomonas aeruginosa cytochrome cbiogenesisrdquo Journal of Proteins and Proteomics vol 3 no 1 pp1ndash7 2012

[26] LThony-Meyer andP Kunzler ldquoTranslocation to the periplasmand signal sequence cleavage of preapocytochrome c depend onsec and lep but not on the ccmgene productsrdquoEuropean Journalof Biochemistry vol 246 no 3 pp 794ndash799 1997

[27] S J Facey and A Kuhn ldquoBiogenesis of bacterial inner-membrane proteinsrdquo Cellular and Molecular Life Sciences vol67 no 14 pp 2343ndash2362 2010

[28] K Diekert A I de Kroon U Ahting et al ldquoApocytochromec requires the TOM complex for translocation across themitochondrial outer membranerdquo The EMBO Journal vol 20no 20 pp 5626ndash5635 2001

[29] N Wiedemann V Kozjak T Prinz et al ldquoBiogenesis of yeastmitochondrial cytochrome c a unique relationship to the TOMmachineryrdquo Journal ofMolecular Biology vol 327 no 2 pp 465ndash474 2003

[30] F-U Hartl N Pfanner and W Neupert ldquoTranslocation inter-mediates on the import pathway of proteins intomitochondriardquoBiochemical Society Transactions vol 15 no 1 pp 95ndash97 1987

[31] B S Glick A Brandt K Cunningham SMuller R L Hallbergand G Schatz ldquoCytochromes c1 and b2 are sorted to theintermembrane space of yeast mitochondria by a stop-transfermechanismrdquo Cell vol 69 no 5 pp 809ndash822 1992

[32] G Howe and S Merchant ldquoRole of heme in the biosynthesis ofcytochrome c6rdquo Journal of Biological Chemistry vol 269 no 8pp 5824ndash5832 1994

[33] S S Nakamoto P Hamel and S Merchant ldquoAssembly ofchloroplast cytochromes b and crdquo Biochimie vol 82 no 6-7 pp603ndash614 2000

[34] C Sanders S Turkarslan D-W Lee and F DaldalldquoCytochrome c biogenesis the Ccm systemrdquo Trends inMicrobiology vol 18 no 6 pp 266ndash274 2010

[35] J M Stevens D A Mavridou R Hamer P Kritsiligkou A DGoddard and S J Ferguson ldquoCytochrome c biogenesis systemIrdquoThe FEBS Journal vol 278 no 22 pp 4170ndash4178 2011

[36] R G Kranz C Richard-Fogal J-S Taylor and E R FrawleyldquoCytochrome c biogenesis mechanisms for covalent modifica-tions and trafficking of heme and for heme-iron redox controlrdquo

14 Scientifica

Microbiology and Molecular Biology Reviews vol 73 no 3 pp510ndash528 2009

[37] J W A Allen ldquoCytochrome c biogenesis in mitochondriamdashsystems III and VrdquoThe FEBS Journal vol 278 no 22 pp 4198ndash4216 2011

[38] D A Mavridou S J Ferguson and J M Stevens ldquoCytochromec assemblyrdquo IUBMB Life vol 65 no 3 pp 209ndash216 2013

[39] I Bertini G Cavallaro and A Rosato ldquoEvolution ofmitochondrial-type cytochrome c domains and of theprotein machinery for their assemblyrdquo Journal of InorganicBiochemistry vol 101 no 11-12 pp 1798ndash1811 2007

[40] B S Goldman and R G Kranz ldquoEvolution and horizontaltransfer of an entire biosynthetic pathway for cytochromec biogenesis helicobacter deinococcus archae and morerdquoMolecular Microbiology vol 27 no 4 pp 871ndash873 1998

[41] C L Richard-Fogal E R Frawley E R Bonner H Zhu BSan Francisco and R G Kranz ldquoA conserved haem redoxand trafficking pathway for cofactor attachmentrdquo The EMBOJournal vol 28 no 16 pp 2349ndash2359 2009

[42] L Thony-Meyer F Fischer P Kunzler D Ritz and H Hen-necke ldquoEscherichia coli genes required for cytochrome cmaturationrdquo Journal of Bacteriology vol 177 no 15 pp 4321ndash4326 1995

[43] C S Beckett J A Loughman K A Karberg G M DonatoW E Goldman and R G Kranz ldquoFour genes are required forthe system II cytochrome c biogenesis pathway in Bordetellapertussis a unique bacterial modelrdquo Molecular Microbiologyvol 38 no 3 pp 465ndash481 2000

[44] N E Le Brun J Bengtsson and L Hederstedt ldquoGenes requiredfor cytochrome c synthesis in Bacillus subtilisrdquo MolecularMicrobiology vol 36 no 3 pp 638ndash650 2000

[45] P Giege J M Grienenberger and G Bonnard ldquoCytochromec biogenesis in mitochondriardquo Mitochondrion vol 8 no 1 pp61ndash73 2008

[46] R E Feissner C L Richard-Fogal E R Frawley J A Lough-man KW Earley and R G Kranz ldquoRecombinant cytochromesc biogenesis systems I and II and analysis of haem deliverypathways in Escherichia colirdquo Molecular Microbiology vol 60no 3 pp 563ndash577 2006

[47] N P Cianciotto P Cornelis and C Baysse ldquoImpact of thebacterial type I cytochrome c maturation system on differentbiological processesrdquoMolecular Microbiology vol 56 no 6 pp1408ndash1415 2005

[48] E Arslan H Schulz R Zufferey P Kunzler and L Thony-Meyer ldquoOverproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in Escherichiacolirdquo Biochemical and Biophysical Research Communicationsvol 251 no 3 pp 744ndash747 1998

[49] C Sanders and H Lill ldquoExpression of prokaryotic and eukary-otic cytochromes c in Escherichia colirdquoBiochimica et BiophysicaActa vol 1459 no 1 pp 131ndash138 2000

[50] M Braun and L Thony-Meyer ldquoBiosynthesis of artificialmicroperoxidases by exploiting the secretion and cytochromec maturation apparatuses of Escherichia colirdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 35 pp 12830ndash12835 2004

[51] B Baert C Baysse S Matthijs and P Cornelis ldquoMultiplephenotypic alterations caused by a c-type cytochrome mat-uration ccmC gene mutation in Pseudomonas aeruginosardquoMicrobiology vol 154 no 1 pp 127ndash138 2008

[52] S Turkarslan C Sanders and F Daldal ldquoExtracytoplasmicprosthetic group ligation to apoproteins maturation of c-typecytochromesrdquo Molecular Microbiology vol 60 no 3 pp 537ndash541 2006

[53] P M Jones and A M George ldquoThe ABC transporter structureand mechanism perspectives on recent researchrdquo Cellular andMolecular Life Sciences vol 61 no 6 pp 682ndash699 2004

[54] O Christensen E M Harvat L Thony-Meyer S J Fergusonand J M Stevens ldquoLoss of ATP hydrolysis activity by CcmABresults in loss of c-type cytochrome synthesis and incompleteprocessing ofCcmErdquoTheFEBS Journal vol 274 no 9 pp 2322ndash2332 2007

[55] J-H Lee E M Harvat J M Stevens S J Ferguson and M HSaier Jr ldquoEvolutionary origins of members of a superfamily ofintegralmembrane cytochrome c biogenesis proteinsrdquoBiochim-ica et Biophysica Acta vol 1768 no 9 pp 2164ndash2181 2007

[56] D L Beckman D R Trawick and R G Kranz ldquoBacterialcytochromes c biogenesisrdquo Genes and Development vol 6 no2 pp 268ndash283 1992

[57] H Schulz R A Fabianek E C Pellicioli H Hennecke andL Thony-Meyer ldquoHeme transfer to the heme chaperone CcmEduring cytochrome c maturation requires the CcmC proteinwhich may function independently of the ABC-transporterCcmABrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 96 no 11 pp 6462ndash6467 1999

[58] Q Ren and L Thony-Meyer ldquoPhysical Interaction ofCcmC with Heme and the Heme Chaperone CcmE duringCytochrome cMaturationrdquo Journal of Biological Chemistry vol276 no 35 pp 32591ndash32596 2001

[59] C Richard-Fogal and R G Kranz ldquoThe CcmChemeCcmEcomplex in heme trafficking and cytochrome c biosynthesisrdquoJournal of Molecular Biology vol 401 no 3 pp 350ndash362 2010

[60] V K Viswanathan S Kurtz L L Pedersen et al ldquoThecytochrome C maturation locus of Legionella pneumophilapromotes iron assimilation and intracellular infection andcontains a strain-specific insertion sequence elementrdquo Infectionand Immunity vol 70 no 4 pp 1842ndash1852 2002

[61] U Ahuja and L Thony-Meyer ldquoCcmD is involved in complexformation between CcmC and the heme chaperone CcmE dur-ing cytochrome c maturationrdquo Journal of Biological Chemistryvol 280 no 1 pp 236ndash243 2005

[62] C L Richard-Fogal E R Frawley and R G Kranz ldquoTopologyand function of CcmD in cytochrome Cmaturationrdquo Journal ofBacteriology vol 190 no 10 pp 3489ndash3493 2008

[63] H Schulz H Hennecke and L Thony-Meyer ldquoPrototype ofa heme chaperone essential for cytochrome c maturationrdquoScience vol 281 no 5380 pp 1197ndash1200 1998

[64] F Arnesano L Banci P D Barker et al ldquoSolution structure andcharacterization of the heme chaperone CcmErdquo Biochemistryvol 41 no 46 pp 13587ndash13594 2002

[65] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[66] L Thony-Meyer ldquoA heme chaperone for cytochrome c biosyn-thesisrdquo Biochemistry vol 42 no 45 pp 13099ndash13105 2003

[67] E M Harvat C Redfield J M Stevens and S J FergusonldquoProbing the heme-binding site of the cytochrome cmaturationprotein CcmErdquoBiochemistry vol 48 no 8 pp 1820ndash1828 2009

[68] D Lee K Pervushin D Bischof M Braun and L Thony-Meyer ldquoUnusual heme-histidine bond in the active site of a

Scientifica 15

chaperonerdquo Journal of the American Chemical Society vol 127no 11 pp 3716ndash3717 2005

[69] A D Goddard J M Stevens F Rao et al ldquoc-Type cytochromebiogenesis can occur via a natural Ccm system lacking CcmHCcmG and the heme-binding histidine of CcmErdquo Journal ofBiological Chemistry vol 285 no 30 pp 22882ndash22889 2010

[70] J M Aramini K Hamilton P Rossi et al ldquoSolution NMRstructure backbone dynamics and heme-binding propertiesof a novel cytochrome c maturation protein CcmE fromDesulfovibrio vulgarisrdquo Biochemistry vol 51 no 18 pp 3705ndash3707 2012

[71] T Uchida J M Stevens O Daltrop et al ldquoThe interactionof covalently bound heme with the cytochrome c maturationprotein CcmErdquo Journal of Biological Chemistry vol 279 no 50pp 51981ndash51988 2004

[72] I Garcıa-Rubio M Braun I Gromov L Thony-Meyer andA Schweiger ldquoAxial coordination of heme in ferric CcmEchaperone characterized by EPR spectroscopyrdquo BiophysicalJournal vol 92 no 4 pp 1361ndash1373 2007

[73] L Thony-Meyer ldquoHaem-polypeptide interactions duringcytochrome c maturationrdquo Biochimica et Biophysica Acta vol1459 no 2-3 pp 316ndash324 2000

[74] J A Keightley D Sanders T R Todaro A Pastuszyn and J AFee ldquoCloning expression in Escherichia coli of the cytochromec552 gene from Thermus thermophilus HB8 Evidence forgenetic linkage to an ATP- binding cassette protein and initialcharacterization of the cycA gene productsrdquo Journal of Biologi-cal Chemistry vol 273 no 20 pp 12006ndash12016 1998

[75] E M Monika B S Goldman D L Beckman and R GKranz ldquoA thioreduction pathway tethered to the membranefor periplasmic cytochromes c biogenesis In vitro and in vivostudiesrdquo Journal of Molecular Biology vol 271 no 5 pp 679ndash692 1997

[76] M Throne-Holst L Thony-Meyer and L HederstedtldquoEscherichia coli ccm in-frame deletion mutants can produceperiplasmic cytochrome b but not cytochrome crdquo FEBS Lettersvol 410 no 2-3 pp 351ndash355 1997

[77] U Ahuja and L Thony-Meyer ldquoDynamic features of a hemedelivery system for cytochrome c maturationrdquo Journal of Bio-logical Chemistry vol 278 no 52 pp 52061ndash52070 2003

[78] J W A Allen P D Barker O Daltrop et al ldquoWhy isnrsquot ldquostan-dardrdquo heme good enough for c-type and di-type cytochromesrdquoDalton Transactions no 21 pp 3410ndash3418 2005

[79] K Inaba and K Ito ldquoStructure and mechanisms of the DsbB-DsbA disulfide bond generation machinerdquo Biochimica et Bio-physica Acta vol 1783 no 4 pp 520ndash529 2008

[80] H Kadokura F Katzen and J Beckwith ldquoProtein disulfidebond formation in prokaryotesrdquoAnnual Review of Biochemistryvol 72 pp 111ndash135 2003

[81] S R Shouldice B Heras P M Walden M Totsika M ASchembri and J L Martin ldquoStructure and function of DsbA akey bacterial oxidative folding catalystrdquoAntioxidants and RedoxSignaling vol 14 no 9 pp 1729ndash1760 2011

[82] R Metheringham L Griffiths H Crooke S Forsythe and JCole ldquoAn essential role for DsbA in cytochrome c synthesis andformate-dependent nitrite reduction by Escherichia coli K-12rdquoArchives of Microbiology vol 164 no 4 pp 301ndash307 1995

[83] Y Sambongi and S J Ferguson ldquoMutants of Escherichia colilacking disulphide oxidoreductases DsbA and DsbB cannotsynthesise an exogenous monohaem c-type cytochrome exceptin the presence of disulphide compoundsrdquo FEBS Letters vol398 no 2-3 pp 265ndash268 1996

[84] D AMavridou S J Ferguson and JM Stevens ldquoThe interplaybetween the disulfide bond formation pathway and cytochromec maturation in Escherichia colirdquo FEBS Letters vol 586 no 12pp 1702ndash1707 2012

[85] C U Stirnimann A Rozhkova U Grauschopf M G GrutterR Glockshuber and G Capitani ldquoStructural basis and kineticsof DsbD-dependent cytochrome c maturationrdquo Structure vol13 no 7 pp 985ndash993 2005

[86] N Ouyang Y-G Gao H-Y Hu and Z-X Xia ldquoCrystalstructures of E coli CcmGand itsmutants reveal key roles of theN-terminal120573-sheet and the fingerprint regionrdquoProteins vol 65no 4 pp 1021ndash1031 2006

[87] M A Edeling L W Guddat R A Fabianek et al ldquoCrys-tallization and preliminary diffraction studies of native andselenomethionine CcmG (CycY DsbE)rdquoActa CrystallographicaD vol 57 no 9 pp 1293ndash1295 2001

[88] R A Fabianek M Huber-Wunderlich R Glockshuber PKunzler H Hennecke and L Thony-Meyer ldquoCharacterizationof the Bradyrhizobium japonicum CycY protein a membrane-anchored periplasmic thioredoxin that may play a role as areductant in the biogenesis of c-type cytochromesrdquo Journal ofBiological Chemistry vol 272 no 7 pp 4467ndash4473 1997

[89] G B Kallis and A Holmgren ldquoDifferential reactivity of thefunctional sulfhydryl groups of cysteine-32 and cysteine-32present in the reduced form of thioredoxin from Escherichiacolirdquo Journal of Biological Chemistry vol 255 no 21 pp 10261ndash10265 1980

[90] P T Chivers andR T Raines ldquoGeneral acidbase catalysis in theactive site of Escherichia coli thioredoxinrdquoBiochemistry vol 36no 50 pp 15810ndash15816 1997

[91] X M Zheng J Hong H Y Li D H Lin and H Y HuldquoBiochemical properties and catalytic domain structure of theCcmH protein from Escherichia colirdquo Biochim Biophys Actavol 1824 no 12 pp 1394ndash1400 2012

[92] UAhujaA Rozhkova RGlockshuber LThony-Meyer andOEinsle ldquoHelix swapping leads to dimerization of the N-terminaldomain of the c-type cytochrome maturation protein CcmHfrom Escherichia colirdquo FEBS Letters vol 582 no 18 pp 2779ndash2786 2008

[93] R A Fabianek T Hofer and L Thony-Meyer ldquoCharacteriza-tion of the Escherichia coli CcmH protein reveals new insightsinto the redox pathway required for cytochrome c maturationrdquoArchives of Microbiology vol 171 no 2 pp 92ndash100 1999

[94] E Reid J Cole and D J Eaves ldquoThe Escherichia coli CcmGprotein fulfils a specific role in cytochrome c assemblyrdquo Bio-chemical Journal vol 355 no 1 pp 51ndash58 2001

[95] Q Ren U Ahuja and LThony-Meyer ldquoA bacterial cytochromec heme lyase CcmF forms a complex with the heme chaperoneCcmE and CcmH but not with apocytochrome crdquo Journal ofBiological Chemistry vol 277 no 10 pp 7657ndash7663 2002

[96] E H Meyer P Giege E Gelhaye et al ldquoAtCCMH an essentialcomponent of the c-type cytochrome maturation pathway inArabidopsis mitochondria interacts with apocytochrome crdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 44 pp 16113ndash16118 2005

[97] C Rios-Velazquez R Coller and T J Donohue ldquoFeatures ofRhodobacter sphaeroides CcmFHrdquo Journal of Bacteriology vol185 no 2 pp 422ndash431 2003

[98] C Sanders M Deshmukh D Astor R G Kranz and F DaldalldquoOverproduction of CcmG and CcmFHRc fully suppresses thec-type cytochrome biogenesis defect of Rhodobacter capsulatus

16 Scientifica

CcmI-null mutantsrdquo Journal of Bacteriology vol 187 no 12 pp4245ndash4256 2005

[99] A F Verissimo H Yang X Wu C Sanders and F DaldalldquoCcmI subunit of CcmFHI heme ligation complex functionsas an apocytochrome c chaperone during c-type cytochromematurationrdquo Journal of Biological Chemistry vol 286 no 47 pp40452ndash40463 2011

[100] K Bryson L J McGuffin R L Marsden J J Ward J SSodhi and D T Jones ldquoProtein structure prediction servers atUniversity College Londonrdquo Nucleic Acids Research vol 33 no2 pp W36ndashW38 2005

[101] D T Jones ldquoProtein secondary structure prediction basedon position-specific scoring matricesrdquo Journal of MolecularBiology vol 292 no 2 pp 195ndash202 1999

[102] C Sanders C Boulay and F Daldal ldquoMembrane-spanning andperiplasmic segments of CcmI have distinct functions duringcytochrome c biogenesis in Rhodobacter capsulatusrdquo Journal ofBacteriology vol 189 no 3 pp 789ndash800 2007

[103] D Han K Kim J Oh J Park and Y Kim ldquoTPR domainof NrfG mediates complex formation between heme lyaseand formate-dependent nitrite reductase in Escherichia coliO157H7rdquo Proteins vol 70 no 3 pp 900ndash914 2008

[104] D J Eaves J Grove W Staudenmann et al ldquoInvolvement ofproducts of the nrfEFG genes in the covalent attachment ofhaem c to a novel cysteine-lysine motif in the cytochrome c552nitrite reductase from Escherichia colirdquo Molecular Microbiol-ogy vol 28 no 1 pp 205ndash216 1998

[105] J Nilsson B Persson and G Von Heijne ldquoConsensus predic-tions of membrane protein topologyrdquo FEBS Letters vol 486 no3 pp 267ndash269 2000

[106] E di Silvio A di Matteo F Malatesta and C Travaglini-Allocatelli ldquoRecognition and binding of apocytochrome c toP aeruginosa CcmI a component of cytochrome c maturationmachineryrdquo Biochim Biophys Acta vol 1834 no 8 pp 1554ndash1561 2013

[107] N Zeytuni and R Zarivach ldquoStructural and functional dis-cussion of the tetra-trico-peptide repeat a protein interactionmodulerdquo Structure vol 20 no 3 pp 397ndash405 2012

[108] C Sanders S Turkarslan D-W Lee O Onder R G Kranz andF Daldal ldquoThe cytochrome c maturation components CcmFCcmH and CcmI form a membrane-integral multisubunitheme ligation complexrdquo Journal of Biological Chemistry vol283 no 44 pp 29715ndash29722 2008

[109] A F Verissimo M A Mohtar and F Daldal ldquoThe hemechaperone ApoCcmE forms a ternary complex with CcmI andapocytochrome crdquoThe Journal of Biological Chemistry vol 288no 9 pp 6272ndash6283 2013

[110] P D Barker J C Ferrer M Mylrajan et al ldquoTransmutation of aheme proteinrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 90 no 14 pp 6542ndash65461993

[111] J Simon and L Hederstedt ldquoComposition and function ofcytochrome c biogenesis system IIrdquoThe FEBS Journal vol 278no 22 pp 4179ndash4188 2011

[112] T R H M Kouwen and J M van Dijl ldquoInterchangeablemodules in bacterial thiol-disulfide exchange pathwaysrdquo Trendsin Microbiology vol 17 no 1 pp 6ndash12 2009

[113] A Crow A Lewin O Hecht et al ldquoCrystal structure andbiophysical properties of Bacillus subtilis BdbD An oxidizingthioldisulfide oxidoreductase containing a novel metal siterdquoJournal of Biological Chemistry vol 284 no 35 pp 23719ndash23733 2009

[114] M C Moller and L Hederstedt ldquoExtracytoplasmic processesimpaired by inactivation of trxA (thioredoxin gene) in Bacillussubtilisrdquo Journal of Bacteriology vol 190 no 13 pp 4660ndash46652008

[115] A Lewin A Crow A Oubrie and N E Le Brun ldquoMolecularbasis for specificity of the extracytoplasmic thioredoxin ResArdquoJournal of Biological Chemistry vol 281 no 46 pp 35467ndash35477 2006

[116] C L Colbert QWu P J A Erbel K H Gardner and J Deisen-hofer ldquoMechanism of substrate specificity in Bacillus subtilisResA a thioredoxin-like protein involved in cytochrome cmaturationrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 103 no 12 pp 4410ndash44152006

[117] UAhuja P Kjelgaard B L Schulz LThony-Meyer andLHed-erstedt ldquoHaem-delivery proteins in cytochrome c maturationsystem IIrdquoMolecular Microbiology vol 73 no 6 pp 1058ndash10712009

[118] M L D Page P P Hamel S T Gabilly et al ldquoA homologof prokaryotic thiol disulfide transporter CcdA is required forthe assembly of the cytochrome b6f complex in Arabidopsischloroplastsrdquo Journal of Biological Chemistry vol 279 no 31pp 32474ndash32482 2004

[119] A Rozhkova C U Stirnimann P Frei et al ldquoStructural basisand kinetics of inter- and intramolecular disulfide exchange inthe redox catalyst DsbDrdquoThe EMBO Journal vol 23 no 8 pp1709ndash1719 2004

[120] T Schiott M Throne-Holst and L Hederstedt ldquoBacillussubtilis CcdA-defective mutants are blocked in a late step ofcytochrome C biogenesisrdquo Journal of Bacteriology vol 179 no14 pp 4523ndash4529 1997

[121] R E Feissner C S Beckett J A Loughman and R G KranzldquoMutations in cytochrome assembly and periplasmic redoxpathways in Bordetella pertussisrdquo Journal of Bacteriology vol187 no 12 pp 3941ndash3949 2005

[122] L S ErlendssonMMoller and L Hederstedt ldquoBacillus subtilisStoA is a thiol-disulfide oxidoreductase important for sporecortex synthesisrdquo Journal of Bacteriology vol 186 no 18 pp6230ndash6238 2004

[123] L S Erlendsson R M Acheson L Hederstedt and N E LeBrun ldquoBacillus subtilis ResA is a thiol-disulfide oxidoreductaseinvolved in cytochrome c synthesisrdquo Journal of BiologicalChemistry vol 278 no 20 pp 17852ndash17858 2003

[124] L S Erlendsson and L Hederstedt ldquoMutations in the thiol-disulfide oxidoreductases BdbC and BdbD can suppresscytochrome c deficiency of CcdA-defective Bacillus subtiliscellsrdquo Journal of Bacteriology vol 184 no 5 pp 1423ndash1429 2002

[125] M E Dumont J F Ernst D M Hampsey and F ShermanldquoIdentification and sequence of the gene encoding cytochromec heme lyase in the yeast Saccharomyces cerevisiaerdquoThe EMBOJournal vol 6 no 1 pp 235ndash241 1987

[126] M E Dumont J F Ernst and F Sherman ldquoCoupling of hemeattachment to import of cytochrome c into yeast mitochondriaStudies with heme lyase-deficient mitochondria and alteredapocytochromes crdquo Journal of Biological Chemistry vol 263 no31 pp 15928ndash15937 1988

[127] B San Francisco E C Bretsnyder and R G Kranz ldquoHumanmitochondrial holocytochrome c synthasersquos hemebindingmat-uration determinants and complex formationwith cytochromecrdquo Proceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 9 pp E788ndashE797 2012

Scientifica 17

[128] L Schaefer A Ballabio and H Y Zoghbi ldquoCloning andcharacterization of a putative human holocytochrome c-typesynthetase gene (HCCS) isolated from the critical region formicrophthalmia with linear skin defects (MLS)rdquoGenomics vol34 no 2 pp 166ndash172 1996

[129] S K Prakash T A Cormier A E McCall et al ldquoLoss ofholocytochrome c-type synthetase causes the male lethality ofX-linked dominant microphthalmia with linear skin defects(MLS) syndromerdquo Human Molecular Genetics vol 11 no 25pp 3237ndash3248 2002

[130] I Wimplinger M Morleo G Rosenberger et al ldquoMutations ofthe mitochondrial holocytochrome c-type synthase in X-linkeddominant microphthalmia with linear skin defects syndromerdquoTheAmerican Journal of HumanGenetics vol 79 no 5 pp 878ndash889 2006

[131] S Kiryu-Seo KGamo T Tachibana K Tanaka andHKiyamaldquoUnique anti-apoptotic activity of EAAC1 in injured motorneuronsrdquoTheEMBO Journal vol 25 no 14 pp 3411ndash3421 2006

[132] R Lill R A Stuart M E Drygas F E Nargang and W Neu-pert ldquoImport of cytochrome c heme lyase into mitochondriaa novel pathway into the intermembrane spacerdquo The EMBOJournal vol 11 no 2 pp 449ndash456 1992

[133] H Steiner A Zollner A Haid W Neupert and R LillldquoBiogenesis of mitochondrial heme lyases in yeast Importand folding in the intermembrane spacerdquo Journal of BiologicalChemistry vol 270 no 39 pp 22842ndash22849 1995

[134] A Zollner G Rodel and A Haid ldquoMolecular cloning andcharacterization of the Saccharomyces cerevisiae CYT2 geneencoding cytochrome-c1-heme lyaserdquo European Journal of Bio-chemistry vol 207 no 3 pp 1093ndash1100 1992

[135] V Corvest D A Murrey D G Bernard D B Knaff B Guiardand P PHamel ldquoc-Type cytochrome assembly in Saccharomycescerevisiae a key residue for apocytochrome c1lyase interactionrdquoGenetics vol 186 no 2 pp 561ndash571 2010

[136] V Corvest D A Murrey M Hirasawa D B Knaff B Guiardand P P Hamel ldquoThe flavoprotein Cyc2p a mitochondrialcytochrome c assembly factor is a NAD(P)H-dependent haemreductaserdquo Molecular Microbiology vol 83 no 5 pp 968ndash9802012

[137] D G Bernard S Quevillon-Cheruel S Merchant B Guiardand P P Hamel ldquoCyc2p a membrane-bound flavopro-tein involved in the maturation of mitochondrial c-typecytochromesrdquo Journal of Biological Chemistry vol 280 no 48pp 39852ndash39859 2005

[138] J G Kleingardner and K L Bren ldquoComparing substratespecificity between cytochrome cmaturation and cytochrome cheme lyase systems for cytochrome c biogenesisrdquo Metallomicsvol 3 no 4 pp 396ndash403 2011

[139] C Travaglini-Allocatelli S Gianni VMorea A Tramontano TSoulimane and M Brunori ldquoExploring the cytochrome c fold-ing mechanism cytochrome c552 fromThermus thermophilusfolds through an on-pathway intermediaterdquo Journal of BiologicalChemistry vol 278 no 42 pp 41136ndash41140 2003

[140] L Zhang and L Guarente ldquoHeme binds to a short sequencethat serves a regulatory function in diverse proteinsrdquoTheEMBOJournal vol 14 no 2 pp 313ndash320 1995

[141] S Hira T Tomita T Matsui K Igarashi and M Ikeda-SaitoldquoBach1 a heme-dependent transcription factor reveals pres-ence of multiple heme binding sites with distinct coordinationstructurerdquo IUBMB Life vol 59 no 8-9 pp 542ndash551 2007

[142] T K Kuhl A Wiszligbrock N Goradia et al ldquoAnalysis of Fe(III)heme binding to cysteine-containing heme-regulatory motifs

in proteinsrdquo ACS Chemical Biology vol 8 no 8 pp 1785ndash17932013

[143] R L Moore J M Stevens and S J Ferguson ldquoMitochondrialcytochrome c synthase CP motifs are not necessary for hemeattachment to apocytochrome crdquo FEBS Letters vol 585 no 21pp 3415ndash3419 2011

[144] S Gianni M Brunori and C Travaglini-Allocatelli ldquoRefoldingkinetics of cytochrome c551 reveals a mechanistic differencebetween urea and guanidinerdquo Protein Science vol 10 no 8 pp1685ndash1688 2001

[145] S Gianni C Travaglini-Allocatelli F Cutruzzola M BrunoriM C R Shastry and H Roder ldquoParallel pathways incytochrome c551 foldingrdquo Journal of Molecular Biology vol 330no 5 pp 1145ndash1152 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Microbiology

6 Scientifica

helix a periplasmic-orientedN-terminus and a cytoplasmic-oriented C-terminus [62] Immunoprecipitation experimentsindicate that CcmD interacts with the CcmA

2CcmB

1CcmC

1

complex even if it is not essential for heme transfer andattachment from CcmC to CcmE CcmD is strictly requiredfor the release of holoCcmE from the ABC transporter [6162]

CcmE is a heme-binding protein discovered as an essen-tial System I component as early as the late 1990rsquos [63]CcmE is a monotopic membrane protein anchored to themembrane via its N-terminal TM segment and exposing itsactive site to the periplasm it is the only Ccm componentof the heme trafficking and delivery module of System I forwhich a three-dimensional structure is available ([21] PDB1SR3 [64] PDB 1LM0) The 3D structure of the apo-state(without bound heme) consists of a six-stranded antiparallel120573-sheet reminiscent of the classical OB-fold [65] with N-and C-terminal extensions CcmE can be considered a ldquohemechaperonerdquo as it protects the cell from a potentially dangerouscompound by sequestering free heme in the periplasm [66]it is thought to act as an intermediate in the heme deliverypathway of Cytc maturation The structure of apoCcmEshowed no recognizable heme-binding cavities and in theabsence of a 3D structure of CcmEwith bound heme (holoC-cmE) the heme-binding region could only be predicted byin silico modeling It is generally believed that the hemein holoCcmE is solvent exposed but recent mutagenesisexperiments challenged this view [67] The unusual covalentbond between the nitrogen atomof a histidine residue presentin the conserved VLAKHDE motif located in a solventexposed environment (H130 in E coli CcmE) and a 120573-carbonof one of the heme vinyl groups has been described in greatdetail by NMR spectroscopy [68] Recently it was shown thatCcmE proteins from the proteobacteria D desulfuricans andD vulgaris contain the unusual CXXXY heme-bindingmotifwhere the Cys residue replaces the canonical His bindingresidue NMR solution structure of D vulgaris CcmE (PDB2KCT) revealed that the proteins adopt the same OB-foldcharacteristic of the CcmE superfamily Contrary to whatreported for theD desulfuricansCcmE [69] the homologousprotein from D vulgaris binds ferric heme noncovalentlythrough the conserved C127 residue [70] An additionalconserved residue in CcmE proteins is Tyr134 which wasshown to provide a coordination bond to the heme ironof holoCcmE [71 72] once it is released from CcmABCDcomplex [36] as discussed below

22 System I Heme Translocation and Delivery PathwayMechanisms We still do not know how the b heme istranslocated from the cytoplasm (where it is synthesized)to the periplasm where Cyt c maturation occurs Differ-ent mechanisms such as translocation through a proteinchannel or free diffusion across the membrane have beenproposed [73] The CcmAB proteins show structural featurestypical of the ABC transporters and for these reasons theywere originally hypothesized to be involved in the heme btranslocation process [57 63 74 75] However it is nowclear that an alternative process must exist since it has

been shown that periplasmic b-type cytochromes can beproduced in the absence of Ccm proteins [76] and thatinactivation of the ATPase activity of CcmA does not abolishheme accumulation in the periplasm [46 54] We have nowevidence that CcmC has the ability to bind heme at its WWDdomain present in the second periplasmic domain but itis still not clear if this membrane protein acts as a proteinchannel for heme translocation or simply collects it in theperiplasm [77]

Another important aspect concerns the oxidation state ofthe heme iron during translocation and delivery processesindeed this property of the heme iron may determine thereaction mechanism by which the unusual CcmE H130nitrogen is covalently linked to the vinyl 120573-carbon of theheme (see [36] for a detailed discussion of this topic) Basedon mutagenesis studies on CcmC [59] a model has beenpresented whereby oxidized heme is bound to CcmC onlyin the presence of CcmE forming a ternary complex BothCcmC and CcmE provide critical residues for heme-bindingthe two conserved His residues (H60 and H180 coordinatingthe heme iron) and theWWDdomain ofCcmCandHis130 ofCcmE forming the unusual covalent bond with heme vinyl-2 [59] The ATPase activity of CcmA is then required torelease holoCcmE from the CcmABCD complex a processthat depends also on the presence of CcmD [46 54] It shouldbe noticed that purified holoCcmE alone or in the CcmCDEcomplex [36 59] contains the heme iron in the oxidized statean observation that is apparently in contrast with the fact thatthe heme must be in its reduced state before attachment toapoCyt can occur Although the oxidation state of the hemeiron is currently debated [36 78] it is possible that CcmFwhich was recently shown to contain a heme b cofactor mayact as specific heme oxidoreductase (see Section 25)

23 System I ApoCyt Thioreduction Pathway ComponentsThe periplasm can be considered a relatively oxidizing envi-ronment due to the presence of an efficient oxidative systemcomposed of the DsbAB proteins [79 80] DsbA is a highlyoxidizing protein (1198641015840

0= 120mV) that is responsible for

the introduction of disulfide bonds into extracytoplasmicproteins [81] On the basis of the results obtained on E colidsbA deletion mutants that are unable to synthesize c-typecytochromes [82 83] it was generally accepted that formationof the intramolecular disulfide bond in apoCyt was a nec-essary step in the Cyt c biogenesis However only reducedapoCyt is clearly competent for heme ligation It is possiblethat this seemingly paradoxical thioreduction process hasevolved in order to protect the apoCyt from proteolyticdegradation aggregation andor formation of intermolec-ular disulfide bonds with thiols from other molecules (seealso Section 31 for a discussion about this aspect in SystemII) Recently however an analysis of c-type cytochromesproduction in several E coli dsb genes deletion strains ledto the hypothesis that DsbA is not necessary for Cyt cmaturation and that heme ligation to apoCyt and apoCytoxidation pathways is alternative competing processes [84]

In gram-negative bacteria a thioreduction pathway hasevolved to specifically reduce the oxidized apoCyt substrate

Scientifica 7

which includes the Ccm proteins CcmG and CcmH Thenecessary reducing power is transferred from the cytoplasmicthioredoxin (TRX) to CcmG via DsbD a large membraneprotein organized in three structural domains an N-terminalperiplasmic domain with a IgG-like fold (nDsbD) a C-terminal periplasmic domain with a thioredoxin-like (TRX-like) fold (cDsbD) and a central domain composed ofeight TM helices [85] Each of these domains contains apair of Cys residues and transfer electrons via a cascadeof disulfide exchange reactions making DsbD a ldquoredox-hubrdquo in the periplasm performing disulfide bond exchangereactions with different oxidized proteins [79] In particulara combination of X-ray crystallography experiments andkinetic analyses showed that electrons are transferred fromthe cytoplasmic TRX to the membrane domain of DsbDfollowed by reduction of cDsbD and finally of nDsbD whichis the direct electron donor to CcmG [85]

CcmG is a membrane-anchored protein linked to themembrane via an N-terminal TM helix and exposing itssoluble TRX-like domain in the periplasm The 3D structureof the TRX-like domain of CcmG from different bacteriahas been solved by X-ray crystallography (E coli PDB 1Z5Y[85] PDB 2B1K [86] B japonicum PDB 1KNG [87] Paeruginosa PDB 3KH7 3KH9 [22]) and is generally wellconserved as proved by the low RMSD (08 A between Pa-CcmG and Ec-CcmG 135 A between Pa-CcmG and Bj-CcmG) Although all these proteins adopt a TRX-like foldand contain the redox-active motif CXXC in the first 120572-helixthey are inactive in the classic insulin reduction assay [7588] CcmG proteins are therefore considered specific thiol-oxidoreductase able to recognize and selectively interactonly with their upstream and downstream binding partnersin the thioreduction process leading to reduced apoCytcLooking at the 3D structure of the periplasmic domain ofthe prototypical Pa-CcmG it is possible to identify the 120573120572120573and 120573120573120572 structural motifs of the TRX fold linked by a short120572-helix and forming a four-stranded 120573-sheet surrounded bythree helices the protein contains an additional N-terminalextension (residues 26ndash62) and a central insert (residues 102ndash123) The redox-active motif of Pa-CcmG (CPSC) is locatedin the first 120572-helix of the TRX fold as usually observed in allTRX-like proteins As for any molecular machinery whereeach component must recognize and interact with more thanone target (ie the substrate and the other components ofthe apparatus) an open question concerns the mechanismwhereby CcmG is able to recognize its different partnersThe availability of the crystal structures of Pa-CcmG bothin the oxidized (22 A resolution) and reduced state (18 Aresolution) [22] allowed highlighting the structural similaritybetween the two redox states (Rmsd of the C120572 atoms inthe two redox forms is 019 A) and therefore to excludestructural rearrangement as the mechanism used by Pa-CcmG to discriminate between reduced (such as the nDsbDdomain) and oxidized partners (Pa-CcmH andor apoCyt)

The standard redox potential of Pa-CcmG (11986410158400= 0213V

at pH 70 [22] as well as that of Ec-CcmG (11986410158400= 0212V

[86]) indicates that these proteins act as mild reductants inthe thioreductive pathway of Cytc biogenesis However the

Figure 5 Three-dimensional structure of Pa-CcmH shown inribbon representation The figure shows the three-helix bundleforming the characteristic fold of Pa-CcmHThe active site disulfidebond between residues Cys25 andCys28 in the long loop connectinghelices 120572-helix1 and 120572-helix 2 is highlighted in yellow

function of thiol-oxidoreductases obviously depends on thepKa values of their activesite Cys residues The pKa of CysX(613 plusmn 005) and CysY (105 plusmn 007) are consistent with thepKa values measured in different TRXs where the active N-terminal Cys residue has a pKa close to pH 70 whereas theC-terminal Cys has a much higher pKa [89 90] Such a largedifference between the two pKa values in the TRX family isfunctionally relevant because it allows the N-terminal Cysto perform the nucleophilic attack on the target disulfidewhile the C-terminal Cys is involved in the resolution of theresulting mixed-disulfide [90]

CcmH is the other component of System I involvedin the reduction of apoCyt Notably CcmH proteins fromdifferent bacterial subgroups may display structural vari-ability indeed while in E coli Ec-CcmH is a bipartiteprotein characterized by two soluble domains exposed to theperiplasm and two TM segments CcmH from P aeruginosa(Pa-CcmH) is a one-domain redox-active protein anchoredto the membrane via a single TM helix and homologous tothe N-terminal redox-active domain of Ec-CcmH Surpris-ingly the 3D structure of the soluble periplasmic domainof Pa-CcmH revealed that it adopts a peculiar three-helixbundle fold strikingly different from that of canonical thiol-oxidoreductases (Figure 5 PDB 2HL7 [23])TheN-terminaldomain of Ec-CcmH was also shown to have the same 3Dstructure although helix-swapping and dimerization havebeen observed in this case (PDB 2KW0 [91 92]) Theconserved redox-active motif (LRCPKC) is located in theloop connecting helices 1 and 2 close to the activesite thecrystal structure reveals the presence of a small pocket on thesurface of Pa-CcmH surrounded by conserved hydrophobicand polar residues which could represent the recognition sitefor the heme-binding motif of apoCyt

Concerning the functional properties of this unusualthiol-oxidoreductase it is interesting to note that its standardredox potential (1198641015840

0= 0215V) [23] is similar to that ob-

tained for Pa-CcmG This observation stands against thelinear redox cascade hypothesis whereby CcmG reducesCcmH While in the canonical redox-active CXXC motif

8 Scientifica

S SSS

SH

SH

Scheme 1 Scheme 2

SH

SH

SHSHS

S

CcmG 7477

CcmG 7477

CcmH 2528

CcmH 2528

apoCytox

apoCytred

S

SHCcmG 74

77

SH

SH

HSHS

CcmG 7477

S

S

SS

3

1

2CcmG 7477

SH

SHCcmH 25

28SH

HSS S

CcmH 2528

SHS

CcmH2528

apoCytox

apoCytred

+

Figure 6 Alternative thioreduction pathways whichmay be operative in System I and hypothesized on the basis of structural and functionalcharacterization of the redox-active Ccm proteins from P aeruginosa [22 23 25] Scheme 1 is a linear redox cascade whereby CcmG is thedirect reductant of CcmH which reduces oxidized apoCyt Scheme 2 envisages a more complex scenario involving the formation of a mixed-disulfide complex between CcmH and apoCyt (Step 1) This complex is the substrate for the attack by reduced CcmG (Step 2) that liberatesreduced apoCyt The resulting disulfide bond between CcmH and CcmG is then resolved by the free Cys thiol of CcmG (probably Cys77 inPa-CcmG) Adapted from [25]

of the TRX family the N-terminal Cys is always solventexposed in CcmH proteins the arrangement of the two Cysresidues is reversed the N-terminal Cys residue is buriedwhereas the C-terminal Cys residue is solvent exposed Onthe basis of this observation it was suggested that differentfrom the canonical TRX redox mechanism CcmH proteinsperform the nucleophilic attack on the apoCyt disulfide viatheir C-terminal Cys residue [23] This mechanism which isin agreement with the mechanism proposed earlier for Ec-CcmH on the basis of mutational-complementation studies[93 94] is substantiated by the peculiar pKa values of theactive site Cys residues of Pa-CcmH which were found tobe similar for both cysteines (84 plusmn 01 and 86 plusmn 01 [23])Again this is different from what is generally observed in thecase of TRX proteins where the pKa value of the Cys residueperforming the initial nucleophilic attack is significantlylower than the pKa value of theCys residue responsible for theresolution of the intermediate mixed-disulfide It is temptingto speculate that the unusual pKa values of the Pa-CcmHactive site thiols may ensure the necessary specificity of thiscomponent of the Ccm apparatus toward the CXXCH motifof the apoCyt substrate

24 System I ApoCyt Thioreduction Pathway MechanismAlthough we know that CcmG and CcmH are the redox-active components of System I involved in the thioreductivepathway of Cyt c biogenesis not only an acceptedmechanismfor the reduction of apoCyt disulfide bond is still lackingbut also the absolute requirement of such a process is nowdebated [38 84] Focusing our attention on the reductionof the apoCyt internal disulfide at least two mechanismscan been hypothesized which involve either a linear redoxcascade of disulfide exchange reactions or a nonlinear redox

process involving transient formation of a mixed-disulfidecomplex as depicted in Figure 6 and Schemes 1 and 2respectively

Both the thiol-disulfide exchange mechanisms depictedin Figure 6 suggest that CcmH is the direct reductant ofthe apoCytc disulfide however even if immunoprecipitationexperiments failed to detect the formation of a mixed-disulfide complex between apoCyt and CcmH proteins [95]some in vitro evidence supporting the formation of sucha complex has been presented In particular it has beenshown that Rhodobacter capsulatus and Arabidopsis thalianaCcmH homologues (Rc-CcmH and At-CcmH) are able toreduce the CXXCH motif of an apoCyt-mimicking peptide[75 96] In the latter case yeast two-hybrid experimentscarried out on At-CcmH indeed revealed an interactionbetween the protein and a peptide mimicking the A thalianaCyt c sequence In the case of Pa-CcmH FRET kineticexperiments employing a Trp-containing fluorescent variantof the protein and a dansylated nonapeptide encompassingthe heme-binding motif of P aeruginosa cytochrome c551(dans-KGCVACHAI) [23] allowed to directly observe theformation of themixed-disulfide complex and tomeasure theoff-rate constant of the bound peptide The results of these invitro binding experiments allowed to calculate an equilibriumdissociation constant which combines an adequate affinity(low 120583M) with the need to release efficiently reduced apoCytto other component(s) of the System I maturase complex[23] More recently the results obtained by FRET bindingexperiments carried out with single Cys-containing mutantsof Pa-CcmH and Pa-CcmG [25] substantiated the hypothesisdepicted in Scheme 2 (Figure 6) Altogether these structuraland functional results suggest that the thioreduction pathwaymechanism leading to reduced apoCyt is better describedby Scheme 2 and that reducing equivalents might not be

Scientifica 9

transferred directly from CcmG to apoCyt as depicted inScheme 1 According to Scheme 2 reduced CcmH (a non-TRX-like thiol-oxidoreductase) specifically recognizes andreduces oxidized apoCyt via the formation of a mixed-disulfide complex which is subsequently resolved by CcmGThe resulting disulfide bond between CcmH and CcmG isthen resolved by the free Cys thiol of CcmG (probably Cys77in Pa-CcmG)

However further in vitro experiments with CcmH andapoCyt single Cys-containing mutants are needed to unveilthe details of the thioreduction of oxidized apoCyt by CcmHIn particular it would be crucial to identify the Cys residueof apoCyt that remains free in the apoCyt-CcmH mixed-disulfide complex intermediate (see Scheme 2 and Section 26below) and available to thioether bond formation with oneof the heme vinyl groups Clearly structure determinationof the trapped mixed-disulfide complexes between CcmHCcmG and apoCyt (or apoCyt peptides) would providekey information for our understanding of this specializedthioreduction pathway mechanism

25 System I ApoCyt Chaperoning and Heme AttachmentComponents The reduced heme-binding motif of apoCyt isnow available to the heme ligation reaction However themolecularmechanismwhereby the Ccmmachinery catalyzesor promotes the formation of the heme-apoCyt covalentbonds is still largely obscure representing themost importantgoal in the field Past observations and recent experimentssuggest that CcmF and CcmI possibly together with CcmHare involved in these final steps [16 34 36]

CcmF is a large integral membrane protein of more than600 residues belonging to the heme handling protein family(HHP [55]) and predicted to contain 10ndash15 TM helices (notethat some discrepancy exists as to the number of TM helicespredicted by computer programs and those predicted onthe basis of phoA and lacZ fusion experiments [40 97])a conserved WWD domain and a larger domain devoidof any recognizable sequence features both exposed to theperiplasm Only recently E coli CcmF (Ec-CcmF) has beenoverexpressed solubilized from the membrane fraction andspectroscopically characterized in vitro [36 41] Surprisinglythe biochemical characterization of recombinant Ec-CcmFallowed to show that the purified protein contains heme b ascofactor in a 1 1 stoichiometry this observation led to thehypothesis that in addition to its heme lyase function Ec-CcmF may act as a heme oxidoreductase In particular it ispossible that the heme b of Ec-CcmF may act as a reductantfor the oxidized iron of the heme bound to CcmE [41]indeed the in vitro reduction of Ec-CcmF by quinones hasbeen experimentally observed strengthening the hypothesisabout the quinolhemeoxidoreductase function of this elusiveproteinThe structuralmodel proposed for Ec-CcmFpredicts13 TMhelices and notably the location of the four completelyconserved His residues according to the model two of them(His173 andHis303) are located in periplasmic exposed loopsnext to the conserved WWD domain which is believedto provide a platform for the heme bound to holoCcmEwhile His261 is located in one of the TM helices and it is

predicted to act as an axial ligand to the heme b of Ec-CcmFthe other conserved His residue (H491) could provide thesecond axial coordination bond to the heme although thishas not been experimentally addressed This model of Ec-CcmF therefore envisages that this large membrane proteinis characterized by two heme-binding sites one of themis embedded in the membrane and coordinates a heme bprosthetic group necessary to reduce the CcmE-bound hemehosted in the second heme-binding site and constituted by itsWWD domain

It is interesting to note that in plants mitochondria theCcmF ortholog appears to be split into three different pro-teins (At-CcmFN1 At-CcmFN2 and At-CcmFC) possiblyinteracting each other [16] Since each of these proteins issimilar to the corresponding domain in the bacterial CcmFortholog this observation may provide useful informationin the design of engineered fragments of bacterial CcmFproteins amenable to structural analyses

The other System I component which is generallybelieved to be involved in the final steps of Cyt c maturationis CcmI As stated above the ccmI gene is present only insome Ccm operons while in others the corresponding ORFis present within the ccmH gene (as in E coli)The functionalrole of CcmI in Cytc biogenesis is revealed by geneticstudies showing that in R capsulatus and B japonicuminactivation of the ccmI gene leads to inability to synthesizefunctional c-type cytochromes [98 99] In R capsulatus andP aeruginosa the CcmI protein (Rc-CcmI and Pa-CcmIresp) can be described as being composed of two domainsstarting from the N-terminus a first domain composed oftwo TM helices connected by a short cytoplasmic regionand a large periplasmic domain Structural variations maybe observed among CcmI members from different bacteriaindeed multiple sequence alignment indicates that the cyto-plasmic region of Rc-CcmI contains a leucine zipper motifwhich is not present in the putative cytoplasmic region of Pa-CcmI [100ndash102] Surprisingly no crystallographic structureis available up to now for the soluble domain of any CcmIprotein with the exception of the ortholog protein NrfGfrom E coli (Ec-NrfG) [103] This protein is necessary toattach the heme to the unusual heme-binding motif CWSCK(where a Lys residue substitutes the conservedHis) present inNrfA a pentaheme c-type cytochrome [103 104] Accordingto secondary structure prediction methods [105] it hasbeen proposed that the periplasmic domain of Pa-CcmI iscomposed of aN-terminal120572-helical region containing at leastthree TPR motifs connected by a disordered linker to a 120572-120573C-terminal regionMultiple sequence analyses and secondarystructure predictionmethods show that the TPR region of Pa-CcmI can be successfully aligned with many TPR-containingproteins including Ec-NrfG [106]

26 System I ApoCyt Chaperoning and Heme AttachmentMechanisms TPR domain-containing proteins are commonto eukaryotes prokaryotes and archaea these proteins aregenerally involved in the assembly of multiprotein complexesand to the chaperoning of unfolded proteins [103 107] Itis therefore plausible that CcmI (or the TPR C-terminal

10 Scientifica

domain of Ec-CcmH) may act to provide a platform for theunfolded apoCyt chaperoning it to the heme attachment sitepresumably located on the WWD domain of CcmF CcmImay thus be considered a component of amembrane-integralmultisubunit heme ligation complex together with CcmFand CcmH as experimentally observed by affinity purifi-cation experiments carried out with Rc-CcmFHI proteins[97 99 108] According to the proposed function of CcmI acritical requirement is represented by its ability to recognizedifferent protein targets over and above apoCyt such asCcmFandCcmHHowever until now direct evidence has been pre-sented only for the interaction of CcmI with apoCyt but thepossibility remains that CcmFHI proteins interact each othervia their TM helices and not via their periplasmic domainsInterestingly both for Pa-CcmI [106] or Rc-CcmI [99] CDspectroscopy experiments carried out on the CcmIapoCytcomplex highlighted major conformational changes at thesecondary structure level It is tempting to speculate on thebasis of these results that in vivo the folding of apoCyt maybe induced by the interaction with CcmI In the case of Paeruginosa System I proteins the binding process betweenPa-CcmI and its target protein apoCyt c551 (Pa-apoCyt) hasbeen studied both at equilibrium and kinetically [106] the119870119863measured for this interaction (in the 120583M range) appeared

to be low enough to ensure apoCyt delivery to the othercomponents of the Ccmmachinery Clearly a major questionconcerns the molecular determinants of such recognitionprocess interestingly both affinity coprecipitation assays[99] and equilibrium and kinetic binding experiments [106]highlighted the role played by the C-terminal 120572-helix of Cytc Similar observations have been made for the interaction ofEc-NrfGwith a peptidemimickingNrfA its apoCyt substrate[103] in this case isothermal titration calorimetry (ITC)experiments indicate that the TPR-domain of NrfG serves asa binding site for the C-terminal motif of NrfA Altogetherthese observations are in agreement with the fact that TPRproteins generally bind to their targets by recognizing theirC-terminal region [107]

The CcmI chaperoning activity has been experimentallysupported for the first time in the case of Pa-CcmI by citratesynthase tests [106] it has been proposed that the observedability to suppress protein aggregation in vitromay reflect thecapacity of CcmI to avoid apoCyt aggregation in vivo Stillanother piece of the Cyt c biogenesis puzzle has been addedrecently by showing that Rc-CcmI is able to interact withapoCcmE either alone or together with its substrate apoCytc2 forming a stable ternary complex in the absence of heme[109] This unexpected observation obtained by reciprocalcopurification experiments provides supporting evidence forthe existence of a large multisubunit complex composed ofCcmFHI andCcmE possibly interactingwith theCcmABCDcomplex It is interesting to note that while in the case ofthe CcmIapoCyt recognition different studies highlightedthe crucial role of the C-terminal helical region of apoCyt(see above) in the case of the apoCcmE apoCyt recognitionthe N-terminal region of apoCyt seems to represent a criticalregion

It is generally accepted that CcmF is the Ccm compo-nent responsible for heme covalent attachment to apoCyt

however as discussed above it is possible that this largemembrane protein plays such a role only together withother Ccm proteins such as CcmH and CcmI Moreover asrecently discovered by Kranz and coworkers [36 41] CcmFmay also act as a quinoleheme oxidoreductase ensuringthe necessary reduction of the oxidized heme b bound toCcmE Why it is necessary that the heme iron be in itsreduced state rather than in its oxidized state is not completelyclear although it is possible that this is a prerequisite to themechanism of thioether bond formation [110] According tocurrent hypotheses it is likely that the periplasmic WWDdomain of CcmF provides a platform for heme b bindingSanders et al and Verissimo et al [34 109] have presenteda mechanistic view of the heme attachment process whichtakes into account all the available experimental observationson the different Ccmproteins According to thismodel stere-ospecific heme ligation to reduced apoCyt occurs becauseonly the vinyl-4 group is available to form the first thioetherbond with a free cysteine at the apoCyt heme-binding motifsince the vinyl-2 group is involved (at least in the Ec-CcmE)in the covalent bond with His130 of CcmE [67] Howeverexperimental proof for this hypothesis requires a detailedinvestigation of the apoCyt thioreduction process catalyzedby CcmH (see Section 24)

It should be noticed that the mechanisms described sofar for the function(s) played by CcmF (see [34 36 109]) donot envisage a clear role for its large C-terminal periplasmicdomain (residues 510 to 611 in Ec-CcmF) It would be inter-esting to see if this domain apparently devoid of recognizablesequence features may mediate intermolecular recognitionprocesses with one (or more) component(s) involved in thehemeapoCyt ligation process

3 System II

System II is typically found in gram-positive bacteria and inin 120576-proteobacteria it is also present in most 120573- and some120575-proteobacteria in Aquificales and cyanobacteria as wellas in algal and plant chloroplasts System II is composedof three or four membrane-bound proteins CcdA ResACcsA (also known as ResC) and CcsB (also known as ResB)(Figure 3) CcdA andResA are redox-active proteins involvedin the reduction of the disulfide bond in the heme-bindingmotif of apoCyt whereas CcsA and CcsB are responsible forthe heme-apoCyt ligation process and are considered Cyt csynthethases (CCS) BothCcsAwhich is evolutionary relatedto the CcmC and CcmF proteins of System I [55] and CcsBare integral membrane proteins In some 120576-proteobacteriasuch asHelicobacter hepaticus andHelicobacter pylori a singlefusion protein composed of CcsA and CcsB polypeptides ispresent [41 111] Although as discussed below evidence hasbeen put forward to support the hypothesis that the CcsBAcomplex acts a heme translocase we still do not know if theheme is transported across the membrane by component(s)of System II itself or by a different unidentified process

31 System II ApoCyt Thioreduction Pathway After the Secmachinery secretes the newly synthesized apoCyt it readily

Scientifica 11

becomes a substrate for the oxidative system present on theouter surface of the cytoplasmic membrane of the gram-positive bacteria In B subtilis the BdbCD system [112113] is functionally but not structurally equivalent to thewell-characterized DsbAB system present in gram-negativebacteria As discussed above for System I the disulfide ofthe apoCyt heme-binding motif must be reduced in orderto allow thioether bonds formation and heme attachmentResA is the extracytoplasmic membrane-anchored TRX-likeprotein involved in the specific reduction of the apoCytdisulfide bond after the disulfide bond exchange reactionhas occurred oxidized ResA is reduced by CcdA whichreceives its reducing equivalents from a cytoplasmic TRX[114] Although ResA displays a classical TRX-like foldthe analysis of the 3D structure of oxidized and reducedResA from B subtilis (Bs-ResA) showed redox-dependentconformational modifications not observed in other TRX-like proteins Interestingly such modifications occur at thelevel of a cavity proposed to represent the binding site foroxidized apoCyt [24 115 116] Another peculiar feature of Bs-ResA is represented by the unusually similar pKa values of itsthe active site Cys residues (88 and 82 resp) as observedalso in the case of the active site Cys residues of the SystemI Pa-CcmH (see Section 23) However at variance with Pa-CcmH in Bs-ResA the large separation between the twocysteine thiols observed in the structure of the reduced formof the protein can be invoked to account for this result

CcdA is a large membrane protein containing six TMhelices [117 118] homologous to the TM domain of E coliDsbD [85 119] Its role in the Cyt c biogenesis is supportedby the observation that inactivation of CcdA blocks theproduction of c-type cytochromes in B subtilis [120 121]Moreover over and above the reduction of its apoCyt sub-strate Bs-CcdA is able to reduce the disulfide bond of othersecreted proteins such as StoA [122] It is interesting to notethat ResA and CcdA are not essential for Cyt c synthesisin the absence of BdbD or BdbC or if a disulfide reductantis present in the growth medium [123 124] As discussedabove (Section 24) a similar observation was made on therole of the thio-reductive pathway of System I in this casepersistent production of c-type cytochromes was observedin Dsb-inactivated bacterial strains [38] Following theseobservations the question has been put forward as to why theCyt c biogenesis process involves this apparently redundantthio-reduction route only to correct the effects of Bdb orDsb activity Referring to the case of B subtilis [111] it hasbeen proposed that the main reason is that BdbD mustefficiently oxidize newly secreted proteins since the activityof most of them depends on the presence of disulfide bondsAlternatively it can be hypothesized that the intramoleculardisulfide bondof apoCyt protects the protein fromproteolyticdegradation or aggregation or from cross-linking to otherthiol-containing proteins

32 System II Heme Translocation and Attachment to ApoCytRecent experimental evidence is accumulating supportingthe involvement of the heterodimeric membrane complexResBC or of the fusion protein CcsBA in the translocation

of heme from the n-side to the p-side of the bacterialmembrane In particular it has been shown that CcsBAfrom H hepaticus (Hh-CcsBA) is able to reconstitute Cyt cbiogenesis in the periplasm of E coli [36] Moreover it wasalso observed that Hh-CcsBA is able to bind reduced hemevia two conserved histidines flanking the WWD domainand required both for the translocation of the heme andfor the synthetase function of Hh-CcsBA [36] A differentstudy carried out on B subtilis System II proteins providedsupport for heme-binding capability by the ResBC complexaccording to the results obtained on recombinant ResBC theResB component of the heterodimeric CCS complex is ableto covalently bind the heme in the cytoplasm (probably by aCys residue) and to deliver it to an extracytoplamic domain ofResC which is responsible for the covalent ligation to apoCyt[117] It should be noticed however that the transfer of theCCS-bound heme to apoCyt still awaits direct experimentalproof Moreover it has also been reported that in B subtilisthe inactivation of CCS does not affect the presence ofother heme-containing proteins in the periplasm such ascytochrome b562 [111] this observation which is in contrastto the heme-transport hypothesis by CCSs parallels similarconcerns about heme translocation mechanism that arecurrently discussed in the context of System I (see Section 22above) In the case of the Hh-CcsBA synthetase site-directedmutagenesis experiments allowed to assign a heme-bindingrole for the two pairs of conserved His residues accordingto the topological model of the protein obtained by alkalinephosphatase (PhoA) assays and GFP fusions the conservedHis77 and His858 residues are located on TM helices whileHis761 and His897 are located on the periplasmic side [36]These observations together with the results of site-directedmutagenesis experiments allowed the authors to suggest thatHis77 andHis858 form a low affinitymdashmembrane embeddedheme-binding site for ferrous heme which is subsequentlytranslocated to an external heme-binding domain of Hh-CcsBA where it is coordinated by His761 and His897 Thetopological model therefore predicts that this last His pairis part of a periplasmic-located WWD domain (homologousto theWWDdomains found in theCcmCandCcmFproteinsof System I described above)

4 System III

Strikingly different from Systems I and II the Cyt c mat-uration apparatus found in fungi and in metazoan cells(System III) is composed of a single protein known as Holo-Cytochrome c Synthetase (HCCS) or Cytochrome c HemeLyase (CCHL) apparently responsible for all the subprocessesdescribed above (heme transport and chaperoning reductionand chaperoning of apoCyt and catalysis of the thioetherbonds formation between heme and apoCyt) (Figure 4)Surprisingly although this protein has been identified in Scerevisiaemitochondria several years ago [125 126] only veryrecently the human HCCS has been expressed as a recombi-nant protein in E coli and spectroscopically characterized invitro [127] It is worth noticing that humanHCCS is attractinginterest since over and above its role in Cyt c biogenesis it

12 Scientifica

is involved in diseases such as microphthalmia with linearskin defects syndrome (MLS) an X-linked genetic disorder[128ndash130] and in other processes such as Cytc-independentapoptosis in injured motor neurons [131] Different fromanimal cells where a single HCCS is sufficient for thematuration of both soluble and membrane-anchored Cytc(Cyt c and Cy c1 resp) in S cerevisiae two homologs HCCSand HCC1S located in the inner mitochondrial membraneand facing to the IMS space [132 133] are responsible for hemeattachment to apoCyt c and apoCyt c1 respectively [125 134]It should also be noticed that in fungi an additional FAD-containing protein (Cyc2p) is required for Cyt c synthesisCyc2p is a mitochondrial membrane-anchored flavoproteinexposing its redox domain to the IMS which is requiredfor the maturation of Cyt c but not for that of Cyt c1 [135]This protein does not contain the conserved Cys residuestypically found in disulfide reductases and indeed it is notable to reduce oxidized apoCyt in vitro However it has beenrecently shown that Cyc2p is able to catalyze the NAD(P)H-dependent reduction of heme in vitro [136] a necessarystep before the thioether bond formation can occur asdiscussed above (see Section 26) This result together withthe observation that Cyc2p interacts with HCCS and withapoCyt c and c1 lends support to the proposal that Cyc2p isinvolved in the reduction of the heme iron in vivo [136 137]

AlthoughHCCS proteins are crucial for Cyt cmaturationwe still do not know how these proteins recognize theirsubstrates (heme and apoCyt) and how they promote orcatalyze the formation of thioether bonds between the hemevinyl groups and the cysteine thiols of the apoCyt CXXCHmotif

Contrary to the broad specificity of System I whichis able to recognize and attach the heme to prokaryoticand eukaryotic c-type cytochromes [49 50] and even tovery short microperoxidase-like sequences [49 50] mito-chondrial HCCS is characterized by a higher specificityas it does not recognize bacterial apoCyt sequences Theseobservations prompted the investigation of the recognitionprocess between apoCyt and HCCS Recently by using arecombinant yeast HCCS and chimerical apoCyt sequencesexpressed in E coli it was possible to conclude that a crucialrecognition determinant is represented by the N-terminalregion of apoCyt containing the heme-binding motif [35]notice that in the context of System I the same N-terminalregion of the Cyt c sequence has been recently identified to beimportant for the recognition by apoCcmE (see Section 26)Over and above the role played by the intervening residuesin the CXXCH heme-binding motif [135] it has been shownthat a conserved Phe residue occurring in the N-terminalregion before the CXXCH motif is important for HCCSrecognition [35 138] These results support the hypothesisthat this residue known to be a key determinant of Cytcfolding and stability [19 20 139] may also be crucial for Cytcmaturation by HCCS

Another relevant question concerns the ability of HCCSto recognize and bind the heme molecule as the region ofthe protein responsible for heme recognition remains to beidentified Initially it was hypothesized that the recognitionof heme could be mediated by the CP motifs present in

the HCCS protein [140] these short sequences are indeedknown to bind heme in a variety of heme-containing proteins[141 142] However it has been recently shown that CPmotifsof the recombinant S cerevisiae HCCS are not necessaryfor Cyt c production in E coli [143] excluding them as keydeterminants of heme recognition

The long-awaited in vitro characterization of the recom-binant human HCCS allowed for the first time to pro-pose a molecular mechanism underlying Cytc maturationin eukaryotes which can be experimentally tested [127]According to the proposed model the human HCCS activitycan be described as a four step mechanism involving (i)heme-binding (ii) apoCyt recognition (iii) thioether bondsformation and (iv) holoCyt c release In particular theheme is proposed to play the role of a scaffolding moleculemediating the contacts between HCCS and apoCyt Muta-genesis experiments carried out on the recombinant HCCSstrongly suggest that heme-binding (Step 1) depends on thepresence of a specific His residue (His154) acting as anaxial ligand to ferrous heme Residues present at the N-terminus of apoCyt mediate the recognition with HCCS(Step 2) as discussed above it is known that this regionforms structurally conserved 120572-helix in the fold of all c-type cytochromes Unfortunately no information is availableup to now concerning the region of HCCS involved in therecognition and binding to the N-terminal region of apoCytCoordination of the heme iron by the His residue of theapoCyt heme-binding motif CXXCH provides the secondaxial ligand to the heme iron and is probably importantfor the correct positioning of the two apoCyt Cys residuesand formation of the thioether bonds (Step 3) The finalrelease of functional Cyt c (Step 4) clearly requires thedisplacement of the His154Fe2+ coordination bond sucha displacement is probably mediated by formation of thecoordination bond with the Cyt c conserved Met residueandor by the simultaneous folding of Cyt c Again it isinteresting to note that this last hypothesis is in accordancewith in vitro folding studies on c-type cytochromes thathighlighted the late formation of the Met-Fe coordinationduring Cyt c folding [144 145]

Acknowledgments

The author thanks A Di Matteo and E Di Silvio for fruitfuldiscussions and for help in preparing this paper This work issupported by grants from the University of Rome ldquoSapienzardquo(C26A11MBXJ and C26A13MWF4)

References

[1] M G Galinato J G Kleingardner S E Bowman et alldquoHeme-protein vibrational couplings in cytochrome c providea dynamic link that connects the heme-iron and the proteinsurfacerdquo Proceedings of the National Academy of Sciences vol109 no 23 pp 8896ndash8900 2012

[2] H B Gray and J R Winkler ldquoElectron flow through metal-loproteinsrdquo Biochimica et Biophysica Acta vol 1797 no 9 pp1563ndash1572 2010

Scientifica 13

[3] P Ascenzi R Santucci M Coletta and F PolticellildquoCytochromes reactivity of the ldquodark siderdquo of the hemerdquoBiophysical Chemistry vol 152 no 1ndash3 pp 21ndash27 2010

[4] S Yamada N D Bouley Ford G E Keller W C Ford HB Gray and J R Winkler ldquoSnapshots of a protein foldingintermediaterdquo Proceedings of the National Academy of Sciencesvol 110 no 5 pp 1606ndash1610 2013

[5] P Weinkam J Zimmermann F E Romesberg and P GWolynes ldquoThe folding energy landscape and free energy excita-tions of cytochrome crdquo Accounts of Chemical Research vol 43no 5 pp 652ndash660 2010

[6] M Yamanaka M Masanari and Y Sambongi ldquoConfermentof folding ability to a naturally unfolded apocytochrome cthrough introduction of hydrophobic amino acid residuesrdquoBiochemistry vol 50 no 12 pp 2313ndash2320 2011

[7] G W Moore and G W Pettigrew Cytochrome C EvolutionaryStructural and Physicochemical Aspects Springer Berlin Ger-many 1990

[8] I Bertini G Cavallaro and A Rosato ldquoCytochrome c occur-rence and functionsrdquo Chemical Reviews vol 106 no 1 pp 90ndash115 2006

[9] D A Pearce and F Sherman ldquoDegradation of cytochromeoxidase subunits in mutants of yeast lacking cytochrome c andsuppression of the degradation by mutation of yme1rdquo Journal ofBiological Chemistry vol 270 no 36 pp 20879ndash20882 1995

[10] G Silkstone S M Kapetanaki I Husu M H Vos and MT Wilson ldquoNitric oxide binding to the cardiolipin complex offerric cytochrome crdquo Biochemistry vol 51 no 34 pp 6760ndash6766 2012

[11] M Giorgio E Migliaccio F Orsini et al ldquoElectron transferbetween cytochrome c and p66Shc generates reactive oxygenspecies that trigger mitochondrial apoptosisrdquo Cell vol 122 no2 pp 221ndash233 2005

[12] S M Kilbride and J H Prehn ldquoCentral roles of apoptoticproteins in mitochondrial functionrdquo Oncogene vol 32 no 22pp 2703ndash2711 2013

[13] T Noll and G Noll ldquoStrategies for ldquowiringrdquo redox-activeproteins to electrodes and applications in biosensors biofuelcells and nanotechnologyrdquo Chemical Society Reviews vol 40no 7 pp 3564ndash3576 2011

[14] G Layer J Reichelt D Jahn and D W Heinz ldquoStructure andfunction of enzymes in heme biosynthesisrdquo Protein Science vol19 no 6 pp 1137ndash1161 2010

[15] S Severance and IHamza ldquoTrafficking of heme and porphyrinsin metazoardquo Chemical Reviews vol 109 no 10 pp 4596ndash46162009

[16] P Hamel V Corvest P Giege and G Bonnard ldquoBiochemi-cal requirements for the maturation of mitochondrial c-typecytochromesrdquo Biochimica et Biophysica Acta vol 1793 no 1 pp125ndash138 2009

[17] W R Fisher H Taniuchi and C B Anfinsen ldquoOn the role ofheme in the formation of the structure of cytochrome crdquo Journalof Biological Chemistry vol 248 no 9 pp 3188ndash3195 1973

[18] M Yamanaka H Mita Y Yamamoto and Y Sambongi ldquoHemeis not required for aquifex aeolicus cytochrome c555 polypep-tide foldingrdquoBioscience Biotechnology andBiochemistry vol 73no 9 pp 2022ndash2025 2009

[19] C Travaglini-Allocatelli S Gianni andMBrunori ldquoA commonfolding mechanism in the cytochrome c familyrdquo Trends inBiochemical Sciences vol 29 no 10 pp 535ndash541 2004

[20] A Borgia D Bonivento C Travaglini-Allocatelli A DiMatteoand M Brunori ldquoUnveiling a hidden folding intermediate in c-type cytochromes by protein engineeringrdquo Journal of BiologicalChemistry vol 281 no 14 pp 9331ndash9336 2006

[21] E Enggist L Thony-Meyer P Guntert and K PervushinldquoNMR structure of the heme chaperone CcmE reveals a novelfunctional motifrdquo Structure vol 10 no 11 pp 1551ndash1557 2002

[22] A di Matteo N Calosci S Gianni P Jemth M Brunori and CTravaglini-Allocatelli ldquoStructural and functional characteriza-tion of CcmG from pseudomonas aeruginosa a key componentof the bacterial cytochrome c maturation apparatusrdquo Proteinsvol 78 no 10 pp 2213ndash2221 2010

[23] A diMatteo S GianniM E Schinina et al ldquoA strategic proteinin cytochrome c maturation three-dimensional structure ofCcmH and binding to apocytochrome crdquo Journal of BiologicalChemistry vol 282 no 37 pp 27012ndash27019 2007

[24] A Crow R M Acheson N E Le Brun and A OubrieldquoStructural basis of redox-coupled protein substrate selectionby the cytochrome c biosynthesis protein ResArdquo Journal ofBiological Chemistry vol 279 no 22 pp 23654ndash23660 2004

[25] E di Silvio A di Matteo and C Travaglini-Allocatelli ldquoTheRedox pathway of Pseudomonas aeruginosa cytochrome cbiogenesisrdquo Journal of Proteins and Proteomics vol 3 no 1 pp1ndash7 2012

[26] LThony-Meyer andP Kunzler ldquoTranslocation to the periplasmand signal sequence cleavage of preapocytochrome c depend onsec and lep but not on the ccmgene productsrdquoEuropean Journalof Biochemistry vol 246 no 3 pp 794ndash799 1997

[27] S J Facey and A Kuhn ldquoBiogenesis of bacterial inner-membrane proteinsrdquo Cellular and Molecular Life Sciences vol67 no 14 pp 2343ndash2362 2010

[28] K Diekert A I de Kroon U Ahting et al ldquoApocytochromec requires the TOM complex for translocation across themitochondrial outer membranerdquo The EMBO Journal vol 20no 20 pp 5626ndash5635 2001

[29] N Wiedemann V Kozjak T Prinz et al ldquoBiogenesis of yeastmitochondrial cytochrome c a unique relationship to the TOMmachineryrdquo Journal ofMolecular Biology vol 327 no 2 pp 465ndash474 2003

[30] F-U Hartl N Pfanner and W Neupert ldquoTranslocation inter-mediates on the import pathway of proteins intomitochondriardquoBiochemical Society Transactions vol 15 no 1 pp 95ndash97 1987

[31] B S Glick A Brandt K Cunningham SMuller R L Hallbergand G Schatz ldquoCytochromes c1 and b2 are sorted to theintermembrane space of yeast mitochondria by a stop-transfermechanismrdquo Cell vol 69 no 5 pp 809ndash822 1992

[32] G Howe and S Merchant ldquoRole of heme in the biosynthesis ofcytochrome c6rdquo Journal of Biological Chemistry vol 269 no 8pp 5824ndash5832 1994

[33] S S Nakamoto P Hamel and S Merchant ldquoAssembly ofchloroplast cytochromes b and crdquo Biochimie vol 82 no 6-7 pp603ndash614 2000

[34] C Sanders S Turkarslan D-W Lee and F DaldalldquoCytochrome c biogenesis the Ccm systemrdquo Trends inMicrobiology vol 18 no 6 pp 266ndash274 2010

[35] J M Stevens D A Mavridou R Hamer P Kritsiligkou A DGoddard and S J Ferguson ldquoCytochrome c biogenesis systemIrdquoThe FEBS Journal vol 278 no 22 pp 4170ndash4178 2011

[36] R G Kranz C Richard-Fogal J-S Taylor and E R FrawleyldquoCytochrome c biogenesis mechanisms for covalent modifica-tions and trafficking of heme and for heme-iron redox controlrdquo

14 Scientifica

Microbiology and Molecular Biology Reviews vol 73 no 3 pp510ndash528 2009

[37] J W A Allen ldquoCytochrome c biogenesis in mitochondriamdashsystems III and VrdquoThe FEBS Journal vol 278 no 22 pp 4198ndash4216 2011

[38] D A Mavridou S J Ferguson and J M Stevens ldquoCytochromec assemblyrdquo IUBMB Life vol 65 no 3 pp 209ndash216 2013

[39] I Bertini G Cavallaro and A Rosato ldquoEvolution ofmitochondrial-type cytochrome c domains and of theprotein machinery for their assemblyrdquo Journal of InorganicBiochemistry vol 101 no 11-12 pp 1798ndash1811 2007

[40] B S Goldman and R G Kranz ldquoEvolution and horizontaltransfer of an entire biosynthetic pathway for cytochromec biogenesis helicobacter deinococcus archae and morerdquoMolecular Microbiology vol 27 no 4 pp 871ndash873 1998

[41] C L Richard-Fogal E R Frawley E R Bonner H Zhu BSan Francisco and R G Kranz ldquoA conserved haem redoxand trafficking pathway for cofactor attachmentrdquo The EMBOJournal vol 28 no 16 pp 2349ndash2359 2009

[42] L Thony-Meyer F Fischer P Kunzler D Ritz and H Hen-necke ldquoEscherichia coli genes required for cytochrome cmaturationrdquo Journal of Bacteriology vol 177 no 15 pp 4321ndash4326 1995

[43] C S Beckett J A Loughman K A Karberg G M DonatoW E Goldman and R G Kranz ldquoFour genes are required forthe system II cytochrome c biogenesis pathway in Bordetellapertussis a unique bacterial modelrdquo Molecular Microbiologyvol 38 no 3 pp 465ndash481 2000

[44] N E Le Brun J Bengtsson and L Hederstedt ldquoGenes requiredfor cytochrome c synthesis in Bacillus subtilisrdquo MolecularMicrobiology vol 36 no 3 pp 638ndash650 2000

[45] P Giege J M Grienenberger and G Bonnard ldquoCytochromec biogenesis in mitochondriardquo Mitochondrion vol 8 no 1 pp61ndash73 2008

[46] R E Feissner C L Richard-Fogal E R Frawley J A Lough-man KW Earley and R G Kranz ldquoRecombinant cytochromesc biogenesis systems I and II and analysis of haem deliverypathways in Escherichia colirdquo Molecular Microbiology vol 60no 3 pp 563ndash577 2006

[47] N P Cianciotto P Cornelis and C Baysse ldquoImpact of thebacterial type I cytochrome c maturation system on differentbiological processesrdquoMolecular Microbiology vol 56 no 6 pp1408ndash1415 2005

[48] E Arslan H Schulz R Zufferey P Kunzler and L Thony-Meyer ldquoOverproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in Escherichiacolirdquo Biochemical and Biophysical Research Communicationsvol 251 no 3 pp 744ndash747 1998

[49] C Sanders and H Lill ldquoExpression of prokaryotic and eukary-otic cytochromes c in Escherichia colirdquoBiochimica et BiophysicaActa vol 1459 no 1 pp 131ndash138 2000

[50] M Braun and L Thony-Meyer ldquoBiosynthesis of artificialmicroperoxidases by exploiting the secretion and cytochromec maturation apparatuses of Escherichia colirdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 35 pp 12830ndash12835 2004

[51] B Baert C Baysse S Matthijs and P Cornelis ldquoMultiplephenotypic alterations caused by a c-type cytochrome mat-uration ccmC gene mutation in Pseudomonas aeruginosardquoMicrobiology vol 154 no 1 pp 127ndash138 2008

[52] S Turkarslan C Sanders and F Daldal ldquoExtracytoplasmicprosthetic group ligation to apoproteins maturation of c-typecytochromesrdquo Molecular Microbiology vol 60 no 3 pp 537ndash541 2006

[53] P M Jones and A M George ldquoThe ABC transporter structureand mechanism perspectives on recent researchrdquo Cellular andMolecular Life Sciences vol 61 no 6 pp 682ndash699 2004

[54] O Christensen E M Harvat L Thony-Meyer S J Fergusonand J M Stevens ldquoLoss of ATP hydrolysis activity by CcmABresults in loss of c-type cytochrome synthesis and incompleteprocessing ofCcmErdquoTheFEBS Journal vol 274 no 9 pp 2322ndash2332 2007

[55] J-H Lee E M Harvat J M Stevens S J Ferguson and M HSaier Jr ldquoEvolutionary origins of members of a superfamily ofintegralmembrane cytochrome c biogenesis proteinsrdquoBiochim-ica et Biophysica Acta vol 1768 no 9 pp 2164ndash2181 2007

[56] D L Beckman D R Trawick and R G Kranz ldquoBacterialcytochromes c biogenesisrdquo Genes and Development vol 6 no2 pp 268ndash283 1992

[57] H Schulz R A Fabianek E C Pellicioli H Hennecke andL Thony-Meyer ldquoHeme transfer to the heme chaperone CcmEduring cytochrome c maturation requires the CcmC proteinwhich may function independently of the ABC-transporterCcmABrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 96 no 11 pp 6462ndash6467 1999

[58] Q Ren and L Thony-Meyer ldquoPhysical Interaction ofCcmC with Heme and the Heme Chaperone CcmE duringCytochrome cMaturationrdquo Journal of Biological Chemistry vol276 no 35 pp 32591ndash32596 2001

[59] C Richard-Fogal and R G Kranz ldquoThe CcmChemeCcmEcomplex in heme trafficking and cytochrome c biosynthesisrdquoJournal of Molecular Biology vol 401 no 3 pp 350ndash362 2010

[60] V K Viswanathan S Kurtz L L Pedersen et al ldquoThecytochrome C maturation locus of Legionella pneumophilapromotes iron assimilation and intracellular infection andcontains a strain-specific insertion sequence elementrdquo Infectionand Immunity vol 70 no 4 pp 1842ndash1852 2002

[61] U Ahuja and L Thony-Meyer ldquoCcmD is involved in complexformation between CcmC and the heme chaperone CcmE dur-ing cytochrome c maturationrdquo Journal of Biological Chemistryvol 280 no 1 pp 236ndash243 2005

[62] C L Richard-Fogal E R Frawley and R G Kranz ldquoTopologyand function of CcmD in cytochrome Cmaturationrdquo Journal ofBacteriology vol 190 no 10 pp 3489ndash3493 2008

[63] H Schulz H Hennecke and L Thony-Meyer ldquoPrototype ofa heme chaperone essential for cytochrome c maturationrdquoScience vol 281 no 5380 pp 1197ndash1200 1998

[64] F Arnesano L Banci P D Barker et al ldquoSolution structure andcharacterization of the heme chaperone CcmErdquo Biochemistryvol 41 no 46 pp 13587ndash13594 2002

[65] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[66] L Thony-Meyer ldquoA heme chaperone for cytochrome c biosyn-thesisrdquo Biochemistry vol 42 no 45 pp 13099ndash13105 2003

[67] E M Harvat C Redfield J M Stevens and S J FergusonldquoProbing the heme-binding site of the cytochrome cmaturationprotein CcmErdquoBiochemistry vol 48 no 8 pp 1820ndash1828 2009

[68] D Lee K Pervushin D Bischof M Braun and L Thony-Meyer ldquoUnusual heme-histidine bond in the active site of a

Scientifica 15

chaperonerdquo Journal of the American Chemical Society vol 127no 11 pp 3716ndash3717 2005

[69] A D Goddard J M Stevens F Rao et al ldquoc-Type cytochromebiogenesis can occur via a natural Ccm system lacking CcmHCcmG and the heme-binding histidine of CcmErdquo Journal ofBiological Chemistry vol 285 no 30 pp 22882ndash22889 2010

[70] J M Aramini K Hamilton P Rossi et al ldquoSolution NMRstructure backbone dynamics and heme-binding propertiesof a novel cytochrome c maturation protein CcmE fromDesulfovibrio vulgarisrdquo Biochemistry vol 51 no 18 pp 3705ndash3707 2012

[71] T Uchida J M Stevens O Daltrop et al ldquoThe interactionof covalently bound heme with the cytochrome c maturationprotein CcmErdquo Journal of Biological Chemistry vol 279 no 50pp 51981ndash51988 2004

[72] I Garcıa-Rubio M Braun I Gromov L Thony-Meyer andA Schweiger ldquoAxial coordination of heme in ferric CcmEchaperone characterized by EPR spectroscopyrdquo BiophysicalJournal vol 92 no 4 pp 1361ndash1373 2007

[73] L Thony-Meyer ldquoHaem-polypeptide interactions duringcytochrome c maturationrdquo Biochimica et Biophysica Acta vol1459 no 2-3 pp 316ndash324 2000

[74] J A Keightley D Sanders T R Todaro A Pastuszyn and J AFee ldquoCloning expression in Escherichia coli of the cytochromec552 gene from Thermus thermophilus HB8 Evidence forgenetic linkage to an ATP- binding cassette protein and initialcharacterization of the cycA gene productsrdquo Journal of Biologi-cal Chemistry vol 273 no 20 pp 12006ndash12016 1998

[75] E M Monika B S Goldman D L Beckman and R GKranz ldquoA thioreduction pathway tethered to the membranefor periplasmic cytochromes c biogenesis In vitro and in vivostudiesrdquo Journal of Molecular Biology vol 271 no 5 pp 679ndash692 1997

[76] M Throne-Holst L Thony-Meyer and L HederstedtldquoEscherichia coli ccm in-frame deletion mutants can produceperiplasmic cytochrome b but not cytochrome crdquo FEBS Lettersvol 410 no 2-3 pp 351ndash355 1997

[77] U Ahuja and L Thony-Meyer ldquoDynamic features of a hemedelivery system for cytochrome c maturationrdquo Journal of Bio-logical Chemistry vol 278 no 52 pp 52061ndash52070 2003

[78] J W A Allen P D Barker O Daltrop et al ldquoWhy isnrsquot ldquostan-dardrdquo heme good enough for c-type and di-type cytochromesrdquoDalton Transactions no 21 pp 3410ndash3418 2005

[79] K Inaba and K Ito ldquoStructure and mechanisms of the DsbB-DsbA disulfide bond generation machinerdquo Biochimica et Bio-physica Acta vol 1783 no 4 pp 520ndash529 2008

[80] H Kadokura F Katzen and J Beckwith ldquoProtein disulfidebond formation in prokaryotesrdquoAnnual Review of Biochemistryvol 72 pp 111ndash135 2003

[81] S R Shouldice B Heras P M Walden M Totsika M ASchembri and J L Martin ldquoStructure and function of DsbA akey bacterial oxidative folding catalystrdquoAntioxidants and RedoxSignaling vol 14 no 9 pp 1729ndash1760 2011

[82] R Metheringham L Griffiths H Crooke S Forsythe and JCole ldquoAn essential role for DsbA in cytochrome c synthesis andformate-dependent nitrite reduction by Escherichia coli K-12rdquoArchives of Microbiology vol 164 no 4 pp 301ndash307 1995

[83] Y Sambongi and S J Ferguson ldquoMutants of Escherichia colilacking disulphide oxidoreductases DsbA and DsbB cannotsynthesise an exogenous monohaem c-type cytochrome exceptin the presence of disulphide compoundsrdquo FEBS Letters vol398 no 2-3 pp 265ndash268 1996

[84] D AMavridou S J Ferguson and JM Stevens ldquoThe interplaybetween the disulfide bond formation pathway and cytochromec maturation in Escherichia colirdquo FEBS Letters vol 586 no 12pp 1702ndash1707 2012

[85] C U Stirnimann A Rozhkova U Grauschopf M G GrutterR Glockshuber and G Capitani ldquoStructural basis and kineticsof DsbD-dependent cytochrome c maturationrdquo Structure vol13 no 7 pp 985ndash993 2005

[86] N Ouyang Y-G Gao H-Y Hu and Z-X Xia ldquoCrystalstructures of E coli CcmGand itsmutants reveal key roles of theN-terminal120573-sheet and the fingerprint regionrdquoProteins vol 65no 4 pp 1021ndash1031 2006

[87] M A Edeling L W Guddat R A Fabianek et al ldquoCrys-tallization and preliminary diffraction studies of native andselenomethionine CcmG (CycY DsbE)rdquoActa CrystallographicaD vol 57 no 9 pp 1293ndash1295 2001

[88] R A Fabianek M Huber-Wunderlich R Glockshuber PKunzler H Hennecke and L Thony-Meyer ldquoCharacterizationof the Bradyrhizobium japonicum CycY protein a membrane-anchored periplasmic thioredoxin that may play a role as areductant in the biogenesis of c-type cytochromesrdquo Journal ofBiological Chemistry vol 272 no 7 pp 4467ndash4473 1997

[89] G B Kallis and A Holmgren ldquoDifferential reactivity of thefunctional sulfhydryl groups of cysteine-32 and cysteine-32present in the reduced form of thioredoxin from Escherichiacolirdquo Journal of Biological Chemistry vol 255 no 21 pp 10261ndash10265 1980

[90] P T Chivers andR T Raines ldquoGeneral acidbase catalysis in theactive site of Escherichia coli thioredoxinrdquoBiochemistry vol 36no 50 pp 15810ndash15816 1997

[91] X M Zheng J Hong H Y Li D H Lin and H Y HuldquoBiochemical properties and catalytic domain structure of theCcmH protein from Escherichia colirdquo Biochim Biophys Actavol 1824 no 12 pp 1394ndash1400 2012

[92] UAhujaA Rozhkova RGlockshuber LThony-Meyer andOEinsle ldquoHelix swapping leads to dimerization of the N-terminaldomain of the c-type cytochrome maturation protein CcmHfrom Escherichia colirdquo FEBS Letters vol 582 no 18 pp 2779ndash2786 2008

[93] R A Fabianek T Hofer and L Thony-Meyer ldquoCharacteriza-tion of the Escherichia coli CcmH protein reveals new insightsinto the redox pathway required for cytochrome c maturationrdquoArchives of Microbiology vol 171 no 2 pp 92ndash100 1999

[94] E Reid J Cole and D J Eaves ldquoThe Escherichia coli CcmGprotein fulfils a specific role in cytochrome c assemblyrdquo Bio-chemical Journal vol 355 no 1 pp 51ndash58 2001

[95] Q Ren U Ahuja and LThony-Meyer ldquoA bacterial cytochromec heme lyase CcmF forms a complex with the heme chaperoneCcmE and CcmH but not with apocytochrome crdquo Journal ofBiological Chemistry vol 277 no 10 pp 7657ndash7663 2002

[96] E H Meyer P Giege E Gelhaye et al ldquoAtCCMH an essentialcomponent of the c-type cytochrome maturation pathway inArabidopsis mitochondria interacts with apocytochrome crdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 44 pp 16113ndash16118 2005

[97] C Rios-Velazquez R Coller and T J Donohue ldquoFeatures ofRhodobacter sphaeroides CcmFHrdquo Journal of Bacteriology vol185 no 2 pp 422ndash431 2003

[98] C Sanders M Deshmukh D Astor R G Kranz and F DaldalldquoOverproduction of CcmG and CcmFHRc fully suppresses thec-type cytochrome biogenesis defect of Rhodobacter capsulatus

16 Scientifica

CcmI-null mutantsrdquo Journal of Bacteriology vol 187 no 12 pp4245ndash4256 2005

[99] A F Verissimo H Yang X Wu C Sanders and F DaldalldquoCcmI subunit of CcmFHI heme ligation complex functionsas an apocytochrome c chaperone during c-type cytochromematurationrdquo Journal of Biological Chemistry vol 286 no 47 pp40452ndash40463 2011

[100] K Bryson L J McGuffin R L Marsden J J Ward J SSodhi and D T Jones ldquoProtein structure prediction servers atUniversity College Londonrdquo Nucleic Acids Research vol 33 no2 pp W36ndashW38 2005

[101] D T Jones ldquoProtein secondary structure prediction basedon position-specific scoring matricesrdquo Journal of MolecularBiology vol 292 no 2 pp 195ndash202 1999

[102] C Sanders C Boulay and F Daldal ldquoMembrane-spanning andperiplasmic segments of CcmI have distinct functions duringcytochrome c biogenesis in Rhodobacter capsulatusrdquo Journal ofBacteriology vol 189 no 3 pp 789ndash800 2007

[103] D Han K Kim J Oh J Park and Y Kim ldquoTPR domainof NrfG mediates complex formation between heme lyaseand formate-dependent nitrite reductase in Escherichia coliO157H7rdquo Proteins vol 70 no 3 pp 900ndash914 2008

[104] D J Eaves J Grove W Staudenmann et al ldquoInvolvement ofproducts of the nrfEFG genes in the covalent attachment ofhaem c to a novel cysteine-lysine motif in the cytochrome c552nitrite reductase from Escherichia colirdquo Molecular Microbiol-ogy vol 28 no 1 pp 205ndash216 1998

[105] J Nilsson B Persson and G Von Heijne ldquoConsensus predic-tions of membrane protein topologyrdquo FEBS Letters vol 486 no3 pp 267ndash269 2000

[106] E di Silvio A di Matteo F Malatesta and C Travaglini-Allocatelli ldquoRecognition and binding of apocytochrome c toP aeruginosa CcmI a component of cytochrome c maturationmachineryrdquo Biochim Biophys Acta vol 1834 no 8 pp 1554ndash1561 2013

[107] N Zeytuni and R Zarivach ldquoStructural and functional dis-cussion of the tetra-trico-peptide repeat a protein interactionmodulerdquo Structure vol 20 no 3 pp 397ndash405 2012

[108] C Sanders S Turkarslan D-W Lee O Onder R G Kranz andF Daldal ldquoThe cytochrome c maturation components CcmFCcmH and CcmI form a membrane-integral multisubunitheme ligation complexrdquo Journal of Biological Chemistry vol283 no 44 pp 29715ndash29722 2008

[109] A F Verissimo M A Mohtar and F Daldal ldquoThe hemechaperone ApoCcmE forms a ternary complex with CcmI andapocytochrome crdquoThe Journal of Biological Chemistry vol 288no 9 pp 6272ndash6283 2013

[110] P D Barker J C Ferrer M Mylrajan et al ldquoTransmutation of aheme proteinrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 90 no 14 pp 6542ndash65461993

[111] J Simon and L Hederstedt ldquoComposition and function ofcytochrome c biogenesis system IIrdquoThe FEBS Journal vol 278no 22 pp 4179ndash4188 2011

[112] T R H M Kouwen and J M van Dijl ldquoInterchangeablemodules in bacterial thiol-disulfide exchange pathwaysrdquo Trendsin Microbiology vol 17 no 1 pp 6ndash12 2009

[113] A Crow A Lewin O Hecht et al ldquoCrystal structure andbiophysical properties of Bacillus subtilis BdbD An oxidizingthioldisulfide oxidoreductase containing a novel metal siterdquoJournal of Biological Chemistry vol 284 no 35 pp 23719ndash23733 2009

[114] M C Moller and L Hederstedt ldquoExtracytoplasmic processesimpaired by inactivation of trxA (thioredoxin gene) in Bacillussubtilisrdquo Journal of Bacteriology vol 190 no 13 pp 4660ndash46652008

[115] A Lewin A Crow A Oubrie and N E Le Brun ldquoMolecularbasis for specificity of the extracytoplasmic thioredoxin ResArdquoJournal of Biological Chemistry vol 281 no 46 pp 35467ndash35477 2006

[116] C L Colbert QWu P J A Erbel K H Gardner and J Deisen-hofer ldquoMechanism of substrate specificity in Bacillus subtilisResA a thioredoxin-like protein involved in cytochrome cmaturationrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 103 no 12 pp 4410ndash44152006

[117] UAhuja P Kjelgaard B L Schulz LThony-Meyer andLHed-erstedt ldquoHaem-delivery proteins in cytochrome c maturationsystem IIrdquoMolecular Microbiology vol 73 no 6 pp 1058ndash10712009

[118] M L D Page P P Hamel S T Gabilly et al ldquoA homologof prokaryotic thiol disulfide transporter CcdA is required forthe assembly of the cytochrome b6f complex in Arabidopsischloroplastsrdquo Journal of Biological Chemistry vol 279 no 31pp 32474ndash32482 2004

[119] A Rozhkova C U Stirnimann P Frei et al ldquoStructural basisand kinetics of inter- and intramolecular disulfide exchange inthe redox catalyst DsbDrdquoThe EMBO Journal vol 23 no 8 pp1709ndash1719 2004

[120] T Schiott M Throne-Holst and L Hederstedt ldquoBacillussubtilis CcdA-defective mutants are blocked in a late step ofcytochrome C biogenesisrdquo Journal of Bacteriology vol 179 no14 pp 4523ndash4529 1997

[121] R E Feissner C S Beckett J A Loughman and R G KranzldquoMutations in cytochrome assembly and periplasmic redoxpathways in Bordetella pertussisrdquo Journal of Bacteriology vol187 no 12 pp 3941ndash3949 2005

[122] L S ErlendssonMMoller and L Hederstedt ldquoBacillus subtilisStoA is a thiol-disulfide oxidoreductase important for sporecortex synthesisrdquo Journal of Bacteriology vol 186 no 18 pp6230ndash6238 2004

[123] L S Erlendsson R M Acheson L Hederstedt and N E LeBrun ldquoBacillus subtilis ResA is a thiol-disulfide oxidoreductaseinvolved in cytochrome c synthesisrdquo Journal of BiologicalChemistry vol 278 no 20 pp 17852ndash17858 2003

[124] L S Erlendsson and L Hederstedt ldquoMutations in the thiol-disulfide oxidoreductases BdbC and BdbD can suppresscytochrome c deficiency of CcdA-defective Bacillus subtiliscellsrdquo Journal of Bacteriology vol 184 no 5 pp 1423ndash1429 2002

[125] M E Dumont J F Ernst D M Hampsey and F ShermanldquoIdentification and sequence of the gene encoding cytochromec heme lyase in the yeast Saccharomyces cerevisiaerdquoThe EMBOJournal vol 6 no 1 pp 235ndash241 1987

[126] M E Dumont J F Ernst and F Sherman ldquoCoupling of hemeattachment to import of cytochrome c into yeast mitochondriaStudies with heme lyase-deficient mitochondria and alteredapocytochromes crdquo Journal of Biological Chemistry vol 263 no31 pp 15928ndash15937 1988

[127] B San Francisco E C Bretsnyder and R G Kranz ldquoHumanmitochondrial holocytochrome c synthasersquos hemebindingmat-uration determinants and complex formationwith cytochromecrdquo Proceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 9 pp E788ndashE797 2012

Scientifica 17

[128] L Schaefer A Ballabio and H Y Zoghbi ldquoCloning andcharacterization of a putative human holocytochrome c-typesynthetase gene (HCCS) isolated from the critical region formicrophthalmia with linear skin defects (MLS)rdquoGenomics vol34 no 2 pp 166ndash172 1996

[129] S K Prakash T A Cormier A E McCall et al ldquoLoss ofholocytochrome c-type synthetase causes the male lethality ofX-linked dominant microphthalmia with linear skin defects(MLS) syndromerdquo Human Molecular Genetics vol 11 no 25pp 3237ndash3248 2002

[130] I Wimplinger M Morleo G Rosenberger et al ldquoMutations ofthe mitochondrial holocytochrome c-type synthase in X-linkeddominant microphthalmia with linear skin defects syndromerdquoTheAmerican Journal of HumanGenetics vol 79 no 5 pp 878ndash889 2006

[131] S Kiryu-Seo KGamo T Tachibana K Tanaka andHKiyamaldquoUnique anti-apoptotic activity of EAAC1 in injured motorneuronsrdquoTheEMBO Journal vol 25 no 14 pp 3411ndash3421 2006

[132] R Lill R A Stuart M E Drygas F E Nargang and W Neu-pert ldquoImport of cytochrome c heme lyase into mitochondriaa novel pathway into the intermembrane spacerdquo The EMBOJournal vol 11 no 2 pp 449ndash456 1992

[133] H Steiner A Zollner A Haid W Neupert and R LillldquoBiogenesis of mitochondrial heme lyases in yeast Importand folding in the intermembrane spacerdquo Journal of BiologicalChemistry vol 270 no 39 pp 22842ndash22849 1995

[134] A Zollner G Rodel and A Haid ldquoMolecular cloning andcharacterization of the Saccharomyces cerevisiae CYT2 geneencoding cytochrome-c1-heme lyaserdquo European Journal of Bio-chemistry vol 207 no 3 pp 1093ndash1100 1992

[135] V Corvest D A Murrey D G Bernard D B Knaff B Guiardand P PHamel ldquoc-Type cytochrome assembly in Saccharomycescerevisiae a key residue for apocytochrome c1lyase interactionrdquoGenetics vol 186 no 2 pp 561ndash571 2010

[136] V Corvest D A Murrey M Hirasawa D B Knaff B Guiardand P P Hamel ldquoThe flavoprotein Cyc2p a mitochondrialcytochrome c assembly factor is a NAD(P)H-dependent haemreductaserdquo Molecular Microbiology vol 83 no 5 pp 968ndash9802012

[137] D G Bernard S Quevillon-Cheruel S Merchant B Guiardand P P Hamel ldquoCyc2p a membrane-bound flavopro-tein involved in the maturation of mitochondrial c-typecytochromesrdquo Journal of Biological Chemistry vol 280 no 48pp 39852ndash39859 2005

[138] J G Kleingardner and K L Bren ldquoComparing substratespecificity between cytochrome cmaturation and cytochrome cheme lyase systems for cytochrome c biogenesisrdquo Metallomicsvol 3 no 4 pp 396ndash403 2011

[139] C Travaglini-Allocatelli S Gianni VMorea A Tramontano TSoulimane and M Brunori ldquoExploring the cytochrome c fold-ing mechanism cytochrome c552 fromThermus thermophilusfolds through an on-pathway intermediaterdquo Journal of BiologicalChemistry vol 278 no 42 pp 41136ndash41140 2003

[140] L Zhang and L Guarente ldquoHeme binds to a short sequencethat serves a regulatory function in diverse proteinsrdquoTheEMBOJournal vol 14 no 2 pp 313ndash320 1995

[141] S Hira T Tomita T Matsui K Igarashi and M Ikeda-SaitoldquoBach1 a heme-dependent transcription factor reveals pres-ence of multiple heme binding sites with distinct coordinationstructurerdquo IUBMB Life vol 59 no 8-9 pp 542ndash551 2007

[142] T K Kuhl A Wiszligbrock N Goradia et al ldquoAnalysis of Fe(III)heme binding to cysteine-containing heme-regulatory motifs

in proteinsrdquo ACS Chemical Biology vol 8 no 8 pp 1785ndash17932013

[143] R L Moore J M Stevens and S J Ferguson ldquoMitochondrialcytochrome c synthase CP motifs are not necessary for hemeattachment to apocytochrome crdquo FEBS Letters vol 585 no 21pp 3415ndash3419 2011

[144] S Gianni M Brunori and C Travaglini-Allocatelli ldquoRefoldingkinetics of cytochrome c551 reveals a mechanistic differencebetween urea and guanidinerdquo Protein Science vol 10 no 8 pp1685ndash1688 2001

[145] S Gianni C Travaglini-Allocatelli F Cutruzzola M BrunoriM C R Shastry and H Roder ldquoParallel pathways incytochrome c551 foldingrdquo Journal of Molecular Biology vol 330no 5 pp 1145ndash1152 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

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Enzyme Research

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International Journal of

Microbiology

Scientifica 7

which includes the Ccm proteins CcmG and CcmH Thenecessary reducing power is transferred from the cytoplasmicthioredoxin (TRX) to CcmG via DsbD a large membraneprotein organized in three structural domains an N-terminalperiplasmic domain with a IgG-like fold (nDsbD) a C-terminal periplasmic domain with a thioredoxin-like (TRX-like) fold (cDsbD) and a central domain composed ofeight TM helices [85] Each of these domains contains apair of Cys residues and transfer electrons via a cascadeof disulfide exchange reactions making DsbD a ldquoredox-hubrdquo in the periplasm performing disulfide bond exchangereactions with different oxidized proteins [79] In particulara combination of X-ray crystallography experiments andkinetic analyses showed that electrons are transferred fromthe cytoplasmic TRX to the membrane domain of DsbDfollowed by reduction of cDsbD and finally of nDsbD whichis the direct electron donor to CcmG [85]

CcmG is a membrane-anchored protein linked to themembrane via an N-terminal TM helix and exposing itssoluble TRX-like domain in the periplasm The 3D structureof the TRX-like domain of CcmG from different bacteriahas been solved by X-ray crystallography (E coli PDB 1Z5Y[85] PDB 2B1K [86] B japonicum PDB 1KNG [87] Paeruginosa PDB 3KH7 3KH9 [22]) and is generally wellconserved as proved by the low RMSD (08 A between Pa-CcmG and Ec-CcmG 135 A between Pa-CcmG and Bj-CcmG) Although all these proteins adopt a TRX-like foldand contain the redox-active motif CXXC in the first 120572-helixthey are inactive in the classic insulin reduction assay [7588] CcmG proteins are therefore considered specific thiol-oxidoreductase able to recognize and selectively interactonly with their upstream and downstream binding partnersin the thioreduction process leading to reduced apoCytcLooking at the 3D structure of the periplasmic domain ofthe prototypical Pa-CcmG it is possible to identify the 120573120572120573and 120573120573120572 structural motifs of the TRX fold linked by a short120572-helix and forming a four-stranded 120573-sheet surrounded bythree helices the protein contains an additional N-terminalextension (residues 26ndash62) and a central insert (residues 102ndash123) The redox-active motif of Pa-CcmG (CPSC) is locatedin the first 120572-helix of the TRX fold as usually observed in allTRX-like proteins As for any molecular machinery whereeach component must recognize and interact with more thanone target (ie the substrate and the other components ofthe apparatus) an open question concerns the mechanismwhereby CcmG is able to recognize its different partnersThe availability of the crystal structures of Pa-CcmG bothin the oxidized (22 A resolution) and reduced state (18 Aresolution) [22] allowed highlighting the structural similaritybetween the two redox states (Rmsd of the C120572 atoms inthe two redox forms is 019 A) and therefore to excludestructural rearrangement as the mechanism used by Pa-CcmG to discriminate between reduced (such as the nDsbDdomain) and oxidized partners (Pa-CcmH andor apoCyt)

The standard redox potential of Pa-CcmG (11986410158400= 0213V

at pH 70 [22] as well as that of Ec-CcmG (11986410158400= 0212V

[86]) indicates that these proteins act as mild reductants inthe thioreductive pathway of Cytc biogenesis However the

Figure 5 Three-dimensional structure of Pa-CcmH shown inribbon representation The figure shows the three-helix bundleforming the characteristic fold of Pa-CcmHThe active site disulfidebond between residues Cys25 andCys28 in the long loop connectinghelices 120572-helix1 and 120572-helix 2 is highlighted in yellow

function of thiol-oxidoreductases obviously depends on thepKa values of their activesite Cys residues The pKa of CysX(613 plusmn 005) and CysY (105 plusmn 007) are consistent with thepKa values measured in different TRXs where the active N-terminal Cys residue has a pKa close to pH 70 whereas theC-terminal Cys has a much higher pKa [89 90] Such a largedifference between the two pKa values in the TRX family isfunctionally relevant because it allows the N-terminal Cysto perform the nucleophilic attack on the target disulfidewhile the C-terminal Cys is involved in the resolution of theresulting mixed-disulfide [90]

CcmH is the other component of System I involvedin the reduction of apoCyt Notably CcmH proteins fromdifferent bacterial subgroups may display structural vari-ability indeed while in E coli Ec-CcmH is a bipartiteprotein characterized by two soluble domains exposed to theperiplasm and two TM segments CcmH from P aeruginosa(Pa-CcmH) is a one-domain redox-active protein anchoredto the membrane via a single TM helix and homologous tothe N-terminal redox-active domain of Ec-CcmH Surpris-ingly the 3D structure of the soluble periplasmic domainof Pa-CcmH revealed that it adopts a peculiar three-helixbundle fold strikingly different from that of canonical thiol-oxidoreductases (Figure 5 PDB 2HL7 [23])TheN-terminaldomain of Ec-CcmH was also shown to have the same 3Dstructure although helix-swapping and dimerization havebeen observed in this case (PDB 2KW0 [91 92]) Theconserved redox-active motif (LRCPKC) is located in theloop connecting helices 1 and 2 close to the activesite thecrystal structure reveals the presence of a small pocket on thesurface of Pa-CcmH surrounded by conserved hydrophobicand polar residues which could represent the recognition sitefor the heme-binding motif of apoCyt

Concerning the functional properties of this unusualthiol-oxidoreductase it is interesting to note that its standardredox potential (1198641015840

0= 0215V) [23] is similar to that ob-

tained for Pa-CcmG This observation stands against thelinear redox cascade hypothesis whereby CcmG reducesCcmH While in the canonical redox-active CXXC motif

8 Scientifica

S SSS

SH

SH

Scheme 1 Scheme 2

SH

SH

SHSHS

S

CcmG 7477

CcmG 7477

CcmH 2528

CcmH 2528

apoCytox

apoCytred

S

SHCcmG 74

77

SH

SH

HSHS

CcmG 7477

S

S

SS

3

1

2CcmG 7477

SH

SHCcmH 25

28SH

HSS S

CcmH 2528

SHS

CcmH2528

apoCytox

apoCytred

+

Figure 6 Alternative thioreduction pathways whichmay be operative in System I and hypothesized on the basis of structural and functionalcharacterization of the redox-active Ccm proteins from P aeruginosa [22 23 25] Scheme 1 is a linear redox cascade whereby CcmG is thedirect reductant of CcmH which reduces oxidized apoCyt Scheme 2 envisages a more complex scenario involving the formation of a mixed-disulfide complex between CcmH and apoCyt (Step 1) This complex is the substrate for the attack by reduced CcmG (Step 2) that liberatesreduced apoCyt The resulting disulfide bond between CcmH and CcmG is then resolved by the free Cys thiol of CcmG (probably Cys77 inPa-CcmG) Adapted from [25]

of the TRX family the N-terminal Cys is always solventexposed in CcmH proteins the arrangement of the two Cysresidues is reversed the N-terminal Cys residue is buriedwhereas the C-terminal Cys residue is solvent exposed Onthe basis of this observation it was suggested that differentfrom the canonical TRX redox mechanism CcmH proteinsperform the nucleophilic attack on the apoCyt disulfide viatheir C-terminal Cys residue [23] This mechanism which isin agreement with the mechanism proposed earlier for Ec-CcmH on the basis of mutational-complementation studies[93 94] is substantiated by the peculiar pKa values of theactive site Cys residues of Pa-CcmH which were found tobe similar for both cysteines (84 plusmn 01 and 86 plusmn 01 [23])Again this is different from what is generally observed in thecase of TRX proteins where the pKa value of the Cys residueperforming the initial nucleophilic attack is significantlylower than the pKa value of theCys residue responsible for theresolution of the intermediate mixed-disulfide It is temptingto speculate that the unusual pKa values of the Pa-CcmHactive site thiols may ensure the necessary specificity of thiscomponent of the Ccm apparatus toward the CXXCH motifof the apoCyt substrate

24 System I ApoCyt Thioreduction Pathway MechanismAlthough we know that CcmG and CcmH are the redox-active components of System I involved in the thioreductivepathway of Cyt c biogenesis not only an acceptedmechanismfor the reduction of apoCyt disulfide bond is still lackingbut also the absolute requirement of such a process is nowdebated [38 84] Focusing our attention on the reductionof the apoCyt internal disulfide at least two mechanismscan been hypothesized which involve either a linear redoxcascade of disulfide exchange reactions or a nonlinear redox

process involving transient formation of a mixed-disulfidecomplex as depicted in Figure 6 and Schemes 1 and 2respectively

Both the thiol-disulfide exchange mechanisms depictedin Figure 6 suggest that CcmH is the direct reductant ofthe apoCytc disulfide however even if immunoprecipitationexperiments failed to detect the formation of a mixed-disulfide complex between apoCyt and CcmH proteins [95]some in vitro evidence supporting the formation of sucha complex has been presented In particular it has beenshown that Rhodobacter capsulatus and Arabidopsis thalianaCcmH homologues (Rc-CcmH and At-CcmH) are able toreduce the CXXCH motif of an apoCyt-mimicking peptide[75 96] In the latter case yeast two-hybrid experimentscarried out on At-CcmH indeed revealed an interactionbetween the protein and a peptide mimicking the A thalianaCyt c sequence In the case of Pa-CcmH FRET kineticexperiments employing a Trp-containing fluorescent variantof the protein and a dansylated nonapeptide encompassingthe heme-binding motif of P aeruginosa cytochrome c551(dans-KGCVACHAI) [23] allowed to directly observe theformation of themixed-disulfide complex and tomeasure theoff-rate constant of the bound peptide The results of these invitro binding experiments allowed to calculate an equilibriumdissociation constant which combines an adequate affinity(low 120583M) with the need to release efficiently reduced apoCytto other component(s) of the System I maturase complex[23] More recently the results obtained by FRET bindingexperiments carried out with single Cys-containing mutantsof Pa-CcmH and Pa-CcmG [25] substantiated the hypothesisdepicted in Scheme 2 (Figure 6) Altogether these structuraland functional results suggest that the thioreduction pathwaymechanism leading to reduced apoCyt is better describedby Scheme 2 and that reducing equivalents might not be

Scientifica 9

transferred directly from CcmG to apoCyt as depicted inScheme 1 According to Scheme 2 reduced CcmH (a non-TRX-like thiol-oxidoreductase) specifically recognizes andreduces oxidized apoCyt via the formation of a mixed-disulfide complex which is subsequently resolved by CcmGThe resulting disulfide bond between CcmH and CcmG isthen resolved by the free Cys thiol of CcmG (probably Cys77in Pa-CcmG)

However further in vitro experiments with CcmH andapoCyt single Cys-containing mutants are needed to unveilthe details of the thioreduction of oxidized apoCyt by CcmHIn particular it would be crucial to identify the Cys residueof apoCyt that remains free in the apoCyt-CcmH mixed-disulfide complex intermediate (see Scheme 2 and Section 26below) and available to thioether bond formation with oneof the heme vinyl groups Clearly structure determinationof the trapped mixed-disulfide complexes between CcmHCcmG and apoCyt (or apoCyt peptides) would providekey information for our understanding of this specializedthioreduction pathway mechanism

25 System I ApoCyt Chaperoning and Heme AttachmentComponents The reduced heme-binding motif of apoCyt isnow available to the heme ligation reaction However themolecularmechanismwhereby the Ccmmachinery catalyzesor promotes the formation of the heme-apoCyt covalentbonds is still largely obscure representing themost importantgoal in the field Past observations and recent experimentssuggest that CcmF and CcmI possibly together with CcmHare involved in these final steps [16 34 36]

CcmF is a large integral membrane protein of more than600 residues belonging to the heme handling protein family(HHP [55]) and predicted to contain 10ndash15 TM helices (notethat some discrepancy exists as to the number of TM helicespredicted by computer programs and those predicted onthe basis of phoA and lacZ fusion experiments [40 97])a conserved WWD domain and a larger domain devoidof any recognizable sequence features both exposed to theperiplasm Only recently E coli CcmF (Ec-CcmF) has beenoverexpressed solubilized from the membrane fraction andspectroscopically characterized in vitro [36 41] Surprisinglythe biochemical characterization of recombinant Ec-CcmFallowed to show that the purified protein contains heme b ascofactor in a 1 1 stoichiometry this observation led to thehypothesis that in addition to its heme lyase function Ec-CcmF may act as a heme oxidoreductase In particular it ispossible that the heme b of Ec-CcmF may act as a reductantfor the oxidized iron of the heme bound to CcmE [41]indeed the in vitro reduction of Ec-CcmF by quinones hasbeen experimentally observed strengthening the hypothesisabout the quinolhemeoxidoreductase function of this elusiveproteinThe structuralmodel proposed for Ec-CcmFpredicts13 TMhelices and notably the location of the four completelyconserved His residues according to the model two of them(His173 andHis303) are located in periplasmic exposed loopsnext to the conserved WWD domain which is believedto provide a platform for the heme bound to holoCcmEwhile His261 is located in one of the TM helices and it is

predicted to act as an axial ligand to the heme b of Ec-CcmFthe other conserved His residue (H491) could provide thesecond axial coordination bond to the heme although thishas not been experimentally addressed This model of Ec-CcmF therefore envisages that this large membrane proteinis characterized by two heme-binding sites one of themis embedded in the membrane and coordinates a heme bprosthetic group necessary to reduce the CcmE-bound hemehosted in the second heme-binding site and constituted by itsWWD domain

It is interesting to note that in plants mitochondria theCcmF ortholog appears to be split into three different pro-teins (At-CcmFN1 At-CcmFN2 and At-CcmFC) possiblyinteracting each other [16] Since each of these proteins issimilar to the corresponding domain in the bacterial CcmFortholog this observation may provide useful informationin the design of engineered fragments of bacterial CcmFproteins amenable to structural analyses

The other System I component which is generallybelieved to be involved in the final steps of Cyt c maturationis CcmI As stated above the ccmI gene is present only insome Ccm operons while in others the corresponding ORFis present within the ccmH gene (as in E coli)The functionalrole of CcmI in Cytc biogenesis is revealed by geneticstudies showing that in R capsulatus and B japonicuminactivation of the ccmI gene leads to inability to synthesizefunctional c-type cytochromes [98 99] In R capsulatus andP aeruginosa the CcmI protein (Rc-CcmI and Pa-CcmIresp) can be described as being composed of two domainsstarting from the N-terminus a first domain composed oftwo TM helices connected by a short cytoplasmic regionand a large periplasmic domain Structural variations maybe observed among CcmI members from different bacteriaindeed multiple sequence alignment indicates that the cyto-plasmic region of Rc-CcmI contains a leucine zipper motifwhich is not present in the putative cytoplasmic region of Pa-CcmI [100ndash102] Surprisingly no crystallographic structureis available up to now for the soluble domain of any CcmIprotein with the exception of the ortholog protein NrfGfrom E coli (Ec-NrfG) [103] This protein is necessary toattach the heme to the unusual heme-binding motif CWSCK(where a Lys residue substitutes the conservedHis) present inNrfA a pentaheme c-type cytochrome [103 104] Accordingto secondary structure prediction methods [105] it hasbeen proposed that the periplasmic domain of Pa-CcmI iscomposed of aN-terminal120572-helical region containing at leastthree TPR motifs connected by a disordered linker to a 120572-120573C-terminal regionMultiple sequence analyses and secondarystructure predictionmethods show that the TPR region of Pa-CcmI can be successfully aligned with many TPR-containingproteins including Ec-NrfG [106]

26 System I ApoCyt Chaperoning and Heme AttachmentMechanisms TPR domain-containing proteins are commonto eukaryotes prokaryotes and archaea these proteins aregenerally involved in the assembly of multiprotein complexesand to the chaperoning of unfolded proteins [103 107] Itis therefore plausible that CcmI (or the TPR C-terminal

10 Scientifica

domain of Ec-CcmH) may act to provide a platform for theunfolded apoCyt chaperoning it to the heme attachment sitepresumably located on the WWD domain of CcmF CcmImay thus be considered a component of amembrane-integralmultisubunit heme ligation complex together with CcmFand CcmH as experimentally observed by affinity purifi-cation experiments carried out with Rc-CcmFHI proteins[97 99 108] According to the proposed function of CcmI acritical requirement is represented by its ability to recognizedifferent protein targets over and above apoCyt such asCcmFandCcmHHowever until now direct evidence has been pre-sented only for the interaction of CcmI with apoCyt but thepossibility remains that CcmFHI proteins interact each othervia their TM helices and not via their periplasmic domainsInterestingly both for Pa-CcmI [106] or Rc-CcmI [99] CDspectroscopy experiments carried out on the CcmIapoCytcomplex highlighted major conformational changes at thesecondary structure level It is tempting to speculate on thebasis of these results that in vivo the folding of apoCyt maybe induced by the interaction with CcmI In the case of Paeruginosa System I proteins the binding process betweenPa-CcmI and its target protein apoCyt c551 (Pa-apoCyt) hasbeen studied both at equilibrium and kinetically [106] the119870119863measured for this interaction (in the 120583M range) appeared

to be low enough to ensure apoCyt delivery to the othercomponents of the Ccmmachinery Clearly a major questionconcerns the molecular determinants of such recognitionprocess interestingly both affinity coprecipitation assays[99] and equilibrium and kinetic binding experiments [106]highlighted the role played by the C-terminal 120572-helix of Cytc Similar observations have been made for the interaction ofEc-NrfGwith a peptidemimickingNrfA its apoCyt substrate[103] in this case isothermal titration calorimetry (ITC)experiments indicate that the TPR-domain of NrfG serves asa binding site for the C-terminal motif of NrfA Altogetherthese observations are in agreement with the fact that TPRproteins generally bind to their targets by recognizing theirC-terminal region [107]

The CcmI chaperoning activity has been experimentallysupported for the first time in the case of Pa-CcmI by citratesynthase tests [106] it has been proposed that the observedability to suppress protein aggregation in vitromay reflect thecapacity of CcmI to avoid apoCyt aggregation in vivo Stillanother piece of the Cyt c biogenesis puzzle has been addedrecently by showing that Rc-CcmI is able to interact withapoCcmE either alone or together with its substrate apoCytc2 forming a stable ternary complex in the absence of heme[109] This unexpected observation obtained by reciprocalcopurification experiments provides supporting evidence forthe existence of a large multisubunit complex composed ofCcmFHI andCcmE possibly interactingwith theCcmABCDcomplex It is interesting to note that while in the case ofthe CcmIapoCyt recognition different studies highlightedthe crucial role of the C-terminal helical region of apoCyt(see above) in the case of the apoCcmE apoCyt recognitionthe N-terminal region of apoCyt seems to represent a criticalregion

It is generally accepted that CcmF is the Ccm compo-nent responsible for heme covalent attachment to apoCyt

however as discussed above it is possible that this largemembrane protein plays such a role only together withother Ccm proteins such as CcmH and CcmI Moreover asrecently discovered by Kranz and coworkers [36 41] CcmFmay also act as a quinoleheme oxidoreductase ensuringthe necessary reduction of the oxidized heme b bound toCcmE Why it is necessary that the heme iron be in itsreduced state rather than in its oxidized state is not completelyclear although it is possible that this is a prerequisite to themechanism of thioether bond formation [110] According tocurrent hypotheses it is likely that the periplasmic WWDdomain of CcmF provides a platform for heme b bindingSanders et al and Verissimo et al [34 109] have presenteda mechanistic view of the heme attachment process whichtakes into account all the available experimental observationson the different Ccmproteins According to thismodel stere-ospecific heme ligation to reduced apoCyt occurs becauseonly the vinyl-4 group is available to form the first thioetherbond with a free cysteine at the apoCyt heme-binding motifsince the vinyl-2 group is involved (at least in the Ec-CcmE)in the covalent bond with His130 of CcmE [67] Howeverexperimental proof for this hypothesis requires a detailedinvestigation of the apoCyt thioreduction process catalyzedby CcmH (see Section 24)

It should be noticed that the mechanisms described sofar for the function(s) played by CcmF (see [34 36 109]) donot envisage a clear role for its large C-terminal periplasmicdomain (residues 510 to 611 in Ec-CcmF) It would be inter-esting to see if this domain apparently devoid of recognizablesequence features may mediate intermolecular recognitionprocesses with one (or more) component(s) involved in thehemeapoCyt ligation process

3 System II

System II is typically found in gram-positive bacteria and inin 120576-proteobacteria it is also present in most 120573- and some120575-proteobacteria in Aquificales and cyanobacteria as wellas in algal and plant chloroplasts System II is composedof three or four membrane-bound proteins CcdA ResACcsA (also known as ResC) and CcsB (also known as ResB)(Figure 3) CcdA andResA are redox-active proteins involvedin the reduction of the disulfide bond in the heme-bindingmotif of apoCyt whereas CcsA and CcsB are responsible forthe heme-apoCyt ligation process and are considered Cyt csynthethases (CCS) BothCcsAwhich is evolutionary relatedto the CcmC and CcmF proteins of System I [55] and CcsBare integral membrane proteins In some 120576-proteobacteriasuch asHelicobacter hepaticus andHelicobacter pylori a singlefusion protein composed of CcsA and CcsB polypeptides ispresent [41 111] Although as discussed below evidence hasbeen put forward to support the hypothesis that the CcsBAcomplex acts a heme translocase we still do not know if theheme is transported across the membrane by component(s)of System II itself or by a different unidentified process

31 System II ApoCyt Thioreduction Pathway After the Secmachinery secretes the newly synthesized apoCyt it readily

Scientifica 11

becomes a substrate for the oxidative system present on theouter surface of the cytoplasmic membrane of the gram-positive bacteria In B subtilis the BdbCD system [112113] is functionally but not structurally equivalent to thewell-characterized DsbAB system present in gram-negativebacteria As discussed above for System I the disulfide ofthe apoCyt heme-binding motif must be reduced in orderto allow thioether bonds formation and heme attachmentResA is the extracytoplasmic membrane-anchored TRX-likeprotein involved in the specific reduction of the apoCytdisulfide bond after the disulfide bond exchange reactionhas occurred oxidized ResA is reduced by CcdA whichreceives its reducing equivalents from a cytoplasmic TRX[114] Although ResA displays a classical TRX-like foldthe analysis of the 3D structure of oxidized and reducedResA from B subtilis (Bs-ResA) showed redox-dependentconformational modifications not observed in other TRX-like proteins Interestingly such modifications occur at thelevel of a cavity proposed to represent the binding site foroxidized apoCyt [24 115 116] Another peculiar feature of Bs-ResA is represented by the unusually similar pKa values of itsthe active site Cys residues (88 and 82 resp) as observedalso in the case of the active site Cys residues of the SystemI Pa-CcmH (see Section 23) However at variance with Pa-CcmH in Bs-ResA the large separation between the twocysteine thiols observed in the structure of the reduced formof the protein can be invoked to account for this result

CcdA is a large membrane protein containing six TMhelices [117 118] homologous to the TM domain of E coliDsbD [85 119] Its role in the Cyt c biogenesis is supportedby the observation that inactivation of CcdA blocks theproduction of c-type cytochromes in B subtilis [120 121]Moreover over and above the reduction of its apoCyt sub-strate Bs-CcdA is able to reduce the disulfide bond of othersecreted proteins such as StoA [122] It is interesting to notethat ResA and CcdA are not essential for Cyt c synthesisin the absence of BdbD or BdbC or if a disulfide reductantis present in the growth medium [123 124] As discussedabove (Section 24) a similar observation was made on therole of the thio-reductive pathway of System I in this casepersistent production of c-type cytochromes was observedin Dsb-inactivated bacterial strains [38] Following theseobservations the question has been put forward as to why theCyt c biogenesis process involves this apparently redundantthio-reduction route only to correct the effects of Bdb orDsb activity Referring to the case of B subtilis [111] it hasbeen proposed that the main reason is that BdbD mustefficiently oxidize newly secreted proteins since the activityof most of them depends on the presence of disulfide bondsAlternatively it can be hypothesized that the intramoleculardisulfide bondof apoCyt protects the protein fromproteolyticdegradation or aggregation or from cross-linking to otherthiol-containing proteins

32 System II Heme Translocation and Attachment to ApoCytRecent experimental evidence is accumulating supportingthe involvement of the heterodimeric membrane complexResBC or of the fusion protein CcsBA in the translocation

of heme from the n-side to the p-side of the bacterialmembrane In particular it has been shown that CcsBAfrom H hepaticus (Hh-CcsBA) is able to reconstitute Cyt cbiogenesis in the periplasm of E coli [36] Moreover it wasalso observed that Hh-CcsBA is able to bind reduced hemevia two conserved histidines flanking the WWD domainand required both for the translocation of the heme andfor the synthetase function of Hh-CcsBA [36] A differentstudy carried out on B subtilis System II proteins providedsupport for heme-binding capability by the ResBC complexaccording to the results obtained on recombinant ResBC theResB component of the heterodimeric CCS complex is ableto covalently bind the heme in the cytoplasm (probably by aCys residue) and to deliver it to an extracytoplamic domain ofResC which is responsible for the covalent ligation to apoCyt[117] It should be noticed however that the transfer of theCCS-bound heme to apoCyt still awaits direct experimentalproof Moreover it has also been reported that in B subtilisthe inactivation of CCS does not affect the presence ofother heme-containing proteins in the periplasm such ascytochrome b562 [111] this observation which is in contrastto the heme-transport hypothesis by CCSs parallels similarconcerns about heme translocation mechanism that arecurrently discussed in the context of System I (see Section 22above) In the case of the Hh-CcsBA synthetase site-directedmutagenesis experiments allowed to assign a heme-bindingrole for the two pairs of conserved His residues accordingto the topological model of the protein obtained by alkalinephosphatase (PhoA) assays and GFP fusions the conservedHis77 and His858 residues are located on TM helices whileHis761 and His897 are located on the periplasmic side [36]These observations together with the results of site-directedmutagenesis experiments allowed the authors to suggest thatHis77 andHis858 form a low affinitymdashmembrane embeddedheme-binding site for ferrous heme which is subsequentlytranslocated to an external heme-binding domain of Hh-CcsBA where it is coordinated by His761 and His897 Thetopological model therefore predicts that this last His pairis part of a periplasmic-located WWD domain (homologousto theWWDdomains found in theCcmCandCcmFproteinsof System I described above)

4 System III

Strikingly different from Systems I and II the Cyt c mat-uration apparatus found in fungi and in metazoan cells(System III) is composed of a single protein known as Holo-Cytochrome c Synthetase (HCCS) or Cytochrome c HemeLyase (CCHL) apparently responsible for all the subprocessesdescribed above (heme transport and chaperoning reductionand chaperoning of apoCyt and catalysis of the thioetherbonds formation between heme and apoCyt) (Figure 4)Surprisingly although this protein has been identified in Scerevisiaemitochondria several years ago [125 126] only veryrecently the human HCCS has been expressed as a recombi-nant protein in E coli and spectroscopically characterized invitro [127] It is worth noticing that humanHCCS is attractinginterest since over and above its role in Cyt c biogenesis it

12 Scientifica

is involved in diseases such as microphthalmia with linearskin defects syndrome (MLS) an X-linked genetic disorder[128ndash130] and in other processes such as Cytc-independentapoptosis in injured motor neurons [131] Different fromanimal cells where a single HCCS is sufficient for thematuration of both soluble and membrane-anchored Cytc(Cyt c and Cy c1 resp) in S cerevisiae two homologs HCCSand HCC1S located in the inner mitochondrial membraneand facing to the IMS space [132 133] are responsible for hemeattachment to apoCyt c and apoCyt c1 respectively [125 134]It should also be noticed that in fungi an additional FAD-containing protein (Cyc2p) is required for Cyt c synthesisCyc2p is a mitochondrial membrane-anchored flavoproteinexposing its redox domain to the IMS which is requiredfor the maturation of Cyt c but not for that of Cyt c1 [135]This protein does not contain the conserved Cys residuestypically found in disulfide reductases and indeed it is notable to reduce oxidized apoCyt in vitro However it has beenrecently shown that Cyc2p is able to catalyze the NAD(P)H-dependent reduction of heme in vitro [136] a necessarystep before the thioether bond formation can occur asdiscussed above (see Section 26) This result together withthe observation that Cyc2p interacts with HCCS and withapoCyt c and c1 lends support to the proposal that Cyc2p isinvolved in the reduction of the heme iron in vivo [136 137]

AlthoughHCCS proteins are crucial for Cyt cmaturationwe still do not know how these proteins recognize theirsubstrates (heme and apoCyt) and how they promote orcatalyze the formation of thioether bonds between the hemevinyl groups and the cysteine thiols of the apoCyt CXXCHmotif

Contrary to the broad specificity of System I whichis able to recognize and attach the heme to prokaryoticand eukaryotic c-type cytochromes [49 50] and even tovery short microperoxidase-like sequences [49 50] mito-chondrial HCCS is characterized by a higher specificityas it does not recognize bacterial apoCyt sequences Theseobservations prompted the investigation of the recognitionprocess between apoCyt and HCCS Recently by using arecombinant yeast HCCS and chimerical apoCyt sequencesexpressed in E coli it was possible to conclude that a crucialrecognition determinant is represented by the N-terminalregion of apoCyt containing the heme-binding motif [35]notice that in the context of System I the same N-terminalregion of the Cyt c sequence has been recently identified to beimportant for the recognition by apoCcmE (see Section 26)Over and above the role played by the intervening residuesin the CXXCH heme-binding motif [135] it has been shownthat a conserved Phe residue occurring in the N-terminalregion before the CXXCH motif is important for HCCSrecognition [35 138] These results support the hypothesisthat this residue known to be a key determinant of Cytcfolding and stability [19 20 139] may also be crucial for Cytcmaturation by HCCS

Another relevant question concerns the ability of HCCSto recognize and bind the heme molecule as the region ofthe protein responsible for heme recognition remains to beidentified Initially it was hypothesized that the recognitionof heme could be mediated by the CP motifs present in

the HCCS protein [140] these short sequences are indeedknown to bind heme in a variety of heme-containing proteins[141 142] However it has been recently shown that CPmotifsof the recombinant S cerevisiae HCCS are not necessaryfor Cyt c production in E coli [143] excluding them as keydeterminants of heme recognition

The long-awaited in vitro characterization of the recom-binant human HCCS allowed for the first time to pro-pose a molecular mechanism underlying Cytc maturationin eukaryotes which can be experimentally tested [127]According to the proposed model the human HCCS activitycan be described as a four step mechanism involving (i)heme-binding (ii) apoCyt recognition (iii) thioether bondsformation and (iv) holoCyt c release In particular theheme is proposed to play the role of a scaffolding moleculemediating the contacts between HCCS and apoCyt Muta-genesis experiments carried out on the recombinant HCCSstrongly suggest that heme-binding (Step 1) depends on thepresence of a specific His residue (His154) acting as anaxial ligand to ferrous heme Residues present at the N-terminus of apoCyt mediate the recognition with HCCS(Step 2) as discussed above it is known that this regionforms structurally conserved 120572-helix in the fold of all c-type cytochromes Unfortunately no information is availableup to now concerning the region of HCCS involved in therecognition and binding to the N-terminal region of apoCytCoordination of the heme iron by the His residue of theapoCyt heme-binding motif CXXCH provides the secondaxial ligand to the heme iron and is probably importantfor the correct positioning of the two apoCyt Cys residuesand formation of the thioether bonds (Step 3) The finalrelease of functional Cyt c (Step 4) clearly requires thedisplacement of the His154Fe2+ coordination bond sucha displacement is probably mediated by formation of thecoordination bond with the Cyt c conserved Met residueandor by the simultaneous folding of Cyt c Again it isinteresting to note that this last hypothesis is in accordancewith in vitro folding studies on c-type cytochromes thathighlighted the late formation of the Met-Fe coordinationduring Cyt c folding [144 145]

Acknowledgments

The author thanks A Di Matteo and E Di Silvio for fruitfuldiscussions and for help in preparing this paper This work issupported by grants from the University of Rome ldquoSapienzardquo(C26A11MBXJ and C26A13MWF4)

References

[1] M G Galinato J G Kleingardner S E Bowman et alldquoHeme-protein vibrational couplings in cytochrome c providea dynamic link that connects the heme-iron and the proteinsurfacerdquo Proceedings of the National Academy of Sciences vol109 no 23 pp 8896ndash8900 2012

[2] H B Gray and J R Winkler ldquoElectron flow through metal-loproteinsrdquo Biochimica et Biophysica Acta vol 1797 no 9 pp1563ndash1572 2010

Scientifica 13

[3] P Ascenzi R Santucci M Coletta and F PolticellildquoCytochromes reactivity of the ldquodark siderdquo of the hemerdquoBiophysical Chemistry vol 152 no 1ndash3 pp 21ndash27 2010

[4] S Yamada N D Bouley Ford G E Keller W C Ford HB Gray and J R Winkler ldquoSnapshots of a protein foldingintermediaterdquo Proceedings of the National Academy of Sciencesvol 110 no 5 pp 1606ndash1610 2013

[5] P Weinkam J Zimmermann F E Romesberg and P GWolynes ldquoThe folding energy landscape and free energy excita-tions of cytochrome crdquo Accounts of Chemical Research vol 43no 5 pp 652ndash660 2010

[6] M Yamanaka M Masanari and Y Sambongi ldquoConfermentof folding ability to a naturally unfolded apocytochrome cthrough introduction of hydrophobic amino acid residuesrdquoBiochemistry vol 50 no 12 pp 2313ndash2320 2011

[7] G W Moore and G W Pettigrew Cytochrome C EvolutionaryStructural and Physicochemical Aspects Springer Berlin Ger-many 1990

[8] I Bertini G Cavallaro and A Rosato ldquoCytochrome c occur-rence and functionsrdquo Chemical Reviews vol 106 no 1 pp 90ndash115 2006

[9] D A Pearce and F Sherman ldquoDegradation of cytochromeoxidase subunits in mutants of yeast lacking cytochrome c andsuppression of the degradation by mutation of yme1rdquo Journal ofBiological Chemistry vol 270 no 36 pp 20879ndash20882 1995

[10] G Silkstone S M Kapetanaki I Husu M H Vos and MT Wilson ldquoNitric oxide binding to the cardiolipin complex offerric cytochrome crdquo Biochemistry vol 51 no 34 pp 6760ndash6766 2012

[11] M Giorgio E Migliaccio F Orsini et al ldquoElectron transferbetween cytochrome c and p66Shc generates reactive oxygenspecies that trigger mitochondrial apoptosisrdquo Cell vol 122 no2 pp 221ndash233 2005

[12] S M Kilbride and J H Prehn ldquoCentral roles of apoptoticproteins in mitochondrial functionrdquo Oncogene vol 32 no 22pp 2703ndash2711 2013

[13] T Noll and G Noll ldquoStrategies for ldquowiringrdquo redox-activeproteins to electrodes and applications in biosensors biofuelcells and nanotechnologyrdquo Chemical Society Reviews vol 40no 7 pp 3564ndash3576 2011

[14] G Layer J Reichelt D Jahn and D W Heinz ldquoStructure andfunction of enzymes in heme biosynthesisrdquo Protein Science vol19 no 6 pp 1137ndash1161 2010

[15] S Severance and IHamza ldquoTrafficking of heme and porphyrinsin metazoardquo Chemical Reviews vol 109 no 10 pp 4596ndash46162009

[16] P Hamel V Corvest P Giege and G Bonnard ldquoBiochemi-cal requirements for the maturation of mitochondrial c-typecytochromesrdquo Biochimica et Biophysica Acta vol 1793 no 1 pp125ndash138 2009

[17] W R Fisher H Taniuchi and C B Anfinsen ldquoOn the role ofheme in the formation of the structure of cytochrome crdquo Journalof Biological Chemistry vol 248 no 9 pp 3188ndash3195 1973

[18] M Yamanaka H Mita Y Yamamoto and Y Sambongi ldquoHemeis not required for aquifex aeolicus cytochrome c555 polypep-tide foldingrdquoBioscience Biotechnology andBiochemistry vol 73no 9 pp 2022ndash2025 2009

[19] C Travaglini-Allocatelli S Gianni andMBrunori ldquoA commonfolding mechanism in the cytochrome c familyrdquo Trends inBiochemical Sciences vol 29 no 10 pp 535ndash541 2004

[20] A Borgia D Bonivento C Travaglini-Allocatelli A DiMatteoand M Brunori ldquoUnveiling a hidden folding intermediate in c-type cytochromes by protein engineeringrdquo Journal of BiologicalChemistry vol 281 no 14 pp 9331ndash9336 2006

[21] E Enggist L Thony-Meyer P Guntert and K PervushinldquoNMR structure of the heme chaperone CcmE reveals a novelfunctional motifrdquo Structure vol 10 no 11 pp 1551ndash1557 2002

[22] A di Matteo N Calosci S Gianni P Jemth M Brunori and CTravaglini-Allocatelli ldquoStructural and functional characteriza-tion of CcmG from pseudomonas aeruginosa a key componentof the bacterial cytochrome c maturation apparatusrdquo Proteinsvol 78 no 10 pp 2213ndash2221 2010

[23] A diMatteo S GianniM E Schinina et al ldquoA strategic proteinin cytochrome c maturation three-dimensional structure ofCcmH and binding to apocytochrome crdquo Journal of BiologicalChemistry vol 282 no 37 pp 27012ndash27019 2007

[24] A Crow R M Acheson N E Le Brun and A OubrieldquoStructural basis of redox-coupled protein substrate selectionby the cytochrome c biosynthesis protein ResArdquo Journal ofBiological Chemistry vol 279 no 22 pp 23654ndash23660 2004

[25] E di Silvio A di Matteo and C Travaglini-Allocatelli ldquoTheRedox pathway of Pseudomonas aeruginosa cytochrome cbiogenesisrdquo Journal of Proteins and Proteomics vol 3 no 1 pp1ndash7 2012

[26] LThony-Meyer andP Kunzler ldquoTranslocation to the periplasmand signal sequence cleavage of preapocytochrome c depend onsec and lep but not on the ccmgene productsrdquoEuropean Journalof Biochemistry vol 246 no 3 pp 794ndash799 1997

[27] S J Facey and A Kuhn ldquoBiogenesis of bacterial inner-membrane proteinsrdquo Cellular and Molecular Life Sciences vol67 no 14 pp 2343ndash2362 2010

[28] K Diekert A I de Kroon U Ahting et al ldquoApocytochromec requires the TOM complex for translocation across themitochondrial outer membranerdquo The EMBO Journal vol 20no 20 pp 5626ndash5635 2001

[29] N Wiedemann V Kozjak T Prinz et al ldquoBiogenesis of yeastmitochondrial cytochrome c a unique relationship to the TOMmachineryrdquo Journal ofMolecular Biology vol 327 no 2 pp 465ndash474 2003

[30] F-U Hartl N Pfanner and W Neupert ldquoTranslocation inter-mediates on the import pathway of proteins intomitochondriardquoBiochemical Society Transactions vol 15 no 1 pp 95ndash97 1987

[31] B S Glick A Brandt K Cunningham SMuller R L Hallbergand G Schatz ldquoCytochromes c1 and b2 are sorted to theintermembrane space of yeast mitochondria by a stop-transfermechanismrdquo Cell vol 69 no 5 pp 809ndash822 1992

[32] G Howe and S Merchant ldquoRole of heme in the biosynthesis ofcytochrome c6rdquo Journal of Biological Chemistry vol 269 no 8pp 5824ndash5832 1994

[33] S S Nakamoto P Hamel and S Merchant ldquoAssembly ofchloroplast cytochromes b and crdquo Biochimie vol 82 no 6-7 pp603ndash614 2000

[34] C Sanders S Turkarslan D-W Lee and F DaldalldquoCytochrome c biogenesis the Ccm systemrdquo Trends inMicrobiology vol 18 no 6 pp 266ndash274 2010

[35] J M Stevens D A Mavridou R Hamer P Kritsiligkou A DGoddard and S J Ferguson ldquoCytochrome c biogenesis systemIrdquoThe FEBS Journal vol 278 no 22 pp 4170ndash4178 2011

[36] R G Kranz C Richard-Fogal J-S Taylor and E R FrawleyldquoCytochrome c biogenesis mechanisms for covalent modifica-tions and trafficking of heme and for heme-iron redox controlrdquo

14 Scientifica

Microbiology and Molecular Biology Reviews vol 73 no 3 pp510ndash528 2009

[37] J W A Allen ldquoCytochrome c biogenesis in mitochondriamdashsystems III and VrdquoThe FEBS Journal vol 278 no 22 pp 4198ndash4216 2011

[38] D A Mavridou S J Ferguson and J M Stevens ldquoCytochromec assemblyrdquo IUBMB Life vol 65 no 3 pp 209ndash216 2013

[39] I Bertini G Cavallaro and A Rosato ldquoEvolution ofmitochondrial-type cytochrome c domains and of theprotein machinery for their assemblyrdquo Journal of InorganicBiochemistry vol 101 no 11-12 pp 1798ndash1811 2007

[40] B S Goldman and R G Kranz ldquoEvolution and horizontaltransfer of an entire biosynthetic pathway for cytochromec biogenesis helicobacter deinococcus archae and morerdquoMolecular Microbiology vol 27 no 4 pp 871ndash873 1998

[41] C L Richard-Fogal E R Frawley E R Bonner H Zhu BSan Francisco and R G Kranz ldquoA conserved haem redoxand trafficking pathway for cofactor attachmentrdquo The EMBOJournal vol 28 no 16 pp 2349ndash2359 2009

[42] L Thony-Meyer F Fischer P Kunzler D Ritz and H Hen-necke ldquoEscherichia coli genes required for cytochrome cmaturationrdquo Journal of Bacteriology vol 177 no 15 pp 4321ndash4326 1995

[43] C S Beckett J A Loughman K A Karberg G M DonatoW E Goldman and R G Kranz ldquoFour genes are required forthe system II cytochrome c biogenesis pathway in Bordetellapertussis a unique bacterial modelrdquo Molecular Microbiologyvol 38 no 3 pp 465ndash481 2000

[44] N E Le Brun J Bengtsson and L Hederstedt ldquoGenes requiredfor cytochrome c synthesis in Bacillus subtilisrdquo MolecularMicrobiology vol 36 no 3 pp 638ndash650 2000

[45] P Giege J M Grienenberger and G Bonnard ldquoCytochromec biogenesis in mitochondriardquo Mitochondrion vol 8 no 1 pp61ndash73 2008

[46] R E Feissner C L Richard-Fogal E R Frawley J A Lough-man KW Earley and R G Kranz ldquoRecombinant cytochromesc biogenesis systems I and II and analysis of haem deliverypathways in Escherichia colirdquo Molecular Microbiology vol 60no 3 pp 563ndash577 2006

[47] N P Cianciotto P Cornelis and C Baysse ldquoImpact of thebacterial type I cytochrome c maturation system on differentbiological processesrdquoMolecular Microbiology vol 56 no 6 pp1408ndash1415 2005

[48] E Arslan H Schulz R Zufferey P Kunzler and L Thony-Meyer ldquoOverproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in Escherichiacolirdquo Biochemical and Biophysical Research Communicationsvol 251 no 3 pp 744ndash747 1998

[49] C Sanders and H Lill ldquoExpression of prokaryotic and eukary-otic cytochromes c in Escherichia colirdquoBiochimica et BiophysicaActa vol 1459 no 1 pp 131ndash138 2000

[50] M Braun and L Thony-Meyer ldquoBiosynthesis of artificialmicroperoxidases by exploiting the secretion and cytochromec maturation apparatuses of Escherichia colirdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 35 pp 12830ndash12835 2004

[51] B Baert C Baysse S Matthijs and P Cornelis ldquoMultiplephenotypic alterations caused by a c-type cytochrome mat-uration ccmC gene mutation in Pseudomonas aeruginosardquoMicrobiology vol 154 no 1 pp 127ndash138 2008

[52] S Turkarslan C Sanders and F Daldal ldquoExtracytoplasmicprosthetic group ligation to apoproteins maturation of c-typecytochromesrdquo Molecular Microbiology vol 60 no 3 pp 537ndash541 2006

[53] P M Jones and A M George ldquoThe ABC transporter structureand mechanism perspectives on recent researchrdquo Cellular andMolecular Life Sciences vol 61 no 6 pp 682ndash699 2004

[54] O Christensen E M Harvat L Thony-Meyer S J Fergusonand J M Stevens ldquoLoss of ATP hydrolysis activity by CcmABresults in loss of c-type cytochrome synthesis and incompleteprocessing ofCcmErdquoTheFEBS Journal vol 274 no 9 pp 2322ndash2332 2007

[55] J-H Lee E M Harvat J M Stevens S J Ferguson and M HSaier Jr ldquoEvolutionary origins of members of a superfamily ofintegralmembrane cytochrome c biogenesis proteinsrdquoBiochim-ica et Biophysica Acta vol 1768 no 9 pp 2164ndash2181 2007

[56] D L Beckman D R Trawick and R G Kranz ldquoBacterialcytochromes c biogenesisrdquo Genes and Development vol 6 no2 pp 268ndash283 1992

[57] H Schulz R A Fabianek E C Pellicioli H Hennecke andL Thony-Meyer ldquoHeme transfer to the heme chaperone CcmEduring cytochrome c maturation requires the CcmC proteinwhich may function independently of the ABC-transporterCcmABrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 96 no 11 pp 6462ndash6467 1999

[58] Q Ren and L Thony-Meyer ldquoPhysical Interaction ofCcmC with Heme and the Heme Chaperone CcmE duringCytochrome cMaturationrdquo Journal of Biological Chemistry vol276 no 35 pp 32591ndash32596 2001

[59] C Richard-Fogal and R G Kranz ldquoThe CcmChemeCcmEcomplex in heme trafficking and cytochrome c biosynthesisrdquoJournal of Molecular Biology vol 401 no 3 pp 350ndash362 2010

[60] V K Viswanathan S Kurtz L L Pedersen et al ldquoThecytochrome C maturation locus of Legionella pneumophilapromotes iron assimilation and intracellular infection andcontains a strain-specific insertion sequence elementrdquo Infectionand Immunity vol 70 no 4 pp 1842ndash1852 2002

[61] U Ahuja and L Thony-Meyer ldquoCcmD is involved in complexformation between CcmC and the heme chaperone CcmE dur-ing cytochrome c maturationrdquo Journal of Biological Chemistryvol 280 no 1 pp 236ndash243 2005

[62] C L Richard-Fogal E R Frawley and R G Kranz ldquoTopologyand function of CcmD in cytochrome Cmaturationrdquo Journal ofBacteriology vol 190 no 10 pp 3489ndash3493 2008

[63] H Schulz H Hennecke and L Thony-Meyer ldquoPrototype ofa heme chaperone essential for cytochrome c maturationrdquoScience vol 281 no 5380 pp 1197ndash1200 1998

[64] F Arnesano L Banci P D Barker et al ldquoSolution structure andcharacterization of the heme chaperone CcmErdquo Biochemistryvol 41 no 46 pp 13587ndash13594 2002

[65] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[66] L Thony-Meyer ldquoA heme chaperone for cytochrome c biosyn-thesisrdquo Biochemistry vol 42 no 45 pp 13099ndash13105 2003

[67] E M Harvat C Redfield J M Stevens and S J FergusonldquoProbing the heme-binding site of the cytochrome cmaturationprotein CcmErdquoBiochemistry vol 48 no 8 pp 1820ndash1828 2009

[68] D Lee K Pervushin D Bischof M Braun and L Thony-Meyer ldquoUnusual heme-histidine bond in the active site of a

Scientifica 15

chaperonerdquo Journal of the American Chemical Society vol 127no 11 pp 3716ndash3717 2005

[69] A D Goddard J M Stevens F Rao et al ldquoc-Type cytochromebiogenesis can occur via a natural Ccm system lacking CcmHCcmG and the heme-binding histidine of CcmErdquo Journal ofBiological Chemistry vol 285 no 30 pp 22882ndash22889 2010

[70] J M Aramini K Hamilton P Rossi et al ldquoSolution NMRstructure backbone dynamics and heme-binding propertiesof a novel cytochrome c maturation protein CcmE fromDesulfovibrio vulgarisrdquo Biochemistry vol 51 no 18 pp 3705ndash3707 2012

[71] T Uchida J M Stevens O Daltrop et al ldquoThe interactionof covalently bound heme with the cytochrome c maturationprotein CcmErdquo Journal of Biological Chemistry vol 279 no 50pp 51981ndash51988 2004

[72] I Garcıa-Rubio M Braun I Gromov L Thony-Meyer andA Schweiger ldquoAxial coordination of heme in ferric CcmEchaperone characterized by EPR spectroscopyrdquo BiophysicalJournal vol 92 no 4 pp 1361ndash1373 2007

[73] L Thony-Meyer ldquoHaem-polypeptide interactions duringcytochrome c maturationrdquo Biochimica et Biophysica Acta vol1459 no 2-3 pp 316ndash324 2000

[74] J A Keightley D Sanders T R Todaro A Pastuszyn and J AFee ldquoCloning expression in Escherichia coli of the cytochromec552 gene from Thermus thermophilus HB8 Evidence forgenetic linkage to an ATP- binding cassette protein and initialcharacterization of the cycA gene productsrdquo Journal of Biologi-cal Chemistry vol 273 no 20 pp 12006ndash12016 1998

[75] E M Monika B S Goldman D L Beckman and R GKranz ldquoA thioreduction pathway tethered to the membranefor periplasmic cytochromes c biogenesis In vitro and in vivostudiesrdquo Journal of Molecular Biology vol 271 no 5 pp 679ndash692 1997

[76] M Throne-Holst L Thony-Meyer and L HederstedtldquoEscherichia coli ccm in-frame deletion mutants can produceperiplasmic cytochrome b but not cytochrome crdquo FEBS Lettersvol 410 no 2-3 pp 351ndash355 1997

[77] U Ahuja and L Thony-Meyer ldquoDynamic features of a hemedelivery system for cytochrome c maturationrdquo Journal of Bio-logical Chemistry vol 278 no 52 pp 52061ndash52070 2003

[78] J W A Allen P D Barker O Daltrop et al ldquoWhy isnrsquot ldquostan-dardrdquo heme good enough for c-type and di-type cytochromesrdquoDalton Transactions no 21 pp 3410ndash3418 2005

[79] K Inaba and K Ito ldquoStructure and mechanisms of the DsbB-DsbA disulfide bond generation machinerdquo Biochimica et Bio-physica Acta vol 1783 no 4 pp 520ndash529 2008

[80] H Kadokura F Katzen and J Beckwith ldquoProtein disulfidebond formation in prokaryotesrdquoAnnual Review of Biochemistryvol 72 pp 111ndash135 2003

[81] S R Shouldice B Heras P M Walden M Totsika M ASchembri and J L Martin ldquoStructure and function of DsbA akey bacterial oxidative folding catalystrdquoAntioxidants and RedoxSignaling vol 14 no 9 pp 1729ndash1760 2011

[82] R Metheringham L Griffiths H Crooke S Forsythe and JCole ldquoAn essential role for DsbA in cytochrome c synthesis andformate-dependent nitrite reduction by Escherichia coli K-12rdquoArchives of Microbiology vol 164 no 4 pp 301ndash307 1995

[83] Y Sambongi and S J Ferguson ldquoMutants of Escherichia colilacking disulphide oxidoreductases DsbA and DsbB cannotsynthesise an exogenous monohaem c-type cytochrome exceptin the presence of disulphide compoundsrdquo FEBS Letters vol398 no 2-3 pp 265ndash268 1996

[84] D AMavridou S J Ferguson and JM Stevens ldquoThe interplaybetween the disulfide bond formation pathway and cytochromec maturation in Escherichia colirdquo FEBS Letters vol 586 no 12pp 1702ndash1707 2012

[85] C U Stirnimann A Rozhkova U Grauschopf M G GrutterR Glockshuber and G Capitani ldquoStructural basis and kineticsof DsbD-dependent cytochrome c maturationrdquo Structure vol13 no 7 pp 985ndash993 2005

[86] N Ouyang Y-G Gao H-Y Hu and Z-X Xia ldquoCrystalstructures of E coli CcmGand itsmutants reveal key roles of theN-terminal120573-sheet and the fingerprint regionrdquoProteins vol 65no 4 pp 1021ndash1031 2006

[87] M A Edeling L W Guddat R A Fabianek et al ldquoCrys-tallization and preliminary diffraction studies of native andselenomethionine CcmG (CycY DsbE)rdquoActa CrystallographicaD vol 57 no 9 pp 1293ndash1295 2001

[88] R A Fabianek M Huber-Wunderlich R Glockshuber PKunzler H Hennecke and L Thony-Meyer ldquoCharacterizationof the Bradyrhizobium japonicum CycY protein a membrane-anchored periplasmic thioredoxin that may play a role as areductant in the biogenesis of c-type cytochromesrdquo Journal ofBiological Chemistry vol 272 no 7 pp 4467ndash4473 1997

[89] G B Kallis and A Holmgren ldquoDifferential reactivity of thefunctional sulfhydryl groups of cysteine-32 and cysteine-32present in the reduced form of thioredoxin from Escherichiacolirdquo Journal of Biological Chemistry vol 255 no 21 pp 10261ndash10265 1980

[90] P T Chivers andR T Raines ldquoGeneral acidbase catalysis in theactive site of Escherichia coli thioredoxinrdquoBiochemistry vol 36no 50 pp 15810ndash15816 1997

[91] X M Zheng J Hong H Y Li D H Lin and H Y HuldquoBiochemical properties and catalytic domain structure of theCcmH protein from Escherichia colirdquo Biochim Biophys Actavol 1824 no 12 pp 1394ndash1400 2012

[92] UAhujaA Rozhkova RGlockshuber LThony-Meyer andOEinsle ldquoHelix swapping leads to dimerization of the N-terminaldomain of the c-type cytochrome maturation protein CcmHfrom Escherichia colirdquo FEBS Letters vol 582 no 18 pp 2779ndash2786 2008

[93] R A Fabianek T Hofer and L Thony-Meyer ldquoCharacteriza-tion of the Escherichia coli CcmH protein reveals new insightsinto the redox pathway required for cytochrome c maturationrdquoArchives of Microbiology vol 171 no 2 pp 92ndash100 1999

[94] E Reid J Cole and D J Eaves ldquoThe Escherichia coli CcmGprotein fulfils a specific role in cytochrome c assemblyrdquo Bio-chemical Journal vol 355 no 1 pp 51ndash58 2001

[95] Q Ren U Ahuja and LThony-Meyer ldquoA bacterial cytochromec heme lyase CcmF forms a complex with the heme chaperoneCcmE and CcmH but not with apocytochrome crdquo Journal ofBiological Chemistry vol 277 no 10 pp 7657ndash7663 2002

[96] E H Meyer P Giege E Gelhaye et al ldquoAtCCMH an essentialcomponent of the c-type cytochrome maturation pathway inArabidopsis mitochondria interacts with apocytochrome crdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 44 pp 16113ndash16118 2005

[97] C Rios-Velazquez R Coller and T J Donohue ldquoFeatures ofRhodobacter sphaeroides CcmFHrdquo Journal of Bacteriology vol185 no 2 pp 422ndash431 2003

[98] C Sanders M Deshmukh D Astor R G Kranz and F DaldalldquoOverproduction of CcmG and CcmFHRc fully suppresses thec-type cytochrome biogenesis defect of Rhodobacter capsulatus

16 Scientifica

CcmI-null mutantsrdquo Journal of Bacteriology vol 187 no 12 pp4245ndash4256 2005

[99] A F Verissimo H Yang X Wu C Sanders and F DaldalldquoCcmI subunit of CcmFHI heme ligation complex functionsas an apocytochrome c chaperone during c-type cytochromematurationrdquo Journal of Biological Chemistry vol 286 no 47 pp40452ndash40463 2011

[100] K Bryson L J McGuffin R L Marsden J J Ward J SSodhi and D T Jones ldquoProtein structure prediction servers atUniversity College Londonrdquo Nucleic Acids Research vol 33 no2 pp W36ndashW38 2005

[101] D T Jones ldquoProtein secondary structure prediction basedon position-specific scoring matricesrdquo Journal of MolecularBiology vol 292 no 2 pp 195ndash202 1999

[102] C Sanders C Boulay and F Daldal ldquoMembrane-spanning andperiplasmic segments of CcmI have distinct functions duringcytochrome c biogenesis in Rhodobacter capsulatusrdquo Journal ofBacteriology vol 189 no 3 pp 789ndash800 2007

[103] D Han K Kim J Oh J Park and Y Kim ldquoTPR domainof NrfG mediates complex formation between heme lyaseand formate-dependent nitrite reductase in Escherichia coliO157H7rdquo Proteins vol 70 no 3 pp 900ndash914 2008

[104] D J Eaves J Grove W Staudenmann et al ldquoInvolvement ofproducts of the nrfEFG genes in the covalent attachment ofhaem c to a novel cysteine-lysine motif in the cytochrome c552nitrite reductase from Escherichia colirdquo Molecular Microbiol-ogy vol 28 no 1 pp 205ndash216 1998

[105] J Nilsson B Persson and G Von Heijne ldquoConsensus predic-tions of membrane protein topologyrdquo FEBS Letters vol 486 no3 pp 267ndash269 2000

[106] E di Silvio A di Matteo F Malatesta and C Travaglini-Allocatelli ldquoRecognition and binding of apocytochrome c toP aeruginosa CcmI a component of cytochrome c maturationmachineryrdquo Biochim Biophys Acta vol 1834 no 8 pp 1554ndash1561 2013

[107] N Zeytuni and R Zarivach ldquoStructural and functional dis-cussion of the tetra-trico-peptide repeat a protein interactionmodulerdquo Structure vol 20 no 3 pp 397ndash405 2012

[108] C Sanders S Turkarslan D-W Lee O Onder R G Kranz andF Daldal ldquoThe cytochrome c maturation components CcmFCcmH and CcmI form a membrane-integral multisubunitheme ligation complexrdquo Journal of Biological Chemistry vol283 no 44 pp 29715ndash29722 2008

[109] A F Verissimo M A Mohtar and F Daldal ldquoThe hemechaperone ApoCcmE forms a ternary complex with CcmI andapocytochrome crdquoThe Journal of Biological Chemistry vol 288no 9 pp 6272ndash6283 2013

[110] P D Barker J C Ferrer M Mylrajan et al ldquoTransmutation of aheme proteinrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 90 no 14 pp 6542ndash65461993

[111] J Simon and L Hederstedt ldquoComposition and function ofcytochrome c biogenesis system IIrdquoThe FEBS Journal vol 278no 22 pp 4179ndash4188 2011

[112] T R H M Kouwen and J M van Dijl ldquoInterchangeablemodules in bacterial thiol-disulfide exchange pathwaysrdquo Trendsin Microbiology vol 17 no 1 pp 6ndash12 2009

[113] A Crow A Lewin O Hecht et al ldquoCrystal structure andbiophysical properties of Bacillus subtilis BdbD An oxidizingthioldisulfide oxidoreductase containing a novel metal siterdquoJournal of Biological Chemistry vol 284 no 35 pp 23719ndash23733 2009

[114] M C Moller and L Hederstedt ldquoExtracytoplasmic processesimpaired by inactivation of trxA (thioredoxin gene) in Bacillussubtilisrdquo Journal of Bacteriology vol 190 no 13 pp 4660ndash46652008

[115] A Lewin A Crow A Oubrie and N E Le Brun ldquoMolecularbasis for specificity of the extracytoplasmic thioredoxin ResArdquoJournal of Biological Chemistry vol 281 no 46 pp 35467ndash35477 2006

[116] C L Colbert QWu P J A Erbel K H Gardner and J Deisen-hofer ldquoMechanism of substrate specificity in Bacillus subtilisResA a thioredoxin-like protein involved in cytochrome cmaturationrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 103 no 12 pp 4410ndash44152006

[117] UAhuja P Kjelgaard B L Schulz LThony-Meyer andLHed-erstedt ldquoHaem-delivery proteins in cytochrome c maturationsystem IIrdquoMolecular Microbiology vol 73 no 6 pp 1058ndash10712009

[118] M L D Page P P Hamel S T Gabilly et al ldquoA homologof prokaryotic thiol disulfide transporter CcdA is required forthe assembly of the cytochrome b6f complex in Arabidopsischloroplastsrdquo Journal of Biological Chemistry vol 279 no 31pp 32474ndash32482 2004

[119] A Rozhkova C U Stirnimann P Frei et al ldquoStructural basisand kinetics of inter- and intramolecular disulfide exchange inthe redox catalyst DsbDrdquoThe EMBO Journal vol 23 no 8 pp1709ndash1719 2004

[120] T Schiott M Throne-Holst and L Hederstedt ldquoBacillussubtilis CcdA-defective mutants are blocked in a late step ofcytochrome C biogenesisrdquo Journal of Bacteriology vol 179 no14 pp 4523ndash4529 1997

[121] R E Feissner C S Beckett J A Loughman and R G KranzldquoMutations in cytochrome assembly and periplasmic redoxpathways in Bordetella pertussisrdquo Journal of Bacteriology vol187 no 12 pp 3941ndash3949 2005

[122] L S ErlendssonMMoller and L Hederstedt ldquoBacillus subtilisStoA is a thiol-disulfide oxidoreductase important for sporecortex synthesisrdquo Journal of Bacteriology vol 186 no 18 pp6230ndash6238 2004

[123] L S Erlendsson R M Acheson L Hederstedt and N E LeBrun ldquoBacillus subtilis ResA is a thiol-disulfide oxidoreductaseinvolved in cytochrome c synthesisrdquo Journal of BiologicalChemistry vol 278 no 20 pp 17852ndash17858 2003

[124] L S Erlendsson and L Hederstedt ldquoMutations in the thiol-disulfide oxidoreductases BdbC and BdbD can suppresscytochrome c deficiency of CcdA-defective Bacillus subtiliscellsrdquo Journal of Bacteriology vol 184 no 5 pp 1423ndash1429 2002

[125] M E Dumont J F Ernst D M Hampsey and F ShermanldquoIdentification and sequence of the gene encoding cytochromec heme lyase in the yeast Saccharomyces cerevisiaerdquoThe EMBOJournal vol 6 no 1 pp 235ndash241 1987

[126] M E Dumont J F Ernst and F Sherman ldquoCoupling of hemeattachment to import of cytochrome c into yeast mitochondriaStudies with heme lyase-deficient mitochondria and alteredapocytochromes crdquo Journal of Biological Chemistry vol 263 no31 pp 15928ndash15937 1988

[127] B San Francisco E C Bretsnyder and R G Kranz ldquoHumanmitochondrial holocytochrome c synthasersquos hemebindingmat-uration determinants and complex formationwith cytochromecrdquo Proceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 9 pp E788ndashE797 2012

Scientifica 17

[128] L Schaefer A Ballabio and H Y Zoghbi ldquoCloning andcharacterization of a putative human holocytochrome c-typesynthetase gene (HCCS) isolated from the critical region formicrophthalmia with linear skin defects (MLS)rdquoGenomics vol34 no 2 pp 166ndash172 1996

[129] S K Prakash T A Cormier A E McCall et al ldquoLoss ofholocytochrome c-type synthetase causes the male lethality ofX-linked dominant microphthalmia with linear skin defects(MLS) syndromerdquo Human Molecular Genetics vol 11 no 25pp 3237ndash3248 2002

[130] I Wimplinger M Morleo G Rosenberger et al ldquoMutations ofthe mitochondrial holocytochrome c-type synthase in X-linkeddominant microphthalmia with linear skin defects syndromerdquoTheAmerican Journal of HumanGenetics vol 79 no 5 pp 878ndash889 2006

[131] S Kiryu-Seo KGamo T Tachibana K Tanaka andHKiyamaldquoUnique anti-apoptotic activity of EAAC1 in injured motorneuronsrdquoTheEMBO Journal vol 25 no 14 pp 3411ndash3421 2006

[132] R Lill R A Stuart M E Drygas F E Nargang and W Neu-pert ldquoImport of cytochrome c heme lyase into mitochondriaa novel pathway into the intermembrane spacerdquo The EMBOJournal vol 11 no 2 pp 449ndash456 1992

[133] H Steiner A Zollner A Haid W Neupert and R LillldquoBiogenesis of mitochondrial heme lyases in yeast Importand folding in the intermembrane spacerdquo Journal of BiologicalChemistry vol 270 no 39 pp 22842ndash22849 1995

[134] A Zollner G Rodel and A Haid ldquoMolecular cloning andcharacterization of the Saccharomyces cerevisiae CYT2 geneencoding cytochrome-c1-heme lyaserdquo European Journal of Bio-chemistry vol 207 no 3 pp 1093ndash1100 1992

[135] V Corvest D A Murrey D G Bernard D B Knaff B Guiardand P PHamel ldquoc-Type cytochrome assembly in Saccharomycescerevisiae a key residue for apocytochrome c1lyase interactionrdquoGenetics vol 186 no 2 pp 561ndash571 2010

[136] V Corvest D A Murrey M Hirasawa D B Knaff B Guiardand P P Hamel ldquoThe flavoprotein Cyc2p a mitochondrialcytochrome c assembly factor is a NAD(P)H-dependent haemreductaserdquo Molecular Microbiology vol 83 no 5 pp 968ndash9802012

[137] D G Bernard S Quevillon-Cheruel S Merchant B Guiardand P P Hamel ldquoCyc2p a membrane-bound flavopro-tein involved in the maturation of mitochondrial c-typecytochromesrdquo Journal of Biological Chemistry vol 280 no 48pp 39852ndash39859 2005

[138] J G Kleingardner and K L Bren ldquoComparing substratespecificity between cytochrome cmaturation and cytochrome cheme lyase systems for cytochrome c biogenesisrdquo Metallomicsvol 3 no 4 pp 396ndash403 2011

[139] C Travaglini-Allocatelli S Gianni VMorea A Tramontano TSoulimane and M Brunori ldquoExploring the cytochrome c fold-ing mechanism cytochrome c552 fromThermus thermophilusfolds through an on-pathway intermediaterdquo Journal of BiologicalChemistry vol 278 no 42 pp 41136ndash41140 2003

[140] L Zhang and L Guarente ldquoHeme binds to a short sequencethat serves a regulatory function in diverse proteinsrdquoTheEMBOJournal vol 14 no 2 pp 313ndash320 1995

[141] S Hira T Tomita T Matsui K Igarashi and M Ikeda-SaitoldquoBach1 a heme-dependent transcription factor reveals pres-ence of multiple heme binding sites with distinct coordinationstructurerdquo IUBMB Life vol 59 no 8-9 pp 542ndash551 2007

[142] T K Kuhl A Wiszligbrock N Goradia et al ldquoAnalysis of Fe(III)heme binding to cysteine-containing heme-regulatory motifs

in proteinsrdquo ACS Chemical Biology vol 8 no 8 pp 1785ndash17932013

[143] R L Moore J M Stevens and S J Ferguson ldquoMitochondrialcytochrome c synthase CP motifs are not necessary for hemeattachment to apocytochrome crdquo FEBS Letters vol 585 no 21pp 3415ndash3419 2011

[144] S Gianni M Brunori and C Travaglini-Allocatelli ldquoRefoldingkinetics of cytochrome c551 reveals a mechanistic differencebetween urea and guanidinerdquo Protein Science vol 10 no 8 pp1685ndash1688 2001

[145] S Gianni C Travaglini-Allocatelli F Cutruzzola M BrunoriM C R Shastry and H Roder ldquoParallel pathways incytochrome c551 foldingrdquo Journal of Molecular Biology vol 330no 5 pp 1145ndash1152 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

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Molecular Biology International

GenomicsInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

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International Journal of

Microbiology

8 Scientifica

S SSS

SH

SH

Scheme 1 Scheme 2

SH

SH

SHSHS

S

CcmG 7477

CcmG 7477

CcmH 2528

CcmH 2528

apoCytox

apoCytred

S

SHCcmG 74

77

SH

SH

HSHS

CcmG 7477

S

S

SS

3

1

2CcmG 7477

SH

SHCcmH 25

28SH

HSS S

CcmH 2528

SHS

CcmH2528

apoCytox

apoCytred

+

Figure 6 Alternative thioreduction pathways whichmay be operative in System I and hypothesized on the basis of structural and functionalcharacterization of the redox-active Ccm proteins from P aeruginosa [22 23 25] Scheme 1 is a linear redox cascade whereby CcmG is thedirect reductant of CcmH which reduces oxidized apoCyt Scheme 2 envisages a more complex scenario involving the formation of a mixed-disulfide complex between CcmH and apoCyt (Step 1) This complex is the substrate for the attack by reduced CcmG (Step 2) that liberatesreduced apoCyt The resulting disulfide bond between CcmH and CcmG is then resolved by the free Cys thiol of CcmG (probably Cys77 inPa-CcmG) Adapted from [25]

of the TRX family the N-terminal Cys is always solventexposed in CcmH proteins the arrangement of the two Cysresidues is reversed the N-terminal Cys residue is buriedwhereas the C-terminal Cys residue is solvent exposed Onthe basis of this observation it was suggested that differentfrom the canonical TRX redox mechanism CcmH proteinsperform the nucleophilic attack on the apoCyt disulfide viatheir C-terminal Cys residue [23] This mechanism which isin agreement with the mechanism proposed earlier for Ec-CcmH on the basis of mutational-complementation studies[93 94] is substantiated by the peculiar pKa values of theactive site Cys residues of Pa-CcmH which were found tobe similar for both cysteines (84 plusmn 01 and 86 plusmn 01 [23])Again this is different from what is generally observed in thecase of TRX proteins where the pKa value of the Cys residueperforming the initial nucleophilic attack is significantlylower than the pKa value of theCys residue responsible for theresolution of the intermediate mixed-disulfide It is temptingto speculate that the unusual pKa values of the Pa-CcmHactive site thiols may ensure the necessary specificity of thiscomponent of the Ccm apparatus toward the CXXCH motifof the apoCyt substrate

24 System I ApoCyt Thioreduction Pathway MechanismAlthough we know that CcmG and CcmH are the redox-active components of System I involved in the thioreductivepathway of Cyt c biogenesis not only an acceptedmechanismfor the reduction of apoCyt disulfide bond is still lackingbut also the absolute requirement of such a process is nowdebated [38 84] Focusing our attention on the reductionof the apoCyt internal disulfide at least two mechanismscan been hypothesized which involve either a linear redoxcascade of disulfide exchange reactions or a nonlinear redox

process involving transient formation of a mixed-disulfidecomplex as depicted in Figure 6 and Schemes 1 and 2respectively

Both the thiol-disulfide exchange mechanisms depictedin Figure 6 suggest that CcmH is the direct reductant ofthe apoCytc disulfide however even if immunoprecipitationexperiments failed to detect the formation of a mixed-disulfide complex between apoCyt and CcmH proteins [95]some in vitro evidence supporting the formation of sucha complex has been presented In particular it has beenshown that Rhodobacter capsulatus and Arabidopsis thalianaCcmH homologues (Rc-CcmH and At-CcmH) are able toreduce the CXXCH motif of an apoCyt-mimicking peptide[75 96] In the latter case yeast two-hybrid experimentscarried out on At-CcmH indeed revealed an interactionbetween the protein and a peptide mimicking the A thalianaCyt c sequence In the case of Pa-CcmH FRET kineticexperiments employing a Trp-containing fluorescent variantof the protein and a dansylated nonapeptide encompassingthe heme-binding motif of P aeruginosa cytochrome c551(dans-KGCVACHAI) [23] allowed to directly observe theformation of themixed-disulfide complex and tomeasure theoff-rate constant of the bound peptide The results of these invitro binding experiments allowed to calculate an equilibriumdissociation constant which combines an adequate affinity(low 120583M) with the need to release efficiently reduced apoCytto other component(s) of the System I maturase complex[23] More recently the results obtained by FRET bindingexperiments carried out with single Cys-containing mutantsof Pa-CcmH and Pa-CcmG [25] substantiated the hypothesisdepicted in Scheme 2 (Figure 6) Altogether these structuraland functional results suggest that the thioreduction pathwaymechanism leading to reduced apoCyt is better describedby Scheme 2 and that reducing equivalents might not be

Scientifica 9

transferred directly from CcmG to apoCyt as depicted inScheme 1 According to Scheme 2 reduced CcmH (a non-TRX-like thiol-oxidoreductase) specifically recognizes andreduces oxidized apoCyt via the formation of a mixed-disulfide complex which is subsequently resolved by CcmGThe resulting disulfide bond between CcmH and CcmG isthen resolved by the free Cys thiol of CcmG (probably Cys77in Pa-CcmG)

However further in vitro experiments with CcmH andapoCyt single Cys-containing mutants are needed to unveilthe details of the thioreduction of oxidized apoCyt by CcmHIn particular it would be crucial to identify the Cys residueof apoCyt that remains free in the apoCyt-CcmH mixed-disulfide complex intermediate (see Scheme 2 and Section 26below) and available to thioether bond formation with oneof the heme vinyl groups Clearly structure determinationof the trapped mixed-disulfide complexes between CcmHCcmG and apoCyt (or apoCyt peptides) would providekey information for our understanding of this specializedthioreduction pathway mechanism

25 System I ApoCyt Chaperoning and Heme AttachmentComponents The reduced heme-binding motif of apoCyt isnow available to the heme ligation reaction However themolecularmechanismwhereby the Ccmmachinery catalyzesor promotes the formation of the heme-apoCyt covalentbonds is still largely obscure representing themost importantgoal in the field Past observations and recent experimentssuggest that CcmF and CcmI possibly together with CcmHare involved in these final steps [16 34 36]

CcmF is a large integral membrane protein of more than600 residues belonging to the heme handling protein family(HHP [55]) and predicted to contain 10ndash15 TM helices (notethat some discrepancy exists as to the number of TM helicespredicted by computer programs and those predicted onthe basis of phoA and lacZ fusion experiments [40 97])a conserved WWD domain and a larger domain devoidof any recognizable sequence features both exposed to theperiplasm Only recently E coli CcmF (Ec-CcmF) has beenoverexpressed solubilized from the membrane fraction andspectroscopically characterized in vitro [36 41] Surprisinglythe biochemical characterization of recombinant Ec-CcmFallowed to show that the purified protein contains heme b ascofactor in a 1 1 stoichiometry this observation led to thehypothesis that in addition to its heme lyase function Ec-CcmF may act as a heme oxidoreductase In particular it ispossible that the heme b of Ec-CcmF may act as a reductantfor the oxidized iron of the heme bound to CcmE [41]indeed the in vitro reduction of Ec-CcmF by quinones hasbeen experimentally observed strengthening the hypothesisabout the quinolhemeoxidoreductase function of this elusiveproteinThe structuralmodel proposed for Ec-CcmFpredicts13 TMhelices and notably the location of the four completelyconserved His residues according to the model two of them(His173 andHis303) are located in periplasmic exposed loopsnext to the conserved WWD domain which is believedto provide a platform for the heme bound to holoCcmEwhile His261 is located in one of the TM helices and it is

predicted to act as an axial ligand to the heme b of Ec-CcmFthe other conserved His residue (H491) could provide thesecond axial coordination bond to the heme although thishas not been experimentally addressed This model of Ec-CcmF therefore envisages that this large membrane proteinis characterized by two heme-binding sites one of themis embedded in the membrane and coordinates a heme bprosthetic group necessary to reduce the CcmE-bound hemehosted in the second heme-binding site and constituted by itsWWD domain

It is interesting to note that in plants mitochondria theCcmF ortholog appears to be split into three different pro-teins (At-CcmFN1 At-CcmFN2 and At-CcmFC) possiblyinteracting each other [16] Since each of these proteins issimilar to the corresponding domain in the bacterial CcmFortholog this observation may provide useful informationin the design of engineered fragments of bacterial CcmFproteins amenable to structural analyses

The other System I component which is generallybelieved to be involved in the final steps of Cyt c maturationis CcmI As stated above the ccmI gene is present only insome Ccm operons while in others the corresponding ORFis present within the ccmH gene (as in E coli)The functionalrole of CcmI in Cytc biogenesis is revealed by geneticstudies showing that in R capsulatus and B japonicuminactivation of the ccmI gene leads to inability to synthesizefunctional c-type cytochromes [98 99] In R capsulatus andP aeruginosa the CcmI protein (Rc-CcmI and Pa-CcmIresp) can be described as being composed of two domainsstarting from the N-terminus a first domain composed oftwo TM helices connected by a short cytoplasmic regionand a large periplasmic domain Structural variations maybe observed among CcmI members from different bacteriaindeed multiple sequence alignment indicates that the cyto-plasmic region of Rc-CcmI contains a leucine zipper motifwhich is not present in the putative cytoplasmic region of Pa-CcmI [100ndash102] Surprisingly no crystallographic structureis available up to now for the soluble domain of any CcmIprotein with the exception of the ortholog protein NrfGfrom E coli (Ec-NrfG) [103] This protein is necessary toattach the heme to the unusual heme-binding motif CWSCK(where a Lys residue substitutes the conservedHis) present inNrfA a pentaheme c-type cytochrome [103 104] Accordingto secondary structure prediction methods [105] it hasbeen proposed that the periplasmic domain of Pa-CcmI iscomposed of aN-terminal120572-helical region containing at leastthree TPR motifs connected by a disordered linker to a 120572-120573C-terminal regionMultiple sequence analyses and secondarystructure predictionmethods show that the TPR region of Pa-CcmI can be successfully aligned with many TPR-containingproteins including Ec-NrfG [106]

26 System I ApoCyt Chaperoning and Heme AttachmentMechanisms TPR domain-containing proteins are commonto eukaryotes prokaryotes and archaea these proteins aregenerally involved in the assembly of multiprotein complexesand to the chaperoning of unfolded proteins [103 107] Itis therefore plausible that CcmI (or the TPR C-terminal

10 Scientifica

domain of Ec-CcmH) may act to provide a platform for theunfolded apoCyt chaperoning it to the heme attachment sitepresumably located on the WWD domain of CcmF CcmImay thus be considered a component of amembrane-integralmultisubunit heme ligation complex together with CcmFand CcmH as experimentally observed by affinity purifi-cation experiments carried out with Rc-CcmFHI proteins[97 99 108] According to the proposed function of CcmI acritical requirement is represented by its ability to recognizedifferent protein targets over and above apoCyt such asCcmFandCcmHHowever until now direct evidence has been pre-sented only for the interaction of CcmI with apoCyt but thepossibility remains that CcmFHI proteins interact each othervia their TM helices and not via their periplasmic domainsInterestingly both for Pa-CcmI [106] or Rc-CcmI [99] CDspectroscopy experiments carried out on the CcmIapoCytcomplex highlighted major conformational changes at thesecondary structure level It is tempting to speculate on thebasis of these results that in vivo the folding of apoCyt maybe induced by the interaction with CcmI In the case of Paeruginosa System I proteins the binding process betweenPa-CcmI and its target protein apoCyt c551 (Pa-apoCyt) hasbeen studied both at equilibrium and kinetically [106] the119870119863measured for this interaction (in the 120583M range) appeared

to be low enough to ensure apoCyt delivery to the othercomponents of the Ccmmachinery Clearly a major questionconcerns the molecular determinants of such recognitionprocess interestingly both affinity coprecipitation assays[99] and equilibrium and kinetic binding experiments [106]highlighted the role played by the C-terminal 120572-helix of Cytc Similar observations have been made for the interaction ofEc-NrfGwith a peptidemimickingNrfA its apoCyt substrate[103] in this case isothermal titration calorimetry (ITC)experiments indicate that the TPR-domain of NrfG serves asa binding site for the C-terminal motif of NrfA Altogetherthese observations are in agreement with the fact that TPRproteins generally bind to their targets by recognizing theirC-terminal region [107]

The CcmI chaperoning activity has been experimentallysupported for the first time in the case of Pa-CcmI by citratesynthase tests [106] it has been proposed that the observedability to suppress protein aggregation in vitromay reflect thecapacity of CcmI to avoid apoCyt aggregation in vivo Stillanother piece of the Cyt c biogenesis puzzle has been addedrecently by showing that Rc-CcmI is able to interact withapoCcmE either alone or together with its substrate apoCytc2 forming a stable ternary complex in the absence of heme[109] This unexpected observation obtained by reciprocalcopurification experiments provides supporting evidence forthe existence of a large multisubunit complex composed ofCcmFHI andCcmE possibly interactingwith theCcmABCDcomplex It is interesting to note that while in the case ofthe CcmIapoCyt recognition different studies highlightedthe crucial role of the C-terminal helical region of apoCyt(see above) in the case of the apoCcmE apoCyt recognitionthe N-terminal region of apoCyt seems to represent a criticalregion

It is generally accepted that CcmF is the Ccm compo-nent responsible for heme covalent attachment to apoCyt

however as discussed above it is possible that this largemembrane protein plays such a role only together withother Ccm proteins such as CcmH and CcmI Moreover asrecently discovered by Kranz and coworkers [36 41] CcmFmay also act as a quinoleheme oxidoreductase ensuringthe necessary reduction of the oxidized heme b bound toCcmE Why it is necessary that the heme iron be in itsreduced state rather than in its oxidized state is not completelyclear although it is possible that this is a prerequisite to themechanism of thioether bond formation [110] According tocurrent hypotheses it is likely that the periplasmic WWDdomain of CcmF provides a platform for heme b bindingSanders et al and Verissimo et al [34 109] have presenteda mechanistic view of the heme attachment process whichtakes into account all the available experimental observationson the different Ccmproteins According to thismodel stere-ospecific heme ligation to reduced apoCyt occurs becauseonly the vinyl-4 group is available to form the first thioetherbond with a free cysteine at the apoCyt heme-binding motifsince the vinyl-2 group is involved (at least in the Ec-CcmE)in the covalent bond with His130 of CcmE [67] Howeverexperimental proof for this hypothesis requires a detailedinvestigation of the apoCyt thioreduction process catalyzedby CcmH (see Section 24)

It should be noticed that the mechanisms described sofar for the function(s) played by CcmF (see [34 36 109]) donot envisage a clear role for its large C-terminal periplasmicdomain (residues 510 to 611 in Ec-CcmF) It would be inter-esting to see if this domain apparently devoid of recognizablesequence features may mediate intermolecular recognitionprocesses with one (or more) component(s) involved in thehemeapoCyt ligation process

3 System II

System II is typically found in gram-positive bacteria and inin 120576-proteobacteria it is also present in most 120573- and some120575-proteobacteria in Aquificales and cyanobacteria as wellas in algal and plant chloroplasts System II is composedof three or four membrane-bound proteins CcdA ResACcsA (also known as ResC) and CcsB (also known as ResB)(Figure 3) CcdA andResA are redox-active proteins involvedin the reduction of the disulfide bond in the heme-bindingmotif of apoCyt whereas CcsA and CcsB are responsible forthe heme-apoCyt ligation process and are considered Cyt csynthethases (CCS) BothCcsAwhich is evolutionary relatedto the CcmC and CcmF proteins of System I [55] and CcsBare integral membrane proteins In some 120576-proteobacteriasuch asHelicobacter hepaticus andHelicobacter pylori a singlefusion protein composed of CcsA and CcsB polypeptides ispresent [41 111] Although as discussed below evidence hasbeen put forward to support the hypothesis that the CcsBAcomplex acts a heme translocase we still do not know if theheme is transported across the membrane by component(s)of System II itself or by a different unidentified process

31 System II ApoCyt Thioreduction Pathway After the Secmachinery secretes the newly synthesized apoCyt it readily

Scientifica 11

becomes a substrate for the oxidative system present on theouter surface of the cytoplasmic membrane of the gram-positive bacteria In B subtilis the BdbCD system [112113] is functionally but not structurally equivalent to thewell-characterized DsbAB system present in gram-negativebacteria As discussed above for System I the disulfide ofthe apoCyt heme-binding motif must be reduced in orderto allow thioether bonds formation and heme attachmentResA is the extracytoplasmic membrane-anchored TRX-likeprotein involved in the specific reduction of the apoCytdisulfide bond after the disulfide bond exchange reactionhas occurred oxidized ResA is reduced by CcdA whichreceives its reducing equivalents from a cytoplasmic TRX[114] Although ResA displays a classical TRX-like foldthe analysis of the 3D structure of oxidized and reducedResA from B subtilis (Bs-ResA) showed redox-dependentconformational modifications not observed in other TRX-like proteins Interestingly such modifications occur at thelevel of a cavity proposed to represent the binding site foroxidized apoCyt [24 115 116] Another peculiar feature of Bs-ResA is represented by the unusually similar pKa values of itsthe active site Cys residues (88 and 82 resp) as observedalso in the case of the active site Cys residues of the SystemI Pa-CcmH (see Section 23) However at variance with Pa-CcmH in Bs-ResA the large separation between the twocysteine thiols observed in the structure of the reduced formof the protein can be invoked to account for this result

CcdA is a large membrane protein containing six TMhelices [117 118] homologous to the TM domain of E coliDsbD [85 119] Its role in the Cyt c biogenesis is supportedby the observation that inactivation of CcdA blocks theproduction of c-type cytochromes in B subtilis [120 121]Moreover over and above the reduction of its apoCyt sub-strate Bs-CcdA is able to reduce the disulfide bond of othersecreted proteins such as StoA [122] It is interesting to notethat ResA and CcdA are not essential for Cyt c synthesisin the absence of BdbD or BdbC or if a disulfide reductantis present in the growth medium [123 124] As discussedabove (Section 24) a similar observation was made on therole of the thio-reductive pathway of System I in this casepersistent production of c-type cytochromes was observedin Dsb-inactivated bacterial strains [38] Following theseobservations the question has been put forward as to why theCyt c biogenesis process involves this apparently redundantthio-reduction route only to correct the effects of Bdb orDsb activity Referring to the case of B subtilis [111] it hasbeen proposed that the main reason is that BdbD mustefficiently oxidize newly secreted proteins since the activityof most of them depends on the presence of disulfide bondsAlternatively it can be hypothesized that the intramoleculardisulfide bondof apoCyt protects the protein fromproteolyticdegradation or aggregation or from cross-linking to otherthiol-containing proteins

32 System II Heme Translocation and Attachment to ApoCytRecent experimental evidence is accumulating supportingthe involvement of the heterodimeric membrane complexResBC or of the fusion protein CcsBA in the translocation

of heme from the n-side to the p-side of the bacterialmembrane In particular it has been shown that CcsBAfrom H hepaticus (Hh-CcsBA) is able to reconstitute Cyt cbiogenesis in the periplasm of E coli [36] Moreover it wasalso observed that Hh-CcsBA is able to bind reduced hemevia two conserved histidines flanking the WWD domainand required both for the translocation of the heme andfor the synthetase function of Hh-CcsBA [36] A differentstudy carried out on B subtilis System II proteins providedsupport for heme-binding capability by the ResBC complexaccording to the results obtained on recombinant ResBC theResB component of the heterodimeric CCS complex is ableto covalently bind the heme in the cytoplasm (probably by aCys residue) and to deliver it to an extracytoplamic domain ofResC which is responsible for the covalent ligation to apoCyt[117] It should be noticed however that the transfer of theCCS-bound heme to apoCyt still awaits direct experimentalproof Moreover it has also been reported that in B subtilisthe inactivation of CCS does not affect the presence ofother heme-containing proteins in the periplasm such ascytochrome b562 [111] this observation which is in contrastto the heme-transport hypothesis by CCSs parallels similarconcerns about heme translocation mechanism that arecurrently discussed in the context of System I (see Section 22above) In the case of the Hh-CcsBA synthetase site-directedmutagenesis experiments allowed to assign a heme-bindingrole for the two pairs of conserved His residues accordingto the topological model of the protein obtained by alkalinephosphatase (PhoA) assays and GFP fusions the conservedHis77 and His858 residues are located on TM helices whileHis761 and His897 are located on the periplasmic side [36]These observations together with the results of site-directedmutagenesis experiments allowed the authors to suggest thatHis77 andHis858 form a low affinitymdashmembrane embeddedheme-binding site for ferrous heme which is subsequentlytranslocated to an external heme-binding domain of Hh-CcsBA where it is coordinated by His761 and His897 Thetopological model therefore predicts that this last His pairis part of a periplasmic-located WWD domain (homologousto theWWDdomains found in theCcmCandCcmFproteinsof System I described above)

4 System III

Strikingly different from Systems I and II the Cyt c mat-uration apparatus found in fungi and in metazoan cells(System III) is composed of a single protein known as Holo-Cytochrome c Synthetase (HCCS) or Cytochrome c HemeLyase (CCHL) apparently responsible for all the subprocessesdescribed above (heme transport and chaperoning reductionand chaperoning of apoCyt and catalysis of the thioetherbonds formation between heme and apoCyt) (Figure 4)Surprisingly although this protein has been identified in Scerevisiaemitochondria several years ago [125 126] only veryrecently the human HCCS has been expressed as a recombi-nant protein in E coli and spectroscopically characterized invitro [127] It is worth noticing that humanHCCS is attractinginterest since over and above its role in Cyt c biogenesis it

12 Scientifica

is involved in diseases such as microphthalmia with linearskin defects syndrome (MLS) an X-linked genetic disorder[128ndash130] and in other processes such as Cytc-independentapoptosis in injured motor neurons [131] Different fromanimal cells where a single HCCS is sufficient for thematuration of both soluble and membrane-anchored Cytc(Cyt c and Cy c1 resp) in S cerevisiae two homologs HCCSand HCC1S located in the inner mitochondrial membraneand facing to the IMS space [132 133] are responsible for hemeattachment to apoCyt c and apoCyt c1 respectively [125 134]It should also be noticed that in fungi an additional FAD-containing protein (Cyc2p) is required for Cyt c synthesisCyc2p is a mitochondrial membrane-anchored flavoproteinexposing its redox domain to the IMS which is requiredfor the maturation of Cyt c but not for that of Cyt c1 [135]This protein does not contain the conserved Cys residuestypically found in disulfide reductases and indeed it is notable to reduce oxidized apoCyt in vitro However it has beenrecently shown that Cyc2p is able to catalyze the NAD(P)H-dependent reduction of heme in vitro [136] a necessarystep before the thioether bond formation can occur asdiscussed above (see Section 26) This result together withthe observation that Cyc2p interacts with HCCS and withapoCyt c and c1 lends support to the proposal that Cyc2p isinvolved in the reduction of the heme iron in vivo [136 137]

AlthoughHCCS proteins are crucial for Cyt cmaturationwe still do not know how these proteins recognize theirsubstrates (heme and apoCyt) and how they promote orcatalyze the formation of thioether bonds between the hemevinyl groups and the cysteine thiols of the apoCyt CXXCHmotif

Contrary to the broad specificity of System I whichis able to recognize and attach the heme to prokaryoticand eukaryotic c-type cytochromes [49 50] and even tovery short microperoxidase-like sequences [49 50] mito-chondrial HCCS is characterized by a higher specificityas it does not recognize bacterial apoCyt sequences Theseobservations prompted the investigation of the recognitionprocess between apoCyt and HCCS Recently by using arecombinant yeast HCCS and chimerical apoCyt sequencesexpressed in E coli it was possible to conclude that a crucialrecognition determinant is represented by the N-terminalregion of apoCyt containing the heme-binding motif [35]notice that in the context of System I the same N-terminalregion of the Cyt c sequence has been recently identified to beimportant for the recognition by apoCcmE (see Section 26)Over and above the role played by the intervening residuesin the CXXCH heme-binding motif [135] it has been shownthat a conserved Phe residue occurring in the N-terminalregion before the CXXCH motif is important for HCCSrecognition [35 138] These results support the hypothesisthat this residue known to be a key determinant of Cytcfolding and stability [19 20 139] may also be crucial for Cytcmaturation by HCCS

Another relevant question concerns the ability of HCCSto recognize and bind the heme molecule as the region ofthe protein responsible for heme recognition remains to beidentified Initially it was hypothesized that the recognitionof heme could be mediated by the CP motifs present in

the HCCS protein [140] these short sequences are indeedknown to bind heme in a variety of heme-containing proteins[141 142] However it has been recently shown that CPmotifsof the recombinant S cerevisiae HCCS are not necessaryfor Cyt c production in E coli [143] excluding them as keydeterminants of heme recognition

The long-awaited in vitro characterization of the recom-binant human HCCS allowed for the first time to pro-pose a molecular mechanism underlying Cytc maturationin eukaryotes which can be experimentally tested [127]According to the proposed model the human HCCS activitycan be described as a four step mechanism involving (i)heme-binding (ii) apoCyt recognition (iii) thioether bondsformation and (iv) holoCyt c release In particular theheme is proposed to play the role of a scaffolding moleculemediating the contacts between HCCS and apoCyt Muta-genesis experiments carried out on the recombinant HCCSstrongly suggest that heme-binding (Step 1) depends on thepresence of a specific His residue (His154) acting as anaxial ligand to ferrous heme Residues present at the N-terminus of apoCyt mediate the recognition with HCCS(Step 2) as discussed above it is known that this regionforms structurally conserved 120572-helix in the fold of all c-type cytochromes Unfortunately no information is availableup to now concerning the region of HCCS involved in therecognition and binding to the N-terminal region of apoCytCoordination of the heme iron by the His residue of theapoCyt heme-binding motif CXXCH provides the secondaxial ligand to the heme iron and is probably importantfor the correct positioning of the two apoCyt Cys residuesand formation of the thioether bonds (Step 3) The finalrelease of functional Cyt c (Step 4) clearly requires thedisplacement of the His154Fe2+ coordination bond sucha displacement is probably mediated by formation of thecoordination bond with the Cyt c conserved Met residueandor by the simultaneous folding of Cyt c Again it isinteresting to note that this last hypothesis is in accordancewith in vitro folding studies on c-type cytochromes thathighlighted the late formation of the Met-Fe coordinationduring Cyt c folding [144 145]

Acknowledgments

The author thanks A Di Matteo and E Di Silvio for fruitfuldiscussions and for help in preparing this paper This work issupported by grants from the University of Rome ldquoSapienzardquo(C26A11MBXJ and C26A13MWF4)

References

[1] M G Galinato J G Kleingardner S E Bowman et alldquoHeme-protein vibrational couplings in cytochrome c providea dynamic link that connects the heme-iron and the proteinsurfacerdquo Proceedings of the National Academy of Sciences vol109 no 23 pp 8896ndash8900 2012

[2] H B Gray and J R Winkler ldquoElectron flow through metal-loproteinsrdquo Biochimica et Biophysica Acta vol 1797 no 9 pp1563ndash1572 2010

Scientifica 13

[3] P Ascenzi R Santucci M Coletta and F PolticellildquoCytochromes reactivity of the ldquodark siderdquo of the hemerdquoBiophysical Chemistry vol 152 no 1ndash3 pp 21ndash27 2010

[4] S Yamada N D Bouley Ford G E Keller W C Ford HB Gray and J R Winkler ldquoSnapshots of a protein foldingintermediaterdquo Proceedings of the National Academy of Sciencesvol 110 no 5 pp 1606ndash1610 2013

[5] P Weinkam J Zimmermann F E Romesberg and P GWolynes ldquoThe folding energy landscape and free energy excita-tions of cytochrome crdquo Accounts of Chemical Research vol 43no 5 pp 652ndash660 2010

[6] M Yamanaka M Masanari and Y Sambongi ldquoConfermentof folding ability to a naturally unfolded apocytochrome cthrough introduction of hydrophobic amino acid residuesrdquoBiochemistry vol 50 no 12 pp 2313ndash2320 2011

[7] G W Moore and G W Pettigrew Cytochrome C EvolutionaryStructural and Physicochemical Aspects Springer Berlin Ger-many 1990

[8] I Bertini G Cavallaro and A Rosato ldquoCytochrome c occur-rence and functionsrdquo Chemical Reviews vol 106 no 1 pp 90ndash115 2006

[9] D A Pearce and F Sherman ldquoDegradation of cytochromeoxidase subunits in mutants of yeast lacking cytochrome c andsuppression of the degradation by mutation of yme1rdquo Journal ofBiological Chemistry vol 270 no 36 pp 20879ndash20882 1995

[10] G Silkstone S M Kapetanaki I Husu M H Vos and MT Wilson ldquoNitric oxide binding to the cardiolipin complex offerric cytochrome crdquo Biochemistry vol 51 no 34 pp 6760ndash6766 2012

[11] M Giorgio E Migliaccio F Orsini et al ldquoElectron transferbetween cytochrome c and p66Shc generates reactive oxygenspecies that trigger mitochondrial apoptosisrdquo Cell vol 122 no2 pp 221ndash233 2005

[12] S M Kilbride and J H Prehn ldquoCentral roles of apoptoticproteins in mitochondrial functionrdquo Oncogene vol 32 no 22pp 2703ndash2711 2013

[13] T Noll and G Noll ldquoStrategies for ldquowiringrdquo redox-activeproteins to electrodes and applications in biosensors biofuelcells and nanotechnologyrdquo Chemical Society Reviews vol 40no 7 pp 3564ndash3576 2011

[14] G Layer J Reichelt D Jahn and D W Heinz ldquoStructure andfunction of enzymes in heme biosynthesisrdquo Protein Science vol19 no 6 pp 1137ndash1161 2010

[15] S Severance and IHamza ldquoTrafficking of heme and porphyrinsin metazoardquo Chemical Reviews vol 109 no 10 pp 4596ndash46162009

[16] P Hamel V Corvest P Giege and G Bonnard ldquoBiochemi-cal requirements for the maturation of mitochondrial c-typecytochromesrdquo Biochimica et Biophysica Acta vol 1793 no 1 pp125ndash138 2009

[17] W R Fisher H Taniuchi and C B Anfinsen ldquoOn the role ofheme in the formation of the structure of cytochrome crdquo Journalof Biological Chemistry vol 248 no 9 pp 3188ndash3195 1973

[18] M Yamanaka H Mita Y Yamamoto and Y Sambongi ldquoHemeis not required for aquifex aeolicus cytochrome c555 polypep-tide foldingrdquoBioscience Biotechnology andBiochemistry vol 73no 9 pp 2022ndash2025 2009

[19] C Travaglini-Allocatelli S Gianni andMBrunori ldquoA commonfolding mechanism in the cytochrome c familyrdquo Trends inBiochemical Sciences vol 29 no 10 pp 535ndash541 2004

[20] A Borgia D Bonivento C Travaglini-Allocatelli A DiMatteoand M Brunori ldquoUnveiling a hidden folding intermediate in c-type cytochromes by protein engineeringrdquo Journal of BiologicalChemistry vol 281 no 14 pp 9331ndash9336 2006

[21] E Enggist L Thony-Meyer P Guntert and K PervushinldquoNMR structure of the heme chaperone CcmE reveals a novelfunctional motifrdquo Structure vol 10 no 11 pp 1551ndash1557 2002

[22] A di Matteo N Calosci S Gianni P Jemth M Brunori and CTravaglini-Allocatelli ldquoStructural and functional characteriza-tion of CcmG from pseudomonas aeruginosa a key componentof the bacterial cytochrome c maturation apparatusrdquo Proteinsvol 78 no 10 pp 2213ndash2221 2010

[23] A diMatteo S GianniM E Schinina et al ldquoA strategic proteinin cytochrome c maturation three-dimensional structure ofCcmH and binding to apocytochrome crdquo Journal of BiologicalChemistry vol 282 no 37 pp 27012ndash27019 2007

[24] A Crow R M Acheson N E Le Brun and A OubrieldquoStructural basis of redox-coupled protein substrate selectionby the cytochrome c biosynthesis protein ResArdquo Journal ofBiological Chemistry vol 279 no 22 pp 23654ndash23660 2004

[25] E di Silvio A di Matteo and C Travaglini-Allocatelli ldquoTheRedox pathway of Pseudomonas aeruginosa cytochrome cbiogenesisrdquo Journal of Proteins and Proteomics vol 3 no 1 pp1ndash7 2012

[26] LThony-Meyer andP Kunzler ldquoTranslocation to the periplasmand signal sequence cleavage of preapocytochrome c depend onsec and lep but not on the ccmgene productsrdquoEuropean Journalof Biochemistry vol 246 no 3 pp 794ndash799 1997

[27] S J Facey and A Kuhn ldquoBiogenesis of bacterial inner-membrane proteinsrdquo Cellular and Molecular Life Sciences vol67 no 14 pp 2343ndash2362 2010

[28] K Diekert A I de Kroon U Ahting et al ldquoApocytochromec requires the TOM complex for translocation across themitochondrial outer membranerdquo The EMBO Journal vol 20no 20 pp 5626ndash5635 2001

[29] N Wiedemann V Kozjak T Prinz et al ldquoBiogenesis of yeastmitochondrial cytochrome c a unique relationship to the TOMmachineryrdquo Journal ofMolecular Biology vol 327 no 2 pp 465ndash474 2003

[30] F-U Hartl N Pfanner and W Neupert ldquoTranslocation inter-mediates on the import pathway of proteins intomitochondriardquoBiochemical Society Transactions vol 15 no 1 pp 95ndash97 1987

[31] B S Glick A Brandt K Cunningham SMuller R L Hallbergand G Schatz ldquoCytochromes c1 and b2 are sorted to theintermembrane space of yeast mitochondria by a stop-transfermechanismrdquo Cell vol 69 no 5 pp 809ndash822 1992

[32] G Howe and S Merchant ldquoRole of heme in the biosynthesis ofcytochrome c6rdquo Journal of Biological Chemistry vol 269 no 8pp 5824ndash5832 1994

[33] S S Nakamoto P Hamel and S Merchant ldquoAssembly ofchloroplast cytochromes b and crdquo Biochimie vol 82 no 6-7 pp603ndash614 2000

[34] C Sanders S Turkarslan D-W Lee and F DaldalldquoCytochrome c biogenesis the Ccm systemrdquo Trends inMicrobiology vol 18 no 6 pp 266ndash274 2010

[35] J M Stevens D A Mavridou R Hamer P Kritsiligkou A DGoddard and S J Ferguson ldquoCytochrome c biogenesis systemIrdquoThe FEBS Journal vol 278 no 22 pp 4170ndash4178 2011

[36] R G Kranz C Richard-Fogal J-S Taylor and E R FrawleyldquoCytochrome c biogenesis mechanisms for covalent modifica-tions and trafficking of heme and for heme-iron redox controlrdquo

14 Scientifica

Microbiology and Molecular Biology Reviews vol 73 no 3 pp510ndash528 2009

[37] J W A Allen ldquoCytochrome c biogenesis in mitochondriamdashsystems III and VrdquoThe FEBS Journal vol 278 no 22 pp 4198ndash4216 2011

[38] D A Mavridou S J Ferguson and J M Stevens ldquoCytochromec assemblyrdquo IUBMB Life vol 65 no 3 pp 209ndash216 2013

[39] I Bertini G Cavallaro and A Rosato ldquoEvolution ofmitochondrial-type cytochrome c domains and of theprotein machinery for their assemblyrdquo Journal of InorganicBiochemistry vol 101 no 11-12 pp 1798ndash1811 2007

[40] B S Goldman and R G Kranz ldquoEvolution and horizontaltransfer of an entire biosynthetic pathway for cytochromec biogenesis helicobacter deinococcus archae and morerdquoMolecular Microbiology vol 27 no 4 pp 871ndash873 1998

[41] C L Richard-Fogal E R Frawley E R Bonner H Zhu BSan Francisco and R G Kranz ldquoA conserved haem redoxand trafficking pathway for cofactor attachmentrdquo The EMBOJournal vol 28 no 16 pp 2349ndash2359 2009

[42] L Thony-Meyer F Fischer P Kunzler D Ritz and H Hen-necke ldquoEscherichia coli genes required for cytochrome cmaturationrdquo Journal of Bacteriology vol 177 no 15 pp 4321ndash4326 1995

[43] C S Beckett J A Loughman K A Karberg G M DonatoW E Goldman and R G Kranz ldquoFour genes are required forthe system II cytochrome c biogenesis pathway in Bordetellapertussis a unique bacterial modelrdquo Molecular Microbiologyvol 38 no 3 pp 465ndash481 2000

[44] N E Le Brun J Bengtsson and L Hederstedt ldquoGenes requiredfor cytochrome c synthesis in Bacillus subtilisrdquo MolecularMicrobiology vol 36 no 3 pp 638ndash650 2000

[45] P Giege J M Grienenberger and G Bonnard ldquoCytochromec biogenesis in mitochondriardquo Mitochondrion vol 8 no 1 pp61ndash73 2008

[46] R E Feissner C L Richard-Fogal E R Frawley J A Lough-man KW Earley and R G Kranz ldquoRecombinant cytochromesc biogenesis systems I and II and analysis of haem deliverypathways in Escherichia colirdquo Molecular Microbiology vol 60no 3 pp 563ndash577 2006

[47] N P Cianciotto P Cornelis and C Baysse ldquoImpact of thebacterial type I cytochrome c maturation system on differentbiological processesrdquoMolecular Microbiology vol 56 no 6 pp1408ndash1415 2005

[48] E Arslan H Schulz R Zufferey P Kunzler and L Thony-Meyer ldquoOverproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in Escherichiacolirdquo Biochemical and Biophysical Research Communicationsvol 251 no 3 pp 744ndash747 1998

[49] C Sanders and H Lill ldquoExpression of prokaryotic and eukary-otic cytochromes c in Escherichia colirdquoBiochimica et BiophysicaActa vol 1459 no 1 pp 131ndash138 2000

[50] M Braun and L Thony-Meyer ldquoBiosynthesis of artificialmicroperoxidases by exploiting the secretion and cytochromec maturation apparatuses of Escherichia colirdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 35 pp 12830ndash12835 2004

[51] B Baert C Baysse S Matthijs and P Cornelis ldquoMultiplephenotypic alterations caused by a c-type cytochrome mat-uration ccmC gene mutation in Pseudomonas aeruginosardquoMicrobiology vol 154 no 1 pp 127ndash138 2008

[52] S Turkarslan C Sanders and F Daldal ldquoExtracytoplasmicprosthetic group ligation to apoproteins maturation of c-typecytochromesrdquo Molecular Microbiology vol 60 no 3 pp 537ndash541 2006

[53] P M Jones and A M George ldquoThe ABC transporter structureand mechanism perspectives on recent researchrdquo Cellular andMolecular Life Sciences vol 61 no 6 pp 682ndash699 2004

[54] O Christensen E M Harvat L Thony-Meyer S J Fergusonand J M Stevens ldquoLoss of ATP hydrolysis activity by CcmABresults in loss of c-type cytochrome synthesis and incompleteprocessing ofCcmErdquoTheFEBS Journal vol 274 no 9 pp 2322ndash2332 2007

[55] J-H Lee E M Harvat J M Stevens S J Ferguson and M HSaier Jr ldquoEvolutionary origins of members of a superfamily ofintegralmembrane cytochrome c biogenesis proteinsrdquoBiochim-ica et Biophysica Acta vol 1768 no 9 pp 2164ndash2181 2007

[56] D L Beckman D R Trawick and R G Kranz ldquoBacterialcytochromes c biogenesisrdquo Genes and Development vol 6 no2 pp 268ndash283 1992

[57] H Schulz R A Fabianek E C Pellicioli H Hennecke andL Thony-Meyer ldquoHeme transfer to the heme chaperone CcmEduring cytochrome c maturation requires the CcmC proteinwhich may function independently of the ABC-transporterCcmABrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 96 no 11 pp 6462ndash6467 1999

[58] Q Ren and L Thony-Meyer ldquoPhysical Interaction ofCcmC with Heme and the Heme Chaperone CcmE duringCytochrome cMaturationrdquo Journal of Biological Chemistry vol276 no 35 pp 32591ndash32596 2001

[59] C Richard-Fogal and R G Kranz ldquoThe CcmChemeCcmEcomplex in heme trafficking and cytochrome c biosynthesisrdquoJournal of Molecular Biology vol 401 no 3 pp 350ndash362 2010

[60] V K Viswanathan S Kurtz L L Pedersen et al ldquoThecytochrome C maturation locus of Legionella pneumophilapromotes iron assimilation and intracellular infection andcontains a strain-specific insertion sequence elementrdquo Infectionand Immunity vol 70 no 4 pp 1842ndash1852 2002

[61] U Ahuja and L Thony-Meyer ldquoCcmD is involved in complexformation between CcmC and the heme chaperone CcmE dur-ing cytochrome c maturationrdquo Journal of Biological Chemistryvol 280 no 1 pp 236ndash243 2005

[62] C L Richard-Fogal E R Frawley and R G Kranz ldquoTopologyand function of CcmD in cytochrome Cmaturationrdquo Journal ofBacteriology vol 190 no 10 pp 3489ndash3493 2008

[63] H Schulz H Hennecke and L Thony-Meyer ldquoPrototype ofa heme chaperone essential for cytochrome c maturationrdquoScience vol 281 no 5380 pp 1197ndash1200 1998

[64] F Arnesano L Banci P D Barker et al ldquoSolution structure andcharacterization of the heme chaperone CcmErdquo Biochemistryvol 41 no 46 pp 13587ndash13594 2002

[65] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[66] L Thony-Meyer ldquoA heme chaperone for cytochrome c biosyn-thesisrdquo Biochemistry vol 42 no 45 pp 13099ndash13105 2003

[67] E M Harvat C Redfield J M Stevens and S J FergusonldquoProbing the heme-binding site of the cytochrome cmaturationprotein CcmErdquoBiochemistry vol 48 no 8 pp 1820ndash1828 2009

[68] D Lee K Pervushin D Bischof M Braun and L Thony-Meyer ldquoUnusual heme-histidine bond in the active site of a

Scientifica 15

chaperonerdquo Journal of the American Chemical Society vol 127no 11 pp 3716ndash3717 2005

[69] A D Goddard J M Stevens F Rao et al ldquoc-Type cytochromebiogenesis can occur via a natural Ccm system lacking CcmHCcmG and the heme-binding histidine of CcmErdquo Journal ofBiological Chemistry vol 285 no 30 pp 22882ndash22889 2010

[70] J M Aramini K Hamilton P Rossi et al ldquoSolution NMRstructure backbone dynamics and heme-binding propertiesof a novel cytochrome c maturation protein CcmE fromDesulfovibrio vulgarisrdquo Biochemistry vol 51 no 18 pp 3705ndash3707 2012

[71] T Uchida J M Stevens O Daltrop et al ldquoThe interactionof covalently bound heme with the cytochrome c maturationprotein CcmErdquo Journal of Biological Chemistry vol 279 no 50pp 51981ndash51988 2004

[72] I Garcıa-Rubio M Braun I Gromov L Thony-Meyer andA Schweiger ldquoAxial coordination of heme in ferric CcmEchaperone characterized by EPR spectroscopyrdquo BiophysicalJournal vol 92 no 4 pp 1361ndash1373 2007

[73] L Thony-Meyer ldquoHaem-polypeptide interactions duringcytochrome c maturationrdquo Biochimica et Biophysica Acta vol1459 no 2-3 pp 316ndash324 2000

[74] J A Keightley D Sanders T R Todaro A Pastuszyn and J AFee ldquoCloning expression in Escherichia coli of the cytochromec552 gene from Thermus thermophilus HB8 Evidence forgenetic linkage to an ATP- binding cassette protein and initialcharacterization of the cycA gene productsrdquo Journal of Biologi-cal Chemistry vol 273 no 20 pp 12006ndash12016 1998

[75] E M Monika B S Goldman D L Beckman and R GKranz ldquoA thioreduction pathway tethered to the membranefor periplasmic cytochromes c biogenesis In vitro and in vivostudiesrdquo Journal of Molecular Biology vol 271 no 5 pp 679ndash692 1997

[76] M Throne-Holst L Thony-Meyer and L HederstedtldquoEscherichia coli ccm in-frame deletion mutants can produceperiplasmic cytochrome b but not cytochrome crdquo FEBS Lettersvol 410 no 2-3 pp 351ndash355 1997

[77] U Ahuja and L Thony-Meyer ldquoDynamic features of a hemedelivery system for cytochrome c maturationrdquo Journal of Bio-logical Chemistry vol 278 no 52 pp 52061ndash52070 2003

[78] J W A Allen P D Barker O Daltrop et al ldquoWhy isnrsquot ldquostan-dardrdquo heme good enough for c-type and di-type cytochromesrdquoDalton Transactions no 21 pp 3410ndash3418 2005

[79] K Inaba and K Ito ldquoStructure and mechanisms of the DsbB-DsbA disulfide bond generation machinerdquo Biochimica et Bio-physica Acta vol 1783 no 4 pp 520ndash529 2008

[80] H Kadokura F Katzen and J Beckwith ldquoProtein disulfidebond formation in prokaryotesrdquoAnnual Review of Biochemistryvol 72 pp 111ndash135 2003

[81] S R Shouldice B Heras P M Walden M Totsika M ASchembri and J L Martin ldquoStructure and function of DsbA akey bacterial oxidative folding catalystrdquoAntioxidants and RedoxSignaling vol 14 no 9 pp 1729ndash1760 2011

[82] R Metheringham L Griffiths H Crooke S Forsythe and JCole ldquoAn essential role for DsbA in cytochrome c synthesis andformate-dependent nitrite reduction by Escherichia coli K-12rdquoArchives of Microbiology vol 164 no 4 pp 301ndash307 1995

[83] Y Sambongi and S J Ferguson ldquoMutants of Escherichia colilacking disulphide oxidoreductases DsbA and DsbB cannotsynthesise an exogenous monohaem c-type cytochrome exceptin the presence of disulphide compoundsrdquo FEBS Letters vol398 no 2-3 pp 265ndash268 1996

[84] D AMavridou S J Ferguson and JM Stevens ldquoThe interplaybetween the disulfide bond formation pathway and cytochromec maturation in Escherichia colirdquo FEBS Letters vol 586 no 12pp 1702ndash1707 2012

[85] C U Stirnimann A Rozhkova U Grauschopf M G GrutterR Glockshuber and G Capitani ldquoStructural basis and kineticsof DsbD-dependent cytochrome c maturationrdquo Structure vol13 no 7 pp 985ndash993 2005

[86] N Ouyang Y-G Gao H-Y Hu and Z-X Xia ldquoCrystalstructures of E coli CcmGand itsmutants reveal key roles of theN-terminal120573-sheet and the fingerprint regionrdquoProteins vol 65no 4 pp 1021ndash1031 2006

[87] M A Edeling L W Guddat R A Fabianek et al ldquoCrys-tallization and preliminary diffraction studies of native andselenomethionine CcmG (CycY DsbE)rdquoActa CrystallographicaD vol 57 no 9 pp 1293ndash1295 2001

[88] R A Fabianek M Huber-Wunderlich R Glockshuber PKunzler H Hennecke and L Thony-Meyer ldquoCharacterizationof the Bradyrhizobium japonicum CycY protein a membrane-anchored periplasmic thioredoxin that may play a role as areductant in the biogenesis of c-type cytochromesrdquo Journal ofBiological Chemistry vol 272 no 7 pp 4467ndash4473 1997

[89] G B Kallis and A Holmgren ldquoDifferential reactivity of thefunctional sulfhydryl groups of cysteine-32 and cysteine-32present in the reduced form of thioredoxin from Escherichiacolirdquo Journal of Biological Chemistry vol 255 no 21 pp 10261ndash10265 1980

[90] P T Chivers andR T Raines ldquoGeneral acidbase catalysis in theactive site of Escherichia coli thioredoxinrdquoBiochemistry vol 36no 50 pp 15810ndash15816 1997

[91] X M Zheng J Hong H Y Li D H Lin and H Y HuldquoBiochemical properties and catalytic domain structure of theCcmH protein from Escherichia colirdquo Biochim Biophys Actavol 1824 no 12 pp 1394ndash1400 2012

[92] UAhujaA Rozhkova RGlockshuber LThony-Meyer andOEinsle ldquoHelix swapping leads to dimerization of the N-terminaldomain of the c-type cytochrome maturation protein CcmHfrom Escherichia colirdquo FEBS Letters vol 582 no 18 pp 2779ndash2786 2008

[93] R A Fabianek T Hofer and L Thony-Meyer ldquoCharacteriza-tion of the Escherichia coli CcmH protein reveals new insightsinto the redox pathway required for cytochrome c maturationrdquoArchives of Microbiology vol 171 no 2 pp 92ndash100 1999

[94] E Reid J Cole and D J Eaves ldquoThe Escherichia coli CcmGprotein fulfils a specific role in cytochrome c assemblyrdquo Bio-chemical Journal vol 355 no 1 pp 51ndash58 2001

[95] Q Ren U Ahuja and LThony-Meyer ldquoA bacterial cytochromec heme lyase CcmF forms a complex with the heme chaperoneCcmE and CcmH but not with apocytochrome crdquo Journal ofBiological Chemistry vol 277 no 10 pp 7657ndash7663 2002

[96] E H Meyer P Giege E Gelhaye et al ldquoAtCCMH an essentialcomponent of the c-type cytochrome maturation pathway inArabidopsis mitochondria interacts with apocytochrome crdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 44 pp 16113ndash16118 2005

[97] C Rios-Velazquez R Coller and T J Donohue ldquoFeatures ofRhodobacter sphaeroides CcmFHrdquo Journal of Bacteriology vol185 no 2 pp 422ndash431 2003

[98] C Sanders M Deshmukh D Astor R G Kranz and F DaldalldquoOverproduction of CcmG and CcmFHRc fully suppresses thec-type cytochrome biogenesis defect of Rhodobacter capsulatus

16 Scientifica

CcmI-null mutantsrdquo Journal of Bacteriology vol 187 no 12 pp4245ndash4256 2005

[99] A F Verissimo H Yang X Wu C Sanders and F DaldalldquoCcmI subunit of CcmFHI heme ligation complex functionsas an apocytochrome c chaperone during c-type cytochromematurationrdquo Journal of Biological Chemistry vol 286 no 47 pp40452ndash40463 2011

[100] K Bryson L J McGuffin R L Marsden J J Ward J SSodhi and D T Jones ldquoProtein structure prediction servers atUniversity College Londonrdquo Nucleic Acids Research vol 33 no2 pp W36ndashW38 2005

[101] D T Jones ldquoProtein secondary structure prediction basedon position-specific scoring matricesrdquo Journal of MolecularBiology vol 292 no 2 pp 195ndash202 1999

[102] C Sanders C Boulay and F Daldal ldquoMembrane-spanning andperiplasmic segments of CcmI have distinct functions duringcytochrome c biogenesis in Rhodobacter capsulatusrdquo Journal ofBacteriology vol 189 no 3 pp 789ndash800 2007

[103] D Han K Kim J Oh J Park and Y Kim ldquoTPR domainof NrfG mediates complex formation between heme lyaseand formate-dependent nitrite reductase in Escherichia coliO157H7rdquo Proteins vol 70 no 3 pp 900ndash914 2008

[104] D J Eaves J Grove W Staudenmann et al ldquoInvolvement ofproducts of the nrfEFG genes in the covalent attachment ofhaem c to a novel cysteine-lysine motif in the cytochrome c552nitrite reductase from Escherichia colirdquo Molecular Microbiol-ogy vol 28 no 1 pp 205ndash216 1998

[105] J Nilsson B Persson and G Von Heijne ldquoConsensus predic-tions of membrane protein topologyrdquo FEBS Letters vol 486 no3 pp 267ndash269 2000

[106] E di Silvio A di Matteo F Malatesta and C Travaglini-Allocatelli ldquoRecognition and binding of apocytochrome c toP aeruginosa CcmI a component of cytochrome c maturationmachineryrdquo Biochim Biophys Acta vol 1834 no 8 pp 1554ndash1561 2013

[107] N Zeytuni and R Zarivach ldquoStructural and functional dis-cussion of the tetra-trico-peptide repeat a protein interactionmodulerdquo Structure vol 20 no 3 pp 397ndash405 2012

[108] C Sanders S Turkarslan D-W Lee O Onder R G Kranz andF Daldal ldquoThe cytochrome c maturation components CcmFCcmH and CcmI form a membrane-integral multisubunitheme ligation complexrdquo Journal of Biological Chemistry vol283 no 44 pp 29715ndash29722 2008

[109] A F Verissimo M A Mohtar and F Daldal ldquoThe hemechaperone ApoCcmE forms a ternary complex with CcmI andapocytochrome crdquoThe Journal of Biological Chemistry vol 288no 9 pp 6272ndash6283 2013

[110] P D Barker J C Ferrer M Mylrajan et al ldquoTransmutation of aheme proteinrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 90 no 14 pp 6542ndash65461993

[111] J Simon and L Hederstedt ldquoComposition and function ofcytochrome c biogenesis system IIrdquoThe FEBS Journal vol 278no 22 pp 4179ndash4188 2011

[112] T R H M Kouwen and J M van Dijl ldquoInterchangeablemodules in bacterial thiol-disulfide exchange pathwaysrdquo Trendsin Microbiology vol 17 no 1 pp 6ndash12 2009

[113] A Crow A Lewin O Hecht et al ldquoCrystal structure andbiophysical properties of Bacillus subtilis BdbD An oxidizingthioldisulfide oxidoreductase containing a novel metal siterdquoJournal of Biological Chemistry vol 284 no 35 pp 23719ndash23733 2009

[114] M C Moller and L Hederstedt ldquoExtracytoplasmic processesimpaired by inactivation of trxA (thioredoxin gene) in Bacillussubtilisrdquo Journal of Bacteriology vol 190 no 13 pp 4660ndash46652008

[115] A Lewin A Crow A Oubrie and N E Le Brun ldquoMolecularbasis for specificity of the extracytoplasmic thioredoxin ResArdquoJournal of Biological Chemistry vol 281 no 46 pp 35467ndash35477 2006

[116] C L Colbert QWu P J A Erbel K H Gardner and J Deisen-hofer ldquoMechanism of substrate specificity in Bacillus subtilisResA a thioredoxin-like protein involved in cytochrome cmaturationrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 103 no 12 pp 4410ndash44152006

[117] UAhuja P Kjelgaard B L Schulz LThony-Meyer andLHed-erstedt ldquoHaem-delivery proteins in cytochrome c maturationsystem IIrdquoMolecular Microbiology vol 73 no 6 pp 1058ndash10712009

[118] M L D Page P P Hamel S T Gabilly et al ldquoA homologof prokaryotic thiol disulfide transporter CcdA is required forthe assembly of the cytochrome b6f complex in Arabidopsischloroplastsrdquo Journal of Biological Chemistry vol 279 no 31pp 32474ndash32482 2004

[119] A Rozhkova C U Stirnimann P Frei et al ldquoStructural basisand kinetics of inter- and intramolecular disulfide exchange inthe redox catalyst DsbDrdquoThe EMBO Journal vol 23 no 8 pp1709ndash1719 2004

[120] T Schiott M Throne-Holst and L Hederstedt ldquoBacillussubtilis CcdA-defective mutants are blocked in a late step ofcytochrome C biogenesisrdquo Journal of Bacteriology vol 179 no14 pp 4523ndash4529 1997

[121] R E Feissner C S Beckett J A Loughman and R G KranzldquoMutations in cytochrome assembly and periplasmic redoxpathways in Bordetella pertussisrdquo Journal of Bacteriology vol187 no 12 pp 3941ndash3949 2005

[122] L S ErlendssonMMoller and L Hederstedt ldquoBacillus subtilisStoA is a thiol-disulfide oxidoreductase important for sporecortex synthesisrdquo Journal of Bacteriology vol 186 no 18 pp6230ndash6238 2004

[123] L S Erlendsson R M Acheson L Hederstedt and N E LeBrun ldquoBacillus subtilis ResA is a thiol-disulfide oxidoreductaseinvolved in cytochrome c synthesisrdquo Journal of BiologicalChemistry vol 278 no 20 pp 17852ndash17858 2003

[124] L S Erlendsson and L Hederstedt ldquoMutations in the thiol-disulfide oxidoreductases BdbC and BdbD can suppresscytochrome c deficiency of CcdA-defective Bacillus subtiliscellsrdquo Journal of Bacteriology vol 184 no 5 pp 1423ndash1429 2002

[125] M E Dumont J F Ernst D M Hampsey and F ShermanldquoIdentification and sequence of the gene encoding cytochromec heme lyase in the yeast Saccharomyces cerevisiaerdquoThe EMBOJournal vol 6 no 1 pp 235ndash241 1987

[126] M E Dumont J F Ernst and F Sherman ldquoCoupling of hemeattachment to import of cytochrome c into yeast mitochondriaStudies with heme lyase-deficient mitochondria and alteredapocytochromes crdquo Journal of Biological Chemistry vol 263 no31 pp 15928ndash15937 1988

[127] B San Francisco E C Bretsnyder and R G Kranz ldquoHumanmitochondrial holocytochrome c synthasersquos hemebindingmat-uration determinants and complex formationwith cytochromecrdquo Proceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 9 pp E788ndashE797 2012

Scientifica 17

[128] L Schaefer A Ballabio and H Y Zoghbi ldquoCloning andcharacterization of a putative human holocytochrome c-typesynthetase gene (HCCS) isolated from the critical region formicrophthalmia with linear skin defects (MLS)rdquoGenomics vol34 no 2 pp 166ndash172 1996

[129] S K Prakash T A Cormier A E McCall et al ldquoLoss ofholocytochrome c-type synthetase causes the male lethality ofX-linked dominant microphthalmia with linear skin defects(MLS) syndromerdquo Human Molecular Genetics vol 11 no 25pp 3237ndash3248 2002

[130] I Wimplinger M Morleo G Rosenberger et al ldquoMutations ofthe mitochondrial holocytochrome c-type synthase in X-linkeddominant microphthalmia with linear skin defects syndromerdquoTheAmerican Journal of HumanGenetics vol 79 no 5 pp 878ndash889 2006

[131] S Kiryu-Seo KGamo T Tachibana K Tanaka andHKiyamaldquoUnique anti-apoptotic activity of EAAC1 in injured motorneuronsrdquoTheEMBO Journal vol 25 no 14 pp 3411ndash3421 2006

[132] R Lill R A Stuart M E Drygas F E Nargang and W Neu-pert ldquoImport of cytochrome c heme lyase into mitochondriaa novel pathway into the intermembrane spacerdquo The EMBOJournal vol 11 no 2 pp 449ndash456 1992

[133] H Steiner A Zollner A Haid W Neupert and R LillldquoBiogenesis of mitochondrial heme lyases in yeast Importand folding in the intermembrane spacerdquo Journal of BiologicalChemistry vol 270 no 39 pp 22842ndash22849 1995

[134] A Zollner G Rodel and A Haid ldquoMolecular cloning andcharacterization of the Saccharomyces cerevisiae CYT2 geneencoding cytochrome-c1-heme lyaserdquo European Journal of Bio-chemistry vol 207 no 3 pp 1093ndash1100 1992

[135] V Corvest D A Murrey D G Bernard D B Knaff B Guiardand P PHamel ldquoc-Type cytochrome assembly in Saccharomycescerevisiae a key residue for apocytochrome c1lyase interactionrdquoGenetics vol 186 no 2 pp 561ndash571 2010

[136] V Corvest D A Murrey M Hirasawa D B Knaff B Guiardand P P Hamel ldquoThe flavoprotein Cyc2p a mitochondrialcytochrome c assembly factor is a NAD(P)H-dependent haemreductaserdquo Molecular Microbiology vol 83 no 5 pp 968ndash9802012

[137] D G Bernard S Quevillon-Cheruel S Merchant B Guiardand P P Hamel ldquoCyc2p a membrane-bound flavopro-tein involved in the maturation of mitochondrial c-typecytochromesrdquo Journal of Biological Chemistry vol 280 no 48pp 39852ndash39859 2005

[138] J G Kleingardner and K L Bren ldquoComparing substratespecificity between cytochrome cmaturation and cytochrome cheme lyase systems for cytochrome c biogenesisrdquo Metallomicsvol 3 no 4 pp 396ndash403 2011

[139] C Travaglini-Allocatelli S Gianni VMorea A Tramontano TSoulimane and M Brunori ldquoExploring the cytochrome c fold-ing mechanism cytochrome c552 fromThermus thermophilusfolds through an on-pathway intermediaterdquo Journal of BiologicalChemistry vol 278 no 42 pp 41136ndash41140 2003

[140] L Zhang and L Guarente ldquoHeme binds to a short sequencethat serves a regulatory function in diverse proteinsrdquoTheEMBOJournal vol 14 no 2 pp 313ndash320 1995

[141] S Hira T Tomita T Matsui K Igarashi and M Ikeda-SaitoldquoBach1 a heme-dependent transcription factor reveals pres-ence of multiple heme binding sites with distinct coordinationstructurerdquo IUBMB Life vol 59 no 8-9 pp 542ndash551 2007

[142] T K Kuhl A Wiszligbrock N Goradia et al ldquoAnalysis of Fe(III)heme binding to cysteine-containing heme-regulatory motifs

in proteinsrdquo ACS Chemical Biology vol 8 no 8 pp 1785ndash17932013

[143] R L Moore J M Stevens and S J Ferguson ldquoMitochondrialcytochrome c synthase CP motifs are not necessary for hemeattachment to apocytochrome crdquo FEBS Letters vol 585 no 21pp 3415ndash3419 2011

[144] S Gianni M Brunori and C Travaglini-Allocatelli ldquoRefoldingkinetics of cytochrome c551 reveals a mechanistic differencebetween urea and guanidinerdquo Protein Science vol 10 no 8 pp1685ndash1688 2001

[145] S Gianni C Travaglini-Allocatelli F Cutruzzola M BrunoriM C R Shastry and H Roder ldquoParallel pathways incytochrome c551 foldingrdquo Journal of Molecular Biology vol 330no 5 pp 1145ndash1152 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

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Molecular Biology International

GenomicsInternational Journal of

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BioinformaticsAdvances in

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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Evolutionary BiologyInternational Journal of

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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

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International Journal of

Microbiology

Scientifica 9

transferred directly from CcmG to apoCyt as depicted inScheme 1 According to Scheme 2 reduced CcmH (a non-TRX-like thiol-oxidoreductase) specifically recognizes andreduces oxidized apoCyt via the formation of a mixed-disulfide complex which is subsequently resolved by CcmGThe resulting disulfide bond between CcmH and CcmG isthen resolved by the free Cys thiol of CcmG (probably Cys77in Pa-CcmG)

However further in vitro experiments with CcmH andapoCyt single Cys-containing mutants are needed to unveilthe details of the thioreduction of oxidized apoCyt by CcmHIn particular it would be crucial to identify the Cys residueof apoCyt that remains free in the apoCyt-CcmH mixed-disulfide complex intermediate (see Scheme 2 and Section 26below) and available to thioether bond formation with oneof the heme vinyl groups Clearly structure determinationof the trapped mixed-disulfide complexes between CcmHCcmG and apoCyt (or apoCyt peptides) would providekey information for our understanding of this specializedthioreduction pathway mechanism

25 System I ApoCyt Chaperoning and Heme AttachmentComponents The reduced heme-binding motif of apoCyt isnow available to the heme ligation reaction However themolecularmechanismwhereby the Ccmmachinery catalyzesor promotes the formation of the heme-apoCyt covalentbonds is still largely obscure representing themost importantgoal in the field Past observations and recent experimentssuggest that CcmF and CcmI possibly together with CcmHare involved in these final steps [16 34 36]

CcmF is a large integral membrane protein of more than600 residues belonging to the heme handling protein family(HHP [55]) and predicted to contain 10ndash15 TM helices (notethat some discrepancy exists as to the number of TM helicespredicted by computer programs and those predicted onthe basis of phoA and lacZ fusion experiments [40 97])a conserved WWD domain and a larger domain devoidof any recognizable sequence features both exposed to theperiplasm Only recently E coli CcmF (Ec-CcmF) has beenoverexpressed solubilized from the membrane fraction andspectroscopically characterized in vitro [36 41] Surprisinglythe biochemical characterization of recombinant Ec-CcmFallowed to show that the purified protein contains heme b ascofactor in a 1 1 stoichiometry this observation led to thehypothesis that in addition to its heme lyase function Ec-CcmF may act as a heme oxidoreductase In particular it ispossible that the heme b of Ec-CcmF may act as a reductantfor the oxidized iron of the heme bound to CcmE [41]indeed the in vitro reduction of Ec-CcmF by quinones hasbeen experimentally observed strengthening the hypothesisabout the quinolhemeoxidoreductase function of this elusiveproteinThe structuralmodel proposed for Ec-CcmFpredicts13 TMhelices and notably the location of the four completelyconserved His residues according to the model two of them(His173 andHis303) are located in periplasmic exposed loopsnext to the conserved WWD domain which is believedto provide a platform for the heme bound to holoCcmEwhile His261 is located in one of the TM helices and it is

predicted to act as an axial ligand to the heme b of Ec-CcmFthe other conserved His residue (H491) could provide thesecond axial coordination bond to the heme although thishas not been experimentally addressed This model of Ec-CcmF therefore envisages that this large membrane proteinis characterized by two heme-binding sites one of themis embedded in the membrane and coordinates a heme bprosthetic group necessary to reduce the CcmE-bound hemehosted in the second heme-binding site and constituted by itsWWD domain

It is interesting to note that in plants mitochondria theCcmF ortholog appears to be split into three different pro-teins (At-CcmFN1 At-CcmFN2 and At-CcmFC) possiblyinteracting each other [16] Since each of these proteins issimilar to the corresponding domain in the bacterial CcmFortholog this observation may provide useful informationin the design of engineered fragments of bacterial CcmFproteins amenable to structural analyses

The other System I component which is generallybelieved to be involved in the final steps of Cyt c maturationis CcmI As stated above the ccmI gene is present only insome Ccm operons while in others the corresponding ORFis present within the ccmH gene (as in E coli)The functionalrole of CcmI in Cytc biogenesis is revealed by geneticstudies showing that in R capsulatus and B japonicuminactivation of the ccmI gene leads to inability to synthesizefunctional c-type cytochromes [98 99] In R capsulatus andP aeruginosa the CcmI protein (Rc-CcmI and Pa-CcmIresp) can be described as being composed of two domainsstarting from the N-terminus a first domain composed oftwo TM helices connected by a short cytoplasmic regionand a large periplasmic domain Structural variations maybe observed among CcmI members from different bacteriaindeed multiple sequence alignment indicates that the cyto-plasmic region of Rc-CcmI contains a leucine zipper motifwhich is not present in the putative cytoplasmic region of Pa-CcmI [100ndash102] Surprisingly no crystallographic structureis available up to now for the soluble domain of any CcmIprotein with the exception of the ortholog protein NrfGfrom E coli (Ec-NrfG) [103] This protein is necessary toattach the heme to the unusual heme-binding motif CWSCK(where a Lys residue substitutes the conservedHis) present inNrfA a pentaheme c-type cytochrome [103 104] Accordingto secondary structure prediction methods [105] it hasbeen proposed that the periplasmic domain of Pa-CcmI iscomposed of aN-terminal120572-helical region containing at leastthree TPR motifs connected by a disordered linker to a 120572-120573C-terminal regionMultiple sequence analyses and secondarystructure predictionmethods show that the TPR region of Pa-CcmI can be successfully aligned with many TPR-containingproteins including Ec-NrfG [106]

26 System I ApoCyt Chaperoning and Heme AttachmentMechanisms TPR domain-containing proteins are commonto eukaryotes prokaryotes and archaea these proteins aregenerally involved in the assembly of multiprotein complexesand to the chaperoning of unfolded proteins [103 107] Itis therefore plausible that CcmI (or the TPR C-terminal

10 Scientifica

domain of Ec-CcmH) may act to provide a platform for theunfolded apoCyt chaperoning it to the heme attachment sitepresumably located on the WWD domain of CcmF CcmImay thus be considered a component of amembrane-integralmultisubunit heme ligation complex together with CcmFand CcmH as experimentally observed by affinity purifi-cation experiments carried out with Rc-CcmFHI proteins[97 99 108] According to the proposed function of CcmI acritical requirement is represented by its ability to recognizedifferent protein targets over and above apoCyt such asCcmFandCcmHHowever until now direct evidence has been pre-sented only for the interaction of CcmI with apoCyt but thepossibility remains that CcmFHI proteins interact each othervia their TM helices and not via their periplasmic domainsInterestingly both for Pa-CcmI [106] or Rc-CcmI [99] CDspectroscopy experiments carried out on the CcmIapoCytcomplex highlighted major conformational changes at thesecondary structure level It is tempting to speculate on thebasis of these results that in vivo the folding of apoCyt maybe induced by the interaction with CcmI In the case of Paeruginosa System I proteins the binding process betweenPa-CcmI and its target protein apoCyt c551 (Pa-apoCyt) hasbeen studied both at equilibrium and kinetically [106] the119870119863measured for this interaction (in the 120583M range) appeared

to be low enough to ensure apoCyt delivery to the othercomponents of the Ccmmachinery Clearly a major questionconcerns the molecular determinants of such recognitionprocess interestingly both affinity coprecipitation assays[99] and equilibrium and kinetic binding experiments [106]highlighted the role played by the C-terminal 120572-helix of Cytc Similar observations have been made for the interaction ofEc-NrfGwith a peptidemimickingNrfA its apoCyt substrate[103] in this case isothermal titration calorimetry (ITC)experiments indicate that the TPR-domain of NrfG serves asa binding site for the C-terminal motif of NrfA Altogetherthese observations are in agreement with the fact that TPRproteins generally bind to their targets by recognizing theirC-terminal region [107]

The CcmI chaperoning activity has been experimentallysupported for the first time in the case of Pa-CcmI by citratesynthase tests [106] it has been proposed that the observedability to suppress protein aggregation in vitromay reflect thecapacity of CcmI to avoid apoCyt aggregation in vivo Stillanother piece of the Cyt c biogenesis puzzle has been addedrecently by showing that Rc-CcmI is able to interact withapoCcmE either alone or together with its substrate apoCytc2 forming a stable ternary complex in the absence of heme[109] This unexpected observation obtained by reciprocalcopurification experiments provides supporting evidence forthe existence of a large multisubunit complex composed ofCcmFHI andCcmE possibly interactingwith theCcmABCDcomplex It is interesting to note that while in the case ofthe CcmIapoCyt recognition different studies highlightedthe crucial role of the C-terminal helical region of apoCyt(see above) in the case of the apoCcmE apoCyt recognitionthe N-terminal region of apoCyt seems to represent a criticalregion

It is generally accepted that CcmF is the Ccm compo-nent responsible for heme covalent attachment to apoCyt

however as discussed above it is possible that this largemembrane protein plays such a role only together withother Ccm proteins such as CcmH and CcmI Moreover asrecently discovered by Kranz and coworkers [36 41] CcmFmay also act as a quinoleheme oxidoreductase ensuringthe necessary reduction of the oxidized heme b bound toCcmE Why it is necessary that the heme iron be in itsreduced state rather than in its oxidized state is not completelyclear although it is possible that this is a prerequisite to themechanism of thioether bond formation [110] According tocurrent hypotheses it is likely that the periplasmic WWDdomain of CcmF provides a platform for heme b bindingSanders et al and Verissimo et al [34 109] have presenteda mechanistic view of the heme attachment process whichtakes into account all the available experimental observationson the different Ccmproteins According to thismodel stere-ospecific heme ligation to reduced apoCyt occurs becauseonly the vinyl-4 group is available to form the first thioetherbond with a free cysteine at the apoCyt heme-binding motifsince the vinyl-2 group is involved (at least in the Ec-CcmE)in the covalent bond with His130 of CcmE [67] Howeverexperimental proof for this hypothesis requires a detailedinvestigation of the apoCyt thioreduction process catalyzedby CcmH (see Section 24)

It should be noticed that the mechanisms described sofar for the function(s) played by CcmF (see [34 36 109]) donot envisage a clear role for its large C-terminal periplasmicdomain (residues 510 to 611 in Ec-CcmF) It would be inter-esting to see if this domain apparently devoid of recognizablesequence features may mediate intermolecular recognitionprocesses with one (or more) component(s) involved in thehemeapoCyt ligation process

3 System II

System II is typically found in gram-positive bacteria and inin 120576-proteobacteria it is also present in most 120573- and some120575-proteobacteria in Aquificales and cyanobacteria as wellas in algal and plant chloroplasts System II is composedof three or four membrane-bound proteins CcdA ResACcsA (also known as ResC) and CcsB (also known as ResB)(Figure 3) CcdA andResA are redox-active proteins involvedin the reduction of the disulfide bond in the heme-bindingmotif of apoCyt whereas CcsA and CcsB are responsible forthe heme-apoCyt ligation process and are considered Cyt csynthethases (CCS) BothCcsAwhich is evolutionary relatedto the CcmC and CcmF proteins of System I [55] and CcsBare integral membrane proteins In some 120576-proteobacteriasuch asHelicobacter hepaticus andHelicobacter pylori a singlefusion protein composed of CcsA and CcsB polypeptides ispresent [41 111] Although as discussed below evidence hasbeen put forward to support the hypothesis that the CcsBAcomplex acts a heme translocase we still do not know if theheme is transported across the membrane by component(s)of System II itself or by a different unidentified process

31 System II ApoCyt Thioreduction Pathway After the Secmachinery secretes the newly synthesized apoCyt it readily

Scientifica 11

becomes a substrate for the oxidative system present on theouter surface of the cytoplasmic membrane of the gram-positive bacteria In B subtilis the BdbCD system [112113] is functionally but not structurally equivalent to thewell-characterized DsbAB system present in gram-negativebacteria As discussed above for System I the disulfide ofthe apoCyt heme-binding motif must be reduced in orderto allow thioether bonds formation and heme attachmentResA is the extracytoplasmic membrane-anchored TRX-likeprotein involved in the specific reduction of the apoCytdisulfide bond after the disulfide bond exchange reactionhas occurred oxidized ResA is reduced by CcdA whichreceives its reducing equivalents from a cytoplasmic TRX[114] Although ResA displays a classical TRX-like foldthe analysis of the 3D structure of oxidized and reducedResA from B subtilis (Bs-ResA) showed redox-dependentconformational modifications not observed in other TRX-like proteins Interestingly such modifications occur at thelevel of a cavity proposed to represent the binding site foroxidized apoCyt [24 115 116] Another peculiar feature of Bs-ResA is represented by the unusually similar pKa values of itsthe active site Cys residues (88 and 82 resp) as observedalso in the case of the active site Cys residues of the SystemI Pa-CcmH (see Section 23) However at variance with Pa-CcmH in Bs-ResA the large separation between the twocysteine thiols observed in the structure of the reduced formof the protein can be invoked to account for this result

CcdA is a large membrane protein containing six TMhelices [117 118] homologous to the TM domain of E coliDsbD [85 119] Its role in the Cyt c biogenesis is supportedby the observation that inactivation of CcdA blocks theproduction of c-type cytochromes in B subtilis [120 121]Moreover over and above the reduction of its apoCyt sub-strate Bs-CcdA is able to reduce the disulfide bond of othersecreted proteins such as StoA [122] It is interesting to notethat ResA and CcdA are not essential for Cyt c synthesisin the absence of BdbD or BdbC or if a disulfide reductantis present in the growth medium [123 124] As discussedabove (Section 24) a similar observation was made on therole of the thio-reductive pathway of System I in this casepersistent production of c-type cytochromes was observedin Dsb-inactivated bacterial strains [38] Following theseobservations the question has been put forward as to why theCyt c biogenesis process involves this apparently redundantthio-reduction route only to correct the effects of Bdb orDsb activity Referring to the case of B subtilis [111] it hasbeen proposed that the main reason is that BdbD mustefficiently oxidize newly secreted proteins since the activityof most of them depends on the presence of disulfide bondsAlternatively it can be hypothesized that the intramoleculardisulfide bondof apoCyt protects the protein fromproteolyticdegradation or aggregation or from cross-linking to otherthiol-containing proteins

32 System II Heme Translocation and Attachment to ApoCytRecent experimental evidence is accumulating supportingthe involvement of the heterodimeric membrane complexResBC or of the fusion protein CcsBA in the translocation

of heme from the n-side to the p-side of the bacterialmembrane In particular it has been shown that CcsBAfrom H hepaticus (Hh-CcsBA) is able to reconstitute Cyt cbiogenesis in the periplasm of E coli [36] Moreover it wasalso observed that Hh-CcsBA is able to bind reduced hemevia two conserved histidines flanking the WWD domainand required both for the translocation of the heme andfor the synthetase function of Hh-CcsBA [36] A differentstudy carried out on B subtilis System II proteins providedsupport for heme-binding capability by the ResBC complexaccording to the results obtained on recombinant ResBC theResB component of the heterodimeric CCS complex is ableto covalently bind the heme in the cytoplasm (probably by aCys residue) and to deliver it to an extracytoplamic domain ofResC which is responsible for the covalent ligation to apoCyt[117] It should be noticed however that the transfer of theCCS-bound heme to apoCyt still awaits direct experimentalproof Moreover it has also been reported that in B subtilisthe inactivation of CCS does not affect the presence ofother heme-containing proteins in the periplasm such ascytochrome b562 [111] this observation which is in contrastto the heme-transport hypothesis by CCSs parallels similarconcerns about heme translocation mechanism that arecurrently discussed in the context of System I (see Section 22above) In the case of the Hh-CcsBA synthetase site-directedmutagenesis experiments allowed to assign a heme-bindingrole for the two pairs of conserved His residues accordingto the topological model of the protein obtained by alkalinephosphatase (PhoA) assays and GFP fusions the conservedHis77 and His858 residues are located on TM helices whileHis761 and His897 are located on the periplasmic side [36]These observations together with the results of site-directedmutagenesis experiments allowed the authors to suggest thatHis77 andHis858 form a low affinitymdashmembrane embeddedheme-binding site for ferrous heme which is subsequentlytranslocated to an external heme-binding domain of Hh-CcsBA where it is coordinated by His761 and His897 Thetopological model therefore predicts that this last His pairis part of a periplasmic-located WWD domain (homologousto theWWDdomains found in theCcmCandCcmFproteinsof System I described above)

4 System III

Strikingly different from Systems I and II the Cyt c mat-uration apparatus found in fungi and in metazoan cells(System III) is composed of a single protein known as Holo-Cytochrome c Synthetase (HCCS) or Cytochrome c HemeLyase (CCHL) apparently responsible for all the subprocessesdescribed above (heme transport and chaperoning reductionand chaperoning of apoCyt and catalysis of the thioetherbonds formation between heme and apoCyt) (Figure 4)Surprisingly although this protein has been identified in Scerevisiaemitochondria several years ago [125 126] only veryrecently the human HCCS has been expressed as a recombi-nant protein in E coli and spectroscopically characterized invitro [127] It is worth noticing that humanHCCS is attractinginterest since over and above its role in Cyt c biogenesis it

12 Scientifica

is involved in diseases such as microphthalmia with linearskin defects syndrome (MLS) an X-linked genetic disorder[128ndash130] and in other processes such as Cytc-independentapoptosis in injured motor neurons [131] Different fromanimal cells where a single HCCS is sufficient for thematuration of both soluble and membrane-anchored Cytc(Cyt c and Cy c1 resp) in S cerevisiae two homologs HCCSand HCC1S located in the inner mitochondrial membraneand facing to the IMS space [132 133] are responsible for hemeattachment to apoCyt c and apoCyt c1 respectively [125 134]It should also be noticed that in fungi an additional FAD-containing protein (Cyc2p) is required for Cyt c synthesisCyc2p is a mitochondrial membrane-anchored flavoproteinexposing its redox domain to the IMS which is requiredfor the maturation of Cyt c but not for that of Cyt c1 [135]This protein does not contain the conserved Cys residuestypically found in disulfide reductases and indeed it is notable to reduce oxidized apoCyt in vitro However it has beenrecently shown that Cyc2p is able to catalyze the NAD(P)H-dependent reduction of heme in vitro [136] a necessarystep before the thioether bond formation can occur asdiscussed above (see Section 26) This result together withthe observation that Cyc2p interacts with HCCS and withapoCyt c and c1 lends support to the proposal that Cyc2p isinvolved in the reduction of the heme iron in vivo [136 137]

AlthoughHCCS proteins are crucial for Cyt cmaturationwe still do not know how these proteins recognize theirsubstrates (heme and apoCyt) and how they promote orcatalyze the formation of thioether bonds between the hemevinyl groups and the cysteine thiols of the apoCyt CXXCHmotif

Contrary to the broad specificity of System I whichis able to recognize and attach the heme to prokaryoticand eukaryotic c-type cytochromes [49 50] and even tovery short microperoxidase-like sequences [49 50] mito-chondrial HCCS is characterized by a higher specificityas it does not recognize bacterial apoCyt sequences Theseobservations prompted the investigation of the recognitionprocess between apoCyt and HCCS Recently by using arecombinant yeast HCCS and chimerical apoCyt sequencesexpressed in E coli it was possible to conclude that a crucialrecognition determinant is represented by the N-terminalregion of apoCyt containing the heme-binding motif [35]notice that in the context of System I the same N-terminalregion of the Cyt c sequence has been recently identified to beimportant for the recognition by apoCcmE (see Section 26)Over and above the role played by the intervening residuesin the CXXCH heme-binding motif [135] it has been shownthat a conserved Phe residue occurring in the N-terminalregion before the CXXCH motif is important for HCCSrecognition [35 138] These results support the hypothesisthat this residue known to be a key determinant of Cytcfolding and stability [19 20 139] may also be crucial for Cytcmaturation by HCCS

Another relevant question concerns the ability of HCCSto recognize and bind the heme molecule as the region ofthe protein responsible for heme recognition remains to beidentified Initially it was hypothesized that the recognitionof heme could be mediated by the CP motifs present in

the HCCS protein [140] these short sequences are indeedknown to bind heme in a variety of heme-containing proteins[141 142] However it has been recently shown that CPmotifsof the recombinant S cerevisiae HCCS are not necessaryfor Cyt c production in E coli [143] excluding them as keydeterminants of heme recognition

The long-awaited in vitro characterization of the recom-binant human HCCS allowed for the first time to pro-pose a molecular mechanism underlying Cytc maturationin eukaryotes which can be experimentally tested [127]According to the proposed model the human HCCS activitycan be described as a four step mechanism involving (i)heme-binding (ii) apoCyt recognition (iii) thioether bondsformation and (iv) holoCyt c release In particular theheme is proposed to play the role of a scaffolding moleculemediating the contacts between HCCS and apoCyt Muta-genesis experiments carried out on the recombinant HCCSstrongly suggest that heme-binding (Step 1) depends on thepresence of a specific His residue (His154) acting as anaxial ligand to ferrous heme Residues present at the N-terminus of apoCyt mediate the recognition with HCCS(Step 2) as discussed above it is known that this regionforms structurally conserved 120572-helix in the fold of all c-type cytochromes Unfortunately no information is availableup to now concerning the region of HCCS involved in therecognition and binding to the N-terminal region of apoCytCoordination of the heme iron by the His residue of theapoCyt heme-binding motif CXXCH provides the secondaxial ligand to the heme iron and is probably importantfor the correct positioning of the two apoCyt Cys residuesand formation of the thioether bonds (Step 3) The finalrelease of functional Cyt c (Step 4) clearly requires thedisplacement of the His154Fe2+ coordination bond sucha displacement is probably mediated by formation of thecoordination bond with the Cyt c conserved Met residueandor by the simultaneous folding of Cyt c Again it isinteresting to note that this last hypothesis is in accordancewith in vitro folding studies on c-type cytochromes thathighlighted the late formation of the Met-Fe coordinationduring Cyt c folding [144 145]

Acknowledgments

The author thanks A Di Matteo and E Di Silvio for fruitfuldiscussions and for help in preparing this paper This work issupported by grants from the University of Rome ldquoSapienzardquo(C26A11MBXJ and C26A13MWF4)

References

[1] M G Galinato J G Kleingardner S E Bowman et alldquoHeme-protein vibrational couplings in cytochrome c providea dynamic link that connects the heme-iron and the proteinsurfacerdquo Proceedings of the National Academy of Sciences vol109 no 23 pp 8896ndash8900 2012

[2] H B Gray and J R Winkler ldquoElectron flow through metal-loproteinsrdquo Biochimica et Biophysica Acta vol 1797 no 9 pp1563ndash1572 2010

Scientifica 13

[3] P Ascenzi R Santucci M Coletta and F PolticellildquoCytochromes reactivity of the ldquodark siderdquo of the hemerdquoBiophysical Chemistry vol 152 no 1ndash3 pp 21ndash27 2010

[4] S Yamada N D Bouley Ford G E Keller W C Ford HB Gray and J R Winkler ldquoSnapshots of a protein foldingintermediaterdquo Proceedings of the National Academy of Sciencesvol 110 no 5 pp 1606ndash1610 2013

[5] P Weinkam J Zimmermann F E Romesberg and P GWolynes ldquoThe folding energy landscape and free energy excita-tions of cytochrome crdquo Accounts of Chemical Research vol 43no 5 pp 652ndash660 2010

[6] M Yamanaka M Masanari and Y Sambongi ldquoConfermentof folding ability to a naturally unfolded apocytochrome cthrough introduction of hydrophobic amino acid residuesrdquoBiochemistry vol 50 no 12 pp 2313ndash2320 2011

[7] G W Moore and G W Pettigrew Cytochrome C EvolutionaryStructural and Physicochemical Aspects Springer Berlin Ger-many 1990

[8] I Bertini G Cavallaro and A Rosato ldquoCytochrome c occur-rence and functionsrdquo Chemical Reviews vol 106 no 1 pp 90ndash115 2006

[9] D A Pearce and F Sherman ldquoDegradation of cytochromeoxidase subunits in mutants of yeast lacking cytochrome c andsuppression of the degradation by mutation of yme1rdquo Journal ofBiological Chemistry vol 270 no 36 pp 20879ndash20882 1995

[10] G Silkstone S M Kapetanaki I Husu M H Vos and MT Wilson ldquoNitric oxide binding to the cardiolipin complex offerric cytochrome crdquo Biochemistry vol 51 no 34 pp 6760ndash6766 2012

[11] M Giorgio E Migliaccio F Orsini et al ldquoElectron transferbetween cytochrome c and p66Shc generates reactive oxygenspecies that trigger mitochondrial apoptosisrdquo Cell vol 122 no2 pp 221ndash233 2005

[12] S M Kilbride and J H Prehn ldquoCentral roles of apoptoticproteins in mitochondrial functionrdquo Oncogene vol 32 no 22pp 2703ndash2711 2013

[13] T Noll and G Noll ldquoStrategies for ldquowiringrdquo redox-activeproteins to electrodes and applications in biosensors biofuelcells and nanotechnologyrdquo Chemical Society Reviews vol 40no 7 pp 3564ndash3576 2011

[14] G Layer J Reichelt D Jahn and D W Heinz ldquoStructure andfunction of enzymes in heme biosynthesisrdquo Protein Science vol19 no 6 pp 1137ndash1161 2010

[15] S Severance and IHamza ldquoTrafficking of heme and porphyrinsin metazoardquo Chemical Reviews vol 109 no 10 pp 4596ndash46162009

[16] P Hamel V Corvest P Giege and G Bonnard ldquoBiochemi-cal requirements for the maturation of mitochondrial c-typecytochromesrdquo Biochimica et Biophysica Acta vol 1793 no 1 pp125ndash138 2009

[17] W R Fisher H Taniuchi and C B Anfinsen ldquoOn the role ofheme in the formation of the structure of cytochrome crdquo Journalof Biological Chemistry vol 248 no 9 pp 3188ndash3195 1973

[18] M Yamanaka H Mita Y Yamamoto and Y Sambongi ldquoHemeis not required for aquifex aeolicus cytochrome c555 polypep-tide foldingrdquoBioscience Biotechnology andBiochemistry vol 73no 9 pp 2022ndash2025 2009

[19] C Travaglini-Allocatelli S Gianni andMBrunori ldquoA commonfolding mechanism in the cytochrome c familyrdquo Trends inBiochemical Sciences vol 29 no 10 pp 535ndash541 2004

[20] A Borgia D Bonivento C Travaglini-Allocatelli A DiMatteoand M Brunori ldquoUnveiling a hidden folding intermediate in c-type cytochromes by protein engineeringrdquo Journal of BiologicalChemistry vol 281 no 14 pp 9331ndash9336 2006

[21] E Enggist L Thony-Meyer P Guntert and K PervushinldquoNMR structure of the heme chaperone CcmE reveals a novelfunctional motifrdquo Structure vol 10 no 11 pp 1551ndash1557 2002

[22] A di Matteo N Calosci S Gianni P Jemth M Brunori and CTravaglini-Allocatelli ldquoStructural and functional characteriza-tion of CcmG from pseudomonas aeruginosa a key componentof the bacterial cytochrome c maturation apparatusrdquo Proteinsvol 78 no 10 pp 2213ndash2221 2010

[23] A diMatteo S GianniM E Schinina et al ldquoA strategic proteinin cytochrome c maturation three-dimensional structure ofCcmH and binding to apocytochrome crdquo Journal of BiologicalChemistry vol 282 no 37 pp 27012ndash27019 2007

[24] A Crow R M Acheson N E Le Brun and A OubrieldquoStructural basis of redox-coupled protein substrate selectionby the cytochrome c biosynthesis protein ResArdquo Journal ofBiological Chemistry vol 279 no 22 pp 23654ndash23660 2004

[25] E di Silvio A di Matteo and C Travaglini-Allocatelli ldquoTheRedox pathway of Pseudomonas aeruginosa cytochrome cbiogenesisrdquo Journal of Proteins and Proteomics vol 3 no 1 pp1ndash7 2012

[26] LThony-Meyer andP Kunzler ldquoTranslocation to the periplasmand signal sequence cleavage of preapocytochrome c depend onsec and lep but not on the ccmgene productsrdquoEuropean Journalof Biochemistry vol 246 no 3 pp 794ndash799 1997

[27] S J Facey and A Kuhn ldquoBiogenesis of bacterial inner-membrane proteinsrdquo Cellular and Molecular Life Sciences vol67 no 14 pp 2343ndash2362 2010

[28] K Diekert A I de Kroon U Ahting et al ldquoApocytochromec requires the TOM complex for translocation across themitochondrial outer membranerdquo The EMBO Journal vol 20no 20 pp 5626ndash5635 2001

[29] N Wiedemann V Kozjak T Prinz et al ldquoBiogenesis of yeastmitochondrial cytochrome c a unique relationship to the TOMmachineryrdquo Journal ofMolecular Biology vol 327 no 2 pp 465ndash474 2003

[30] F-U Hartl N Pfanner and W Neupert ldquoTranslocation inter-mediates on the import pathway of proteins intomitochondriardquoBiochemical Society Transactions vol 15 no 1 pp 95ndash97 1987

[31] B S Glick A Brandt K Cunningham SMuller R L Hallbergand G Schatz ldquoCytochromes c1 and b2 are sorted to theintermembrane space of yeast mitochondria by a stop-transfermechanismrdquo Cell vol 69 no 5 pp 809ndash822 1992

[32] G Howe and S Merchant ldquoRole of heme in the biosynthesis ofcytochrome c6rdquo Journal of Biological Chemistry vol 269 no 8pp 5824ndash5832 1994

[33] S S Nakamoto P Hamel and S Merchant ldquoAssembly ofchloroplast cytochromes b and crdquo Biochimie vol 82 no 6-7 pp603ndash614 2000

[34] C Sanders S Turkarslan D-W Lee and F DaldalldquoCytochrome c biogenesis the Ccm systemrdquo Trends inMicrobiology vol 18 no 6 pp 266ndash274 2010

[35] J M Stevens D A Mavridou R Hamer P Kritsiligkou A DGoddard and S J Ferguson ldquoCytochrome c biogenesis systemIrdquoThe FEBS Journal vol 278 no 22 pp 4170ndash4178 2011

[36] R G Kranz C Richard-Fogal J-S Taylor and E R FrawleyldquoCytochrome c biogenesis mechanisms for covalent modifica-tions and trafficking of heme and for heme-iron redox controlrdquo

14 Scientifica

Microbiology and Molecular Biology Reviews vol 73 no 3 pp510ndash528 2009

[37] J W A Allen ldquoCytochrome c biogenesis in mitochondriamdashsystems III and VrdquoThe FEBS Journal vol 278 no 22 pp 4198ndash4216 2011

[38] D A Mavridou S J Ferguson and J M Stevens ldquoCytochromec assemblyrdquo IUBMB Life vol 65 no 3 pp 209ndash216 2013

[39] I Bertini G Cavallaro and A Rosato ldquoEvolution ofmitochondrial-type cytochrome c domains and of theprotein machinery for their assemblyrdquo Journal of InorganicBiochemistry vol 101 no 11-12 pp 1798ndash1811 2007

[40] B S Goldman and R G Kranz ldquoEvolution and horizontaltransfer of an entire biosynthetic pathway for cytochromec biogenesis helicobacter deinococcus archae and morerdquoMolecular Microbiology vol 27 no 4 pp 871ndash873 1998

[41] C L Richard-Fogal E R Frawley E R Bonner H Zhu BSan Francisco and R G Kranz ldquoA conserved haem redoxand trafficking pathway for cofactor attachmentrdquo The EMBOJournal vol 28 no 16 pp 2349ndash2359 2009

[42] L Thony-Meyer F Fischer P Kunzler D Ritz and H Hen-necke ldquoEscherichia coli genes required for cytochrome cmaturationrdquo Journal of Bacteriology vol 177 no 15 pp 4321ndash4326 1995

[43] C S Beckett J A Loughman K A Karberg G M DonatoW E Goldman and R G Kranz ldquoFour genes are required forthe system II cytochrome c biogenesis pathway in Bordetellapertussis a unique bacterial modelrdquo Molecular Microbiologyvol 38 no 3 pp 465ndash481 2000

[44] N E Le Brun J Bengtsson and L Hederstedt ldquoGenes requiredfor cytochrome c synthesis in Bacillus subtilisrdquo MolecularMicrobiology vol 36 no 3 pp 638ndash650 2000

[45] P Giege J M Grienenberger and G Bonnard ldquoCytochromec biogenesis in mitochondriardquo Mitochondrion vol 8 no 1 pp61ndash73 2008

[46] R E Feissner C L Richard-Fogal E R Frawley J A Lough-man KW Earley and R G Kranz ldquoRecombinant cytochromesc biogenesis systems I and II and analysis of haem deliverypathways in Escherichia colirdquo Molecular Microbiology vol 60no 3 pp 563ndash577 2006

[47] N P Cianciotto P Cornelis and C Baysse ldquoImpact of thebacterial type I cytochrome c maturation system on differentbiological processesrdquoMolecular Microbiology vol 56 no 6 pp1408ndash1415 2005

[48] E Arslan H Schulz R Zufferey P Kunzler and L Thony-Meyer ldquoOverproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in Escherichiacolirdquo Biochemical and Biophysical Research Communicationsvol 251 no 3 pp 744ndash747 1998

[49] C Sanders and H Lill ldquoExpression of prokaryotic and eukary-otic cytochromes c in Escherichia colirdquoBiochimica et BiophysicaActa vol 1459 no 1 pp 131ndash138 2000

[50] M Braun and L Thony-Meyer ldquoBiosynthesis of artificialmicroperoxidases by exploiting the secretion and cytochromec maturation apparatuses of Escherichia colirdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 35 pp 12830ndash12835 2004

[51] B Baert C Baysse S Matthijs and P Cornelis ldquoMultiplephenotypic alterations caused by a c-type cytochrome mat-uration ccmC gene mutation in Pseudomonas aeruginosardquoMicrobiology vol 154 no 1 pp 127ndash138 2008

[52] S Turkarslan C Sanders and F Daldal ldquoExtracytoplasmicprosthetic group ligation to apoproteins maturation of c-typecytochromesrdquo Molecular Microbiology vol 60 no 3 pp 537ndash541 2006

[53] P M Jones and A M George ldquoThe ABC transporter structureand mechanism perspectives on recent researchrdquo Cellular andMolecular Life Sciences vol 61 no 6 pp 682ndash699 2004

[54] O Christensen E M Harvat L Thony-Meyer S J Fergusonand J M Stevens ldquoLoss of ATP hydrolysis activity by CcmABresults in loss of c-type cytochrome synthesis and incompleteprocessing ofCcmErdquoTheFEBS Journal vol 274 no 9 pp 2322ndash2332 2007

[55] J-H Lee E M Harvat J M Stevens S J Ferguson and M HSaier Jr ldquoEvolutionary origins of members of a superfamily ofintegralmembrane cytochrome c biogenesis proteinsrdquoBiochim-ica et Biophysica Acta vol 1768 no 9 pp 2164ndash2181 2007

[56] D L Beckman D R Trawick and R G Kranz ldquoBacterialcytochromes c biogenesisrdquo Genes and Development vol 6 no2 pp 268ndash283 1992

[57] H Schulz R A Fabianek E C Pellicioli H Hennecke andL Thony-Meyer ldquoHeme transfer to the heme chaperone CcmEduring cytochrome c maturation requires the CcmC proteinwhich may function independently of the ABC-transporterCcmABrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 96 no 11 pp 6462ndash6467 1999

[58] Q Ren and L Thony-Meyer ldquoPhysical Interaction ofCcmC with Heme and the Heme Chaperone CcmE duringCytochrome cMaturationrdquo Journal of Biological Chemistry vol276 no 35 pp 32591ndash32596 2001

[59] C Richard-Fogal and R G Kranz ldquoThe CcmChemeCcmEcomplex in heme trafficking and cytochrome c biosynthesisrdquoJournal of Molecular Biology vol 401 no 3 pp 350ndash362 2010

[60] V K Viswanathan S Kurtz L L Pedersen et al ldquoThecytochrome C maturation locus of Legionella pneumophilapromotes iron assimilation and intracellular infection andcontains a strain-specific insertion sequence elementrdquo Infectionand Immunity vol 70 no 4 pp 1842ndash1852 2002

[61] U Ahuja and L Thony-Meyer ldquoCcmD is involved in complexformation between CcmC and the heme chaperone CcmE dur-ing cytochrome c maturationrdquo Journal of Biological Chemistryvol 280 no 1 pp 236ndash243 2005

[62] C L Richard-Fogal E R Frawley and R G Kranz ldquoTopologyand function of CcmD in cytochrome Cmaturationrdquo Journal ofBacteriology vol 190 no 10 pp 3489ndash3493 2008

[63] H Schulz H Hennecke and L Thony-Meyer ldquoPrototype ofa heme chaperone essential for cytochrome c maturationrdquoScience vol 281 no 5380 pp 1197ndash1200 1998

[64] F Arnesano L Banci P D Barker et al ldquoSolution structure andcharacterization of the heme chaperone CcmErdquo Biochemistryvol 41 no 46 pp 13587ndash13594 2002

[65] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[66] L Thony-Meyer ldquoA heme chaperone for cytochrome c biosyn-thesisrdquo Biochemistry vol 42 no 45 pp 13099ndash13105 2003

[67] E M Harvat C Redfield J M Stevens and S J FergusonldquoProbing the heme-binding site of the cytochrome cmaturationprotein CcmErdquoBiochemistry vol 48 no 8 pp 1820ndash1828 2009

[68] D Lee K Pervushin D Bischof M Braun and L Thony-Meyer ldquoUnusual heme-histidine bond in the active site of a

Scientifica 15

chaperonerdquo Journal of the American Chemical Society vol 127no 11 pp 3716ndash3717 2005

[69] A D Goddard J M Stevens F Rao et al ldquoc-Type cytochromebiogenesis can occur via a natural Ccm system lacking CcmHCcmG and the heme-binding histidine of CcmErdquo Journal ofBiological Chemistry vol 285 no 30 pp 22882ndash22889 2010

[70] J M Aramini K Hamilton P Rossi et al ldquoSolution NMRstructure backbone dynamics and heme-binding propertiesof a novel cytochrome c maturation protein CcmE fromDesulfovibrio vulgarisrdquo Biochemistry vol 51 no 18 pp 3705ndash3707 2012

[71] T Uchida J M Stevens O Daltrop et al ldquoThe interactionof covalently bound heme with the cytochrome c maturationprotein CcmErdquo Journal of Biological Chemistry vol 279 no 50pp 51981ndash51988 2004

[72] I Garcıa-Rubio M Braun I Gromov L Thony-Meyer andA Schweiger ldquoAxial coordination of heme in ferric CcmEchaperone characterized by EPR spectroscopyrdquo BiophysicalJournal vol 92 no 4 pp 1361ndash1373 2007

[73] L Thony-Meyer ldquoHaem-polypeptide interactions duringcytochrome c maturationrdquo Biochimica et Biophysica Acta vol1459 no 2-3 pp 316ndash324 2000

[74] J A Keightley D Sanders T R Todaro A Pastuszyn and J AFee ldquoCloning expression in Escherichia coli of the cytochromec552 gene from Thermus thermophilus HB8 Evidence forgenetic linkage to an ATP- binding cassette protein and initialcharacterization of the cycA gene productsrdquo Journal of Biologi-cal Chemistry vol 273 no 20 pp 12006ndash12016 1998

[75] E M Monika B S Goldman D L Beckman and R GKranz ldquoA thioreduction pathway tethered to the membranefor periplasmic cytochromes c biogenesis In vitro and in vivostudiesrdquo Journal of Molecular Biology vol 271 no 5 pp 679ndash692 1997

[76] M Throne-Holst L Thony-Meyer and L HederstedtldquoEscherichia coli ccm in-frame deletion mutants can produceperiplasmic cytochrome b but not cytochrome crdquo FEBS Lettersvol 410 no 2-3 pp 351ndash355 1997

[77] U Ahuja and L Thony-Meyer ldquoDynamic features of a hemedelivery system for cytochrome c maturationrdquo Journal of Bio-logical Chemistry vol 278 no 52 pp 52061ndash52070 2003

[78] J W A Allen P D Barker O Daltrop et al ldquoWhy isnrsquot ldquostan-dardrdquo heme good enough for c-type and di-type cytochromesrdquoDalton Transactions no 21 pp 3410ndash3418 2005

[79] K Inaba and K Ito ldquoStructure and mechanisms of the DsbB-DsbA disulfide bond generation machinerdquo Biochimica et Bio-physica Acta vol 1783 no 4 pp 520ndash529 2008

[80] H Kadokura F Katzen and J Beckwith ldquoProtein disulfidebond formation in prokaryotesrdquoAnnual Review of Biochemistryvol 72 pp 111ndash135 2003

[81] S R Shouldice B Heras P M Walden M Totsika M ASchembri and J L Martin ldquoStructure and function of DsbA akey bacterial oxidative folding catalystrdquoAntioxidants and RedoxSignaling vol 14 no 9 pp 1729ndash1760 2011

[82] R Metheringham L Griffiths H Crooke S Forsythe and JCole ldquoAn essential role for DsbA in cytochrome c synthesis andformate-dependent nitrite reduction by Escherichia coli K-12rdquoArchives of Microbiology vol 164 no 4 pp 301ndash307 1995

[83] Y Sambongi and S J Ferguson ldquoMutants of Escherichia colilacking disulphide oxidoreductases DsbA and DsbB cannotsynthesise an exogenous monohaem c-type cytochrome exceptin the presence of disulphide compoundsrdquo FEBS Letters vol398 no 2-3 pp 265ndash268 1996

[84] D AMavridou S J Ferguson and JM Stevens ldquoThe interplaybetween the disulfide bond formation pathway and cytochromec maturation in Escherichia colirdquo FEBS Letters vol 586 no 12pp 1702ndash1707 2012

[85] C U Stirnimann A Rozhkova U Grauschopf M G GrutterR Glockshuber and G Capitani ldquoStructural basis and kineticsof DsbD-dependent cytochrome c maturationrdquo Structure vol13 no 7 pp 985ndash993 2005

[86] N Ouyang Y-G Gao H-Y Hu and Z-X Xia ldquoCrystalstructures of E coli CcmGand itsmutants reveal key roles of theN-terminal120573-sheet and the fingerprint regionrdquoProteins vol 65no 4 pp 1021ndash1031 2006

[87] M A Edeling L W Guddat R A Fabianek et al ldquoCrys-tallization and preliminary diffraction studies of native andselenomethionine CcmG (CycY DsbE)rdquoActa CrystallographicaD vol 57 no 9 pp 1293ndash1295 2001

[88] R A Fabianek M Huber-Wunderlich R Glockshuber PKunzler H Hennecke and L Thony-Meyer ldquoCharacterizationof the Bradyrhizobium japonicum CycY protein a membrane-anchored periplasmic thioredoxin that may play a role as areductant in the biogenesis of c-type cytochromesrdquo Journal ofBiological Chemistry vol 272 no 7 pp 4467ndash4473 1997

[89] G B Kallis and A Holmgren ldquoDifferential reactivity of thefunctional sulfhydryl groups of cysteine-32 and cysteine-32present in the reduced form of thioredoxin from Escherichiacolirdquo Journal of Biological Chemistry vol 255 no 21 pp 10261ndash10265 1980

[90] P T Chivers andR T Raines ldquoGeneral acidbase catalysis in theactive site of Escherichia coli thioredoxinrdquoBiochemistry vol 36no 50 pp 15810ndash15816 1997

[91] X M Zheng J Hong H Y Li D H Lin and H Y HuldquoBiochemical properties and catalytic domain structure of theCcmH protein from Escherichia colirdquo Biochim Biophys Actavol 1824 no 12 pp 1394ndash1400 2012

[92] UAhujaA Rozhkova RGlockshuber LThony-Meyer andOEinsle ldquoHelix swapping leads to dimerization of the N-terminaldomain of the c-type cytochrome maturation protein CcmHfrom Escherichia colirdquo FEBS Letters vol 582 no 18 pp 2779ndash2786 2008

[93] R A Fabianek T Hofer and L Thony-Meyer ldquoCharacteriza-tion of the Escherichia coli CcmH protein reveals new insightsinto the redox pathway required for cytochrome c maturationrdquoArchives of Microbiology vol 171 no 2 pp 92ndash100 1999

[94] E Reid J Cole and D J Eaves ldquoThe Escherichia coli CcmGprotein fulfils a specific role in cytochrome c assemblyrdquo Bio-chemical Journal vol 355 no 1 pp 51ndash58 2001

[95] Q Ren U Ahuja and LThony-Meyer ldquoA bacterial cytochromec heme lyase CcmF forms a complex with the heme chaperoneCcmE and CcmH but not with apocytochrome crdquo Journal ofBiological Chemistry vol 277 no 10 pp 7657ndash7663 2002

[96] E H Meyer P Giege E Gelhaye et al ldquoAtCCMH an essentialcomponent of the c-type cytochrome maturation pathway inArabidopsis mitochondria interacts with apocytochrome crdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 44 pp 16113ndash16118 2005

[97] C Rios-Velazquez R Coller and T J Donohue ldquoFeatures ofRhodobacter sphaeroides CcmFHrdquo Journal of Bacteriology vol185 no 2 pp 422ndash431 2003

[98] C Sanders M Deshmukh D Astor R G Kranz and F DaldalldquoOverproduction of CcmG and CcmFHRc fully suppresses thec-type cytochrome biogenesis defect of Rhodobacter capsulatus

16 Scientifica

CcmI-null mutantsrdquo Journal of Bacteriology vol 187 no 12 pp4245ndash4256 2005

[99] A F Verissimo H Yang X Wu C Sanders and F DaldalldquoCcmI subunit of CcmFHI heme ligation complex functionsas an apocytochrome c chaperone during c-type cytochromematurationrdquo Journal of Biological Chemistry vol 286 no 47 pp40452ndash40463 2011

[100] K Bryson L J McGuffin R L Marsden J J Ward J SSodhi and D T Jones ldquoProtein structure prediction servers atUniversity College Londonrdquo Nucleic Acids Research vol 33 no2 pp W36ndashW38 2005

[101] D T Jones ldquoProtein secondary structure prediction basedon position-specific scoring matricesrdquo Journal of MolecularBiology vol 292 no 2 pp 195ndash202 1999

[102] C Sanders C Boulay and F Daldal ldquoMembrane-spanning andperiplasmic segments of CcmI have distinct functions duringcytochrome c biogenesis in Rhodobacter capsulatusrdquo Journal ofBacteriology vol 189 no 3 pp 789ndash800 2007

[103] D Han K Kim J Oh J Park and Y Kim ldquoTPR domainof NrfG mediates complex formation between heme lyaseand formate-dependent nitrite reductase in Escherichia coliO157H7rdquo Proteins vol 70 no 3 pp 900ndash914 2008

[104] D J Eaves J Grove W Staudenmann et al ldquoInvolvement ofproducts of the nrfEFG genes in the covalent attachment ofhaem c to a novel cysteine-lysine motif in the cytochrome c552nitrite reductase from Escherichia colirdquo Molecular Microbiol-ogy vol 28 no 1 pp 205ndash216 1998

[105] J Nilsson B Persson and G Von Heijne ldquoConsensus predic-tions of membrane protein topologyrdquo FEBS Letters vol 486 no3 pp 267ndash269 2000

[106] E di Silvio A di Matteo F Malatesta and C Travaglini-Allocatelli ldquoRecognition and binding of apocytochrome c toP aeruginosa CcmI a component of cytochrome c maturationmachineryrdquo Biochim Biophys Acta vol 1834 no 8 pp 1554ndash1561 2013

[107] N Zeytuni and R Zarivach ldquoStructural and functional dis-cussion of the tetra-trico-peptide repeat a protein interactionmodulerdquo Structure vol 20 no 3 pp 397ndash405 2012

[108] C Sanders S Turkarslan D-W Lee O Onder R G Kranz andF Daldal ldquoThe cytochrome c maturation components CcmFCcmH and CcmI form a membrane-integral multisubunitheme ligation complexrdquo Journal of Biological Chemistry vol283 no 44 pp 29715ndash29722 2008

[109] A F Verissimo M A Mohtar and F Daldal ldquoThe hemechaperone ApoCcmE forms a ternary complex with CcmI andapocytochrome crdquoThe Journal of Biological Chemistry vol 288no 9 pp 6272ndash6283 2013

[110] P D Barker J C Ferrer M Mylrajan et al ldquoTransmutation of aheme proteinrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 90 no 14 pp 6542ndash65461993

[111] J Simon and L Hederstedt ldquoComposition and function ofcytochrome c biogenesis system IIrdquoThe FEBS Journal vol 278no 22 pp 4179ndash4188 2011

[112] T R H M Kouwen and J M van Dijl ldquoInterchangeablemodules in bacterial thiol-disulfide exchange pathwaysrdquo Trendsin Microbiology vol 17 no 1 pp 6ndash12 2009

[113] A Crow A Lewin O Hecht et al ldquoCrystal structure andbiophysical properties of Bacillus subtilis BdbD An oxidizingthioldisulfide oxidoreductase containing a novel metal siterdquoJournal of Biological Chemistry vol 284 no 35 pp 23719ndash23733 2009

[114] M C Moller and L Hederstedt ldquoExtracytoplasmic processesimpaired by inactivation of trxA (thioredoxin gene) in Bacillussubtilisrdquo Journal of Bacteriology vol 190 no 13 pp 4660ndash46652008

[115] A Lewin A Crow A Oubrie and N E Le Brun ldquoMolecularbasis for specificity of the extracytoplasmic thioredoxin ResArdquoJournal of Biological Chemistry vol 281 no 46 pp 35467ndash35477 2006

[116] C L Colbert QWu P J A Erbel K H Gardner and J Deisen-hofer ldquoMechanism of substrate specificity in Bacillus subtilisResA a thioredoxin-like protein involved in cytochrome cmaturationrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 103 no 12 pp 4410ndash44152006

[117] UAhuja P Kjelgaard B L Schulz LThony-Meyer andLHed-erstedt ldquoHaem-delivery proteins in cytochrome c maturationsystem IIrdquoMolecular Microbiology vol 73 no 6 pp 1058ndash10712009

[118] M L D Page P P Hamel S T Gabilly et al ldquoA homologof prokaryotic thiol disulfide transporter CcdA is required forthe assembly of the cytochrome b6f complex in Arabidopsischloroplastsrdquo Journal of Biological Chemistry vol 279 no 31pp 32474ndash32482 2004

[119] A Rozhkova C U Stirnimann P Frei et al ldquoStructural basisand kinetics of inter- and intramolecular disulfide exchange inthe redox catalyst DsbDrdquoThe EMBO Journal vol 23 no 8 pp1709ndash1719 2004

[120] T Schiott M Throne-Holst and L Hederstedt ldquoBacillussubtilis CcdA-defective mutants are blocked in a late step ofcytochrome C biogenesisrdquo Journal of Bacteriology vol 179 no14 pp 4523ndash4529 1997

[121] R E Feissner C S Beckett J A Loughman and R G KranzldquoMutations in cytochrome assembly and periplasmic redoxpathways in Bordetella pertussisrdquo Journal of Bacteriology vol187 no 12 pp 3941ndash3949 2005

[122] L S ErlendssonMMoller and L Hederstedt ldquoBacillus subtilisStoA is a thiol-disulfide oxidoreductase important for sporecortex synthesisrdquo Journal of Bacteriology vol 186 no 18 pp6230ndash6238 2004

[123] L S Erlendsson R M Acheson L Hederstedt and N E LeBrun ldquoBacillus subtilis ResA is a thiol-disulfide oxidoreductaseinvolved in cytochrome c synthesisrdquo Journal of BiologicalChemistry vol 278 no 20 pp 17852ndash17858 2003

[124] L S Erlendsson and L Hederstedt ldquoMutations in the thiol-disulfide oxidoreductases BdbC and BdbD can suppresscytochrome c deficiency of CcdA-defective Bacillus subtiliscellsrdquo Journal of Bacteriology vol 184 no 5 pp 1423ndash1429 2002

[125] M E Dumont J F Ernst D M Hampsey and F ShermanldquoIdentification and sequence of the gene encoding cytochromec heme lyase in the yeast Saccharomyces cerevisiaerdquoThe EMBOJournal vol 6 no 1 pp 235ndash241 1987

[126] M E Dumont J F Ernst and F Sherman ldquoCoupling of hemeattachment to import of cytochrome c into yeast mitochondriaStudies with heme lyase-deficient mitochondria and alteredapocytochromes crdquo Journal of Biological Chemistry vol 263 no31 pp 15928ndash15937 1988

[127] B San Francisco E C Bretsnyder and R G Kranz ldquoHumanmitochondrial holocytochrome c synthasersquos hemebindingmat-uration determinants and complex formationwith cytochromecrdquo Proceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 9 pp E788ndashE797 2012

Scientifica 17

[128] L Schaefer A Ballabio and H Y Zoghbi ldquoCloning andcharacterization of a putative human holocytochrome c-typesynthetase gene (HCCS) isolated from the critical region formicrophthalmia with linear skin defects (MLS)rdquoGenomics vol34 no 2 pp 166ndash172 1996

[129] S K Prakash T A Cormier A E McCall et al ldquoLoss ofholocytochrome c-type synthetase causes the male lethality ofX-linked dominant microphthalmia with linear skin defects(MLS) syndromerdquo Human Molecular Genetics vol 11 no 25pp 3237ndash3248 2002

[130] I Wimplinger M Morleo G Rosenberger et al ldquoMutations ofthe mitochondrial holocytochrome c-type synthase in X-linkeddominant microphthalmia with linear skin defects syndromerdquoTheAmerican Journal of HumanGenetics vol 79 no 5 pp 878ndash889 2006

[131] S Kiryu-Seo KGamo T Tachibana K Tanaka andHKiyamaldquoUnique anti-apoptotic activity of EAAC1 in injured motorneuronsrdquoTheEMBO Journal vol 25 no 14 pp 3411ndash3421 2006

[132] R Lill R A Stuart M E Drygas F E Nargang and W Neu-pert ldquoImport of cytochrome c heme lyase into mitochondriaa novel pathway into the intermembrane spacerdquo The EMBOJournal vol 11 no 2 pp 449ndash456 1992

[133] H Steiner A Zollner A Haid W Neupert and R LillldquoBiogenesis of mitochondrial heme lyases in yeast Importand folding in the intermembrane spacerdquo Journal of BiologicalChemistry vol 270 no 39 pp 22842ndash22849 1995

[134] A Zollner G Rodel and A Haid ldquoMolecular cloning andcharacterization of the Saccharomyces cerevisiae CYT2 geneencoding cytochrome-c1-heme lyaserdquo European Journal of Bio-chemistry vol 207 no 3 pp 1093ndash1100 1992

[135] V Corvest D A Murrey D G Bernard D B Knaff B Guiardand P PHamel ldquoc-Type cytochrome assembly in Saccharomycescerevisiae a key residue for apocytochrome c1lyase interactionrdquoGenetics vol 186 no 2 pp 561ndash571 2010

[136] V Corvest D A Murrey M Hirasawa D B Knaff B Guiardand P P Hamel ldquoThe flavoprotein Cyc2p a mitochondrialcytochrome c assembly factor is a NAD(P)H-dependent haemreductaserdquo Molecular Microbiology vol 83 no 5 pp 968ndash9802012

[137] D G Bernard S Quevillon-Cheruel S Merchant B Guiardand P P Hamel ldquoCyc2p a membrane-bound flavopro-tein involved in the maturation of mitochondrial c-typecytochromesrdquo Journal of Biological Chemistry vol 280 no 48pp 39852ndash39859 2005

[138] J G Kleingardner and K L Bren ldquoComparing substratespecificity between cytochrome cmaturation and cytochrome cheme lyase systems for cytochrome c biogenesisrdquo Metallomicsvol 3 no 4 pp 396ndash403 2011

[139] C Travaglini-Allocatelli S Gianni VMorea A Tramontano TSoulimane and M Brunori ldquoExploring the cytochrome c fold-ing mechanism cytochrome c552 fromThermus thermophilusfolds through an on-pathway intermediaterdquo Journal of BiologicalChemistry vol 278 no 42 pp 41136ndash41140 2003

[140] L Zhang and L Guarente ldquoHeme binds to a short sequencethat serves a regulatory function in diverse proteinsrdquoTheEMBOJournal vol 14 no 2 pp 313ndash320 1995

[141] S Hira T Tomita T Matsui K Igarashi and M Ikeda-SaitoldquoBach1 a heme-dependent transcription factor reveals pres-ence of multiple heme binding sites with distinct coordinationstructurerdquo IUBMB Life vol 59 no 8-9 pp 542ndash551 2007

[142] T K Kuhl A Wiszligbrock N Goradia et al ldquoAnalysis of Fe(III)heme binding to cysteine-containing heme-regulatory motifs

in proteinsrdquo ACS Chemical Biology vol 8 no 8 pp 1785ndash17932013

[143] R L Moore J M Stevens and S J Ferguson ldquoMitochondrialcytochrome c synthase CP motifs are not necessary for hemeattachment to apocytochrome crdquo FEBS Letters vol 585 no 21pp 3415ndash3419 2011

[144] S Gianni M Brunori and C Travaglini-Allocatelli ldquoRefoldingkinetics of cytochrome c551 reveals a mechanistic differencebetween urea and guanidinerdquo Protein Science vol 10 no 8 pp1685ndash1688 2001

[145] S Gianni C Travaglini-Allocatelli F Cutruzzola M BrunoriM C R Shastry and H Roder ldquoParallel pathways incytochrome c551 foldingrdquo Journal of Molecular Biology vol 330no 5 pp 1145ndash1152 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Advances in

Virolog y

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Nucleic AcidsJournal of

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Stem CellsInternational

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Enzyme Research

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International Journal of

Microbiology

10 Scientifica

domain of Ec-CcmH) may act to provide a platform for theunfolded apoCyt chaperoning it to the heme attachment sitepresumably located on the WWD domain of CcmF CcmImay thus be considered a component of amembrane-integralmultisubunit heme ligation complex together with CcmFand CcmH as experimentally observed by affinity purifi-cation experiments carried out with Rc-CcmFHI proteins[97 99 108] According to the proposed function of CcmI acritical requirement is represented by its ability to recognizedifferent protein targets over and above apoCyt such asCcmFandCcmHHowever until now direct evidence has been pre-sented only for the interaction of CcmI with apoCyt but thepossibility remains that CcmFHI proteins interact each othervia their TM helices and not via their periplasmic domainsInterestingly both for Pa-CcmI [106] or Rc-CcmI [99] CDspectroscopy experiments carried out on the CcmIapoCytcomplex highlighted major conformational changes at thesecondary structure level It is tempting to speculate on thebasis of these results that in vivo the folding of apoCyt maybe induced by the interaction with CcmI In the case of Paeruginosa System I proteins the binding process betweenPa-CcmI and its target protein apoCyt c551 (Pa-apoCyt) hasbeen studied both at equilibrium and kinetically [106] the119870119863measured for this interaction (in the 120583M range) appeared

to be low enough to ensure apoCyt delivery to the othercomponents of the Ccmmachinery Clearly a major questionconcerns the molecular determinants of such recognitionprocess interestingly both affinity coprecipitation assays[99] and equilibrium and kinetic binding experiments [106]highlighted the role played by the C-terminal 120572-helix of Cytc Similar observations have been made for the interaction ofEc-NrfGwith a peptidemimickingNrfA its apoCyt substrate[103] in this case isothermal titration calorimetry (ITC)experiments indicate that the TPR-domain of NrfG serves asa binding site for the C-terminal motif of NrfA Altogetherthese observations are in agreement with the fact that TPRproteins generally bind to their targets by recognizing theirC-terminal region [107]

The CcmI chaperoning activity has been experimentallysupported for the first time in the case of Pa-CcmI by citratesynthase tests [106] it has been proposed that the observedability to suppress protein aggregation in vitromay reflect thecapacity of CcmI to avoid apoCyt aggregation in vivo Stillanother piece of the Cyt c biogenesis puzzle has been addedrecently by showing that Rc-CcmI is able to interact withapoCcmE either alone or together with its substrate apoCytc2 forming a stable ternary complex in the absence of heme[109] This unexpected observation obtained by reciprocalcopurification experiments provides supporting evidence forthe existence of a large multisubunit complex composed ofCcmFHI andCcmE possibly interactingwith theCcmABCDcomplex It is interesting to note that while in the case ofthe CcmIapoCyt recognition different studies highlightedthe crucial role of the C-terminal helical region of apoCyt(see above) in the case of the apoCcmE apoCyt recognitionthe N-terminal region of apoCyt seems to represent a criticalregion

It is generally accepted that CcmF is the Ccm compo-nent responsible for heme covalent attachment to apoCyt

however as discussed above it is possible that this largemembrane protein plays such a role only together withother Ccm proteins such as CcmH and CcmI Moreover asrecently discovered by Kranz and coworkers [36 41] CcmFmay also act as a quinoleheme oxidoreductase ensuringthe necessary reduction of the oxidized heme b bound toCcmE Why it is necessary that the heme iron be in itsreduced state rather than in its oxidized state is not completelyclear although it is possible that this is a prerequisite to themechanism of thioether bond formation [110] According tocurrent hypotheses it is likely that the periplasmic WWDdomain of CcmF provides a platform for heme b bindingSanders et al and Verissimo et al [34 109] have presenteda mechanistic view of the heme attachment process whichtakes into account all the available experimental observationson the different Ccmproteins According to thismodel stere-ospecific heme ligation to reduced apoCyt occurs becauseonly the vinyl-4 group is available to form the first thioetherbond with a free cysteine at the apoCyt heme-binding motifsince the vinyl-2 group is involved (at least in the Ec-CcmE)in the covalent bond with His130 of CcmE [67] Howeverexperimental proof for this hypothesis requires a detailedinvestigation of the apoCyt thioreduction process catalyzedby CcmH (see Section 24)

It should be noticed that the mechanisms described sofar for the function(s) played by CcmF (see [34 36 109]) donot envisage a clear role for its large C-terminal periplasmicdomain (residues 510 to 611 in Ec-CcmF) It would be inter-esting to see if this domain apparently devoid of recognizablesequence features may mediate intermolecular recognitionprocesses with one (or more) component(s) involved in thehemeapoCyt ligation process

3 System II

System II is typically found in gram-positive bacteria and inin 120576-proteobacteria it is also present in most 120573- and some120575-proteobacteria in Aquificales and cyanobacteria as wellas in algal and plant chloroplasts System II is composedof three or four membrane-bound proteins CcdA ResACcsA (also known as ResC) and CcsB (also known as ResB)(Figure 3) CcdA andResA are redox-active proteins involvedin the reduction of the disulfide bond in the heme-bindingmotif of apoCyt whereas CcsA and CcsB are responsible forthe heme-apoCyt ligation process and are considered Cyt csynthethases (CCS) BothCcsAwhich is evolutionary relatedto the CcmC and CcmF proteins of System I [55] and CcsBare integral membrane proteins In some 120576-proteobacteriasuch asHelicobacter hepaticus andHelicobacter pylori a singlefusion protein composed of CcsA and CcsB polypeptides ispresent [41 111] Although as discussed below evidence hasbeen put forward to support the hypothesis that the CcsBAcomplex acts a heme translocase we still do not know if theheme is transported across the membrane by component(s)of System II itself or by a different unidentified process

31 System II ApoCyt Thioreduction Pathway After the Secmachinery secretes the newly synthesized apoCyt it readily

Scientifica 11

becomes a substrate for the oxidative system present on theouter surface of the cytoplasmic membrane of the gram-positive bacteria In B subtilis the BdbCD system [112113] is functionally but not structurally equivalent to thewell-characterized DsbAB system present in gram-negativebacteria As discussed above for System I the disulfide ofthe apoCyt heme-binding motif must be reduced in orderto allow thioether bonds formation and heme attachmentResA is the extracytoplasmic membrane-anchored TRX-likeprotein involved in the specific reduction of the apoCytdisulfide bond after the disulfide bond exchange reactionhas occurred oxidized ResA is reduced by CcdA whichreceives its reducing equivalents from a cytoplasmic TRX[114] Although ResA displays a classical TRX-like foldthe analysis of the 3D structure of oxidized and reducedResA from B subtilis (Bs-ResA) showed redox-dependentconformational modifications not observed in other TRX-like proteins Interestingly such modifications occur at thelevel of a cavity proposed to represent the binding site foroxidized apoCyt [24 115 116] Another peculiar feature of Bs-ResA is represented by the unusually similar pKa values of itsthe active site Cys residues (88 and 82 resp) as observedalso in the case of the active site Cys residues of the SystemI Pa-CcmH (see Section 23) However at variance with Pa-CcmH in Bs-ResA the large separation between the twocysteine thiols observed in the structure of the reduced formof the protein can be invoked to account for this result

CcdA is a large membrane protein containing six TMhelices [117 118] homologous to the TM domain of E coliDsbD [85 119] Its role in the Cyt c biogenesis is supportedby the observation that inactivation of CcdA blocks theproduction of c-type cytochromes in B subtilis [120 121]Moreover over and above the reduction of its apoCyt sub-strate Bs-CcdA is able to reduce the disulfide bond of othersecreted proteins such as StoA [122] It is interesting to notethat ResA and CcdA are not essential for Cyt c synthesisin the absence of BdbD or BdbC or if a disulfide reductantis present in the growth medium [123 124] As discussedabove (Section 24) a similar observation was made on therole of the thio-reductive pathway of System I in this casepersistent production of c-type cytochromes was observedin Dsb-inactivated bacterial strains [38] Following theseobservations the question has been put forward as to why theCyt c biogenesis process involves this apparently redundantthio-reduction route only to correct the effects of Bdb orDsb activity Referring to the case of B subtilis [111] it hasbeen proposed that the main reason is that BdbD mustefficiently oxidize newly secreted proteins since the activityof most of them depends on the presence of disulfide bondsAlternatively it can be hypothesized that the intramoleculardisulfide bondof apoCyt protects the protein fromproteolyticdegradation or aggregation or from cross-linking to otherthiol-containing proteins

32 System II Heme Translocation and Attachment to ApoCytRecent experimental evidence is accumulating supportingthe involvement of the heterodimeric membrane complexResBC or of the fusion protein CcsBA in the translocation

of heme from the n-side to the p-side of the bacterialmembrane In particular it has been shown that CcsBAfrom H hepaticus (Hh-CcsBA) is able to reconstitute Cyt cbiogenesis in the periplasm of E coli [36] Moreover it wasalso observed that Hh-CcsBA is able to bind reduced hemevia two conserved histidines flanking the WWD domainand required both for the translocation of the heme andfor the synthetase function of Hh-CcsBA [36] A differentstudy carried out on B subtilis System II proteins providedsupport for heme-binding capability by the ResBC complexaccording to the results obtained on recombinant ResBC theResB component of the heterodimeric CCS complex is ableto covalently bind the heme in the cytoplasm (probably by aCys residue) and to deliver it to an extracytoplamic domain ofResC which is responsible for the covalent ligation to apoCyt[117] It should be noticed however that the transfer of theCCS-bound heme to apoCyt still awaits direct experimentalproof Moreover it has also been reported that in B subtilisthe inactivation of CCS does not affect the presence ofother heme-containing proteins in the periplasm such ascytochrome b562 [111] this observation which is in contrastto the heme-transport hypothesis by CCSs parallels similarconcerns about heme translocation mechanism that arecurrently discussed in the context of System I (see Section 22above) In the case of the Hh-CcsBA synthetase site-directedmutagenesis experiments allowed to assign a heme-bindingrole for the two pairs of conserved His residues accordingto the topological model of the protein obtained by alkalinephosphatase (PhoA) assays and GFP fusions the conservedHis77 and His858 residues are located on TM helices whileHis761 and His897 are located on the periplasmic side [36]These observations together with the results of site-directedmutagenesis experiments allowed the authors to suggest thatHis77 andHis858 form a low affinitymdashmembrane embeddedheme-binding site for ferrous heme which is subsequentlytranslocated to an external heme-binding domain of Hh-CcsBA where it is coordinated by His761 and His897 Thetopological model therefore predicts that this last His pairis part of a periplasmic-located WWD domain (homologousto theWWDdomains found in theCcmCandCcmFproteinsof System I described above)

4 System III

Strikingly different from Systems I and II the Cyt c mat-uration apparatus found in fungi and in metazoan cells(System III) is composed of a single protein known as Holo-Cytochrome c Synthetase (HCCS) or Cytochrome c HemeLyase (CCHL) apparently responsible for all the subprocessesdescribed above (heme transport and chaperoning reductionand chaperoning of apoCyt and catalysis of the thioetherbonds formation between heme and apoCyt) (Figure 4)Surprisingly although this protein has been identified in Scerevisiaemitochondria several years ago [125 126] only veryrecently the human HCCS has been expressed as a recombi-nant protein in E coli and spectroscopically characterized invitro [127] It is worth noticing that humanHCCS is attractinginterest since over and above its role in Cyt c biogenesis it

12 Scientifica

is involved in diseases such as microphthalmia with linearskin defects syndrome (MLS) an X-linked genetic disorder[128ndash130] and in other processes such as Cytc-independentapoptosis in injured motor neurons [131] Different fromanimal cells where a single HCCS is sufficient for thematuration of both soluble and membrane-anchored Cytc(Cyt c and Cy c1 resp) in S cerevisiae two homologs HCCSand HCC1S located in the inner mitochondrial membraneand facing to the IMS space [132 133] are responsible for hemeattachment to apoCyt c and apoCyt c1 respectively [125 134]It should also be noticed that in fungi an additional FAD-containing protein (Cyc2p) is required for Cyt c synthesisCyc2p is a mitochondrial membrane-anchored flavoproteinexposing its redox domain to the IMS which is requiredfor the maturation of Cyt c but not for that of Cyt c1 [135]This protein does not contain the conserved Cys residuestypically found in disulfide reductases and indeed it is notable to reduce oxidized apoCyt in vitro However it has beenrecently shown that Cyc2p is able to catalyze the NAD(P)H-dependent reduction of heme in vitro [136] a necessarystep before the thioether bond formation can occur asdiscussed above (see Section 26) This result together withthe observation that Cyc2p interacts with HCCS and withapoCyt c and c1 lends support to the proposal that Cyc2p isinvolved in the reduction of the heme iron in vivo [136 137]

AlthoughHCCS proteins are crucial for Cyt cmaturationwe still do not know how these proteins recognize theirsubstrates (heme and apoCyt) and how they promote orcatalyze the formation of thioether bonds between the hemevinyl groups and the cysteine thiols of the apoCyt CXXCHmotif

Contrary to the broad specificity of System I whichis able to recognize and attach the heme to prokaryoticand eukaryotic c-type cytochromes [49 50] and even tovery short microperoxidase-like sequences [49 50] mito-chondrial HCCS is characterized by a higher specificityas it does not recognize bacterial apoCyt sequences Theseobservations prompted the investigation of the recognitionprocess between apoCyt and HCCS Recently by using arecombinant yeast HCCS and chimerical apoCyt sequencesexpressed in E coli it was possible to conclude that a crucialrecognition determinant is represented by the N-terminalregion of apoCyt containing the heme-binding motif [35]notice that in the context of System I the same N-terminalregion of the Cyt c sequence has been recently identified to beimportant for the recognition by apoCcmE (see Section 26)Over and above the role played by the intervening residuesin the CXXCH heme-binding motif [135] it has been shownthat a conserved Phe residue occurring in the N-terminalregion before the CXXCH motif is important for HCCSrecognition [35 138] These results support the hypothesisthat this residue known to be a key determinant of Cytcfolding and stability [19 20 139] may also be crucial for Cytcmaturation by HCCS

Another relevant question concerns the ability of HCCSto recognize and bind the heme molecule as the region ofthe protein responsible for heme recognition remains to beidentified Initially it was hypothesized that the recognitionof heme could be mediated by the CP motifs present in

the HCCS protein [140] these short sequences are indeedknown to bind heme in a variety of heme-containing proteins[141 142] However it has been recently shown that CPmotifsof the recombinant S cerevisiae HCCS are not necessaryfor Cyt c production in E coli [143] excluding them as keydeterminants of heme recognition

The long-awaited in vitro characterization of the recom-binant human HCCS allowed for the first time to pro-pose a molecular mechanism underlying Cytc maturationin eukaryotes which can be experimentally tested [127]According to the proposed model the human HCCS activitycan be described as a four step mechanism involving (i)heme-binding (ii) apoCyt recognition (iii) thioether bondsformation and (iv) holoCyt c release In particular theheme is proposed to play the role of a scaffolding moleculemediating the contacts between HCCS and apoCyt Muta-genesis experiments carried out on the recombinant HCCSstrongly suggest that heme-binding (Step 1) depends on thepresence of a specific His residue (His154) acting as anaxial ligand to ferrous heme Residues present at the N-terminus of apoCyt mediate the recognition with HCCS(Step 2) as discussed above it is known that this regionforms structurally conserved 120572-helix in the fold of all c-type cytochromes Unfortunately no information is availableup to now concerning the region of HCCS involved in therecognition and binding to the N-terminal region of apoCytCoordination of the heme iron by the His residue of theapoCyt heme-binding motif CXXCH provides the secondaxial ligand to the heme iron and is probably importantfor the correct positioning of the two apoCyt Cys residuesand formation of the thioether bonds (Step 3) The finalrelease of functional Cyt c (Step 4) clearly requires thedisplacement of the His154Fe2+ coordination bond sucha displacement is probably mediated by formation of thecoordination bond with the Cyt c conserved Met residueandor by the simultaneous folding of Cyt c Again it isinteresting to note that this last hypothesis is in accordancewith in vitro folding studies on c-type cytochromes thathighlighted the late formation of the Met-Fe coordinationduring Cyt c folding [144 145]

Acknowledgments

The author thanks A Di Matteo and E Di Silvio for fruitfuldiscussions and for help in preparing this paper This work issupported by grants from the University of Rome ldquoSapienzardquo(C26A11MBXJ and C26A13MWF4)

References

[1] M G Galinato J G Kleingardner S E Bowman et alldquoHeme-protein vibrational couplings in cytochrome c providea dynamic link that connects the heme-iron and the proteinsurfacerdquo Proceedings of the National Academy of Sciences vol109 no 23 pp 8896ndash8900 2012

[2] H B Gray and J R Winkler ldquoElectron flow through metal-loproteinsrdquo Biochimica et Biophysica Acta vol 1797 no 9 pp1563ndash1572 2010

Scientifica 13

[3] P Ascenzi R Santucci M Coletta and F PolticellildquoCytochromes reactivity of the ldquodark siderdquo of the hemerdquoBiophysical Chemistry vol 152 no 1ndash3 pp 21ndash27 2010

[4] S Yamada N D Bouley Ford G E Keller W C Ford HB Gray and J R Winkler ldquoSnapshots of a protein foldingintermediaterdquo Proceedings of the National Academy of Sciencesvol 110 no 5 pp 1606ndash1610 2013

[5] P Weinkam J Zimmermann F E Romesberg and P GWolynes ldquoThe folding energy landscape and free energy excita-tions of cytochrome crdquo Accounts of Chemical Research vol 43no 5 pp 652ndash660 2010

[6] M Yamanaka M Masanari and Y Sambongi ldquoConfermentof folding ability to a naturally unfolded apocytochrome cthrough introduction of hydrophobic amino acid residuesrdquoBiochemistry vol 50 no 12 pp 2313ndash2320 2011

[7] G W Moore and G W Pettigrew Cytochrome C EvolutionaryStructural and Physicochemical Aspects Springer Berlin Ger-many 1990

[8] I Bertini G Cavallaro and A Rosato ldquoCytochrome c occur-rence and functionsrdquo Chemical Reviews vol 106 no 1 pp 90ndash115 2006

[9] D A Pearce and F Sherman ldquoDegradation of cytochromeoxidase subunits in mutants of yeast lacking cytochrome c andsuppression of the degradation by mutation of yme1rdquo Journal ofBiological Chemistry vol 270 no 36 pp 20879ndash20882 1995

[10] G Silkstone S M Kapetanaki I Husu M H Vos and MT Wilson ldquoNitric oxide binding to the cardiolipin complex offerric cytochrome crdquo Biochemistry vol 51 no 34 pp 6760ndash6766 2012

[11] M Giorgio E Migliaccio F Orsini et al ldquoElectron transferbetween cytochrome c and p66Shc generates reactive oxygenspecies that trigger mitochondrial apoptosisrdquo Cell vol 122 no2 pp 221ndash233 2005

[12] S M Kilbride and J H Prehn ldquoCentral roles of apoptoticproteins in mitochondrial functionrdquo Oncogene vol 32 no 22pp 2703ndash2711 2013

[13] T Noll and G Noll ldquoStrategies for ldquowiringrdquo redox-activeproteins to electrodes and applications in biosensors biofuelcells and nanotechnologyrdquo Chemical Society Reviews vol 40no 7 pp 3564ndash3576 2011

[14] G Layer J Reichelt D Jahn and D W Heinz ldquoStructure andfunction of enzymes in heme biosynthesisrdquo Protein Science vol19 no 6 pp 1137ndash1161 2010

[15] S Severance and IHamza ldquoTrafficking of heme and porphyrinsin metazoardquo Chemical Reviews vol 109 no 10 pp 4596ndash46162009

[16] P Hamel V Corvest P Giege and G Bonnard ldquoBiochemi-cal requirements for the maturation of mitochondrial c-typecytochromesrdquo Biochimica et Biophysica Acta vol 1793 no 1 pp125ndash138 2009

[17] W R Fisher H Taniuchi and C B Anfinsen ldquoOn the role ofheme in the formation of the structure of cytochrome crdquo Journalof Biological Chemistry vol 248 no 9 pp 3188ndash3195 1973

[18] M Yamanaka H Mita Y Yamamoto and Y Sambongi ldquoHemeis not required for aquifex aeolicus cytochrome c555 polypep-tide foldingrdquoBioscience Biotechnology andBiochemistry vol 73no 9 pp 2022ndash2025 2009

[19] C Travaglini-Allocatelli S Gianni andMBrunori ldquoA commonfolding mechanism in the cytochrome c familyrdquo Trends inBiochemical Sciences vol 29 no 10 pp 535ndash541 2004

[20] A Borgia D Bonivento C Travaglini-Allocatelli A DiMatteoand M Brunori ldquoUnveiling a hidden folding intermediate in c-type cytochromes by protein engineeringrdquo Journal of BiologicalChemistry vol 281 no 14 pp 9331ndash9336 2006

[21] E Enggist L Thony-Meyer P Guntert and K PervushinldquoNMR structure of the heme chaperone CcmE reveals a novelfunctional motifrdquo Structure vol 10 no 11 pp 1551ndash1557 2002

[22] A di Matteo N Calosci S Gianni P Jemth M Brunori and CTravaglini-Allocatelli ldquoStructural and functional characteriza-tion of CcmG from pseudomonas aeruginosa a key componentof the bacterial cytochrome c maturation apparatusrdquo Proteinsvol 78 no 10 pp 2213ndash2221 2010

[23] A diMatteo S GianniM E Schinina et al ldquoA strategic proteinin cytochrome c maturation three-dimensional structure ofCcmH and binding to apocytochrome crdquo Journal of BiologicalChemistry vol 282 no 37 pp 27012ndash27019 2007

[24] A Crow R M Acheson N E Le Brun and A OubrieldquoStructural basis of redox-coupled protein substrate selectionby the cytochrome c biosynthesis protein ResArdquo Journal ofBiological Chemistry vol 279 no 22 pp 23654ndash23660 2004

[25] E di Silvio A di Matteo and C Travaglini-Allocatelli ldquoTheRedox pathway of Pseudomonas aeruginosa cytochrome cbiogenesisrdquo Journal of Proteins and Proteomics vol 3 no 1 pp1ndash7 2012

[26] LThony-Meyer andP Kunzler ldquoTranslocation to the periplasmand signal sequence cleavage of preapocytochrome c depend onsec and lep but not on the ccmgene productsrdquoEuropean Journalof Biochemistry vol 246 no 3 pp 794ndash799 1997

[27] S J Facey and A Kuhn ldquoBiogenesis of bacterial inner-membrane proteinsrdquo Cellular and Molecular Life Sciences vol67 no 14 pp 2343ndash2362 2010

[28] K Diekert A I de Kroon U Ahting et al ldquoApocytochromec requires the TOM complex for translocation across themitochondrial outer membranerdquo The EMBO Journal vol 20no 20 pp 5626ndash5635 2001

[29] N Wiedemann V Kozjak T Prinz et al ldquoBiogenesis of yeastmitochondrial cytochrome c a unique relationship to the TOMmachineryrdquo Journal ofMolecular Biology vol 327 no 2 pp 465ndash474 2003

[30] F-U Hartl N Pfanner and W Neupert ldquoTranslocation inter-mediates on the import pathway of proteins intomitochondriardquoBiochemical Society Transactions vol 15 no 1 pp 95ndash97 1987

[31] B S Glick A Brandt K Cunningham SMuller R L Hallbergand G Schatz ldquoCytochromes c1 and b2 are sorted to theintermembrane space of yeast mitochondria by a stop-transfermechanismrdquo Cell vol 69 no 5 pp 809ndash822 1992

[32] G Howe and S Merchant ldquoRole of heme in the biosynthesis ofcytochrome c6rdquo Journal of Biological Chemistry vol 269 no 8pp 5824ndash5832 1994

[33] S S Nakamoto P Hamel and S Merchant ldquoAssembly ofchloroplast cytochromes b and crdquo Biochimie vol 82 no 6-7 pp603ndash614 2000

[34] C Sanders S Turkarslan D-W Lee and F DaldalldquoCytochrome c biogenesis the Ccm systemrdquo Trends inMicrobiology vol 18 no 6 pp 266ndash274 2010

[35] J M Stevens D A Mavridou R Hamer P Kritsiligkou A DGoddard and S J Ferguson ldquoCytochrome c biogenesis systemIrdquoThe FEBS Journal vol 278 no 22 pp 4170ndash4178 2011

[36] R G Kranz C Richard-Fogal J-S Taylor and E R FrawleyldquoCytochrome c biogenesis mechanisms for covalent modifica-tions and trafficking of heme and for heme-iron redox controlrdquo

14 Scientifica

Microbiology and Molecular Biology Reviews vol 73 no 3 pp510ndash528 2009

[37] J W A Allen ldquoCytochrome c biogenesis in mitochondriamdashsystems III and VrdquoThe FEBS Journal vol 278 no 22 pp 4198ndash4216 2011

[38] D A Mavridou S J Ferguson and J M Stevens ldquoCytochromec assemblyrdquo IUBMB Life vol 65 no 3 pp 209ndash216 2013

[39] I Bertini G Cavallaro and A Rosato ldquoEvolution ofmitochondrial-type cytochrome c domains and of theprotein machinery for their assemblyrdquo Journal of InorganicBiochemistry vol 101 no 11-12 pp 1798ndash1811 2007

[40] B S Goldman and R G Kranz ldquoEvolution and horizontaltransfer of an entire biosynthetic pathway for cytochromec biogenesis helicobacter deinococcus archae and morerdquoMolecular Microbiology vol 27 no 4 pp 871ndash873 1998

[41] C L Richard-Fogal E R Frawley E R Bonner H Zhu BSan Francisco and R G Kranz ldquoA conserved haem redoxand trafficking pathway for cofactor attachmentrdquo The EMBOJournal vol 28 no 16 pp 2349ndash2359 2009

[42] L Thony-Meyer F Fischer P Kunzler D Ritz and H Hen-necke ldquoEscherichia coli genes required for cytochrome cmaturationrdquo Journal of Bacteriology vol 177 no 15 pp 4321ndash4326 1995

[43] C S Beckett J A Loughman K A Karberg G M DonatoW E Goldman and R G Kranz ldquoFour genes are required forthe system II cytochrome c biogenesis pathway in Bordetellapertussis a unique bacterial modelrdquo Molecular Microbiologyvol 38 no 3 pp 465ndash481 2000

[44] N E Le Brun J Bengtsson and L Hederstedt ldquoGenes requiredfor cytochrome c synthesis in Bacillus subtilisrdquo MolecularMicrobiology vol 36 no 3 pp 638ndash650 2000

[45] P Giege J M Grienenberger and G Bonnard ldquoCytochromec biogenesis in mitochondriardquo Mitochondrion vol 8 no 1 pp61ndash73 2008

[46] R E Feissner C L Richard-Fogal E R Frawley J A Lough-man KW Earley and R G Kranz ldquoRecombinant cytochromesc biogenesis systems I and II and analysis of haem deliverypathways in Escherichia colirdquo Molecular Microbiology vol 60no 3 pp 563ndash577 2006

[47] N P Cianciotto P Cornelis and C Baysse ldquoImpact of thebacterial type I cytochrome c maturation system on differentbiological processesrdquoMolecular Microbiology vol 56 no 6 pp1408ndash1415 2005

[48] E Arslan H Schulz R Zufferey P Kunzler and L Thony-Meyer ldquoOverproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in Escherichiacolirdquo Biochemical and Biophysical Research Communicationsvol 251 no 3 pp 744ndash747 1998

[49] C Sanders and H Lill ldquoExpression of prokaryotic and eukary-otic cytochromes c in Escherichia colirdquoBiochimica et BiophysicaActa vol 1459 no 1 pp 131ndash138 2000

[50] M Braun and L Thony-Meyer ldquoBiosynthesis of artificialmicroperoxidases by exploiting the secretion and cytochromec maturation apparatuses of Escherichia colirdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 35 pp 12830ndash12835 2004

[51] B Baert C Baysse S Matthijs and P Cornelis ldquoMultiplephenotypic alterations caused by a c-type cytochrome mat-uration ccmC gene mutation in Pseudomonas aeruginosardquoMicrobiology vol 154 no 1 pp 127ndash138 2008

[52] S Turkarslan C Sanders and F Daldal ldquoExtracytoplasmicprosthetic group ligation to apoproteins maturation of c-typecytochromesrdquo Molecular Microbiology vol 60 no 3 pp 537ndash541 2006

[53] P M Jones and A M George ldquoThe ABC transporter structureand mechanism perspectives on recent researchrdquo Cellular andMolecular Life Sciences vol 61 no 6 pp 682ndash699 2004

[54] O Christensen E M Harvat L Thony-Meyer S J Fergusonand J M Stevens ldquoLoss of ATP hydrolysis activity by CcmABresults in loss of c-type cytochrome synthesis and incompleteprocessing ofCcmErdquoTheFEBS Journal vol 274 no 9 pp 2322ndash2332 2007

[55] J-H Lee E M Harvat J M Stevens S J Ferguson and M HSaier Jr ldquoEvolutionary origins of members of a superfamily ofintegralmembrane cytochrome c biogenesis proteinsrdquoBiochim-ica et Biophysica Acta vol 1768 no 9 pp 2164ndash2181 2007

[56] D L Beckman D R Trawick and R G Kranz ldquoBacterialcytochromes c biogenesisrdquo Genes and Development vol 6 no2 pp 268ndash283 1992

[57] H Schulz R A Fabianek E C Pellicioli H Hennecke andL Thony-Meyer ldquoHeme transfer to the heme chaperone CcmEduring cytochrome c maturation requires the CcmC proteinwhich may function independently of the ABC-transporterCcmABrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 96 no 11 pp 6462ndash6467 1999

[58] Q Ren and L Thony-Meyer ldquoPhysical Interaction ofCcmC with Heme and the Heme Chaperone CcmE duringCytochrome cMaturationrdquo Journal of Biological Chemistry vol276 no 35 pp 32591ndash32596 2001

[59] C Richard-Fogal and R G Kranz ldquoThe CcmChemeCcmEcomplex in heme trafficking and cytochrome c biosynthesisrdquoJournal of Molecular Biology vol 401 no 3 pp 350ndash362 2010

[60] V K Viswanathan S Kurtz L L Pedersen et al ldquoThecytochrome C maturation locus of Legionella pneumophilapromotes iron assimilation and intracellular infection andcontains a strain-specific insertion sequence elementrdquo Infectionand Immunity vol 70 no 4 pp 1842ndash1852 2002

[61] U Ahuja and L Thony-Meyer ldquoCcmD is involved in complexformation between CcmC and the heme chaperone CcmE dur-ing cytochrome c maturationrdquo Journal of Biological Chemistryvol 280 no 1 pp 236ndash243 2005

[62] C L Richard-Fogal E R Frawley and R G Kranz ldquoTopologyand function of CcmD in cytochrome Cmaturationrdquo Journal ofBacteriology vol 190 no 10 pp 3489ndash3493 2008

[63] H Schulz H Hennecke and L Thony-Meyer ldquoPrototype ofa heme chaperone essential for cytochrome c maturationrdquoScience vol 281 no 5380 pp 1197ndash1200 1998

[64] F Arnesano L Banci P D Barker et al ldquoSolution structure andcharacterization of the heme chaperone CcmErdquo Biochemistryvol 41 no 46 pp 13587ndash13594 2002

[65] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[66] L Thony-Meyer ldquoA heme chaperone for cytochrome c biosyn-thesisrdquo Biochemistry vol 42 no 45 pp 13099ndash13105 2003

[67] E M Harvat C Redfield J M Stevens and S J FergusonldquoProbing the heme-binding site of the cytochrome cmaturationprotein CcmErdquoBiochemistry vol 48 no 8 pp 1820ndash1828 2009

[68] D Lee K Pervushin D Bischof M Braun and L Thony-Meyer ldquoUnusual heme-histidine bond in the active site of a

Scientifica 15

chaperonerdquo Journal of the American Chemical Society vol 127no 11 pp 3716ndash3717 2005

[69] A D Goddard J M Stevens F Rao et al ldquoc-Type cytochromebiogenesis can occur via a natural Ccm system lacking CcmHCcmG and the heme-binding histidine of CcmErdquo Journal ofBiological Chemistry vol 285 no 30 pp 22882ndash22889 2010

[70] J M Aramini K Hamilton P Rossi et al ldquoSolution NMRstructure backbone dynamics and heme-binding propertiesof a novel cytochrome c maturation protein CcmE fromDesulfovibrio vulgarisrdquo Biochemistry vol 51 no 18 pp 3705ndash3707 2012

[71] T Uchida J M Stevens O Daltrop et al ldquoThe interactionof covalently bound heme with the cytochrome c maturationprotein CcmErdquo Journal of Biological Chemistry vol 279 no 50pp 51981ndash51988 2004

[72] I Garcıa-Rubio M Braun I Gromov L Thony-Meyer andA Schweiger ldquoAxial coordination of heme in ferric CcmEchaperone characterized by EPR spectroscopyrdquo BiophysicalJournal vol 92 no 4 pp 1361ndash1373 2007

[73] L Thony-Meyer ldquoHaem-polypeptide interactions duringcytochrome c maturationrdquo Biochimica et Biophysica Acta vol1459 no 2-3 pp 316ndash324 2000

[74] J A Keightley D Sanders T R Todaro A Pastuszyn and J AFee ldquoCloning expression in Escherichia coli of the cytochromec552 gene from Thermus thermophilus HB8 Evidence forgenetic linkage to an ATP- binding cassette protein and initialcharacterization of the cycA gene productsrdquo Journal of Biologi-cal Chemistry vol 273 no 20 pp 12006ndash12016 1998

[75] E M Monika B S Goldman D L Beckman and R GKranz ldquoA thioreduction pathway tethered to the membranefor periplasmic cytochromes c biogenesis In vitro and in vivostudiesrdquo Journal of Molecular Biology vol 271 no 5 pp 679ndash692 1997

[76] M Throne-Holst L Thony-Meyer and L HederstedtldquoEscherichia coli ccm in-frame deletion mutants can produceperiplasmic cytochrome b but not cytochrome crdquo FEBS Lettersvol 410 no 2-3 pp 351ndash355 1997

[77] U Ahuja and L Thony-Meyer ldquoDynamic features of a hemedelivery system for cytochrome c maturationrdquo Journal of Bio-logical Chemistry vol 278 no 52 pp 52061ndash52070 2003

[78] J W A Allen P D Barker O Daltrop et al ldquoWhy isnrsquot ldquostan-dardrdquo heme good enough for c-type and di-type cytochromesrdquoDalton Transactions no 21 pp 3410ndash3418 2005

[79] K Inaba and K Ito ldquoStructure and mechanisms of the DsbB-DsbA disulfide bond generation machinerdquo Biochimica et Bio-physica Acta vol 1783 no 4 pp 520ndash529 2008

[80] H Kadokura F Katzen and J Beckwith ldquoProtein disulfidebond formation in prokaryotesrdquoAnnual Review of Biochemistryvol 72 pp 111ndash135 2003

[81] S R Shouldice B Heras P M Walden M Totsika M ASchembri and J L Martin ldquoStructure and function of DsbA akey bacterial oxidative folding catalystrdquoAntioxidants and RedoxSignaling vol 14 no 9 pp 1729ndash1760 2011

[82] R Metheringham L Griffiths H Crooke S Forsythe and JCole ldquoAn essential role for DsbA in cytochrome c synthesis andformate-dependent nitrite reduction by Escherichia coli K-12rdquoArchives of Microbiology vol 164 no 4 pp 301ndash307 1995

[83] Y Sambongi and S J Ferguson ldquoMutants of Escherichia colilacking disulphide oxidoreductases DsbA and DsbB cannotsynthesise an exogenous monohaem c-type cytochrome exceptin the presence of disulphide compoundsrdquo FEBS Letters vol398 no 2-3 pp 265ndash268 1996

[84] D AMavridou S J Ferguson and JM Stevens ldquoThe interplaybetween the disulfide bond formation pathway and cytochromec maturation in Escherichia colirdquo FEBS Letters vol 586 no 12pp 1702ndash1707 2012

[85] C U Stirnimann A Rozhkova U Grauschopf M G GrutterR Glockshuber and G Capitani ldquoStructural basis and kineticsof DsbD-dependent cytochrome c maturationrdquo Structure vol13 no 7 pp 985ndash993 2005

[86] N Ouyang Y-G Gao H-Y Hu and Z-X Xia ldquoCrystalstructures of E coli CcmGand itsmutants reveal key roles of theN-terminal120573-sheet and the fingerprint regionrdquoProteins vol 65no 4 pp 1021ndash1031 2006

[87] M A Edeling L W Guddat R A Fabianek et al ldquoCrys-tallization and preliminary diffraction studies of native andselenomethionine CcmG (CycY DsbE)rdquoActa CrystallographicaD vol 57 no 9 pp 1293ndash1295 2001

[88] R A Fabianek M Huber-Wunderlich R Glockshuber PKunzler H Hennecke and L Thony-Meyer ldquoCharacterizationof the Bradyrhizobium japonicum CycY protein a membrane-anchored periplasmic thioredoxin that may play a role as areductant in the biogenesis of c-type cytochromesrdquo Journal ofBiological Chemistry vol 272 no 7 pp 4467ndash4473 1997

[89] G B Kallis and A Holmgren ldquoDifferential reactivity of thefunctional sulfhydryl groups of cysteine-32 and cysteine-32present in the reduced form of thioredoxin from Escherichiacolirdquo Journal of Biological Chemistry vol 255 no 21 pp 10261ndash10265 1980

[90] P T Chivers andR T Raines ldquoGeneral acidbase catalysis in theactive site of Escherichia coli thioredoxinrdquoBiochemistry vol 36no 50 pp 15810ndash15816 1997

[91] X M Zheng J Hong H Y Li D H Lin and H Y HuldquoBiochemical properties and catalytic domain structure of theCcmH protein from Escherichia colirdquo Biochim Biophys Actavol 1824 no 12 pp 1394ndash1400 2012

[92] UAhujaA Rozhkova RGlockshuber LThony-Meyer andOEinsle ldquoHelix swapping leads to dimerization of the N-terminaldomain of the c-type cytochrome maturation protein CcmHfrom Escherichia colirdquo FEBS Letters vol 582 no 18 pp 2779ndash2786 2008

[93] R A Fabianek T Hofer and L Thony-Meyer ldquoCharacteriza-tion of the Escherichia coli CcmH protein reveals new insightsinto the redox pathway required for cytochrome c maturationrdquoArchives of Microbiology vol 171 no 2 pp 92ndash100 1999

[94] E Reid J Cole and D J Eaves ldquoThe Escherichia coli CcmGprotein fulfils a specific role in cytochrome c assemblyrdquo Bio-chemical Journal vol 355 no 1 pp 51ndash58 2001

[95] Q Ren U Ahuja and LThony-Meyer ldquoA bacterial cytochromec heme lyase CcmF forms a complex with the heme chaperoneCcmE and CcmH but not with apocytochrome crdquo Journal ofBiological Chemistry vol 277 no 10 pp 7657ndash7663 2002

[96] E H Meyer P Giege E Gelhaye et al ldquoAtCCMH an essentialcomponent of the c-type cytochrome maturation pathway inArabidopsis mitochondria interacts with apocytochrome crdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 44 pp 16113ndash16118 2005

[97] C Rios-Velazquez R Coller and T J Donohue ldquoFeatures ofRhodobacter sphaeroides CcmFHrdquo Journal of Bacteriology vol185 no 2 pp 422ndash431 2003

[98] C Sanders M Deshmukh D Astor R G Kranz and F DaldalldquoOverproduction of CcmG and CcmFHRc fully suppresses thec-type cytochrome biogenesis defect of Rhodobacter capsulatus

16 Scientifica

CcmI-null mutantsrdquo Journal of Bacteriology vol 187 no 12 pp4245ndash4256 2005

[99] A F Verissimo H Yang X Wu C Sanders and F DaldalldquoCcmI subunit of CcmFHI heme ligation complex functionsas an apocytochrome c chaperone during c-type cytochromematurationrdquo Journal of Biological Chemistry vol 286 no 47 pp40452ndash40463 2011

[100] K Bryson L J McGuffin R L Marsden J J Ward J SSodhi and D T Jones ldquoProtein structure prediction servers atUniversity College Londonrdquo Nucleic Acids Research vol 33 no2 pp W36ndashW38 2005

[101] D T Jones ldquoProtein secondary structure prediction basedon position-specific scoring matricesrdquo Journal of MolecularBiology vol 292 no 2 pp 195ndash202 1999

[102] C Sanders C Boulay and F Daldal ldquoMembrane-spanning andperiplasmic segments of CcmI have distinct functions duringcytochrome c biogenesis in Rhodobacter capsulatusrdquo Journal ofBacteriology vol 189 no 3 pp 789ndash800 2007

[103] D Han K Kim J Oh J Park and Y Kim ldquoTPR domainof NrfG mediates complex formation between heme lyaseand formate-dependent nitrite reductase in Escherichia coliO157H7rdquo Proteins vol 70 no 3 pp 900ndash914 2008

[104] D J Eaves J Grove W Staudenmann et al ldquoInvolvement ofproducts of the nrfEFG genes in the covalent attachment ofhaem c to a novel cysteine-lysine motif in the cytochrome c552nitrite reductase from Escherichia colirdquo Molecular Microbiol-ogy vol 28 no 1 pp 205ndash216 1998

[105] J Nilsson B Persson and G Von Heijne ldquoConsensus predic-tions of membrane protein topologyrdquo FEBS Letters vol 486 no3 pp 267ndash269 2000

[106] E di Silvio A di Matteo F Malatesta and C Travaglini-Allocatelli ldquoRecognition and binding of apocytochrome c toP aeruginosa CcmI a component of cytochrome c maturationmachineryrdquo Biochim Biophys Acta vol 1834 no 8 pp 1554ndash1561 2013

[107] N Zeytuni and R Zarivach ldquoStructural and functional dis-cussion of the tetra-trico-peptide repeat a protein interactionmodulerdquo Structure vol 20 no 3 pp 397ndash405 2012

[108] C Sanders S Turkarslan D-W Lee O Onder R G Kranz andF Daldal ldquoThe cytochrome c maturation components CcmFCcmH and CcmI form a membrane-integral multisubunitheme ligation complexrdquo Journal of Biological Chemistry vol283 no 44 pp 29715ndash29722 2008

[109] A F Verissimo M A Mohtar and F Daldal ldquoThe hemechaperone ApoCcmE forms a ternary complex with CcmI andapocytochrome crdquoThe Journal of Biological Chemistry vol 288no 9 pp 6272ndash6283 2013

[110] P D Barker J C Ferrer M Mylrajan et al ldquoTransmutation of aheme proteinrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 90 no 14 pp 6542ndash65461993

[111] J Simon and L Hederstedt ldquoComposition and function ofcytochrome c biogenesis system IIrdquoThe FEBS Journal vol 278no 22 pp 4179ndash4188 2011

[112] T R H M Kouwen and J M van Dijl ldquoInterchangeablemodules in bacterial thiol-disulfide exchange pathwaysrdquo Trendsin Microbiology vol 17 no 1 pp 6ndash12 2009

[113] A Crow A Lewin O Hecht et al ldquoCrystal structure andbiophysical properties of Bacillus subtilis BdbD An oxidizingthioldisulfide oxidoreductase containing a novel metal siterdquoJournal of Biological Chemistry vol 284 no 35 pp 23719ndash23733 2009

[114] M C Moller and L Hederstedt ldquoExtracytoplasmic processesimpaired by inactivation of trxA (thioredoxin gene) in Bacillussubtilisrdquo Journal of Bacteriology vol 190 no 13 pp 4660ndash46652008

[115] A Lewin A Crow A Oubrie and N E Le Brun ldquoMolecularbasis for specificity of the extracytoplasmic thioredoxin ResArdquoJournal of Biological Chemistry vol 281 no 46 pp 35467ndash35477 2006

[116] C L Colbert QWu P J A Erbel K H Gardner and J Deisen-hofer ldquoMechanism of substrate specificity in Bacillus subtilisResA a thioredoxin-like protein involved in cytochrome cmaturationrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 103 no 12 pp 4410ndash44152006

[117] UAhuja P Kjelgaard B L Schulz LThony-Meyer andLHed-erstedt ldquoHaem-delivery proteins in cytochrome c maturationsystem IIrdquoMolecular Microbiology vol 73 no 6 pp 1058ndash10712009

[118] M L D Page P P Hamel S T Gabilly et al ldquoA homologof prokaryotic thiol disulfide transporter CcdA is required forthe assembly of the cytochrome b6f complex in Arabidopsischloroplastsrdquo Journal of Biological Chemistry vol 279 no 31pp 32474ndash32482 2004

[119] A Rozhkova C U Stirnimann P Frei et al ldquoStructural basisand kinetics of inter- and intramolecular disulfide exchange inthe redox catalyst DsbDrdquoThe EMBO Journal vol 23 no 8 pp1709ndash1719 2004

[120] T Schiott M Throne-Holst and L Hederstedt ldquoBacillussubtilis CcdA-defective mutants are blocked in a late step ofcytochrome C biogenesisrdquo Journal of Bacteriology vol 179 no14 pp 4523ndash4529 1997

[121] R E Feissner C S Beckett J A Loughman and R G KranzldquoMutations in cytochrome assembly and periplasmic redoxpathways in Bordetella pertussisrdquo Journal of Bacteriology vol187 no 12 pp 3941ndash3949 2005

[122] L S ErlendssonMMoller and L Hederstedt ldquoBacillus subtilisStoA is a thiol-disulfide oxidoreductase important for sporecortex synthesisrdquo Journal of Bacteriology vol 186 no 18 pp6230ndash6238 2004

[123] L S Erlendsson R M Acheson L Hederstedt and N E LeBrun ldquoBacillus subtilis ResA is a thiol-disulfide oxidoreductaseinvolved in cytochrome c synthesisrdquo Journal of BiologicalChemistry vol 278 no 20 pp 17852ndash17858 2003

[124] L S Erlendsson and L Hederstedt ldquoMutations in the thiol-disulfide oxidoreductases BdbC and BdbD can suppresscytochrome c deficiency of CcdA-defective Bacillus subtiliscellsrdquo Journal of Bacteriology vol 184 no 5 pp 1423ndash1429 2002

[125] M E Dumont J F Ernst D M Hampsey and F ShermanldquoIdentification and sequence of the gene encoding cytochromec heme lyase in the yeast Saccharomyces cerevisiaerdquoThe EMBOJournal vol 6 no 1 pp 235ndash241 1987

[126] M E Dumont J F Ernst and F Sherman ldquoCoupling of hemeattachment to import of cytochrome c into yeast mitochondriaStudies with heme lyase-deficient mitochondria and alteredapocytochromes crdquo Journal of Biological Chemistry vol 263 no31 pp 15928ndash15937 1988

[127] B San Francisco E C Bretsnyder and R G Kranz ldquoHumanmitochondrial holocytochrome c synthasersquos hemebindingmat-uration determinants and complex formationwith cytochromecrdquo Proceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 9 pp E788ndashE797 2012

Scientifica 17

[128] L Schaefer A Ballabio and H Y Zoghbi ldquoCloning andcharacterization of a putative human holocytochrome c-typesynthetase gene (HCCS) isolated from the critical region formicrophthalmia with linear skin defects (MLS)rdquoGenomics vol34 no 2 pp 166ndash172 1996

[129] S K Prakash T A Cormier A E McCall et al ldquoLoss ofholocytochrome c-type synthetase causes the male lethality ofX-linked dominant microphthalmia with linear skin defects(MLS) syndromerdquo Human Molecular Genetics vol 11 no 25pp 3237ndash3248 2002

[130] I Wimplinger M Morleo G Rosenberger et al ldquoMutations ofthe mitochondrial holocytochrome c-type synthase in X-linkeddominant microphthalmia with linear skin defects syndromerdquoTheAmerican Journal of HumanGenetics vol 79 no 5 pp 878ndash889 2006

[131] S Kiryu-Seo KGamo T Tachibana K Tanaka andHKiyamaldquoUnique anti-apoptotic activity of EAAC1 in injured motorneuronsrdquoTheEMBO Journal vol 25 no 14 pp 3411ndash3421 2006

[132] R Lill R A Stuart M E Drygas F E Nargang and W Neu-pert ldquoImport of cytochrome c heme lyase into mitochondriaa novel pathway into the intermembrane spacerdquo The EMBOJournal vol 11 no 2 pp 449ndash456 1992

[133] H Steiner A Zollner A Haid W Neupert and R LillldquoBiogenesis of mitochondrial heme lyases in yeast Importand folding in the intermembrane spacerdquo Journal of BiologicalChemistry vol 270 no 39 pp 22842ndash22849 1995

[134] A Zollner G Rodel and A Haid ldquoMolecular cloning andcharacterization of the Saccharomyces cerevisiae CYT2 geneencoding cytochrome-c1-heme lyaserdquo European Journal of Bio-chemistry vol 207 no 3 pp 1093ndash1100 1992

[135] V Corvest D A Murrey D G Bernard D B Knaff B Guiardand P PHamel ldquoc-Type cytochrome assembly in Saccharomycescerevisiae a key residue for apocytochrome c1lyase interactionrdquoGenetics vol 186 no 2 pp 561ndash571 2010

[136] V Corvest D A Murrey M Hirasawa D B Knaff B Guiardand P P Hamel ldquoThe flavoprotein Cyc2p a mitochondrialcytochrome c assembly factor is a NAD(P)H-dependent haemreductaserdquo Molecular Microbiology vol 83 no 5 pp 968ndash9802012

[137] D G Bernard S Quevillon-Cheruel S Merchant B Guiardand P P Hamel ldquoCyc2p a membrane-bound flavopro-tein involved in the maturation of mitochondrial c-typecytochromesrdquo Journal of Biological Chemistry vol 280 no 48pp 39852ndash39859 2005

[138] J G Kleingardner and K L Bren ldquoComparing substratespecificity between cytochrome cmaturation and cytochrome cheme lyase systems for cytochrome c biogenesisrdquo Metallomicsvol 3 no 4 pp 396ndash403 2011

[139] C Travaglini-Allocatelli S Gianni VMorea A Tramontano TSoulimane and M Brunori ldquoExploring the cytochrome c fold-ing mechanism cytochrome c552 fromThermus thermophilusfolds through an on-pathway intermediaterdquo Journal of BiologicalChemistry vol 278 no 42 pp 41136ndash41140 2003

[140] L Zhang and L Guarente ldquoHeme binds to a short sequencethat serves a regulatory function in diverse proteinsrdquoTheEMBOJournal vol 14 no 2 pp 313ndash320 1995

[141] S Hira T Tomita T Matsui K Igarashi and M Ikeda-SaitoldquoBach1 a heme-dependent transcription factor reveals pres-ence of multiple heme binding sites with distinct coordinationstructurerdquo IUBMB Life vol 59 no 8-9 pp 542ndash551 2007

[142] T K Kuhl A Wiszligbrock N Goradia et al ldquoAnalysis of Fe(III)heme binding to cysteine-containing heme-regulatory motifs

in proteinsrdquo ACS Chemical Biology vol 8 no 8 pp 1785ndash17932013

[143] R L Moore J M Stevens and S J Ferguson ldquoMitochondrialcytochrome c synthase CP motifs are not necessary for hemeattachment to apocytochrome crdquo FEBS Letters vol 585 no 21pp 3415ndash3419 2011

[144] S Gianni M Brunori and C Travaglini-Allocatelli ldquoRefoldingkinetics of cytochrome c551 reveals a mechanistic differencebetween urea and guanidinerdquo Protein Science vol 10 no 8 pp1685ndash1688 2001

[145] S Gianni C Travaglini-Allocatelli F Cutruzzola M BrunoriM C R Shastry and H Roder ldquoParallel pathways incytochrome c551 foldingrdquo Journal of Molecular Biology vol 330no 5 pp 1145ndash1152 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Scientifica 11

becomes a substrate for the oxidative system present on theouter surface of the cytoplasmic membrane of the gram-positive bacteria In B subtilis the BdbCD system [112113] is functionally but not structurally equivalent to thewell-characterized DsbAB system present in gram-negativebacteria As discussed above for System I the disulfide ofthe apoCyt heme-binding motif must be reduced in orderto allow thioether bonds formation and heme attachmentResA is the extracytoplasmic membrane-anchored TRX-likeprotein involved in the specific reduction of the apoCytdisulfide bond after the disulfide bond exchange reactionhas occurred oxidized ResA is reduced by CcdA whichreceives its reducing equivalents from a cytoplasmic TRX[114] Although ResA displays a classical TRX-like foldthe analysis of the 3D structure of oxidized and reducedResA from B subtilis (Bs-ResA) showed redox-dependentconformational modifications not observed in other TRX-like proteins Interestingly such modifications occur at thelevel of a cavity proposed to represent the binding site foroxidized apoCyt [24 115 116] Another peculiar feature of Bs-ResA is represented by the unusually similar pKa values of itsthe active site Cys residues (88 and 82 resp) as observedalso in the case of the active site Cys residues of the SystemI Pa-CcmH (see Section 23) However at variance with Pa-CcmH in Bs-ResA the large separation between the twocysteine thiols observed in the structure of the reduced formof the protein can be invoked to account for this result

CcdA is a large membrane protein containing six TMhelices [117 118] homologous to the TM domain of E coliDsbD [85 119] Its role in the Cyt c biogenesis is supportedby the observation that inactivation of CcdA blocks theproduction of c-type cytochromes in B subtilis [120 121]Moreover over and above the reduction of its apoCyt sub-strate Bs-CcdA is able to reduce the disulfide bond of othersecreted proteins such as StoA [122] It is interesting to notethat ResA and CcdA are not essential for Cyt c synthesisin the absence of BdbD or BdbC or if a disulfide reductantis present in the growth medium [123 124] As discussedabove (Section 24) a similar observation was made on therole of the thio-reductive pathway of System I in this casepersistent production of c-type cytochromes was observedin Dsb-inactivated bacterial strains [38] Following theseobservations the question has been put forward as to why theCyt c biogenesis process involves this apparently redundantthio-reduction route only to correct the effects of Bdb orDsb activity Referring to the case of B subtilis [111] it hasbeen proposed that the main reason is that BdbD mustefficiently oxidize newly secreted proteins since the activityof most of them depends on the presence of disulfide bondsAlternatively it can be hypothesized that the intramoleculardisulfide bondof apoCyt protects the protein fromproteolyticdegradation or aggregation or from cross-linking to otherthiol-containing proteins

32 System II Heme Translocation and Attachment to ApoCytRecent experimental evidence is accumulating supportingthe involvement of the heterodimeric membrane complexResBC or of the fusion protein CcsBA in the translocation

of heme from the n-side to the p-side of the bacterialmembrane In particular it has been shown that CcsBAfrom H hepaticus (Hh-CcsBA) is able to reconstitute Cyt cbiogenesis in the periplasm of E coli [36] Moreover it wasalso observed that Hh-CcsBA is able to bind reduced hemevia two conserved histidines flanking the WWD domainand required both for the translocation of the heme andfor the synthetase function of Hh-CcsBA [36] A differentstudy carried out on B subtilis System II proteins providedsupport for heme-binding capability by the ResBC complexaccording to the results obtained on recombinant ResBC theResB component of the heterodimeric CCS complex is ableto covalently bind the heme in the cytoplasm (probably by aCys residue) and to deliver it to an extracytoplamic domain ofResC which is responsible for the covalent ligation to apoCyt[117] It should be noticed however that the transfer of theCCS-bound heme to apoCyt still awaits direct experimentalproof Moreover it has also been reported that in B subtilisthe inactivation of CCS does not affect the presence ofother heme-containing proteins in the periplasm such ascytochrome b562 [111] this observation which is in contrastto the heme-transport hypothesis by CCSs parallels similarconcerns about heme translocation mechanism that arecurrently discussed in the context of System I (see Section 22above) In the case of the Hh-CcsBA synthetase site-directedmutagenesis experiments allowed to assign a heme-bindingrole for the two pairs of conserved His residues accordingto the topological model of the protein obtained by alkalinephosphatase (PhoA) assays and GFP fusions the conservedHis77 and His858 residues are located on TM helices whileHis761 and His897 are located on the periplasmic side [36]These observations together with the results of site-directedmutagenesis experiments allowed the authors to suggest thatHis77 andHis858 form a low affinitymdashmembrane embeddedheme-binding site for ferrous heme which is subsequentlytranslocated to an external heme-binding domain of Hh-CcsBA where it is coordinated by His761 and His897 Thetopological model therefore predicts that this last His pairis part of a periplasmic-located WWD domain (homologousto theWWDdomains found in theCcmCandCcmFproteinsof System I described above)

4 System III

Strikingly different from Systems I and II the Cyt c mat-uration apparatus found in fungi and in metazoan cells(System III) is composed of a single protein known as Holo-Cytochrome c Synthetase (HCCS) or Cytochrome c HemeLyase (CCHL) apparently responsible for all the subprocessesdescribed above (heme transport and chaperoning reductionand chaperoning of apoCyt and catalysis of the thioetherbonds formation between heme and apoCyt) (Figure 4)Surprisingly although this protein has been identified in Scerevisiaemitochondria several years ago [125 126] only veryrecently the human HCCS has been expressed as a recombi-nant protein in E coli and spectroscopically characterized invitro [127] It is worth noticing that humanHCCS is attractinginterest since over and above its role in Cyt c biogenesis it

12 Scientifica

is involved in diseases such as microphthalmia with linearskin defects syndrome (MLS) an X-linked genetic disorder[128ndash130] and in other processes such as Cytc-independentapoptosis in injured motor neurons [131] Different fromanimal cells where a single HCCS is sufficient for thematuration of both soluble and membrane-anchored Cytc(Cyt c and Cy c1 resp) in S cerevisiae two homologs HCCSand HCC1S located in the inner mitochondrial membraneand facing to the IMS space [132 133] are responsible for hemeattachment to apoCyt c and apoCyt c1 respectively [125 134]It should also be noticed that in fungi an additional FAD-containing protein (Cyc2p) is required for Cyt c synthesisCyc2p is a mitochondrial membrane-anchored flavoproteinexposing its redox domain to the IMS which is requiredfor the maturation of Cyt c but not for that of Cyt c1 [135]This protein does not contain the conserved Cys residuestypically found in disulfide reductases and indeed it is notable to reduce oxidized apoCyt in vitro However it has beenrecently shown that Cyc2p is able to catalyze the NAD(P)H-dependent reduction of heme in vitro [136] a necessarystep before the thioether bond formation can occur asdiscussed above (see Section 26) This result together withthe observation that Cyc2p interacts with HCCS and withapoCyt c and c1 lends support to the proposal that Cyc2p isinvolved in the reduction of the heme iron in vivo [136 137]

AlthoughHCCS proteins are crucial for Cyt cmaturationwe still do not know how these proteins recognize theirsubstrates (heme and apoCyt) and how they promote orcatalyze the formation of thioether bonds between the hemevinyl groups and the cysteine thiols of the apoCyt CXXCHmotif

Contrary to the broad specificity of System I whichis able to recognize and attach the heme to prokaryoticand eukaryotic c-type cytochromes [49 50] and even tovery short microperoxidase-like sequences [49 50] mito-chondrial HCCS is characterized by a higher specificityas it does not recognize bacterial apoCyt sequences Theseobservations prompted the investigation of the recognitionprocess between apoCyt and HCCS Recently by using arecombinant yeast HCCS and chimerical apoCyt sequencesexpressed in E coli it was possible to conclude that a crucialrecognition determinant is represented by the N-terminalregion of apoCyt containing the heme-binding motif [35]notice that in the context of System I the same N-terminalregion of the Cyt c sequence has been recently identified to beimportant for the recognition by apoCcmE (see Section 26)Over and above the role played by the intervening residuesin the CXXCH heme-binding motif [135] it has been shownthat a conserved Phe residue occurring in the N-terminalregion before the CXXCH motif is important for HCCSrecognition [35 138] These results support the hypothesisthat this residue known to be a key determinant of Cytcfolding and stability [19 20 139] may also be crucial for Cytcmaturation by HCCS

Another relevant question concerns the ability of HCCSto recognize and bind the heme molecule as the region ofthe protein responsible for heme recognition remains to beidentified Initially it was hypothesized that the recognitionof heme could be mediated by the CP motifs present in

the HCCS protein [140] these short sequences are indeedknown to bind heme in a variety of heme-containing proteins[141 142] However it has been recently shown that CPmotifsof the recombinant S cerevisiae HCCS are not necessaryfor Cyt c production in E coli [143] excluding them as keydeterminants of heme recognition

The long-awaited in vitro characterization of the recom-binant human HCCS allowed for the first time to pro-pose a molecular mechanism underlying Cytc maturationin eukaryotes which can be experimentally tested [127]According to the proposed model the human HCCS activitycan be described as a four step mechanism involving (i)heme-binding (ii) apoCyt recognition (iii) thioether bondsformation and (iv) holoCyt c release In particular theheme is proposed to play the role of a scaffolding moleculemediating the contacts between HCCS and apoCyt Muta-genesis experiments carried out on the recombinant HCCSstrongly suggest that heme-binding (Step 1) depends on thepresence of a specific His residue (His154) acting as anaxial ligand to ferrous heme Residues present at the N-terminus of apoCyt mediate the recognition with HCCS(Step 2) as discussed above it is known that this regionforms structurally conserved 120572-helix in the fold of all c-type cytochromes Unfortunately no information is availableup to now concerning the region of HCCS involved in therecognition and binding to the N-terminal region of apoCytCoordination of the heme iron by the His residue of theapoCyt heme-binding motif CXXCH provides the secondaxial ligand to the heme iron and is probably importantfor the correct positioning of the two apoCyt Cys residuesand formation of the thioether bonds (Step 3) The finalrelease of functional Cyt c (Step 4) clearly requires thedisplacement of the His154Fe2+ coordination bond sucha displacement is probably mediated by formation of thecoordination bond with the Cyt c conserved Met residueandor by the simultaneous folding of Cyt c Again it isinteresting to note that this last hypothesis is in accordancewith in vitro folding studies on c-type cytochromes thathighlighted the late formation of the Met-Fe coordinationduring Cyt c folding [144 145]

Acknowledgments

The author thanks A Di Matteo and E Di Silvio for fruitfuldiscussions and for help in preparing this paper This work issupported by grants from the University of Rome ldquoSapienzardquo(C26A11MBXJ and C26A13MWF4)

References

[1] M G Galinato J G Kleingardner S E Bowman et alldquoHeme-protein vibrational couplings in cytochrome c providea dynamic link that connects the heme-iron and the proteinsurfacerdquo Proceedings of the National Academy of Sciences vol109 no 23 pp 8896ndash8900 2012

[2] H B Gray and J R Winkler ldquoElectron flow through metal-loproteinsrdquo Biochimica et Biophysica Acta vol 1797 no 9 pp1563ndash1572 2010

Scientifica 13

[3] P Ascenzi R Santucci M Coletta and F PolticellildquoCytochromes reactivity of the ldquodark siderdquo of the hemerdquoBiophysical Chemistry vol 152 no 1ndash3 pp 21ndash27 2010

[4] S Yamada N D Bouley Ford G E Keller W C Ford HB Gray and J R Winkler ldquoSnapshots of a protein foldingintermediaterdquo Proceedings of the National Academy of Sciencesvol 110 no 5 pp 1606ndash1610 2013

[5] P Weinkam J Zimmermann F E Romesberg and P GWolynes ldquoThe folding energy landscape and free energy excita-tions of cytochrome crdquo Accounts of Chemical Research vol 43no 5 pp 652ndash660 2010

[6] M Yamanaka M Masanari and Y Sambongi ldquoConfermentof folding ability to a naturally unfolded apocytochrome cthrough introduction of hydrophobic amino acid residuesrdquoBiochemistry vol 50 no 12 pp 2313ndash2320 2011

[7] G W Moore and G W Pettigrew Cytochrome C EvolutionaryStructural and Physicochemical Aspects Springer Berlin Ger-many 1990

[8] I Bertini G Cavallaro and A Rosato ldquoCytochrome c occur-rence and functionsrdquo Chemical Reviews vol 106 no 1 pp 90ndash115 2006

[9] D A Pearce and F Sherman ldquoDegradation of cytochromeoxidase subunits in mutants of yeast lacking cytochrome c andsuppression of the degradation by mutation of yme1rdquo Journal ofBiological Chemistry vol 270 no 36 pp 20879ndash20882 1995

[10] G Silkstone S M Kapetanaki I Husu M H Vos and MT Wilson ldquoNitric oxide binding to the cardiolipin complex offerric cytochrome crdquo Biochemistry vol 51 no 34 pp 6760ndash6766 2012

[11] M Giorgio E Migliaccio F Orsini et al ldquoElectron transferbetween cytochrome c and p66Shc generates reactive oxygenspecies that trigger mitochondrial apoptosisrdquo Cell vol 122 no2 pp 221ndash233 2005

[12] S M Kilbride and J H Prehn ldquoCentral roles of apoptoticproteins in mitochondrial functionrdquo Oncogene vol 32 no 22pp 2703ndash2711 2013

[13] T Noll and G Noll ldquoStrategies for ldquowiringrdquo redox-activeproteins to electrodes and applications in biosensors biofuelcells and nanotechnologyrdquo Chemical Society Reviews vol 40no 7 pp 3564ndash3576 2011

[14] G Layer J Reichelt D Jahn and D W Heinz ldquoStructure andfunction of enzymes in heme biosynthesisrdquo Protein Science vol19 no 6 pp 1137ndash1161 2010

[15] S Severance and IHamza ldquoTrafficking of heme and porphyrinsin metazoardquo Chemical Reviews vol 109 no 10 pp 4596ndash46162009

[16] P Hamel V Corvest P Giege and G Bonnard ldquoBiochemi-cal requirements for the maturation of mitochondrial c-typecytochromesrdquo Biochimica et Biophysica Acta vol 1793 no 1 pp125ndash138 2009

[17] W R Fisher H Taniuchi and C B Anfinsen ldquoOn the role ofheme in the formation of the structure of cytochrome crdquo Journalof Biological Chemistry vol 248 no 9 pp 3188ndash3195 1973

[18] M Yamanaka H Mita Y Yamamoto and Y Sambongi ldquoHemeis not required for aquifex aeolicus cytochrome c555 polypep-tide foldingrdquoBioscience Biotechnology andBiochemistry vol 73no 9 pp 2022ndash2025 2009

[19] C Travaglini-Allocatelli S Gianni andMBrunori ldquoA commonfolding mechanism in the cytochrome c familyrdquo Trends inBiochemical Sciences vol 29 no 10 pp 535ndash541 2004

[20] A Borgia D Bonivento C Travaglini-Allocatelli A DiMatteoand M Brunori ldquoUnveiling a hidden folding intermediate in c-type cytochromes by protein engineeringrdquo Journal of BiologicalChemistry vol 281 no 14 pp 9331ndash9336 2006

[21] E Enggist L Thony-Meyer P Guntert and K PervushinldquoNMR structure of the heme chaperone CcmE reveals a novelfunctional motifrdquo Structure vol 10 no 11 pp 1551ndash1557 2002

[22] A di Matteo N Calosci S Gianni P Jemth M Brunori and CTravaglini-Allocatelli ldquoStructural and functional characteriza-tion of CcmG from pseudomonas aeruginosa a key componentof the bacterial cytochrome c maturation apparatusrdquo Proteinsvol 78 no 10 pp 2213ndash2221 2010

[23] A diMatteo S GianniM E Schinina et al ldquoA strategic proteinin cytochrome c maturation three-dimensional structure ofCcmH and binding to apocytochrome crdquo Journal of BiologicalChemistry vol 282 no 37 pp 27012ndash27019 2007

[24] A Crow R M Acheson N E Le Brun and A OubrieldquoStructural basis of redox-coupled protein substrate selectionby the cytochrome c biosynthesis protein ResArdquo Journal ofBiological Chemistry vol 279 no 22 pp 23654ndash23660 2004

[25] E di Silvio A di Matteo and C Travaglini-Allocatelli ldquoTheRedox pathway of Pseudomonas aeruginosa cytochrome cbiogenesisrdquo Journal of Proteins and Proteomics vol 3 no 1 pp1ndash7 2012

[26] LThony-Meyer andP Kunzler ldquoTranslocation to the periplasmand signal sequence cleavage of preapocytochrome c depend onsec and lep but not on the ccmgene productsrdquoEuropean Journalof Biochemistry vol 246 no 3 pp 794ndash799 1997

[27] S J Facey and A Kuhn ldquoBiogenesis of bacterial inner-membrane proteinsrdquo Cellular and Molecular Life Sciences vol67 no 14 pp 2343ndash2362 2010

[28] K Diekert A I de Kroon U Ahting et al ldquoApocytochromec requires the TOM complex for translocation across themitochondrial outer membranerdquo The EMBO Journal vol 20no 20 pp 5626ndash5635 2001

[29] N Wiedemann V Kozjak T Prinz et al ldquoBiogenesis of yeastmitochondrial cytochrome c a unique relationship to the TOMmachineryrdquo Journal ofMolecular Biology vol 327 no 2 pp 465ndash474 2003

[30] F-U Hartl N Pfanner and W Neupert ldquoTranslocation inter-mediates on the import pathway of proteins intomitochondriardquoBiochemical Society Transactions vol 15 no 1 pp 95ndash97 1987

[31] B S Glick A Brandt K Cunningham SMuller R L Hallbergand G Schatz ldquoCytochromes c1 and b2 are sorted to theintermembrane space of yeast mitochondria by a stop-transfermechanismrdquo Cell vol 69 no 5 pp 809ndash822 1992

[32] G Howe and S Merchant ldquoRole of heme in the biosynthesis ofcytochrome c6rdquo Journal of Biological Chemistry vol 269 no 8pp 5824ndash5832 1994

[33] S S Nakamoto P Hamel and S Merchant ldquoAssembly ofchloroplast cytochromes b and crdquo Biochimie vol 82 no 6-7 pp603ndash614 2000

[34] C Sanders S Turkarslan D-W Lee and F DaldalldquoCytochrome c biogenesis the Ccm systemrdquo Trends inMicrobiology vol 18 no 6 pp 266ndash274 2010

[35] J M Stevens D A Mavridou R Hamer P Kritsiligkou A DGoddard and S J Ferguson ldquoCytochrome c biogenesis systemIrdquoThe FEBS Journal vol 278 no 22 pp 4170ndash4178 2011

[36] R G Kranz C Richard-Fogal J-S Taylor and E R FrawleyldquoCytochrome c biogenesis mechanisms for covalent modifica-tions and trafficking of heme and for heme-iron redox controlrdquo

14 Scientifica

Microbiology and Molecular Biology Reviews vol 73 no 3 pp510ndash528 2009

[37] J W A Allen ldquoCytochrome c biogenesis in mitochondriamdashsystems III and VrdquoThe FEBS Journal vol 278 no 22 pp 4198ndash4216 2011

[38] D A Mavridou S J Ferguson and J M Stevens ldquoCytochromec assemblyrdquo IUBMB Life vol 65 no 3 pp 209ndash216 2013

[39] I Bertini G Cavallaro and A Rosato ldquoEvolution ofmitochondrial-type cytochrome c domains and of theprotein machinery for their assemblyrdquo Journal of InorganicBiochemistry vol 101 no 11-12 pp 1798ndash1811 2007

[40] B S Goldman and R G Kranz ldquoEvolution and horizontaltransfer of an entire biosynthetic pathway for cytochromec biogenesis helicobacter deinococcus archae and morerdquoMolecular Microbiology vol 27 no 4 pp 871ndash873 1998

[41] C L Richard-Fogal E R Frawley E R Bonner H Zhu BSan Francisco and R G Kranz ldquoA conserved haem redoxand trafficking pathway for cofactor attachmentrdquo The EMBOJournal vol 28 no 16 pp 2349ndash2359 2009

[42] L Thony-Meyer F Fischer P Kunzler D Ritz and H Hen-necke ldquoEscherichia coli genes required for cytochrome cmaturationrdquo Journal of Bacteriology vol 177 no 15 pp 4321ndash4326 1995

[43] C S Beckett J A Loughman K A Karberg G M DonatoW E Goldman and R G Kranz ldquoFour genes are required forthe system II cytochrome c biogenesis pathway in Bordetellapertussis a unique bacterial modelrdquo Molecular Microbiologyvol 38 no 3 pp 465ndash481 2000

[44] N E Le Brun J Bengtsson and L Hederstedt ldquoGenes requiredfor cytochrome c synthesis in Bacillus subtilisrdquo MolecularMicrobiology vol 36 no 3 pp 638ndash650 2000

[45] P Giege J M Grienenberger and G Bonnard ldquoCytochromec biogenesis in mitochondriardquo Mitochondrion vol 8 no 1 pp61ndash73 2008

[46] R E Feissner C L Richard-Fogal E R Frawley J A Lough-man KW Earley and R G Kranz ldquoRecombinant cytochromesc biogenesis systems I and II and analysis of haem deliverypathways in Escherichia colirdquo Molecular Microbiology vol 60no 3 pp 563ndash577 2006

[47] N P Cianciotto P Cornelis and C Baysse ldquoImpact of thebacterial type I cytochrome c maturation system on differentbiological processesrdquoMolecular Microbiology vol 56 no 6 pp1408ndash1415 2005

[48] E Arslan H Schulz R Zufferey P Kunzler and L Thony-Meyer ldquoOverproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in Escherichiacolirdquo Biochemical and Biophysical Research Communicationsvol 251 no 3 pp 744ndash747 1998

[49] C Sanders and H Lill ldquoExpression of prokaryotic and eukary-otic cytochromes c in Escherichia colirdquoBiochimica et BiophysicaActa vol 1459 no 1 pp 131ndash138 2000

[50] M Braun and L Thony-Meyer ldquoBiosynthesis of artificialmicroperoxidases by exploiting the secretion and cytochromec maturation apparatuses of Escherichia colirdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 35 pp 12830ndash12835 2004

[51] B Baert C Baysse S Matthijs and P Cornelis ldquoMultiplephenotypic alterations caused by a c-type cytochrome mat-uration ccmC gene mutation in Pseudomonas aeruginosardquoMicrobiology vol 154 no 1 pp 127ndash138 2008

[52] S Turkarslan C Sanders and F Daldal ldquoExtracytoplasmicprosthetic group ligation to apoproteins maturation of c-typecytochromesrdquo Molecular Microbiology vol 60 no 3 pp 537ndash541 2006

[53] P M Jones and A M George ldquoThe ABC transporter structureand mechanism perspectives on recent researchrdquo Cellular andMolecular Life Sciences vol 61 no 6 pp 682ndash699 2004

[54] O Christensen E M Harvat L Thony-Meyer S J Fergusonand J M Stevens ldquoLoss of ATP hydrolysis activity by CcmABresults in loss of c-type cytochrome synthesis and incompleteprocessing ofCcmErdquoTheFEBS Journal vol 274 no 9 pp 2322ndash2332 2007

[55] J-H Lee E M Harvat J M Stevens S J Ferguson and M HSaier Jr ldquoEvolutionary origins of members of a superfamily ofintegralmembrane cytochrome c biogenesis proteinsrdquoBiochim-ica et Biophysica Acta vol 1768 no 9 pp 2164ndash2181 2007

[56] D L Beckman D R Trawick and R G Kranz ldquoBacterialcytochromes c biogenesisrdquo Genes and Development vol 6 no2 pp 268ndash283 1992

[57] H Schulz R A Fabianek E C Pellicioli H Hennecke andL Thony-Meyer ldquoHeme transfer to the heme chaperone CcmEduring cytochrome c maturation requires the CcmC proteinwhich may function independently of the ABC-transporterCcmABrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 96 no 11 pp 6462ndash6467 1999

[58] Q Ren and L Thony-Meyer ldquoPhysical Interaction ofCcmC with Heme and the Heme Chaperone CcmE duringCytochrome cMaturationrdquo Journal of Biological Chemistry vol276 no 35 pp 32591ndash32596 2001

[59] C Richard-Fogal and R G Kranz ldquoThe CcmChemeCcmEcomplex in heme trafficking and cytochrome c biosynthesisrdquoJournal of Molecular Biology vol 401 no 3 pp 350ndash362 2010

[60] V K Viswanathan S Kurtz L L Pedersen et al ldquoThecytochrome C maturation locus of Legionella pneumophilapromotes iron assimilation and intracellular infection andcontains a strain-specific insertion sequence elementrdquo Infectionand Immunity vol 70 no 4 pp 1842ndash1852 2002

[61] U Ahuja and L Thony-Meyer ldquoCcmD is involved in complexformation between CcmC and the heme chaperone CcmE dur-ing cytochrome c maturationrdquo Journal of Biological Chemistryvol 280 no 1 pp 236ndash243 2005

[62] C L Richard-Fogal E R Frawley and R G Kranz ldquoTopologyand function of CcmD in cytochrome Cmaturationrdquo Journal ofBacteriology vol 190 no 10 pp 3489ndash3493 2008

[63] H Schulz H Hennecke and L Thony-Meyer ldquoPrototype ofa heme chaperone essential for cytochrome c maturationrdquoScience vol 281 no 5380 pp 1197ndash1200 1998

[64] F Arnesano L Banci P D Barker et al ldquoSolution structure andcharacterization of the heme chaperone CcmErdquo Biochemistryvol 41 no 46 pp 13587ndash13594 2002

[65] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[66] L Thony-Meyer ldquoA heme chaperone for cytochrome c biosyn-thesisrdquo Biochemistry vol 42 no 45 pp 13099ndash13105 2003

[67] E M Harvat C Redfield J M Stevens and S J FergusonldquoProbing the heme-binding site of the cytochrome cmaturationprotein CcmErdquoBiochemistry vol 48 no 8 pp 1820ndash1828 2009

[68] D Lee K Pervushin D Bischof M Braun and L Thony-Meyer ldquoUnusual heme-histidine bond in the active site of a

Scientifica 15

chaperonerdquo Journal of the American Chemical Society vol 127no 11 pp 3716ndash3717 2005

[69] A D Goddard J M Stevens F Rao et al ldquoc-Type cytochromebiogenesis can occur via a natural Ccm system lacking CcmHCcmG and the heme-binding histidine of CcmErdquo Journal ofBiological Chemistry vol 285 no 30 pp 22882ndash22889 2010

[70] J M Aramini K Hamilton P Rossi et al ldquoSolution NMRstructure backbone dynamics and heme-binding propertiesof a novel cytochrome c maturation protein CcmE fromDesulfovibrio vulgarisrdquo Biochemistry vol 51 no 18 pp 3705ndash3707 2012

[71] T Uchida J M Stevens O Daltrop et al ldquoThe interactionof covalently bound heme with the cytochrome c maturationprotein CcmErdquo Journal of Biological Chemistry vol 279 no 50pp 51981ndash51988 2004

[72] I Garcıa-Rubio M Braun I Gromov L Thony-Meyer andA Schweiger ldquoAxial coordination of heme in ferric CcmEchaperone characterized by EPR spectroscopyrdquo BiophysicalJournal vol 92 no 4 pp 1361ndash1373 2007

[73] L Thony-Meyer ldquoHaem-polypeptide interactions duringcytochrome c maturationrdquo Biochimica et Biophysica Acta vol1459 no 2-3 pp 316ndash324 2000

[74] J A Keightley D Sanders T R Todaro A Pastuszyn and J AFee ldquoCloning expression in Escherichia coli of the cytochromec552 gene from Thermus thermophilus HB8 Evidence forgenetic linkage to an ATP- binding cassette protein and initialcharacterization of the cycA gene productsrdquo Journal of Biologi-cal Chemistry vol 273 no 20 pp 12006ndash12016 1998

[75] E M Monika B S Goldman D L Beckman and R GKranz ldquoA thioreduction pathway tethered to the membranefor periplasmic cytochromes c biogenesis In vitro and in vivostudiesrdquo Journal of Molecular Biology vol 271 no 5 pp 679ndash692 1997

[76] M Throne-Holst L Thony-Meyer and L HederstedtldquoEscherichia coli ccm in-frame deletion mutants can produceperiplasmic cytochrome b but not cytochrome crdquo FEBS Lettersvol 410 no 2-3 pp 351ndash355 1997

[77] U Ahuja and L Thony-Meyer ldquoDynamic features of a hemedelivery system for cytochrome c maturationrdquo Journal of Bio-logical Chemistry vol 278 no 52 pp 52061ndash52070 2003

[78] J W A Allen P D Barker O Daltrop et al ldquoWhy isnrsquot ldquostan-dardrdquo heme good enough for c-type and di-type cytochromesrdquoDalton Transactions no 21 pp 3410ndash3418 2005

[79] K Inaba and K Ito ldquoStructure and mechanisms of the DsbB-DsbA disulfide bond generation machinerdquo Biochimica et Bio-physica Acta vol 1783 no 4 pp 520ndash529 2008

[80] H Kadokura F Katzen and J Beckwith ldquoProtein disulfidebond formation in prokaryotesrdquoAnnual Review of Biochemistryvol 72 pp 111ndash135 2003

[81] S R Shouldice B Heras P M Walden M Totsika M ASchembri and J L Martin ldquoStructure and function of DsbA akey bacterial oxidative folding catalystrdquoAntioxidants and RedoxSignaling vol 14 no 9 pp 1729ndash1760 2011

[82] R Metheringham L Griffiths H Crooke S Forsythe and JCole ldquoAn essential role for DsbA in cytochrome c synthesis andformate-dependent nitrite reduction by Escherichia coli K-12rdquoArchives of Microbiology vol 164 no 4 pp 301ndash307 1995

[83] Y Sambongi and S J Ferguson ldquoMutants of Escherichia colilacking disulphide oxidoreductases DsbA and DsbB cannotsynthesise an exogenous monohaem c-type cytochrome exceptin the presence of disulphide compoundsrdquo FEBS Letters vol398 no 2-3 pp 265ndash268 1996

[84] D AMavridou S J Ferguson and JM Stevens ldquoThe interplaybetween the disulfide bond formation pathway and cytochromec maturation in Escherichia colirdquo FEBS Letters vol 586 no 12pp 1702ndash1707 2012

[85] C U Stirnimann A Rozhkova U Grauschopf M G GrutterR Glockshuber and G Capitani ldquoStructural basis and kineticsof DsbD-dependent cytochrome c maturationrdquo Structure vol13 no 7 pp 985ndash993 2005

[86] N Ouyang Y-G Gao H-Y Hu and Z-X Xia ldquoCrystalstructures of E coli CcmGand itsmutants reveal key roles of theN-terminal120573-sheet and the fingerprint regionrdquoProteins vol 65no 4 pp 1021ndash1031 2006

[87] M A Edeling L W Guddat R A Fabianek et al ldquoCrys-tallization and preliminary diffraction studies of native andselenomethionine CcmG (CycY DsbE)rdquoActa CrystallographicaD vol 57 no 9 pp 1293ndash1295 2001

[88] R A Fabianek M Huber-Wunderlich R Glockshuber PKunzler H Hennecke and L Thony-Meyer ldquoCharacterizationof the Bradyrhizobium japonicum CycY protein a membrane-anchored periplasmic thioredoxin that may play a role as areductant in the biogenesis of c-type cytochromesrdquo Journal ofBiological Chemistry vol 272 no 7 pp 4467ndash4473 1997

[89] G B Kallis and A Holmgren ldquoDifferential reactivity of thefunctional sulfhydryl groups of cysteine-32 and cysteine-32present in the reduced form of thioredoxin from Escherichiacolirdquo Journal of Biological Chemistry vol 255 no 21 pp 10261ndash10265 1980

[90] P T Chivers andR T Raines ldquoGeneral acidbase catalysis in theactive site of Escherichia coli thioredoxinrdquoBiochemistry vol 36no 50 pp 15810ndash15816 1997

[91] X M Zheng J Hong H Y Li D H Lin and H Y HuldquoBiochemical properties and catalytic domain structure of theCcmH protein from Escherichia colirdquo Biochim Biophys Actavol 1824 no 12 pp 1394ndash1400 2012

[92] UAhujaA Rozhkova RGlockshuber LThony-Meyer andOEinsle ldquoHelix swapping leads to dimerization of the N-terminaldomain of the c-type cytochrome maturation protein CcmHfrom Escherichia colirdquo FEBS Letters vol 582 no 18 pp 2779ndash2786 2008

[93] R A Fabianek T Hofer and L Thony-Meyer ldquoCharacteriza-tion of the Escherichia coli CcmH protein reveals new insightsinto the redox pathway required for cytochrome c maturationrdquoArchives of Microbiology vol 171 no 2 pp 92ndash100 1999

[94] E Reid J Cole and D J Eaves ldquoThe Escherichia coli CcmGprotein fulfils a specific role in cytochrome c assemblyrdquo Bio-chemical Journal vol 355 no 1 pp 51ndash58 2001

[95] Q Ren U Ahuja and LThony-Meyer ldquoA bacterial cytochromec heme lyase CcmF forms a complex with the heme chaperoneCcmE and CcmH but not with apocytochrome crdquo Journal ofBiological Chemistry vol 277 no 10 pp 7657ndash7663 2002

[96] E H Meyer P Giege E Gelhaye et al ldquoAtCCMH an essentialcomponent of the c-type cytochrome maturation pathway inArabidopsis mitochondria interacts with apocytochrome crdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 44 pp 16113ndash16118 2005

[97] C Rios-Velazquez R Coller and T J Donohue ldquoFeatures ofRhodobacter sphaeroides CcmFHrdquo Journal of Bacteriology vol185 no 2 pp 422ndash431 2003

[98] C Sanders M Deshmukh D Astor R G Kranz and F DaldalldquoOverproduction of CcmG and CcmFHRc fully suppresses thec-type cytochrome biogenesis defect of Rhodobacter capsulatus

16 Scientifica

CcmI-null mutantsrdquo Journal of Bacteriology vol 187 no 12 pp4245ndash4256 2005

[99] A F Verissimo H Yang X Wu C Sanders and F DaldalldquoCcmI subunit of CcmFHI heme ligation complex functionsas an apocytochrome c chaperone during c-type cytochromematurationrdquo Journal of Biological Chemistry vol 286 no 47 pp40452ndash40463 2011

[100] K Bryson L J McGuffin R L Marsden J J Ward J SSodhi and D T Jones ldquoProtein structure prediction servers atUniversity College Londonrdquo Nucleic Acids Research vol 33 no2 pp W36ndashW38 2005

[101] D T Jones ldquoProtein secondary structure prediction basedon position-specific scoring matricesrdquo Journal of MolecularBiology vol 292 no 2 pp 195ndash202 1999

[102] C Sanders C Boulay and F Daldal ldquoMembrane-spanning andperiplasmic segments of CcmI have distinct functions duringcytochrome c biogenesis in Rhodobacter capsulatusrdquo Journal ofBacteriology vol 189 no 3 pp 789ndash800 2007

[103] D Han K Kim J Oh J Park and Y Kim ldquoTPR domainof NrfG mediates complex formation between heme lyaseand formate-dependent nitrite reductase in Escherichia coliO157H7rdquo Proteins vol 70 no 3 pp 900ndash914 2008

[104] D J Eaves J Grove W Staudenmann et al ldquoInvolvement ofproducts of the nrfEFG genes in the covalent attachment ofhaem c to a novel cysteine-lysine motif in the cytochrome c552nitrite reductase from Escherichia colirdquo Molecular Microbiol-ogy vol 28 no 1 pp 205ndash216 1998

[105] J Nilsson B Persson and G Von Heijne ldquoConsensus predic-tions of membrane protein topologyrdquo FEBS Letters vol 486 no3 pp 267ndash269 2000

[106] E di Silvio A di Matteo F Malatesta and C Travaglini-Allocatelli ldquoRecognition and binding of apocytochrome c toP aeruginosa CcmI a component of cytochrome c maturationmachineryrdquo Biochim Biophys Acta vol 1834 no 8 pp 1554ndash1561 2013

[107] N Zeytuni and R Zarivach ldquoStructural and functional dis-cussion of the tetra-trico-peptide repeat a protein interactionmodulerdquo Structure vol 20 no 3 pp 397ndash405 2012

[108] C Sanders S Turkarslan D-W Lee O Onder R G Kranz andF Daldal ldquoThe cytochrome c maturation components CcmFCcmH and CcmI form a membrane-integral multisubunitheme ligation complexrdquo Journal of Biological Chemistry vol283 no 44 pp 29715ndash29722 2008

[109] A F Verissimo M A Mohtar and F Daldal ldquoThe hemechaperone ApoCcmE forms a ternary complex with CcmI andapocytochrome crdquoThe Journal of Biological Chemistry vol 288no 9 pp 6272ndash6283 2013

[110] P D Barker J C Ferrer M Mylrajan et al ldquoTransmutation of aheme proteinrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 90 no 14 pp 6542ndash65461993

[111] J Simon and L Hederstedt ldquoComposition and function ofcytochrome c biogenesis system IIrdquoThe FEBS Journal vol 278no 22 pp 4179ndash4188 2011

[112] T R H M Kouwen and J M van Dijl ldquoInterchangeablemodules in bacterial thiol-disulfide exchange pathwaysrdquo Trendsin Microbiology vol 17 no 1 pp 6ndash12 2009

[113] A Crow A Lewin O Hecht et al ldquoCrystal structure andbiophysical properties of Bacillus subtilis BdbD An oxidizingthioldisulfide oxidoreductase containing a novel metal siterdquoJournal of Biological Chemistry vol 284 no 35 pp 23719ndash23733 2009

[114] M C Moller and L Hederstedt ldquoExtracytoplasmic processesimpaired by inactivation of trxA (thioredoxin gene) in Bacillussubtilisrdquo Journal of Bacteriology vol 190 no 13 pp 4660ndash46652008

[115] A Lewin A Crow A Oubrie and N E Le Brun ldquoMolecularbasis for specificity of the extracytoplasmic thioredoxin ResArdquoJournal of Biological Chemistry vol 281 no 46 pp 35467ndash35477 2006

[116] C L Colbert QWu P J A Erbel K H Gardner and J Deisen-hofer ldquoMechanism of substrate specificity in Bacillus subtilisResA a thioredoxin-like protein involved in cytochrome cmaturationrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 103 no 12 pp 4410ndash44152006

[117] UAhuja P Kjelgaard B L Schulz LThony-Meyer andLHed-erstedt ldquoHaem-delivery proteins in cytochrome c maturationsystem IIrdquoMolecular Microbiology vol 73 no 6 pp 1058ndash10712009

[118] M L D Page P P Hamel S T Gabilly et al ldquoA homologof prokaryotic thiol disulfide transporter CcdA is required forthe assembly of the cytochrome b6f complex in Arabidopsischloroplastsrdquo Journal of Biological Chemistry vol 279 no 31pp 32474ndash32482 2004

[119] A Rozhkova C U Stirnimann P Frei et al ldquoStructural basisand kinetics of inter- and intramolecular disulfide exchange inthe redox catalyst DsbDrdquoThe EMBO Journal vol 23 no 8 pp1709ndash1719 2004

[120] T Schiott M Throne-Holst and L Hederstedt ldquoBacillussubtilis CcdA-defective mutants are blocked in a late step ofcytochrome C biogenesisrdquo Journal of Bacteriology vol 179 no14 pp 4523ndash4529 1997

[121] R E Feissner C S Beckett J A Loughman and R G KranzldquoMutations in cytochrome assembly and periplasmic redoxpathways in Bordetella pertussisrdquo Journal of Bacteriology vol187 no 12 pp 3941ndash3949 2005

[122] L S ErlendssonMMoller and L Hederstedt ldquoBacillus subtilisStoA is a thiol-disulfide oxidoreductase important for sporecortex synthesisrdquo Journal of Bacteriology vol 186 no 18 pp6230ndash6238 2004

[123] L S Erlendsson R M Acheson L Hederstedt and N E LeBrun ldquoBacillus subtilis ResA is a thiol-disulfide oxidoreductaseinvolved in cytochrome c synthesisrdquo Journal of BiologicalChemistry vol 278 no 20 pp 17852ndash17858 2003

[124] L S Erlendsson and L Hederstedt ldquoMutations in the thiol-disulfide oxidoreductases BdbC and BdbD can suppresscytochrome c deficiency of CcdA-defective Bacillus subtiliscellsrdquo Journal of Bacteriology vol 184 no 5 pp 1423ndash1429 2002

[125] M E Dumont J F Ernst D M Hampsey and F ShermanldquoIdentification and sequence of the gene encoding cytochromec heme lyase in the yeast Saccharomyces cerevisiaerdquoThe EMBOJournal vol 6 no 1 pp 235ndash241 1987

[126] M E Dumont J F Ernst and F Sherman ldquoCoupling of hemeattachment to import of cytochrome c into yeast mitochondriaStudies with heme lyase-deficient mitochondria and alteredapocytochromes crdquo Journal of Biological Chemistry vol 263 no31 pp 15928ndash15937 1988

[127] B San Francisco E C Bretsnyder and R G Kranz ldquoHumanmitochondrial holocytochrome c synthasersquos hemebindingmat-uration determinants and complex formationwith cytochromecrdquo Proceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 9 pp E788ndashE797 2012

Scientifica 17

[128] L Schaefer A Ballabio and H Y Zoghbi ldquoCloning andcharacterization of a putative human holocytochrome c-typesynthetase gene (HCCS) isolated from the critical region formicrophthalmia with linear skin defects (MLS)rdquoGenomics vol34 no 2 pp 166ndash172 1996

[129] S K Prakash T A Cormier A E McCall et al ldquoLoss ofholocytochrome c-type synthetase causes the male lethality ofX-linked dominant microphthalmia with linear skin defects(MLS) syndromerdquo Human Molecular Genetics vol 11 no 25pp 3237ndash3248 2002

[130] I Wimplinger M Morleo G Rosenberger et al ldquoMutations ofthe mitochondrial holocytochrome c-type synthase in X-linkeddominant microphthalmia with linear skin defects syndromerdquoTheAmerican Journal of HumanGenetics vol 79 no 5 pp 878ndash889 2006

[131] S Kiryu-Seo KGamo T Tachibana K Tanaka andHKiyamaldquoUnique anti-apoptotic activity of EAAC1 in injured motorneuronsrdquoTheEMBO Journal vol 25 no 14 pp 3411ndash3421 2006

[132] R Lill R A Stuart M E Drygas F E Nargang and W Neu-pert ldquoImport of cytochrome c heme lyase into mitochondriaa novel pathway into the intermembrane spacerdquo The EMBOJournal vol 11 no 2 pp 449ndash456 1992

[133] H Steiner A Zollner A Haid W Neupert and R LillldquoBiogenesis of mitochondrial heme lyases in yeast Importand folding in the intermembrane spacerdquo Journal of BiologicalChemistry vol 270 no 39 pp 22842ndash22849 1995

[134] A Zollner G Rodel and A Haid ldquoMolecular cloning andcharacterization of the Saccharomyces cerevisiae CYT2 geneencoding cytochrome-c1-heme lyaserdquo European Journal of Bio-chemistry vol 207 no 3 pp 1093ndash1100 1992

[135] V Corvest D A Murrey D G Bernard D B Knaff B Guiardand P PHamel ldquoc-Type cytochrome assembly in Saccharomycescerevisiae a key residue for apocytochrome c1lyase interactionrdquoGenetics vol 186 no 2 pp 561ndash571 2010

[136] V Corvest D A Murrey M Hirasawa D B Knaff B Guiardand P P Hamel ldquoThe flavoprotein Cyc2p a mitochondrialcytochrome c assembly factor is a NAD(P)H-dependent haemreductaserdquo Molecular Microbiology vol 83 no 5 pp 968ndash9802012

[137] D G Bernard S Quevillon-Cheruel S Merchant B Guiardand P P Hamel ldquoCyc2p a membrane-bound flavopro-tein involved in the maturation of mitochondrial c-typecytochromesrdquo Journal of Biological Chemistry vol 280 no 48pp 39852ndash39859 2005

[138] J G Kleingardner and K L Bren ldquoComparing substratespecificity between cytochrome cmaturation and cytochrome cheme lyase systems for cytochrome c biogenesisrdquo Metallomicsvol 3 no 4 pp 396ndash403 2011

[139] C Travaglini-Allocatelli S Gianni VMorea A Tramontano TSoulimane and M Brunori ldquoExploring the cytochrome c fold-ing mechanism cytochrome c552 fromThermus thermophilusfolds through an on-pathway intermediaterdquo Journal of BiologicalChemistry vol 278 no 42 pp 41136ndash41140 2003

[140] L Zhang and L Guarente ldquoHeme binds to a short sequencethat serves a regulatory function in diverse proteinsrdquoTheEMBOJournal vol 14 no 2 pp 313ndash320 1995

[141] S Hira T Tomita T Matsui K Igarashi and M Ikeda-SaitoldquoBach1 a heme-dependent transcription factor reveals pres-ence of multiple heme binding sites with distinct coordinationstructurerdquo IUBMB Life vol 59 no 8-9 pp 542ndash551 2007

[142] T K Kuhl A Wiszligbrock N Goradia et al ldquoAnalysis of Fe(III)heme binding to cysteine-containing heme-regulatory motifs

in proteinsrdquo ACS Chemical Biology vol 8 no 8 pp 1785ndash17932013

[143] R L Moore J M Stevens and S J Ferguson ldquoMitochondrialcytochrome c synthase CP motifs are not necessary for hemeattachment to apocytochrome crdquo FEBS Letters vol 585 no 21pp 3415ndash3419 2011

[144] S Gianni M Brunori and C Travaglini-Allocatelli ldquoRefoldingkinetics of cytochrome c551 reveals a mechanistic differencebetween urea and guanidinerdquo Protein Science vol 10 no 8 pp1685ndash1688 2001

[145] S Gianni C Travaglini-Allocatelli F Cutruzzola M BrunoriM C R Shastry and H Roder ldquoParallel pathways incytochrome c551 foldingrdquo Journal of Molecular Biology vol 330no 5 pp 1145ndash1152 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Volume 2014

Zoology

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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Microbiology

12 Scientifica

is involved in diseases such as microphthalmia with linearskin defects syndrome (MLS) an X-linked genetic disorder[128ndash130] and in other processes such as Cytc-independentapoptosis in injured motor neurons [131] Different fromanimal cells where a single HCCS is sufficient for thematuration of both soluble and membrane-anchored Cytc(Cyt c and Cy c1 resp) in S cerevisiae two homologs HCCSand HCC1S located in the inner mitochondrial membraneand facing to the IMS space [132 133] are responsible for hemeattachment to apoCyt c and apoCyt c1 respectively [125 134]It should also be noticed that in fungi an additional FAD-containing protein (Cyc2p) is required for Cyt c synthesisCyc2p is a mitochondrial membrane-anchored flavoproteinexposing its redox domain to the IMS which is requiredfor the maturation of Cyt c but not for that of Cyt c1 [135]This protein does not contain the conserved Cys residuestypically found in disulfide reductases and indeed it is notable to reduce oxidized apoCyt in vitro However it has beenrecently shown that Cyc2p is able to catalyze the NAD(P)H-dependent reduction of heme in vitro [136] a necessarystep before the thioether bond formation can occur asdiscussed above (see Section 26) This result together withthe observation that Cyc2p interacts with HCCS and withapoCyt c and c1 lends support to the proposal that Cyc2p isinvolved in the reduction of the heme iron in vivo [136 137]

AlthoughHCCS proteins are crucial for Cyt cmaturationwe still do not know how these proteins recognize theirsubstrates (heme and apoCyt) and how they promote orcatalyze the formation of thioether bonds between the hemevinyl groups and the cysteine thiols of the apoCyt CXXCHmotif

Contrary to the broad specificity of System I whichis able to recognize and attach the heme to prokaryoticand eukaryotic c-type cytochromes [49 50] and even tovery short microperoxidase-like sequences [49 50] mito-chondrial HCCS is characterized by a higher specificityas it does not recognize bacterial apoCyt sequences Theseobservations prompted the investigation of the recognitionprocess between apoCyt and HCCS Recently by using arecombinant yeast HCCS and chimerical apoCyt sequencesexpressed in E coli it was possible to conclude that a crucialrecognition determinant is represented by the N-terminalregion of apoCyt containing the heme-binding motif [35]notice that in the context of System I the same N-terminalregion of the Cyt c sequence has been recently identified to beimportant for the recognition by apoCcmE (see Section 26)Over and above the role played by the intervening residuesin the CXXCH heme-binding motif [135] it has been shownthat a conserved Phe residue occurring in the N-terminalregion before the CXXCH motif is important for HCCSrecognition [35 138] These results support the hypothesisthat this residue known to be a key determinant of Cytcfolding and stability [19 20 139] may also be crucial for Cytcmaturation by HCCS

Another relevant question concerns the ability of HCCSto recognize and bind the heme molecule as the region ofthe protein responsible for heme recognition remains to beidentified Initially it was hypothesized that the recognitionof heme could be mediated by the CP motifs present in

the HCCS protein [140] these short sequences are indeedknown to bind heme in a variety of heme-containing proteins[141 142] However it has been recently shown that CPmotifsof the recombinant S cerevisiae HCCS are not necessaryfor Cyt c production in E coli [143] excluding them as keydeterminants of heme recognition

The long-awaited in vitro characterization of the recom-binant human HCCS allowed for the first time to pro-pose a molecular mechanism underlying Cytc maturationin eukaryotes which can be experimentally tested [127]According to the proposed model the human HCCS activitycan be described as a four step mechanism involving (i)heme-binding (ii) apoCyt recognition (iii) thioether bondsformation and (iv) holoCyt c release In particular theheme is proposed to play the role of a scaffolding moleculemediating the contacts between HCCS and apoCyt Muta-genesis experiments carried out on the recombinant HCCSstrongly suggest that heme-binding (Step 1) depends on thepresence of a specific His residue (His154) acting as anaxial ligand to ferrous heme Residues present at the N-terminus of apoCyt mediate the recognition with HCCS(Step 2) as discussed above it is known that this regionforms structurally conserved 120572-helix in the fold of all c-type cytochromes Unfortunately no information is availableup to now concerning the region of HCCS involved in therecognition and binding to the N-terminal region of apoCytCoordination of the heme iron by the His residue of theapoCyt heme-binding motif CXXCH provides the secondaxial ligand to the heme iron and is probably importantfor the correct positioning of the two apoCyt Cys residuesand formation of the thioether bonds (Step 3) The finalrelease of functional Cyt c (Step 4) clearly requires thedisplacement of the His154Fe2+ coordination bond sucha displacement is probably mediated by formation of thecoordination bond with the Cyt c conserved Met residueandor by the simultaneous folding of Cyt c Again it isinteresting to note that this last hypothesis is in accordancewith in vitro folding studies on c-type cytochromes thathighlighted the late formation of the Met-Fe coordinationduring Cyt c folding [144 145]

Acknowledgments

The author thanks A Di Matteo and E Di Silvio for fruitfuldiscussions and for help in preparing this paper This work issupported by grants from the University of Rome ldquoSapienzardquo(C26A11MBXJ and C26A13MWF4)

References

[1] M G Galinato J G Kleingardner S E Bowman et alldquoHeme-protein vibrational couplings in cytochrome c providea dynamic link that connects the heme-iron and the proteinsurfacerdquo Proceedings of the National Academy of Sciences vol109 no 23 pp 8896ndash8900 2012

[2] H B Gray and J R Winkler ldquoElectron flow through metal-loproteinsrdquo Biochimica et Biophysica Acta vol 1797 no 9 pp1563ndash1572 2010

Scientifica 13

[3] P Ascenzi R Santucci M Coletta and F PolticellildquoCytochromes reactivity of the ldquodark siderdquo of the hemerdquoBiophysical Chemistry vol 152 no 1ndash3 pp 21ndash27 2010

[4] S Yamada N D Bouley Ford G E Keller W C Ford HB Gray and J R Winkler ldquoSnapshots of a protein foldingintermediaterdquo Proceedings of the National Academy of Sciencesvol 110 no 5 pp 1606ndash1610 2013

[5] P Weinkam J Zimmermann F E Romesberg and P GWolynes ldquoThe folding energy landscape and free energy excita-tions of cytochrome crdquo Accounts of Chemical Research vol 43no 5 pp 652ndash660 2010

[6] M Yamanaka M Masanari and Y Sambongi ldquoConfermentof folding ability to a naturally unfolded apocytochrome cthrough introduction of hydrophobic amino acid residuesrdquoBiochemistry vol 50 no 12 pp 2313ndash2320 2011

[7] G W Moore and G W Pettigrew Cytochrome C EvolutionaryStructural and Physicochemical Aspects Springer Berlin Ger-many 1990

[8] I Bertini G Cavallaro and A Rosato ldquoCytochrome c occur-rence and functionsrdquo Chemical Reviews vol 106 no 1 pp 90ndash115 2006

[9] D A Pearce and F Sherman ldquoDegradation of cytochromeoxidase subunits in mutants of yeast lacking cytochrome c andsuppression of the degradation by mutation of yme1rdquo Journal ofBiological Chemistry vol 270 no 36 pp 20879ndash20882 1995

[10] G Silkstone S M Kapetanaki I Husu M H Vos and MT Wilson ldquoNitric oxide binding to the cardiolipin complex offerric cytochrome crdquo Biochemistry vol 51 no 34 pp 6760ndash6766 2012

[11] M Giorgio E Migliaccio F Orsini et al ldquoElectron transferbetween cytochrome c and p66Shc generates reactive oxygenspecies that trigger mitochondrial apoptosisrdquo Cell vol 122 no2 pp 221ndash233 2005

[12] S M Kilbride and J H Prehn ldquoCentral roles of apoptoticproteins in mitochondrial functionrdquo Oncogene vol 32 no 22pp 2703ndash2711 2013

[13] T Noll and G Noll ldquoStrategies for ldquowiringrdquo redox-activeproteins to electrodes and applications in biosensors biofuelcells and nanotechnologyrdquo Chemical Society Reviews vol 40no 7 pp 3564ndash3576 2011

[14] G Layer J Reichelt D Jahn and D W Heinz ldquoStructure andfunction of enzymes in heme biosynthesisrdquo Protein Science vol19 no 6 pp 1137ndash1161 2010

[15] S Severance and IHamza ldquoTrafficking of heme and porphyrinsin metazoardquo Chemical Reviews vol 109 no 10 pp 4596ndash46162009

[16] P Hamel V Corvest P Giege and G Bonnard ldquoBiochemi-cal requirements for the maturation of mitochondrial c-typecytochromesrdquo Biochimica et Biophysica Acta vol 1793 no 1 pp125ndash138 2009

[17] W R Fisher H Taniuchi and C B Anfinsen ldquoOn the role ofheme in the formation of the structure of cytochrome crdquo Journalof Biological Chemistry vol 248 no 9 pp 3188ndash3195 1973

[18] M Yamanaka H Mita Y Yamamoto and Y Sambongi ldquoHemeis not required for aquifex aeolicus cytochrome c555 polypep-tide foldingrdquoBioscience Biotechnology andBiochemistry vol 73no 9 pp 2022ndash2025 2009

[19] C Travaglini-Allocatelli S Gianni andMBrunori ldquoA commonfolding mechanism in the cytochrome c familyrdquo Trends inBiochemical Sciences vol 29 no 10 pp 535ndash541 2004

[20] A Borgia D Bonivento C Travaglini-Allocatelli A DiMatteoand M Brunori ldquoUnveiling a hidden folding intermediate in c-type cytochromes by protein engineeringrdquo Journal of BiologicalChemistry vol 281 no 14 pp 9331ndash9336 2006

[21] E Enggist L Thony-Meyer P Guntert and K PervushinldquoNMR structure of the heme chaperone CcmE reveals a novelfunctional motifrdquo Structure vol 10 no 11 pp 1551ndash1557 2002

[22] A di Matteo N Calosci S Gianni P Jemth M Brunori and CTravaglini-Allocatelli ldquoStructural and functional characteriza-tion of CcmG from pseudomonas aeruginosa a key componentof the bacterial cytochrome c maturation apparatusrdquo Proteinsvol 78 no 10 pp 2213ndash2221 2010

[23] A diMatteo S GianniM E Schinina et al ldquoA strategic proteinin cytochrome c maturation three-dimensional structure ofCcmH and binding to apocytochrome crdquo Journal of BiologicalChemistry vol 282 no 37 pp 27012ndash27019 2007

[24] A Crow R M Acheson N E Le Brun and A OubrieldquoStructural basis of redox-coupled protein substrate selectionby the cytochrome c biosynthesis protein ResArdquo Journal ofBiological Chemistry vol 279 no 22 pp 23654ndash23660 2004

[25] E di Silvio A di Matteo and C Travaglini-Allocatelli ldquoTheRedox pathway of Pseudomonas aeruginosa cytochrome cbiogenesisrdquo Journal of Proteins and Proteomics vol 3 no 1 pp1ndash7 2012

[26] LThony-Meyer andP Kunzler ldquoTranslocation to the periplasmand signal sequence cleavage of preapocytochrome c depend onsec and lep but not on the ccmgene productsrdquoEuropean Journalof Biochemistry vol 246 no 3 pp 794ndash799 1997

[27] S J Facey and A Kuhn ldquoBiogenesis of bacterial inner-membrane proteinsrdquo Cellular and Molecular Life Sciences vol67 no 14 pp 2343ndash2362 2010

[28] K Diekert A I de Kroon U Ahting et al ldquoApocytochromec requires the TOM complex for translocation across themitochondrial outer membranerdquo The EMBO Journal vol 20no 20 pp 5626ndash5635 2001

[29] N Wiedemann V Kozjak T Prinz et al ldquoBiogenesis of yeastmitochondrial cytochrome c a unique relationship to the TOMmachineryrdquo Journal ofMolecular Biology vol 327 no 2 pp 465ndash474 2003

[30] F-U Hartl N Pfanner and W Neupert ldquoTranslocation inter-mediates on the import pathway of proteins intomitochondriardquoBiochemical Society Transactions vol 15 no 1 pp 95ndash97 1987

[31] B S Glick A Brandt K Cunningham SMuller R L Hallbergand G Schatz ldquoCytochromes c1 and b2 are sorted to theintermembrane space of yeast mitochondria by a stop-transfermechanismrdquo Cell vol 69 no 5 pp 809ndash822 1992

[32] G Howe and S Merchant ldquoRole of heme in the biosynthesis ofcytochrome c6rdquo Journal of Biological Chemistry vol 269 no 8pp 5824ndash5832 1994

[33] S S Nakamoto P Hamel and S Merchant ldquoAssembly ofchloroplast cytochromes b and crdquo Biochimie vol 82 no 6-7 pp603ndash614 2000

[34] C Sanders S Turkarslan D-W Lee and F DaldalldquoCytochrome c biogenesis the Ccm systemrdquo Trends inMicrobiology vol 18 no 6 pp 266ndash274 2010

[35] J M Stevens D A Mavridou R Hamer P Kritsiligkou A DGoddard and S J Ferguson ldquoCytochrome c biogenesis systemIrdquoThe FEBS Journal vol 278 no 22 pp 4170ndash4178 2011

[36] R G Kranz C Richard-Fogal J-S Taylor and E R FrawleyldquoCytochrome c biogenesis mechanisms for covalent modifica-tions and trafficking of heme and for heme-iron redox controlrdquo

14 Scientifica

Microbiology and Molecular Biology Reviews vol 73 no 3 pp510ndash528 2009

[37] J W A Allen ldquoCytochrome c biogenesis in mitochondriamdashsystems III and VrdquoThe FEBS Journal vol 278 no 22 pp 4198ndash4216 2011

[38] D A Mavridou S J Ferguson and J M Stevens ldquoCytochromec assemblyrdquo IUBMB Life vol 65 no 3 pp 209ndash216 2013

[39] I Bertini G Cavallaro and A Rosato ldquoEvolution ofmitochondrial-type cytochrome c domains and of theprotein machinery for their assemblyrdquo Journal of InorganicBiochemistry vol 101 no 11-12 pp 1798ndash1811 2007

[40] B S Goldman and R G Kranz ldquoEvolution and horizontaltransfer of an entire biosynthetic pathway for cytochromec biogenesis helicobacter deinococcus archae and morerdquoMolecular Microbiology vol 27 no 4 pp 871ndash873 1998

[41] C L Richard-Fogal E R Frawley E R Bonner H Zhu BSan Francisco and R G Kranz ldquoA conserved haem redoxand trafficking pathway for cofactor attachmentrdquo The EMBOJournal vol 28 no 16 pp 2349ndash2359 2009

[42] L Thony-Meyer F Fischer P Kunzler D Ritz and H Hen-necke ldquoEscherichia coli genes required for cytochrome cmaturationrdquo Journal of Bacteriology vol 177 no 15 pp 4321ndash4326 1995

[43] C S Beckett J A Loughman K A Karberg G M DonatoW E Goldman and R G Kranz ldquoFour genes are required forthe system II cytochrome c biogenesis pathway in Bordetellapertussis a unique bacterial modelrdquo Molecular Microbiologyvol 38 no 3 pp 465ndash481 2000

[44] N E Le Brun J Bengtsson and L Hederstedt ldquoGenes requiredfor cytochrome c synthesis in Bacillus subtilisrdquo MolecularMicrobiology vol 36 no 3 pp 638ndash650 2000

[45] P Giege J M Grienenberger and G Bonnard ldquoCytochromec biogenesis in mitochondriardquo Mitochondrion vol 8 no 1 pp61ndash73 2008

[46] R E Feissner C L Richard-Fogal E R Frawley J A Lough-man KW Earley and R G Kranz ldquoRecombinant cytochromesc biogenesis systems I and II and analysis of haem deliverypathways in Escherichia colirdquo Molecular Microbiology vol 60no 3 pp 563ndash577 2006

[47] N P Cianciotto P Cornelis and C Baysse ldquoImpact of thebacterial type I cytochrome c maturation system on differentbiological processesrdquoMolecular Microbiology vol 56 no 6 pp1408ndash1415 2005

[48] E Arslan H Schulz R Zufferey P Kunzler and L Thony-Meyer ldquoOverproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in Escherichiacolirdquo Biochemical and Biophysical Research Communicationsvol 251 no 3 pp 744ndash747 1998

[49] C Sanders and H Lill ldquoExpression of prokaryotic and eukary-otic cytochromes c in Escherichia colirdquoBiochimica et BiophysicaActa vol 1459 no 1 pp 131ndash138 2000

[50] M Braun and L Thony-Meyer ldquoBiosynthesis of artificialmicroperoxidases by exploiting the secretion and cytochromec maturation apparatuses of Escherichia colirdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 35 pp 12830ndash12835 2004

[51] B Baert C Baysse S Matthijs and P Cornelis ldquoMultiplephenotypic alterations caused by a c-type cytochrome mat-uration ccmC gene mutation in Pseudomonas aeruginosardquoMicrobiology vol 154 no 1 pp 127ndash138 2008

[52] S Turkarslan C Sanders and F Daldal ldquoExtracytoplasmicprosthetic group ligation to apoproteins maturation of c-typecytochromesrdquo Molecular Microbiology vol 60 no 3 pp 537ndash541 2006

[53] P M Jones and A M George ldquoThe ABC transporter structureand mechanism perspectives on recent researchrdquo Cellular andMolecular Life Sciences vol 61 no 6 pp 682ndash699 2004

[54] O Christensen E M Harvat L Thony-Meyer S J Fergusonand J M Stevens ldquoLoss of ATP hydrolysis activity by CcmABresults in loss of c-type cytochrome synthesis and incompleteprocessing ofCcmErdquoTheFEBS Journal vol 274 no 9 pp 2322ndash2332 2007

[55] J-H Lee E M Harvat J M Stevens S J Ferguson and M HSaier Jr ldquoEvolutionary origins of members of a superfamily ofintegralmembrane cytochrome c biogenesis proteinsrdquoBiochim-ica et Biophysica Acta vol 1768 no 9 pp 2164ndash2181 2007

[56] D L Beckman D R Trawick and R G Kranz ldquoBacterialcytochromes c biogenesisrdquo Genes and Development vol 6 no2 pp 268ndash283 1992

[57] H Schulz R A Fabianek E C Pellicioli H Hennecke andL Thony-Meyer ldquoHeme transfer to the heme chaperone CcmEduring cytochrome c maturation requires the CcmC proteinwhich may function independently of the ABC-transporterCcmABrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 96 no 11 pp 6462ndash6467 1999

[58] Q Ren and L Thony-Meyer ldquoPhysical Interaction ofCcmC with Heme and the Heme Chaperone CcmE duringCytochrome cMaturationrdquo Journal of Biological Chemistry vol276 no 35 pp 32591ndash32596 2001

[59] C Richard-Fogal and R G Kranz ldquoThe CcmChemeCcmEcomplex in heme trafficking and cytochrome c biosynthesisrdquoJournal of Molecular Biology vol 401 no 3 pp 350ndash362 2010

[60] V K Viswanathan S Kurtz L L Pedersen et al ldquoThecytochrome C maturation locus of Legionella pneumophilapromotes iron assimilation and intracellular infection andcontains a strain-specific insertion sequence elementrdquo Infectionand Immunity vol 70 no 4 pp 1842ndash1852 2002

[61] U Ahuja and L Thony-Meyer ldquoCcmD is involved in complexformation between CcmC and the heme chaperone CcmE dur-ing cytochrome c maturationrdquo Journal of Biological Chemistryvol 280 no 1 pp 236ndash243 2005

[62] C L Richard-Fogal E R Frawley and R G Kranz ldquoTopologyand function of CcmD in cytochrome Cmaturationrdquo Journal ofBacteriology vol 190 no 10 pp 3489ndash3493 2008

[63] H Schulz H Hennecke and L Thony-Meyer ldquoPrototype ofa heme chaperone essential for cytochrome c maturationrdquoScience vol 281 no 5380 pp 1197ndash1200 1998

[64] F Arnesano L Banci P D Barker et al ldquoSolution structure andcharacterization of the heme chaperone CcmErdquo Biochemistryvol 41 no 46 pp 13587ndash13594 2002

[65] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[66] L Thony-Meyer ldquoA heme chaperone for cytochrome c biosyn-thesisrdquo Biochemistry vol 42 no 45 pp 13099ndash13105 2003

[67] E M Harvat C Redfield J M Stevens and S J FergusonldquoProbing the heme-binding site of the cytochrome cmaturationprotein CcmErdquoBiochemistry vol 48 no 8 pp 1820ndash1828 2009

[68] D Lee K Pervushin D Bischof M Braun and L Thony-Meyer ldquoUnusual heme-histidine bond in the active site of a

Scientifica 15

chaperonerdquo Journal of the American Chemical Society vol 127no 11 pp 3716ndash3717 2005

[69] A D Goddard J M Stevens F Rao et al ldquoc-Type cytochromebiogenesis can occur via a natural Ccm system lacking CcmHCcmG and the heme-binding histidine of CcmErdquo Journal ofBiological Chemistry vol 285 no 30 pp 22882ndash22889 2010

[70] J M Aramini K Hamilton P Rossi et al ldquoSolution NMRstructure backbone dynamics and heme-binding propertiesof a novel cytochrome c maturation protein CcmE fromDesulfovibrio vulgarisrdquo Biochemistry vol 51 no 18 pp 3705ndash3707 2012

[71] T Uchida J M Stevens O Daltrop et al ldquoThe interactionof covalently bound heme with the cytochrome c maturationprotein CcmErdquo Journal of Biological Chemistry vol 279 no 50pp 51981ndash51988 2004

[72] I Garcıa-Rubio M Braun I Gromov L Thony-Meyer andA Schweiger ldquoAxial coordination of heme in ferric CcmEchaperone characterized by EPR spectroscopyrdquo BiophysicalJournal vol 92 no 4 pp 1361ndash1373 2007

[73] L Thony-Meyer ldquoHaem-polypeptide interactions duringcytochrome c maturationrdquo Biochimica et Biophysica Acta vol1459 no 2-3 pp 316ndash324 2000

[74] J A Keightley D Sanders T R Todaro A Pastuszyn and J AFee ldquoCloning expression in Escherichia coli of the cytochromec552 gene from Thermus thermophilus HB8 Evidence forgenetic linkage to an ATP- binding cassette protein and initialcharacterization of the cycA gene productsrdquo Journal of Biologi-cal Chemistry vol 273 no 20 pp 12006ndash12016 1998

[75] E M Monika B S Goldman D L Beckman and R GKranz ldquoA thioreduction pathway tethered to the membranefor periplasmic cytochromes c biogenesis In vitro and in vivostudiesrdquo Journal of Molecular Biology vol 271 no 5 pp 679ndash692 1997

[76] M Throne-Holst L Thony-Meyer and L HederstedtldquoEscherichia coli ccm in-frame deletion mutants can produceperiplasmic cytochrome b but not cytochrome crdquo FEBS Lettersvol 410 no 2-3 pp 351ndash355 1997

[77] U Ahuja and L Thony-Meyer ldquoDynamic features of a hemedelivery system for cytochrome c maturationrdquo Journal of Bio-logical Chemistry vol 278 no 52 pp 52061ndash52070 2003

[78] J W A Allen P D Barker O Daltrop et al ldquoWhy isnrsquot ldquostan-dardrdquo heme good enough for c-type and di-type cytochromesrdquoDalton Transactions no 21 pp 3410ndash3418 2005

[79] K Inaba and K Ito ldquoStructure and mechanisms of the DsbB-DsbA disulfide bond generation machinerdquo Biochimica et Bio-physica Acta vol 1783 no 4 pp 520ndash529 2008

[80] H Kadokura F Katzen and J Beckwith ldquoProtein disulfidebond formation in prokaryotesrdquoAnnual Review of Biochemistryvol 72 pp 111ndash135 2003

[81] S R Shouldice B Heras P M Walden M Totsika M ASchembri and J L Martin ldquoStructure and function of DsbA akey bacterial oxidative folding catalystrdquoAntioxidants and RedoxSignaling vol 14 no 9 pp 1729ndash1760 2011

[82] R Metheringham L Griffiths H Crooke S Forsythe and JCole ldquoAn essential role for DsbA in cytochrome c synthesis andformate-dependent nitrite reduction by Escherichia coli K-12rdquoArchives of Microbiology vol 164 no 4 pp 301ndash307 1995

[83] Y Sambongi and S J Ferguson ldquoMutants of Escherichia colilacking disulphide oxidoreductases DsbA and DsbB cannotsynthesise an exogenous monohaem c-type cytochrome exceptin the presence of disulphide compoundsrdquo FEBS Letters vol398 no 2-3 pp 265ndash268 1996

[84] D AMavridou S J Ferguson and JM Stevens ldquoThe interplaybetween the disulfide bond formation pathway and cytochromec maturation in Escherichia colirdquo FEBS Letters vol 586 no 12pp 1702ndash1707 2012

[85] C U Stirnimann A Rozhkova U Grauschopf M G GrutterR Glockshuber and G Capitani ldquoStructural basis and kineticsof DsbD-dependent cytochrome c maturationrdquo Structure vol13 no 7 pp 985ndash993 2005

[86] N Ouyang Y-G Gao H-Y Hu and Z-X Xia ldquoCrystalstructures of E coli CcmGand itsmutants reveal key roles of theN-terminal120573-sheet and the fingerprint regionrdquoProteins vol 65no 4 pp 1021ndash1031 2006

[87] M A Edeling L W Guddat R A Fabianek et al ldquoCrys-tallization and preliminary diffraction studies of native andselenomethionine CcmG (CycY DsbE)rdquoActa CrystallographicaD vol 57 no 9 pp 1293ndash1295 2001

[88] R A Fabianek M Huber-Wunderlich R Glockshuber PKunzler H Hennecke and L Thony-Meyer ldquoCharacterizationof the Bradyrhizobium japonicum CycY protein a membrane-anchored periplasmic thioredoxin that may play a role as areductant in the biogenesis of c-type cytochromesrdquo Journal ofBiological Chemistry vol 272 no 7 pp 4467ndash4473 1997

[89] G B Kallis and A Holmgren ldquoDifferential reactivity of thefunctional sulfhydryl groups of cysteine-32 and cysteine-32present in the reduced form of thioredoxin from Escherichiacolirdquo Journal of Biological Chemistry vol 255 no 21 pp 10261ndash10265 1980

[90] P T Chivers andR T Raines ldquoGeneral acidbase catalysis in theactive site of Escherichia coli thioredoxinrdquoBiochemistry vol 36no 50 pp 15810ndash15816 1997

[91] X M Zheng J Hong H Y Li D H Lin and H Y HuldquoBiochemical properties and catalytic domain structure of theCcmH protein from Escherichia colirdquo Biochim Biophys Actavol 1824 no 12 pp 1394ndash1400 2012

[92] UAhujaA Rozhkova RGlockshuber LThony-Meyer andOEinsle ldquoHelix swapping leads to dimerization of the N-terminaldomain of the c-type cytochrome maturation protein CcmHfrom Escherichia colirdquo FEBS Letters vol 582 no 18 pp 2779ndash2786 2008

[93] R A Fabianek T Hofer and L Thony-Meyer ldquoCharacteriza-tion of the Escherichia coli CcmH protein reveals new insightsinto the redox pathway required for cytochrome c maturationrdquoArchives of Microbiology vol 171 no 2 pp 92ndash100 1999

[94] E Reid J Cole and D J Eaves ldquoThe Escherichia coli CcmGprotein fulfils a specific role in cytochrome c assemblyrdquo Bio-chemical Journal vol 355 no 1 pp 51ndash58 2001

[95] Q Ren U Ahuja and LThony-Meyer ldquoA bacterial cytochromec heme lyase CcmF forms a complex with the heme chaperoneCcmE and CcmH but not with apocytochrome crdquo Journal ofBiological Chemistry vol 277 no 10 pp 7657ndash7663 2002

[96] E H Meyer P Giege E Gelhaye et al ldquoAtCCMH an essentialcomponent of the c-type cytochrome maturation pathway inArabidopsis mitochondria interacts with apocytochrome crdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 44 pp 16113ndash16118 2005

[97] C Rios-Velazquez R Coller and T J Donohue ldquoFeatures ofRhodobacter sphaeroides CcmFHrdquo Journal of Bacteriology vol185 no 2 pp 422ndash431 2003

[98] C Sanders M Deshmukh D Astor R G Kranz and F DaldalldquoOverproduction of CcmG and CcmFHRc fully suppresses thec-type cytochrome biogenesis defect of Rhodobacter capsulatus

16 Scientifica

CcmI-null mutantsrdquo Journal of Bacteriology vol 187 no 12 pp4245ndash4256 2005

[99] A F Verissimo H Yang X Wu C Sanders and F DaldalldquoCcmI subunit of CcmFHI heme ligation complex functionsas an apocytochrome c chaperone during c-type cytochromematurationrdquo Journal of Biological Chemistry vol 286 no 47 pp40452ndash40463 2011

[100] K Bryson L J McGuffin R L Marsden J J Ward J SSodhi and D T Jones ldquoProtein structure prediction servers atUniversity College Londonrdquo Nucleic Acids Research vol 33 no2 pp W36ndashW38 2005

[101] D T Jones ldquoProtein secondary structure prediction basedon position-specific scoring matricesrdquo Journal of MolecularBiology vol 292 no 2 pp 195ndash202 1999

[102] C Sanders C Boulay and F Daldal ldquoMembrane-spanning andperiplasmic segments of CcmI have distinct functions duringcytochrome c biogenesis in Rhodobacter capsulatusrdquo Journal ofBacteriology vol 189 no 3 pp 789ndash800 2007

[103] D Han K Kim J Oh J Park and Y Kim ldquoTPR domainof NrfG mediates complex formation between heme lyaseand formate-dependent nitrite reductase in Escherichia coliO157H7rdquo Proteins vol 70 no 3 pp 900ndash914 2008

[104] D J Eaves J Grove W Staudenmann et al ldquoInvolvement ofproducts of the nrfEFG genes in the covalent attachment ofhaem c to a novel cysteine-lysine motif in the cytochrome c552nitrite reductase from Escherichia colirdquo Molecular Microbiol-ogy vol 28 no 1 pp 205ndash216 1998

[105] J Nilsson B Persson and G Von Heijne ldquoConsensus predic-tions of membrane protein topologyrdquo FEBS Letters vol 486 no3 pp 267ndash269 2000

[106] E di Silvio A di Matteo F Malatesta and C Travaglini-Allocatelli ldquoRecognition and binding of apocytochrome c toP aeruginosa CcmI a component of cytochrome c maturationmachineryrdquo Biochim Biophys Acta vol 1834 no 8 pp 1554ndash1561 2013

[107] N Zeytuni and R Zarivach ldquoStructural and functional dis-cussion of the tetra-trico-peptide repeat a protein interactionmodulerdquo Structure vol 20 no 3 pp 397ndash405 2012

[108] C Sanders S Turkarslan D-W Lee O Onder R G Kranz andF Daldal ldquoThe cytochrome c maturation components CcmFCcmH and CcmI form a membrane-integral multisubunitheme ligation complexrdquo Journal of Biological Chemistry vol283 no 44 pp 29715ndash29722 2008

[109] A F Verissimo M A Mohtar and F Daldal ldquoThe hemechaperone ApoCcmE forms a ternary complex with CcmI andapocytochrome crdquoThe Journal of Biological Chemistry vol 288no 9 pp 6272ndash6283 2013

[110] P D Barker J C Ferrer M Mylrajan et al ldquoTransmutation of aheme proteinrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 90 no 14 pp 6542ndash65461993

[111] J Simon and L Hederstedt ldquoComposition and function ofcytochrome c biogenesis system IIrdquoThe FEBS Journal vol 278no 22 pp 4179ndash4188 2011

[112] T R H M Kouwen and J M van Dijl ldquoInterchangeablemodules in bacterial thiol-disulfide exchange pathwaysrdquo Trendsin Microbiology vol 17 no 1 pp 6ndash12 2009

[113] A Crow A Lewin O Hecht et al ldquoCrystal structure andbiophysical properties of Bacillus subtilis BdbD An oxidizingthioldisulfide oxidoreductase containing a novel metal siterdquoJournal of Biological Chemistry vol 284 no 35 pp 23719ndash23733 2009

[114] M C Moller and L Hederstedt ldquoExtracytoplasmic processesimpaired by inactivation of trxA (thioredoxin gene) in Bacillussubtilisrdquo Journal of Bacteriology vol 190 no 13 pp 4660ndash46652008

[115] A Lewin A Crow A Oubrie and N E Le Brun ldquoMolecularbasis for specificity of the extracytoplasmic thioredoxin ResArdquoJournal of Biological Chemistry vol 281 no 46 pp 35467ndash35477 2006

[116] C L Colbert QWu P J A Erbel K H Gardner and J Deisen-hofer ldquoMechanism of substrate specificity in Bacillus subtilisResA a thioredoxin-like protein involved in cytochrome cmaturationrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 103 no 12 pp 4410ndash44152006

[117] UAhuja P Kjelgaard B L Schulz LThony-Meyer andLHed-erstedt ldquoHaem-delivery proteins in cytochrome c maturationsystem IIrdquoMolecular Microbiology vol 73 no 6 pp 1058ndash10712009

[118] M L D Page P P Hamel S T Gabilly et al ldquoA homologof prokaryotic thiol disulfide transporter CcdA is required forthe assembly of the cytochrome b6f complex in Arabidopsischloroplastsrdquo Journal of Biological Chemistry vol 279 no 31pp 32474ndash32482 2004

[119] A Rozhkova C U Stirnimann P Frei et al ldquoStructural basisand kinetics of inter- and intramolecular disulfide exchange inthe redox catalyst DsbDrdquoThe EMBO Journal vol 23 no 8 pp1709ndash1719 2004

[120] T Schiott M Throne-Holst and L Hederstedt ldquoBacillussubtilis CcdA-defective mutants are blocked in a late step ofcytochrome C biogenesisrdquo Journal of Bacteriology vol 179 no14 pp 4523ndash4529 1997

[121] R E Feissner C S Beckett J A Loughman and R G KranzldquoMutations in cytochrome assembly and periplasmic redoxpathways in Bordetella pertussisrdquo Journal of Bacteriology vol187 no 12 pp 3941ndash3949 2005

[122] L S ErlendssonMMoller and L Hederstedt ldquoBacillus subtilisStoA is a thiol-disulfide oxidoreductase important for sporecortex synthesisrdquo Journal of Bacteriology vol 186 no 18 pp6230ndash6238 2004

[123] L S Erlendsson R M Acheson L Hederstedt and N E LeBrun ldquoBacillus subtilis ResA is a thiol-disulfide oxidoreductaseinvolved in cytochrome c synthesisrdquo Journal of BiologicalChemistry vol 278 no 20 pp 17852ndash17858 2003

[124] L S Erlendsson and L Hederstedt ldquoMutations in the thiol-disulfide oxidoreductases BdbC and BdbD can suppresscytochrome c deficiency of CcdA-defective Bacillus subtiliscellsrdquo Journal of Bacteriology vol 184 no 5 pp 1423ndash1429 2002

[125] M E Dumont J F Ernst D M Hampsey and F ShermanldquoIdentification and sequence of the gene encoding cytochromec heme lyase in the yeast Saccharomyces cerevisiaerdquoThe EMBOJournal vol 6 no 1 pp 235ndash241 1987

[126] M E Dumont J F Ernst and F Sherman ldquoCoupling of hemeattachment to import of cytochrome c into yeast mitochondriaStudies with heme lyase-deficient mitochondria and alteredapocytochromes crdquo Journal of Biological Chemistry vol 263 no31 pp 15928ndash15937 1988

[127] B San Francisco E C Bretsnyder and R G Kranz ldquoHumanmitochondrial holocytochrome c synthasersquos hemebindingmat-uration determinants and complex formationwith cytochromecrdquo Proceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 9 pp E788ndashE797 2012

Scientifica 17

[128] L Schaefer A Ballabio and H Y Zoghbi ldquoCloning andcharacterization of a putative human holocytochrome c-typesynthetase gene (HCCS) isolated from the critical region formicrophthalmia with linear skin defects (MLS)rdquoGenomics vol34 no 2 pp 166ndash172 1996

[129] S K Prakash T A Cormier A E McCall et al ldquoLoss ofholocytochrome c-type synthetase causes the male lethality ofX-linked dominant microphthalmia with linear skin defects(MLS) syndromerdquo Human Molecular Genetics vol 11 no 25pp 3237ndash3248 2002

[130] I Wimplinger M Morleo G Rosenberger et al ldquoMutations ofthe mitochondrial holocytochrome c-type synthase in X-linkeddominant microphthalmia with linear skin defects syndromerdquoTheAmerican Journal of HumanGenetics vol 79 no 5 pp 878ndash889 2006

[131] S Kiryu-Seo KGamo T Tachibana K Tanaka andHKiyamaldquoUnique anti-apoptotic activity of EAAC1 in injured motorneuronsrdquoTheEMBO Journal vol 25 no 14 pp 3411ndash3421 2006

[132] R Lill R A Stuart M E Drygas F E Nargang and W Neu-pert ldquoImport of cytochrome c heme lyase into mitochondriaa novel pathway into the intermembrane spacerdquo The EMBOJournal vol 11 no 2 pp 449ndash456 1992

[133] H Steiner A Zollner A Haid W Neupert and R LillldquoBiogenesis of mitochondrial heme lyases in yeast Importand folding in the intermembrane spacerdquo Journal of BiologicalChemistry vol 270 no 39 pp 22842ndash22849 1995

[134] A Zollner G Rodel and A Haid ldquoMolecular cloning andcharacterization of the Saccharomyces cerevisiae CYT2 geneencoding cytochrome-c1-heme lyaserdquo European Journal of Bio-chemistry vol 207 no 3 pp 1093ndash1100 1992

[135] V Corvest D A Murrey D G Bernard D B Knaff B Guiardand P PHamel ldquoc-Type cytochrome assembly in Saccharomycescerevisiae a key residue for apocytochrome c1lyase interactionrdquoGenetics vol 186 no 2 pp 561ndash571 2010

[136] V Corvest D A Murrey M Hirasawa D B Knaff B Guiardand P P Hamel ldquoThe flavoprotein Cyc2p a mitochondrialcytochrome c assembly factor is a NAD(P)H-dependent haemreductaserdquo Molecular Microbiology vol 83 no 5 pp 968ndash9802012

[137] D G Bernard S Quevillon-Cheruel S Merchant B Guiardand P P Hamel ldquoCyc2p a membrane-bound flavopro-tein involved in the maturation of mitochondrial c-typecytochromesrdquo Journal of Biological Chemistry vol 280 no 48pp 39852ndash39859 2005

[138] J G Kleingardner and K L Bren ldquoComparing substratespecificity between cytochrome cmaturation and cytochrome cheme lyase systems for cytochrome c biogenesisrdquo Metallomicsvol 3 no 4 pp 396ndash403 2011

[139] C Travaglini-Allocatelli S Gianni VMorea A Tramontano TSoulimane and M Brunori ldquoExploring the cytochrome c fold-ing mechanism cytochrome c552 fromThermus thermophilusfolds through an on-pathway intermediaterdquo Journal of BiologicalChemistry vol 278 no 42 pp 41136ndash41140 2003

[140] L Zhang and L Guarente ldquoHeme binds to a short sequencethat serves a regulatory function in diverse proteinsrdquoTheEMBOJournal vol 14 no 2 pp 313ndash320 1995

[141] S Hira T Tomita T Matsui K Igarashi and M Ikeda-SaitoldquoBach1 a heme-dependent transcription factor reveals pres-ence of multiple heme binding sites with distinct coordinationstructurerdquo IUBMB Life vol 59 no 8-9 pp 542ndash551 2007

[142] T K Kuhl A Wiszligbrock N Goradia et al ldquoAnalysis of Fe(III)heme binding to cysteine-containing heme-regulatory motifs

in proteinsrdquo ACS Chemical Biology vol 8 no 8 pp 1785ndash17932013

[143] R L Moore J M Stevens and S J Ferguson ldquoMitochondrialcytochrome c synthase CP motifs are not necessary for hemeattachment to apocytochrome crdquo FEBS Letters vol 585 no 21pp 3415ndash3419 2011

[144] S Gianni M Brunori and C Travaglini-Allocatelli ldquoRefoldingkinetics of cytochrome c551 reveals a mechanistic differencebetween urea and guanidinerdquo Protein Science vol 10 no 8 pp1685ndash1688 2001

[145] S Gianni C Travaglini-Allocatelli F Cutruzzola M BrunoriM C R Shastry and H Roder ldquoParallel pathways incytochrome c551 foldingrdquo Journal of Molecular Biology vol 330no 5 pp 1145ndash1152 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Scientifica 13

[3] P Ascenzi R Santucci M Coletta and F PolticellildquoCytochromes reactivity of the ldquodark siderdquo of the hemerdquoBiophysical Chemistry vol 152 no 1ndash3 pp 21ndash27 2010

[4] S Yamada N D Bouley Ford G E Keller W C Ford HB Gray and J R Winkler ldquoSnapshots of a protein foldingintermediaterdquo Proceedings of the National Academy of Sciencesvol 110 no 5 pp 1606ndash1610 2013

[5] P Weinkam J Zimmermann F E Romesberg and P GWolynes ldquoThe folding energy landscape and free energy excita-tions of cytochrome crdquo Accounts of Chemical Research vol 43no 5 pp 652ndash660 2010

[6] M Yamanaka M Masanari and Y Sambongi ldquoConfermentof folding ability to a naturally unfolded apocytochrome cthrough introduction of hydrophobic amino acid residuesrdquoBiochemistry vol 50 no 12 pp 2313ndash2320 2011

[7] G W Moore and G W Pettigrew Cytochrome C EvolutionaryStructural and Physicochemical Aspects Springer Berlin Ger-many 1990

[8] I Bertini G Cavallaro and A Rosato ldquoCytochrome c occur-rence and functionsrdquo Chemical Reviews vol 106 no 1 pp 90ndash115 2006

[9] D A Pearce and F Sherman ldquoDegradation of cytochromeoxidase subunits in mutants of yeast lacking cytochrome c andsuppression of the degradation by mutation of yme1rdquo Journal ofBiological Chemistry vol 270 no 36 pp 20879ndash20882 1995

[10] G Silkstone S M Kapetanaki I Husu M H Vos and MT Wilson ldquoNitric oxide binding to the cardiolipin complex offerric cytochrome crdquo Biochemistry vol 51 no 34 pp 6760ndash6766 2012

[11] M Giorgio E Migliaccio F Orsini et al ldquoElectron transferbetween cytochrome c and p66Shc generates reactive oxygenspecies that trigger mitochondrial apoptosisrdquo Cell vol 122 no2 pp 221ndash233 2005

[12] S M Kilbride and J H Prehn ldquoCentral roles of apoptoticproteins in mitochondrial functionrdquo Oncogene vol 32 no 22pp 2703ndash2711 2013

[13] T Noll and G Noll ldquoStrategies for ldquowiringrdquo redox-activeproteins to electrodes and applications in biosensors biofuelcells and nanotechnologyrdquo Chemical Society Reviews vol 40no 7 pp 3564ndash3576 2011

[14] G Layer J Reichelt D Jahn and D W Heinz ldquoStructure andfunction of enzymes in heme biosynthesisrdquo Protein Science vol19 no 6 pp 1137ndash1161 2010

[15] S Severance and IHamza ldquoTrafficking of heme and porphyrinsin metazoardquo Chemical Reviews vol 109 no 10 pp 4596ndash46162009

[16] P Hamel V Corvest P Giege and G Bonnard ldquoBiochemi-cal requirements for the maturation of mitochondrial c-typecytochromesrdquo Biochimica et Biophysica Acta vol 1793 no 1 pp125ndash138 2009

[17] W R Fisher H Taniuchi and C B Anfinsen ldquoOn the role ofheme in the formation of the structure of cytochrome crdquo Journalof Biological Chemistry vol 248 no 9 pp 3188ndash3195 1973

[18] M Yamanaka H Mita Y Yamamoto and Y Sambongi ldquoHemeis not required for aquifex aeolicus cytochrome c555 polypep-tide foldingrdquoBioscience Biotechnology andBiochemistry vol 73no 9 pp 2022ndash2025 2009

[19] C Travaglini-Allocatelli S Gianni andMBrunori ldquoA commonfolding mechanism in the cytochrome c familyrdquo Trends inBiochemical Sciences vol 29 no 10 pp 535ndash541 2004

[20] A Borgia D Bonivento C Travaglini-Allocatelli A DiMatteoand M Brunori ldquoUnveiling a hidden folding intermediate in c-type cytochromes by protein engineeringrdquo Journal of BiologicalChemistry vol 281 no 14 pp 9331ndash9336 2006

[21] E Enggist L Thony-Meyer P Guntert and K PervushinldquoNMR structure of the heme chaperone CcmE reveals a novelfunctional motifrdquo Structure vol 10 no 11 pp 1551ndash1557 2002

[22] A di Matteo N Calosci S Gianni P Jemth M Brunori and CTravaglini-Allocatelli ldquoStructural and functional characteriza-tion of CcmG from pseudomonas aeruginosa a key componentof the bacterial cytochrome c maturation apparatusrdquo Proteinsvol 78 no 10 pp 2213ndash2221 2010

[23] A diMatteo S GianniM E Schinina et al ldquoA strategic proteinin cytochrome c maturation three-dimensional structure ofCcmH and binding to apocytochrome crdquo Journal of BiologicalChemistry vol 282 no 37 pp 27012ndash27019 2007

[24] A Crow R M Acheson N E Le Brun and A OubrieldquoStructural basis of redox-coupled protein substrate selectionby the cytochrome c biosynthesis protein ResArdquo Journal ofBiological Chemistry vol 279 no 22 pp 23654ndash23660 2004

[25] E di Silvio A di Matteo and C Travaglini-Allocatelli ldquoTheRedox pathway of Pseudomonas aeruginosa cytochrome cbiogenesisrdquo Journal of Proteins and Proteomics vol 3 no 1 pp1ndash7 2012

[26] LThony-Meyer andP Kunzler ldquoTranslocation to the periplasmand signal sequence cleavage of preapocytochrome c depend onsec and lep but not on the ccmgene productsrdquoEuropean Journalof Biochemistry vol 246 no 3 pp 794ndash799 1997

[27] S J Facey and A Kuhn ldquoBiogenesis of bacterial inner-membrane proteinsrdquo Cellular and Molecular Life Sciences vol67 no 14 pp 2343ndash2362 2010

[28] K Diekert A I de Kroon U Ahting et al ldquoApocytochromec requires the TOM complex for translocation across themitochondrial outer membranerdquo The EMBO Journal vol 20no 20 pp 5626ndash5635 2001

[29] N Wiedemann V Kozjak T Prinz et al ldquoBiogenesis of yeastmitochondrial cytochrome c a unique relationship to the TOMmachineryrdquo Journal ofMolecular Biology vol 327 no 2 pp 465ndash474 2003

[30] F-U Hartl N Pfanner and W Neupert ldquoTranslocation inter-mediates on the import pathway of proteins intomitochondriardquoBiochemical Society Transactions vol 15 no 1 pp 95ndash97 1987

[31] B S Glick A Brandt K Cunningham SMuller R L Hallbergand G Schatz ldquoCytochromes c1 and b2 are sorted to theintermembrane space of yeast mitochondria by a stop-transfermechanismrdquo Cell vol 69 no 5 pp 809ndash822 1992

[32] G Howe and S Merchant ldquoRole of heme in the biosynthesis ofcytochrome c6rdquo Journal of Biological Chemistry vol 269 no 8pp 5824ndash5832 1994

[33] S S Nakamoto P Hamel and S Merchant ldquoAssembly ofchloroplast cytochromes b and crdquo Biochimie vol 82 no 6-7 pp603ndash614 2000

[34] C Sanders S Turkarslan D-W Lee and F DaldalldquoCytochrome c biogenesis the Ccm systemrdquo Trends inMicrobiology vol 18 no 6 pp 266ndash274 2010

[35] J M Stevens D A Mavridou R Hamer P Kritsiligkou A DGoddard and S J Ferguson ldquoCytochrome c biogenesis systemIrdquoThe FEBS Journal vol 278 no 22 pp 4170ndash4178 2011

[36] R G Kranz C Richard-Fogal J-S Taylor and E R FrawleyldquoCytochrome c biogenesis mechanisms for covalent modifica-tions and trafficking of heme and for heme-iron redox controlrdquo

14 Scientifica

Microbiology and Molecular Biology Reviews vol 73 no 3 pp510ndash528 2009

[37] J W A Allen ldquoCytochrome c biogenesis in mitochondriamdashsystems III and VrdquoThe FEBS Journal vol 278 no 22 pp 4198ndash4216 2011

[38] D A Mavridou S J Ferguson and J M Stevens ldquoCytochromec assemblyrdquo IUBMB Life vol 65 no 3 pp 209ndash216 2013

[39] I Bertini G Cavallaro and A Rosato ldquoEvolution ofmitochondrial-type cytochrome c domains and of theprotein machinery for their assemblyrdquo Journal of InorganicBiochemistry vol 101 no 11-12 pp 1798ndash1811 2007

[40] B S Goldman and R G Kranz ldquoEvolution and horizontaltransfer of an entire biosynthetic pathway for cytochromec biogenesis helicobacter deinococcus archae and morerdquoMolecular Microbiology vol 27 no 4 pp 871ndash873 1998

[41] C L Richard-Fogal E R Frawley E R Bonner H Zhu BSan Francisco and R G Kranz ldquoA conserved haem redoxand trafficking pathway for cofactor attachmentrdquo The EMBOJournal vol 28 no 16 pp 2349ndash2359 2009

[42] L Thony-Meyer F Fischer P Kunzler D Ritz and H Hen-necke ldquoEscherichia coli genes required for cytochrome cmaturationrdquo Journal of Bacteriology vol 177 no 15 pp 4321ndash4326 1995

[43] C S Beckett J A Loughman K A Karberg G M DonatoW E Goldman and R G Kranz ldquoFour genes are required forthe system II cytochrome c biogenesis pathway in Bordetellapertussis a unique bacterial modelrdquo Molecular Microbiologyvol 38 no 3 pp 465ndash481 2000

[44] N E Le Brun J Bengtsson and L Hederstedt ldquoGenes requiredfor cytochrome c synthesis in Bacillus subtilisrdquo MolecularMicrobiology vol 36 no 3 pp 638ndash650 2000

[45] P Giege J M Grienenberger and G Bonnard ldquoCytochromec biogenesis in mitochondriardquo Mitochondrion vol 8 no 1 pp61ndash73 2008

[46] R E Feissner C L Richard-Fogal E R Frawley J A Lough-man KW Earley and R G Kranz ldquoRecombinant cytochromesc biogenesis systems I and II and analysis of haem deliverypathways in Escherichia colirdquo Molecular Microbiology vol 60no 3 pp 563ndash577 2006

[47] N P Cianciotto P Cornelis and C Baysse ldquoImpact of thebacterial type I cytochrome c maturation system on differentbiological processesrdquoMolecular Microbiology vol 56 no 6 pp1408ndash1415 2005

[48] E Arslan H Schulz R Zufferey P Kunzler and L Thony-Meyer ldquoOverproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in Escherichiacolirdquo Biochemical and Biophysical Research Communicationsvol 251 no 3 pp 744ndash747 1998

[49] C Sanders and H Lill ldquoExpression of prokaryotic and eukary-otic cytochromes c in Escherichia colirdquoBiochimica et BiophysicaActa vol 1459 no 1 pp 131ndash138 2000

[50] M Braun and L Thony-Meyer ldquoBiosynthesis of artificialmicroperoxidases by exploiting the secretion and cytochromec maturation apparatuses of Escherichia colirdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 35 pp 12830ndash12835 2004

[51] B Baert C Baysse S Matthijs and P Cornelis ldquoMultiplephenotypic alterations caused by a c-type cytochrome mat-uration ccmC gene mutation in Pseudomonas aeruginosardquoMicrobiology vol 154 no 1 pp 127ndash138 2008

[52] S Turkarslan C Sanders and F Daldal ldquoExtracytoplasmicprosthetic group ligation to apoproteins maturation of c-typecytochromesrdquo Molecular Microbiology vol 60 no 3 pp 537ndash541 2006

[53] P M Jones and A M George ldquoThe ABC transporter structureand mechanism perspectives on recent researchrdquo Cellular andMolecular Life Sciences vol 61 no 6 pp 682ndash699 2004

[54] O Christensen E M Harvat L Thony-Meyer S J Fergusonand J M Stevens ldquoLoss of ATP hydrolysis activity by CcmABresults in loss of c-type cytochrome synthesis and incompleteprocessing ofCcmErdquoTheFEBS Journal vol 274 no 9 pp 2322ndash2332 2007

[55] J-H Lee E M Harvat J M Stevens S J Ferguson and M HSaier Jr ldquoEvolutionary origins of members of a superfamily ofintegralmembrane cytochrome c biogenesis proteinsrdquoBiochim-ica et Biophysica Acta vol 1768 no 9 pp 2164ndash2181 2007

[56] D L Beckman D R Trawick and R G Kranz ldquoBacterialcytochromes c biogenesisrdquo Genes and Development vol 6 no2 pp 268ndash283 1992

[57] H Schulz R A Fabianek E C Pellicioli H Hennecke andL Thony-Meyer ldquoHeme transfer to the heme chaperone CcmEduring cytochrome c maturation requires the CcmC proteinwhich may function independently of the ABC-transporterCcmABrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 96 no 11 pp 6462ndash6467 1999

[58] Q Ren and L Thony-Meyer ldquoPhysical Interaction ofCcmC with Heme and the Heme Chaperone CcmE duringCytochrome cMaturationrdquo Journal of Biological Chemistry vol276 no 35 pp 32591ndash32596 2001

[59] C Richard-Fogal and R G Kranz ldquoThe CcmChemeCcmEcomplex in heme trafficking and cytochrome c biosynthesisrdquoJournal of Molecular Biology vol 401 no 3 pp 350ndash362 2010

[60] V K Viswanathan S Kurtz L L Pedersen et al ldquoThecytochrome C maturation locus of Legionella pneumophilapromotes iron assimilation and intracellular infection andcontains a strain-specific insertion sequence elementrdquo Infectionand Immunity vol 70 no 4 pp 1842ndash1852 2002

[61] U Ahuja and L Thony-Meyer ldquoCcmD is involved in complexformation between CcmC and the heme chaperone CcmE dur-ing cytochrome c maturationrdquo Journal of Biological Chemistryvol 280 no 1 pp 236ndash243 2005

[62] C L Richard-Fogal E R Frawley and R G Kranz ldquoTopologyand function of CcmD in cytochrome Cmaturationrdquo Journal ofBacteriology vol 190 no 10 pp 3489ndash3493 2008

[63] H Schulz H Hennecke and L Thony-Meyer ldquoPrototype ofa heme chaperone essential for cytochrome c maturationrdquoScience vol 281 no 5380 pp 1197ndash1200 1998

[64] F Arnesano L Banci P D Barker et al ldquoSolution structure andcharacterization of the heme chaperone CcmErdquo Biochemistryvol 41 no 46 pp 13587ndash13594 2002

[65] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[66] L Thony-Meyer ldquoA heme chaperone for cytochrome c biosyn-thesisrdquo Biochemistry vol 42 no 45 pp 13099ndash13105 2003

[67] E M Harvat C Redfield J M Stevens and S J FergusonldquoProbing the heme-binding site of the cytochrome cmaturationprotein CcmErdquoBiochemistry vol 48 no 8 pp 1820ndash1828 2009

[68] D Lee K Pervushin D Bischof M Braun and L Thony-Meyer ldquoUnusual heme-histidine bond in the active site of a

Scientifica 15

chaperonerdquo Journal of the American Chemical Society vol 127no 11 pp 3716ndash3717 2005

[69] A D Goddard J M Stevens F Rao et al ldquoc-Type cytochromebiogenesis can occur via a natural Ccm system lacking CcmHCcmG and the heme-binding histidine of CcmErdquo Journal ofBiological Chemistry vol 285 no 30 pp 22882ndash22889 2010

[70] J M Aramini K Hamilton P Rossi et al ldquoSolution NMRstructure backbone dynamics and heme-binding propertiesof a novel cytochrome c maturation protein CcmE fromDesulfovibrio vulgarisrdquo Biochemistry vol 51 no 18 pp 3705ndash3707 2012

[71] T Uchida J M Stevens O Daltrop et al ldquoThe interactionof covalently bound heme with the cytochrome c maturationprotein CcmErdquo Journal of Biological Chemistry vol 279 no 50pp 51981ndash51988 2004

[72] I Garcıa-Rubio M Braun I Gromov L Thony-Meyer andA Schweiger ldquoAxial coordination of heme in ferric CcmEchaperone characterized by EPR spectroscopyrdquo BiophysicalJournal vol 92 no 4 pp 1361ndash1373 2007

[73] L Thony-Meyer ldquoHaem-polypeptide interactions duringcytochrome c maturationrdquo Biochimica et Biophysica Acta vol1459 no 2-3 pp 316ndash324 2000

[74] J A Keightley D Sanders T R Todaro A Pastuszyn and J AFee ldquoCloning expression in Escherichia coli of the cytochromec552 gene from Thermus thermophilus HB8 Evidence forgenetic linkage to an ATP- binding cassette protein and initialcharacterization of the cycA gene productsrdquo Journal of Biologi-cal Chemistry vol 273 no 20 pp 12006ndash12016 1998

[75] E M Monika B S Goldman D L Beckman and R GKranz ldquoA thioreduction pathway tethered to the membranefor periplasmic cytochromes c biogenesis In vitro and in vivostudiesrdquo Journal of Molecular Biology vol 271 no 5 pp 679ndash692 1997

[76] M Throne-Holst L Thony-Meyer and L HederstedtldquoEscherichia coli ccm in-frame deletion mutants can produceperiplasmic cytochrome b but not cytochrome crdquo FEBS Lettersvol 410 no 2-3 pp 351ndash355 1997

[77] U Ahuja and L Thony-Meyer ldquoDynamic features of a hemedelivery system for cytochrome c maturationrdquo Journal of Bio-logical Chemistry vol 278 no 52 pp 52061ndash52070 2003

[78] J W A Allen P D Barker O Daltrop et al ldquoWhy isnrsquot ldquostan-dardrdquo heme good enough for c-type and di-type cytochromesrdquoDalton Transactions no 21 pp 3410ndash3418 2005

[79] K Inaba and K Ito ldquoStructure and mechanisms of the DsbB-DsbA disulfide bond generation machinerdquo Biochimica et Bio-physica Acta vol 1783 no 4 pp 520ndash529 2008

[80] H Kadokura F Katzen and J Beckwith ldquoProtein disulfidebond formation in prokaryotesrdquoAnnual Review of Biochemistryvol 72 pp 111ndash135 2003

[81] S R Shouldice B Heras P M Walden M Totsika M ASchembri and J L Martin ldquoStructure and function of DsbA akey bacterial oxidative folding catalystrdquoAntioxidants and RedoxSignaling vol 14 no 9 pp 1729ndash1760 2011

[82] R Metheringham L Griffiths H Crooke S Forsythe and JCole ldquoAn essential role for DsbA in cytochrome c synthesis andformate-dependent nitrite reduction by Escherichia coli K-12rdquoArchives of Microbiology vol 164 no 4 pp 301ndash307 1995

[83] Y Sambongi and S J Ferguson ldquoMutants of Escherichia colilacking disulphide oxidoreductases DsbA and DsbB cannotsynthesise an exogenous monohaem c-type cytochrome exceptin the presence of disulphide compoundsrdquo FEBS Letters vol398 no 2-3 pp 265ndash268 1996

[84] D AMavridou S J Ferguson and JM Stevens ldquoThe interplaybetween the disulfide bond formation pathway and cytochromec maturation in Escherichia colirdquo FEBS Letters vol 586 no 12pp 1702ndash1707 2012

[85] C U Stirnimann A Rozhkova U Grauschopf M G GrutterR Glockshuber and G Capitani ldquoStructural basis and kineticsof DsbD-dependent cytochrome c maturationrdquo Structure vol13 no 7 pp 985ndash993 2005

[86] N Ouyang Y-G Gao H-Y Hu and Z-X Xia ldquoCrystalstructures of E coli CcmGand itsmutants reveal key roles of theN-terminal120573-sheet and the fingerprint regionrdquoProteins vol 65no 4 pp 1021ndash1031 2006

[87] M A Edeling L W Guddat R A Fabianek et al ldquoCrys-tallization and preliminary diffraction studies of native andselenomethionine CcmG (CycY DsbE)rdquoActa CrystallographicaD vol 57 no 9 pp 1293ndash1295 2001

[88] R A Fabianek M Huber-Wunderlich R Glockshuber PKunzler H Hennecke and L Thony-Meyer ldquoCharacterizationof the Bradyrhizobium japonicum CycY protein a membrane-anchored periplasmic thioredoxin that may play a role as areductant in the biogenesis of c-type cytochromesrdquo Journal ofBiological Chemistry vol 272 no 7 pp 4467ndash4473 1997

[89] G B Kallis and A Holmgren ldquoDifferential reactivity of thefunctional sulfhydryl groups of cysteine-32 and cysteine-32present in the reduced form of thioredoxin from Escherichiacolirdquo Journal of Biological Chemistry vol 255 no 21 pp 10261ndash10265 1980

[90] P T Chivers andR T Raines ldquoGeneral acidbase catalysis in theactive site of Escherichia coli thioredoxinrdquoBiochemistry vol 36no 50 pp 15810ndash15816 1997

[91] X M Zheng J Hong H Y Li D H Lin and H Y HuldquoBiochemical properties and catalytic domain structure of theCcmH protein from Escherichia colirdquo Biochim Biophys Actavol 1824 no 12 pp 1394ndash1400 2012

[92] UAhujaA Rozhkova RGlockshuber LThony-Meyer andOEinsle ldquoHelix swapping leads to dimerization of the N-terminaldomain of the c-type cytochrome maturation protein CcmHfrom Escherichia colirdquo FEBS Letters vol 582 no 18 pp 2779ndash2786 2008

[93] R A Fabianek T Hofer and L Thony-Meyer ldquoCharacteriza-tion of the Escherichia coli CcmH protein reveals new insightsinto the redox pathway required for cytochrome c maturationrdquoArchives of Microbiology vol 171 no 2 pp 92ndash100 1999

[94] E Reid J Cole and D J Eaves ldquoThe Escherichia coli CcmGprotein fulfils a specific role in cytochrome c assemblyrdquo Bio-chemical Journal vol 355 no 1 pp 51ndash58 2001

[95] Q Ren U Ahuja and LThony-Meyer ldquoA bacterial cytochromec heme lyase CcmF forms a complex with the heme chaperoneCcmE and CcmH but not with apocytochrome crdquo Journal ofBiological Chemistry vol 277 no 10 pp 7657ndash7663 2002

[96] E H Meyer P Giege E Gelhaye et al ldquoAtCCMH an essentialcomponent of the c-type cytochrome maturation pathway inArabidopsis mitochondria interacts with apocytochrome crdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 44 pp 16113ndash16118 2005

[97] C Rios-Velazquez R Coller and T J Donohue ldquoFeatures ofRhodobacter sphaeroides CcmFHrdquo Journal of Bacteriology vol185 no 2 pp 422ndash431 2003

[98] C Sanders M Deshmukh D Astor R G Kranz and F DaldalldquoOverproduction of CcmG and CcmFHRc fully suppresses thec-type cytochrome biogenesis defect of Rhodobacter capsulatus

16 Scientifica

CcmI-null mutantsrdquo Journal of Bacteriology vol 187 no 12 pp4245ndash4256 2005

[99] A F Verissimo H Yang X Wu C Sanders and F DaldalldquoCcmI subunit of CcmFHI heme ligation complex functionsas an apocytochrome c chaperone during c-type cytochromematurationrdquo Journal of Biological Chemistry vol 286 no 47 pp40452ndash40463 2011

[100] K Bryson L J McGuffin R L Marsden J J Ward J SSodhi and D T Jones ldquoProtein structure prediction servers atUniversity College Londonrdquo Nucleic Acids Research vol 33 no2 pp W36ndashW38 2005

[101] D T Jones ldquoProtein secondary structure prediction basedon position-specific scoring matricesrdquo Journal of MolecularBiology vol 292 no 2 pp 195ndash202 1999

[102] C Sanders C Boulay and F Daldal ldquoMembrane-spanning andperiplasmic segments of CcmI have distinct functions duringcytochrome c biogenesis in Rhodobacter capsulatusrdquo Journal ofBacteriology vol 189 no 3 pp 789ndash800 2007

[103] D Han K Kim J Oh J Park and Y Kim ldquoTPR domainof NrfG mediates complex formation between heme lyaseand formate-dependent nitrite reductase in Escherichia coliO157H7rdquo Proteins vol 70 no 3 pp 900ndash914 2008

[104] D J Eaves J Grove W Staudenmann et al ldquoInvolvement ofproducts of the nrfEFG genes in the covalent attachment ofhaem c to a novel cysteine-lysine motif in the cytochrome c552nitrite reductase from Escherichia colirdquo Molecular Microbiol-ogy vol 28 no 1 pp 205ndash216 1998

[105] J Nilsson B Persson and G Von Heijne ldquoConsensus predic-tions of membrane protein topologyrdquo FEBS Letters vol 486 no3 pp 267ndash269 2000

[106] E di Silvio A di Matteo F Malatesta and C Travaglini-Allocatelli ldquoRecognition and binding of apocytochrome c toP aeruginosa CcmI a component of cytochrome c maturationmachineryrdquo Biochim Biophys Acta vol 1834 no 8 pp 1554ndash1561 2013

[107] N Zeytuni and R Zarivach ldquoStructural and functional dis-cussion of the tetra-trico-peptide repeat a protein interactionmodulerdquo Structure vol 20 no 3 pp 397ndash405 2012

[108] C Sanders S Turkarslan D-W Lee O Onder R G Kranz andF Daldal ldquoThe cytochrome c maturation components CcmFCcmH and CcmI form a membrane-integral multisubunitheme ligation complexrdquo Journal of Biological Chemistry vol283 no 44 pp 29715ndash29722 2008

[109] A F Verissimo M A Mohtar and F Daldal ldquoThe hemechaperone ApoCcmE forms a ternary complex with CcmI andapocytochrome crdquoThe Journal of Biological Chemistry vol 288no 9 pp 6272ndash6283 2013

[110] P D Barker J C Ferrer M Mylrajan et al ldquoTransmutation of aheme proteinrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 90 no 14 pp 6542ndash65461993

[111] J Simon and L Hederstedt ldquoComposition and function ofcytochrome c biogenesis system IIrdquoThe FEBS Journal vol 278no 22 pp 4179ndash4188 2011

[112] T R H M Kouwen and J M van Dijl ldquoInterchangeablemodules in bacterial thiol-disulfide exchange pathwaysrdquo Trendsin Microbiology vol 17 no 1 pp 6ndash12 2009

[113] A Crow A Lewin O Hecht et al ldquoCrystal structure andbiophysical properties of Bacillus subtilis BdbD An oxidizingthioldisulfide oxidoreductase containing a novel metal siterdquoJournal of Biological Chemistry vol 284 no 35 pp 23719ndash23733 2009

[114] M C Moller and L Hederstedt ldquoExtracytoplasmic processesimpaired by inactivation of trxA (thioredoxin gene) in Bacillussubtilisrdquo Journal of Bacteriology vol 190 no 13 pp 4660ndash46652008

[115] A Lewin A Crow A Oubrie and N E Le Brun ldquoMolecularbasis for specificity of the extracytoplasmic thioredoxin ResArdquoJournal of Biological Chemistry vol 281 no 46 pp 35467ndash35477 2006

[116] C L Colbert QWu P J A Erbel K H Gardner and J Deisen-hofer ldquoMechanism of substrate specificity in Bacillus subtilisResA a thioredoxin-like protein involved in cytochrome cmaturationrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 103 no 12 pp 4410ndash44152006

[117] UAhuja P Kjelgaard B L Schulz LThony-Meyer andLHed-erstedt ldquoHaem-delivery proteins in cytochrome c maturationsystem IIrdquoMolecular Microbiology vol 73 no 6 pp 1058ndash10712009

[118] M L D Page P P Hamel S T Gabilly et al ldquoA homologof prokaryotic thiol disulfide transporter CcdA is required forthe assembly of the cytochrome b6f complex in Arabidopsischloroplastsrdquo Journal of Biological Chemistry vol 279 no 31pp 32474ndash32482 2004

[119] A Rozhkova C U Stirnimann P Frei et al ldquoStructural basisand kinetics of inter- and intramolecular disulfide exchange inthe redox catalyst DsbDrdquoThe EMBO Journal vol 23 no 8 pp1709ndash1719 2004

[120] T Schiott M Throne-Holst and L Hederstedt ldquoBacillussubtilis CcdA-defective mutants are blocked in a late step ofcytochrome C biogenesisrdquo Journal of Bacteriology vol 179 no14 pp 4523ndash4529 1997

[121] R E Feissner C S Beckett J A Loughman and R G KranzldquoMutations in cytochrome assembly and periplasmic redoxpathways in Bordetella pertussisrdquo Journal of Bacteriology vol187 no 12 pp 3941ndash3949 2005

[122] L S ErlendssonMMoller and L Hederstedt ldquoBacillus subtilisStoA is a thiol-disulfide oxidoreductase important for sporecortex synthesisrdquo Journal of Bacteriology vol 186 no 18 pp6230ndash6238 2004

[123] L S Erlendsson R M Acheson L Hederstedt and N E LeBrun ldquoBacillus subtilis ResA is a thiol-disulfide oxidoreductaseinvolved in cytochrome c synthesisrdquo Journal of BiologicalChemistry vol 278 no 20 pp 17852ndash17858 2003

[124] L S Erlendsson and L Hederstedt ldquoMutations in the thiol-disulfide oxidoreductases BdbC and BdbD can suppresscytochrome c deficiency of CcdA-defective Bacillus subtiliscellsrdquo Journal of Bacteriology vol 184 no 5 pp 1423ndash1429 2002

[125] M E Dumont J F Ernst D M Hampsey and F ShermanldquoIdentification and sequence of the gene encoding cytochromec heme lyase in the yeast Saccharomyces cerevisiaerdquoThe EMBOJournal vol 6 no 1 pp 235ndash241 1987

[126] M E Dumont J F Ernst and F Sherman ldquoCoupling of hemeattachment to import of cytochrome c into yeast mitochondriaStudies with heme lyase-deficient mitochondria and alteredapocytochromes crdquo Journal of Biological Chemistry vol 263 no31 pp 15928ndash15937 1988

[127] B San Francisco E C Bretsnyder and R G Kranz ldquoHumanmitochondrial holocytochrome c synthasersquos hemebindingmat-uration determinants and complex formationwith cytochromecrdquo Proceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 9 pp E788ndashE797 2012

Scientifica 17

[128] L Schaefer A Ballabio and H Y Zoghbi ldquoCloning andcharacterization of a putative human holocytochrome c-typesynthetase gene (HCCS) isolated from the critical region formicrophthalmia with linear skin defects (MLS)rdquoGenomics vol34 no 2 pp 166ndash172 1996

[129] S K Prakash T A Cormier A E McCall et al ldquoLoss ofholocytochrome c-type synthetase causes the male lethality ofX-linked dominant microphthalmia with linear skin defects(MLS) syndromerdquo Human Molecular Genetics vol 11 no 25pp 3237ndash3248 2002

[130] I Wimplinger M Morleo G Rosenberger et al ldquoMutations ofthe mitochondrial holocytochrome c-type synthase in X-linkeddominant microphthalmia with linear skin defects syndromerdquoTheAmerican Journal of HumanGenetics vol 79 no 5 pp 878ndash889 2006

[131] S Kiryu-Seo KGamo T Tachibana K Tanaka andHKiyamaldquoUnique anti-apoptotic activity of EAAC1 in injured motorneuronsrdquoTheEMBO Journal vol 25 no 14 pp 3411ndash3421 2006

[132] R Lill R A Stuart M E Drygas F E Nargang and W Neu-pert ldquoImport of cytochrome c heme lyase into mitochondriaa novel pathway into the intermembrane spacerdquo The EMBOJournal vol 11 no 2 pp 449ndash456 1992

[133] H Steiner A Zollner A Haid W Neupert and R LillldquoBiogenesis of mitochondrial heme lyases in yeast Importand folding in the intermembrane spacerdquo Journal of BiologicalChemistry vol 270 no 39 pp 22842ndash22849 1995

[134] A Zollner G Rodel and A Haid ldquoMolecular cloning andcharacterization of the Saccharomyces cerevisiae CYT2 geneencoding cytochrome-c1-heme lyaserdquo European Journal of Bio-chemistry vol 207 no 3 pp 1093ndash1100 1992

[135] V Corvest D A Murrey D G Bernard D B Knaff B Guiardand P PHamel ldquoc-Type cytochrome assembly in Saccharomycescerevisiae a key residue for apocytochrome c1lyase interactionrdquoGenetics vol 186 no 2 pp 561ndash571 2010

[136] V Corvest D A Murrey M Hirasawa D B Knaff B Guiardand P P Hamel ldquoThe flavoprotein Cyc2p a mitochondrialcytochrome c assembly factor is a NAD(P)H-dependent haemreductaserdquo Molecular Microbiology vol 83 no 5 pp 968ndash9802012

[137] D G Bernard S Quevillon-Cheruel S Merchant B Guiardand P P Hamel ldquoCyc2p a membrane-bound flavopro-tein involved in the maturation of mitochondrial c-typecytochromesrdquo Journal of Biological Chemistry vol 280 no 48pp 39852ndash39859 2005

[138] J G Kleingardner and K L Bren ldquoComparing substratespecificity between cytochrome cmaturation and cytochrome cheme lyase systems for cytochrome c biogenesisrdquo Metallomicsvol 3 no 4 pp 396ndash403 2011

[139] C Travaglini-Allocatelli S Gianni VMorea A Tramontano TSoulimane and M Brunori ldquoExploring the cytochrome c fold-ing mechanism cytochrome c552 fromThermus thermophilusfolds through an on-pathway intermediaterdquo Journal of BiologicalChemistry vol 278 no 42 pp 41136ndash41140 2003

[140] L Zhang and L Guarente ldquoHeme binds to a short sequencethat serves a regulatory function in diverse proteinsrdquoTheEMBOJournal vol 14 no 2 pp 313ndash320 1995

[141] S Hira T Tomita T Matsui K Igarashi and M Ikeda-SaitoldquoBach1 a heme-dependent transcription factor reveals pres-ence of multiple heme binding sites with distinct coordinationstructurerdquo IUBMB Life vol 59 no 8-9 pp 542ndash551 2007

[142] T K Kuhl A Wiszligbrock N Goradia et al ldquoAnalysis of Fe(III)heme binding to cysteine-containing heme-regulatory motifs

in proteinsrdquo ACS Chemical Biology vol 8 no 8 pp 1785ndash17932013

[143] R L Moore J M Stevens and S J Ferguson ldquoMitochondrialcytochrome c synthase CP motifs are not necessary for hemeattachment to apocytochrome crdquo FEBS Letters vol 585 no 21pp 3415ndash3419 2011

[144] S Gianni M Brunori and C Travaglini-Allocatelli ldquoRefoldingkinetics of cytochrome c551 reveals a mechanistic differencebetween urea and guanidinerdquo Protein Science vol 10 no 8 pp1685ndash1688 2001

[145] S Gianni C Travaglini-Allocatelli F Cutruzzola M BrunoriM C R Shastry and H Roder ldquoParallel pathways incytochrome c551 foldingrdquo Journal of Molecular Biology vol 330no 5 pp 1145ndash1152 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

14 Scientifica

Microbiology and Molecular Biology Reviews vol 73 no 3 pp510ndash528 2009

[37] J W A Allen ldquoCytochrome c biogenesis in mitochondriamdashsystems III and VrdquoThe FEBS Journal vol 278 no 22 pp 4198ndash4216 2011

[38] D A Mavridou S J Ferguson and J M Stevens ldquoCytochromec assemblyrdquo IUBMB Life vol 65 no 3 pp 209ndash216 2013

[39] I Bertini G Cavallaro and A Rosato ldquoEvolution ofmitochondrial-type cytochrome c domains and of theprotein machinery for their assemblyrdquo Journal of InorganicBiochemistry vol 101 no 11-12 pp 1798ndash1811 2007

[40] B S Goldman and R G Kranz ldquoEvolution and horizontaltransfer of an entire biosynthetic pathway for cytochromec biogenesis helicobacter deinococcus archae and morerdquoMolecular Microbiology vol 27 no 4 pp 871ndash873 1998

[41] C L Richard-Fogal E R Frawley E R Bonner H Zhu BSan Francisco and R G Kranz ldquoA conserved haem redoxand trafficking pathway for cofactor attachmentrdquo The EMBOJournal vol 28 no 16 pp 2349ndash2359 2009

[42] L Thony-Meyer F Fischer P Kunzler D Ritz and H Hen-necke ldquoEscherichia coli genes required for cytochrome cmaturationrdquo Journal of Bacteriology vol 177 no 15 pp 4321ndash4326 1995

[43] C S Beckett J A Loughman K A Karberg G M DonatoW E Goldman and R G Kranz ldquoFour genes are required forthe system II cytochrome c biogenesis pathway in Bordetellapertussis a unique bacterial modelrdquo Molecular Microbiologyvol 38 no 3 pp 465ndash481 2000

[44] N E Le Brun J Bengtsson and L Hederstedt ldquoGenes requiredfor cytochrome c synthesis in Bacillus subtilisrdquo MolecularMicrobiology vol 36 no 3 pp 638ndash650 2000

[45] P Giege J M Grienenberger and G Bonnard ldquoCytochromec biogenesis in mitochondriardquo Mitochondrion vol 8 no 1 pp61ndash73 2008

[46] R E Feissner C L Richard-Fogal E R Frawley J A Lough-man KW Earley and R G Kranz ldquoRecombinant cytochromesc biogenesis systems I and II and analysis of haem deliverypathways in Escherichia colirdquo Molecular Microbiology vol 60no 3 pp 563ndash577 2006

[47] N P Cianciotto P Cornelis and C Baysse ldquoImpact of thebacterial type I cytochrome c maturation system on differentbiological processesrdquoMolecular Microbiology vol 56 no 6 pp1408ndash1415 2005

[48] E Arslan H Schulz R Zufferey P Kunzler and L Thony-Meyer ldquoOverproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in Escherichiacolirdquo Biochemical and Biophysical Research Communicationsvol 251 no 3 pp 744ndash747 1998

[49] C Sanders and H Lill ldquoExpression of prokaryotic and eukary-otic cytochromes c in Escherichia colirdquoBiochimica et BiophysicaActa vol 1459 no 1 pp 131ndash138 2000

[50] M Braun and L Thony-Meyer ldquoBiosynthesis of artificialmicroperoxidases by exploiting the secretion and cytochromec maturation apparatuses of Escherichia colirdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 35 pp 12830ndash12835 2004

[51] B Baert C Baysse S Matthijs and P Cornelis ldquoMultiplephenotypic alterations caused by a c-type cytochrome mat-uration ccmC gene mutation in Pseudomonas aeruginosardquoMicrobiology vol 154 no 1 pp 127ndash138 2008

[52] S Turkarslan C Sanders and F Daldal ldquoExtracytoplasmicprosthetic group ligation to apoproteins maturation of c-typecytochromesrdquo Molecular Microbiology vol 60 no 3 pp 537ndash541 2006

[53] P M Jones and A M George ldquoThe ABC transporter structureand mechanism perspectives on recent researchrdquo Cellular andMolecular Life Sciences vol 61 no 6 pp 682ndash699 2004

[54] O Christensen E M Harvat L Thony-Meyer S J Fergusonand J M Stevens ldquoLoss of ATP hydrolysis activity by CcmABresults in loss of c-type cytochrome synthesis and incompleteprocessing ofCcmErdquoTheFEBS Journal vol 274 no 9 pp 2322ndash2332 2007

[55] J-H Lee E M Harvat J M Stevens S J Ferguson and M HSaier Jr ldquoEvolutionary origins of members of a superfamily ofintegralmembrane cytochrome c biogenesis proteinsrdquoBiochim-ica et Biophysica Acta vol 1768 no 9 pp 2164ndash2181 2007

[56] D L Beckman D R Trawick and R G Kranz ldquoBacterialcytochromes c biogenesisrdquo Genes and Development vol 6 no2 pp 268ndash283 1992

[57] H Schulz R A Fabianek E C Pellicioli H Hennecke andL Thony-Meyer ldquoHeme transfer to the heme chaperone CcmEduring cytochrome c maturation requires the CcmC proteinwhich may function independently of the ABC-transporterCcmABrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 96 no 11 pp 6462ndash6467 1999

[58] Q Ren and L Thony-Meyer ldquoPhysical Interaction ofCcmC with Heme and the Heme Chaperone CcmE duringCytochrome cMaturationrdquo Journal of Biological Chemistry vol276 no 35 pp 32591ndash32596 2001

[59] C Richard-Fogal and R G Kranz ldquoThe CcmChemeCcmEcomplex in heme trafficking and cytochrome c biosynthesisrdquoJournal of Molecular Biology vol 401 no 3 pp 350ndash362 2010

[60] V K Viswanathan S Kurtz L L Pedersen et al ldquoThecytochrome C maturation locus of Legionella pneumophilapromotes iron assimilation and intracellular infection andcontains a strain-specific insertion sequence elementrdquo Infectionand Immunity vol 70 no 4 pp 1842ndash1852 2002

[61] U Ahuja and L Thony-Meyer ldquoCcmD is involved in complexformation between CcmC and the heme chaperone CcmE dur-ing cytochrome c maturationrdquo Journal of Biological Chemistryvol 280 no 1 pp 236ndash243 2005

[62] C L Richard-Fogal E R Frawley and R G Kranz ldquoTopologyand function of CcmD in cytochrome Cmaturationrdquo Journal ofBacteriology vol 190 no 10 pp 3489ndash3493 2008

[63] H Schulz H Hennecke and L Thony-Meyer ldquoPrototype ofa heme chaperone essential for cytochrome c maturationrdquoScience vol 281 no 5380 pp 1197ndash1200 1998

[64] F Arnesano L Banci P D Barker et al ldquoSolution structure andcharacterization of the heme chaperone CcmErdquo Biochemistryvol 41 no 46 pp 13587ndash13594 2002

[65] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[66] L Thony-Meyer ldquoA heme chaperone for cytochrome c biosyn-thesisrdquo Biochemistry vol 42 no 45 pp 13099ndash13105 2003

[67] E M Harvat C Redfield J M Stevens and S J FergusonldquoProbing the heme-binding site of the cytochrome cmaturationprotein CcmErdquoBiochemistry vol 48 no 8 pp 1820ndash1828 2009

[68] D Lee K Pervushin D Bischof M Braun and L Thony-Meyer ldquoUnusual heme-histidine bond in the active site of a

Scientifica 15

chaperonerdquo Journal of the American Chemical Society vol 127no 11 pp 3716ndash3717 2005

[69] A D Goddard J M Stevens F Rao et al ldquoc-Type cytochromebiogenesis can occur via a natural Ccm system lacking CcmHCcmG and the heme-binding histidine of CcmErdquo Journal ofBiological Chemistry vol 285 no 30 pp 22882ndash22889 2010

[70] J M Aramini K Hamilton P Rossi et al ldquoSolution NMRstructure backbone dynamics and heme-binding propertiesof a novel cytochrome c maturation protein CcmE fromDesulfovibrio vulgarisrdquo Biochemistry vol 51 no 18 pp 3705ndash3707 2012

[71] T Uchida J M Stevens O Daltrop et al ldquoThe interactionof covalently bound heme with the cytochrome c maturationprotein CcmErdquo Journal of Biological Chemistry vol 279 no 50pp 51981ndash51988 2004

[72] I Garcıa-Rubio M Braun I Gromov L Thony-Meyer andA Schweiger ldquoAxial coordination of heme in ferric CcmEchaperone characterized by EPR spectroscopyrdquo BiophysicalJournal vol 92 no 4 pp 1361ndash1373 2007

[73] L Thony-Meyer ldquoHaem-polypeptide interactions duringcytochrome c maturationrdquo Biochimica et Biophysica Acta vol1459 no 2-3 pp 316ndash324 2000

[74] J A Keightley D Sanders T R Todaro A Pastuszyn and J AFee ldquoCloning expression in Escherichia coli of the cytochromec552 gene from Thermus thermophilus HB8 Evidence forgenetic linkage to an ATP- binding cassette protein and initialcharacterization of the cycA gene productsrdquo Journal of Biologi-cal Chemistry vol 273 no 20 pp 12006ndash12016 1998

[75] E M Monika B S Goldman D L Beckman and R GKranz ldquoA thioreduction pathway tethered to the membranefor periplasmic cytochromes c biogenesis In vitro and in vivostudiesrdquo Journal of Molecular Biology vol 271 no 5 pp 679ndash692 1997

[76] M Throne-Holst L Thony-Meyer and L HederstedtldquoEscherichia coli ccm in-frame deletion mutants can produceperiplasmic cytochrome b but not cytochrome crdquo FEBS Lettersvol 410 no 2-3 pp 351ndash355 1997

[77] U Ahuja and L Thony-Meyer ldquoDynamic features of a hemedelivery system for cytochrome c maturationrdquo Journal of Bio-logical Chemistry vol 278 no 52 pp 52061ndash52070 2003

[78] J W A Allen P D Barker O Daltrop et al ldquoWhy isnrsquot ldquostan-dardrdquo heme good enough for c-type and di-type cytochromesrdquoDalton Transactions no 21 pp 3410ndash3418 2005

[79] K Inaba and K Ito ldquoStructure and mechanisms of the DsbB-DsbA disulfide bond generation machinerdquo Biochimica et Bio-physica Acta vol 1783 no 4 pp 520ndash529 2008

[80] H Kadokura F Katzen and J Beckwith ldquoProtein disulfidebond formation in prokaryotesrdquoAnnual Review of Biochemistryvol 72 pp 111ndash135 2003

[81] S R Shouldice B Heras P M Walden M Totsika M ASchembri and J L Martin ldquoStructure and function of DsbA akey bacterial oxidative folding catalystrdquoAntioxidants and RedoxSignaling vol 14 no 9 pp 1729ndash1760 2011

[82] R Metheringham L Griffiths H Crooke S Forsythe and JCole ldquoAn essential role for DsbA in cytochrome c synthesis andformate-dependent nitrite reduction by Escherichia coli K-12rdquoArchives of Microbiology vol 164 no 4 pp 301ndash307 1995

[83] Y Sambongi and S J Ferguson ldquoMutants of Escherichia colilacking disulphide oxidoreductases DsbA and DsbB cannotsynthesise an exogenous monohaem c-type cytochrome exceptin the presence of disulphide compoundsrdquo FEBS Letters vol398 no 2-3 pp 265ndash268 1996

[84] D AMavridou S J Ferguson and JM Stevens ldquoThe interplaybetween the disulfide bond formation pathway and cytochromec maturation in Escherichia colirdquo FEBS Letters vol 586 no 12pp 1702ndash1707 2012

[85] C U Stirnimann A Rozhkova U Grauschopf M G GrutterR Glockshuber and G Capitani ldquoStructural basis and kineticsof DsbD-dependent cytochrome c maturationrdquo Structure vol13 no 7 pp 985ndash993 2005

[86] N Ouyang Y-G Gao H-Y Hu and Z-X Xia ldquoCrystalstructures of E coli CcmGand itsmutants reveal key roles of theN-terminal120573-sheet and the fingerprint regionrdquoProteins vol 65no 4 pp 1021ndash1031 2006

[87] M A Edeling L W Guddat R A Fabianek et al ldquoCrys-tallization and preliminary diffraction studies of native andselenomethionine CcmG (CycY DsbE)rdquoActa CrystallographicaD vol 57 no 9 pp 1293ndash1295 2001

[88] R A Fabianek M Huber-Wunderlich R Glockshuber PKunzler H Hennecke and L Thony-Meyer ldquoCharacterizationof the Bradyrhizobium japonicum CycY protein a membrane-anchored periplasmic thioredoxin that may play a role as areductant in the biogenesis of c-type cytochromesrdquo Journal ofBiological Chemistry vol 272 no 7 pp 4467ndash4473 1997

[89] G B Kallis and A Holmgren ldquoDifferential reactivity of thefunctional sulfhydryl groups of cysteine-32 and cysteine-32present in the reduced form of thioredoxin from Escherichiacolirdquo Journal of Biological Chemistry vol 255 no 21 pp 10261ndash10265 1980

[90] P T Chivers andR T Raines ldquoGeneral acidbase catalysis in theactive site of Escherichia coli thioredoxinrdquoBiochemistry vol 36no 50 pp 15810ndash15816 1997

[91] X M Zheng J Hong H Y Li D H Lin and H Y HuldquoBiochemical properties and catalytic domain structure of theCcmH protein from Escherichia colirdquo Biochim Biophys Actavol 1824 no 12 pp 1394ndash1400 2012

[92] UAhujaA Rozhkova RGlockshuber LThony-Meyer andOEinsle ldquoHelix swapping leads to dimerization of the N-terminaldomain of the c-type cytochrome maturation protein CcmHfrom Escherichia colirdquo FEBS Letters vol 582 no 18 pp 2779ndash2786 2008

[93] R A Fabianek T Hofer and L Thony-Meyer ldquoCharacteriza-tion of the Escherichia coli CcmH protein reveals new insightsinto the redox pathway required for cytochrome c maturationrdquoArchives of Microbiology vol 171 no 2 pp 92ndash100 1999

[94] E Reid J Cole and D J Eaves ldquoThe Escherichia coli CcmGprotein fulfils a specific role in cytochrome c assemblyrdquo Bio-chemical Journal vol 355 no 1 pp 51ndash58 2001

[95] Q Ren U Ahuja and LThony-Meyer ldquoA bacterial cytochromec heme lyase CcmF forms a complex with the heme chaperoneCcmE and CcmH but not with apocytochrome crdquo Journal ofBiological Chemistry vol 277 no 10 pp 7657ndash7663 2002

[96] E H Meyer P Giege E Gelhaye et al ldquoAtCCMH an essentialcomponent of the c-type cytochrome maturation pathway inArabidopsis mitochondria interacts with apocytochrome crdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 44 pp 16113ndash16118 2005

[97] C Rios-Velazquez R Coller and T J Donohue ldquoFeatures ofRhodobacter sphaeroides CcmFHrdquo Journal of Bacteriology vol185 no 2 pp 422ndash431 2003

[98] C Sanders M Deshmukh D Astor R G Kranz and F DaldalldquoOverproduction of CcmG and CcmFHRc fully suppresses thec-type cytochrome biogenesis defect of Rhodobacter capsulatus

16 Scientifica

CcmI-null mutantsrdquo Journal of Bacteriology vol 187 no 12 pp4245ndash4256 2005

[99] A F Verissimo H Yang X Wu C Sanders and F DaldalldquoCcmI subunit of CcmFHI heme ligation complex functionsas an apocytochrome c chaperone during c-type cytochromematurationrdquo Journal of Biological Chemistry vol 286 no 47 pp40452ndash40463 2011

[100] K Bryson L J McGuffin R L Marsden J J Ward J SSodhi and D T Jones ldquoProtein structure prediction servers atUniversity College Londonrdquo Nucleic Acids Research vol 33 no2 pp W36ndashW38 2005

[101] D T Jones ldquoProtein secondary structure prediction basedon position-specific scoring matricesrdquo Journal of MolecularBiology vol 292 no 2 pp 195ndash202 1999

[102] C Sanders C Boulay and F Daldal ldquoMembrane-spanning andperiplasmic segments of CcmI have distinct functions duringcytochrome c biogenesis in Rhodobacter capsulatusrdquo Journal ofBacteriology vol 189 no 3 pp 789ndash800 2007

[103] D Han K Kim J Oh J Park and Y Kim ldquoTPR domainof NrfG mediates complex formation between heme lyaseand formate-dependent nitrite reductase in Escherichia coliO157H7rdquo Proteins vol 70 no 3 pp 900ndash914 2008

[104] D J Eaves J Grove W Staudenmann et al ldquoInvolvement ofproducts of the nrfEFG genes in the covalent attachment ofhaem c to a novel cysteine-lysine motif in the cytochrome c552nitrite reductase from Escherichia colirdquo Molecular Microbiol-ogy vol 28 no 1 pp 205ndash216 1998

[105] J Nilsson B Persson and G Von Heijne ldquoConsensus predic-tions of membrane protein topologyrdquo FEBS Letters vol 486 no3 pp 267ndash269 2000

[106] E di Silvio A di Matteo F Malatesta and C Travaglini-Allocatelli ldquoRecognition and binding of apocytochrome c toP aeruginosa CcmI a component of cytochrome c maturationmachineryrdquo Biochim Biophys Acta vol 1834 no 8 pp 1554ndash1561 2013

[107] N Zeytuni and R Zarivach ldquoStructural and functional dis-cussion of the tetra-trico-peptide repeat a protein interactionmodulerdquo Structure vol 20 no 3 pp 397ndash405 2012

[108] C Sanders S Turkarslan D-W Lee O Onder R G Kranz andF Daldal ldquoThe cytochrome c maturation components CcmFCcmH and CcmI form a membrane-integral multisubunitheme ligation complexrdquo Journal of Biological Chemistry vol283 no 44 pp 29715ndash29722 2008

[109] A F Verissimo M A Mohtar and F Daldal ldquoThe hemechaperone ApoCcmE forms a ternary complex with CcmI andapocytochrome crdquoThe Journal of Biological Chemistry vol 288no 9 pp 6272ndash6283 2013

[110] P D Barker J C Ferrer M Mylrajan et al ldquoTransmutation of aheme proteinrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 90 no 14 pp 6542ndash65461993

[111] J Simon and L Hederstedt ldquoComposition and function ofcytochrome c biogenesis system IIrdquoThe FEBS Journal vol 278no 22 pp 4179ndash4188 2011

[112] T R H M Kouwen and J M van Dijl ldquoInterchangeablemodules in bacterial thiol-disulfide exchange pathwaysrdquo Trendsin Microbiology vol 17 no 1 pp 6ndash12 2009

[113] A Crow A Lewin O Hecht et al ldquoCrystal structure andbiophysical properties of Bacillus subtilis BdbD An oxidizingthioldisulfide oxidoreductase containing a novel metal siterdquoJournal of Biological Chemistry vol 284 no 35 pp 23719ndash23733 2009

[114] M C Moller and L Hederstedt ldquoExtracytoplasmic processesimpaired by inactivation of trxA (thioredoxin gene) in Bacillussubtilisrdquo Journal of Bacteriology vol 190 no 13 pp 4660ndash46652008

[115] A Lewin A Crow A Oubrie and N E Le Brun ldquoMolecularbasis for specificity of the extracytoplasmic thioredoxin ResArdquoJournal of Biological Chemistry vol 281 no 46 pp 35467ndash35477 2006

[116] C L Colbert QWu P J A Erbel K H Gardner and J Deisen-hofer ldquoMechanism of substrate specificity in Bacillus subtilisResA a thioredoxin-like protein involved in cytochrome cmaturationrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 103 no 12 pp 4410ndash44152006

[117] UAhuja P Kjelgaard B L Schulz LThony-Meyer andLHed-erstedt ldquoHaem-delivery proteins in cytochrome c maturationsystem IIrdquoMolecular Microbiology vol 73 no 6 pp 1058ndash10712009

[118] M L D Page P P Hamel S T Gabilly et al ldquoA homologof prokaryotic thiol disulfide transporter CcdA is required forthe assembly of the cytochrome b6f complex in Arabidopsischloroplastsrdquo Journal of Biological Chemistry vol 279 no 31pp 32474ndash32482 2004

[119] A Rozhkova C U Stirnimann P Frei et al ldquoStructural basisand kinetics of inter- and intramolecular disulfide exchange inthe redox catalyst DsbDrdquoThe EMBO Journal vol 23 no 8 pp1709ndash1719 2004

[120] T Schiott M Throne-Holst and L Hederstedt ldquoBacillussubtilis CcdA-defective mutants are blocked in a late step ofcytochrome C biogenesisrdquo Journal of Bacteriology vol 179 no14 pp 4523ndash4529 1997

[121] R E Feissner C S Beckett J A Loughman and R G KranzldquoMutations in cytochrome assembly and periplasmic redoxpathways in Bordetella pertussisrdquo Journal of Bacteriology vol187 no 12 pp 3941ndash3949 2005

[122] L S ErlendssonMMoller and L Hederstedt ldquoBacillus subtilisStoA is a thiol-disulfide oxidoreductase important for sporecortex synthesisrdquo Journal of Bacteriology vol 186 no 18 pp6230ndash6238 2004

[123] L S Erlendsson R M Acheson L Hederstedt and N E LeBrun ldquoBacillus subtilis ResA is a thiol-disulfide oxidoreductaseinvolved in cytochrome c synthesisrdquo Journal of BiologicalChemistry vol 278 no 20 pp 17852ndash17858 2003

[124] L S Erlendsson and L Hederstedt ldquoMutations in the thiol-disulfide oxidoreductases BdbC and BdbD can suppresscytochrome c deficiency of CcdA-defective Bacillus subtiliscellsrdquo Journal of Bacteriology vol 184 no 5 pp 1423ndash1429 2002

[125] M E Dumont J F Ernst D M Hampsey and F ShermanldquoIdentification and sequence of the gene encoding cytochromec heme lyase in the yeast Saccharomyces cerevisiaerdquoThe EMBOJournal vol 6 no 1 pp 235ndash241 1987

[126] M E Dumont J F Ernst and F Sherman ldquoCoupling of hemeattachment to import of cytochrome c into yeast mitochondriaStudies with heme lyase-deficient mitochondria and alteredapocytochromes crdquo Journal of Biological Chemistry vol 263 no31 pp 15928ndash15937 1988

[127] B San Francisco E C Bretsnyder and R G Kranz ldquoHumanmitochondrial holocytochrome c synthasersquos hemebindingmat-uration determinants and complex formationwith cytochromecrdquo Proceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 9 pp E788ndashE797 2012

Scientifica 17

[128] L Schaefer A Ballabio and H Y Zoghbi ldquoCloning andcharacterization of a putative human holocytochrome c-typesynthetase gene (HCCS) isolated from the critical region formicrophthalmia with linear skin defects (MLS)rdquoGenomics vol34 no 2 pp 166ndash172 1996

[129] S K Prakash T A Cormier A E McCall et al ldquoLoss ofholocytochrome c-type synthetase causes the male lethality ofX-linked dominant microphthalmia with linear skin defects(MLS) syndromerdquo Human Molecular Genetics vol 11 no 25pp 3237ndash3248 2002

[130] I Wimplinger M Morleo G Rosenberger et al ldquoMutations ofthe mitochondrial holocytochrome c-type synthase in X-linkeddominant microphthalmia with linear skin defects syndromerdquoTheAmerican Journal of HumanGenetics vol 79 no 5 pp 878ndash889 2006

[131] S Kiryu-Seo KGamo T Tachibana K Tanaka andHKiyamaldquoUnique anti-apoptotic activity of EAAC1 in injured motorneuronsrdquoTheEMBO Journal vol 25 no 14 pp 3411ndash3421 2006

[132] R Lill R A Stuart M E Drygas F E Nargang and W Neu-pert ldquoImport of cytochrome c heme lyase into mitochondriaa novel pathway into the intermembrane spacerdquo The EMBOJournal vol 11 no 2 pp 449ndash456 1992

[133] H Steiner A Zollner A Haid W Neupert and R LillldquoBiogenesis of mitochondrial heme lyases in yeast Importand folding in the intermembrane spacerdquo Journal of BiologicalChemistry vol 270 no 39 pp 22842ndash22849 1995

[134] A Zollner G Rodel and A Haid ldquoMolecular cloning andcharacterization of the Saccharomyces cerevisiae CYT2 geneencoding cytochrome-c1-heme lyaserdquo European Journal of Bio-chemistry vol 207 no 3 pp 1093ndash1100 1992

[135] V Corvest D A Murrey D G Bernard D B Knaff B Guiardand P PHamel ldquoc-Type cytochrome assembly in Saccharomycescerevisiae a key residue for apocytochrome c1lyase interactionrdquoGenetics vol 186 no 2 pp 561ndash571 2010

[136] V Corvest D A Murrey M Hirasawa D B Knaff B Guiardand P P Hamel ldquoThe flavoprotein Cyc2p a mitochondrialcytochrome c assembly factor is a NAD(P)H-dependent haemreductaserdquo Molecular Microbiology vol 83 no 5 pp 968ndash9802012

[137] D G Bernard S Quevillon-Cheruel S Merchant B Guiardand P P Hamel ldquoCyc2p a membrane-bound flavopro-tein involved in the maturation of mitochondrial c-typecytochromesrdquo Journal of Biological Chemistry vol 280 no 48pp 39852ndash39859 2005

[138] J G Kleingardner and K L Bren ldquoComparing substratespecificity between cytochrome cmaturation and cytochrome cheme lyase systems for cytochrome c biogenesisrdquo Metallomicsvol 3 no 4 pp 396ndash403 2011

[139] C Travaglini-Allocatelli S Gianni VMorea A Tramontano TSoulimane and M Brunori ldquoExploring the cytochrome c fold-ing mechanism cytochrome c552 fromThermus thermophilusfolds through an on-pathway intermediaterdquo Journal of BiologicalChemistry vol 278 no 42 pp 41136ndash41140 2003

[140] L Zhang and L Guarente ldquoHeme binds to a short sequencethat serves a regulatory function in diverse proteinsrdquoTheEMBOJournal vol 14 no 2 pp 313ndash320 1995

[141] S Hira T Tomita T Matsui K Igarashi and M Ikeda-SaitoldquoBach1 a heme-dependent transcription factor reveals pres-ence of multiple heme binding sites with distinct coordinationstructurerdquo IUBMB Life vol 59 no 8-9 pp 542ndash551 2007

[142] T K Kuhl A Wiszligbrock N Goradia et al ldquoAnalysis of Fe(III)heme binding to cysteine-containing heme-regulatory motifs

in proteinsrdquo ACS Chemical Biology vol 8 no 8 pp 1785ndash17932013

[143] R L Moore J M Stevens and S J Ferguson ldquoMitochondrialcytochrome c synthase CP motifs are not necessary for hemeattachment to apocytochrome crdquo FEBS Letters vol 585 no 21pp 3415ndash3419 2011

[144] S Gianni M Brunori and C Travaglini-Allocatelli ldquoRefoldingkinetics of cytochrome c551 reveals a mechanistic differencebetween urea and guanidinerdquo Protein Science vol 10 no 8 pp1685ndash1688 2001

[145] S Gianni C Travaglini-Allocatelli F Cutruzzola M BrunoriM C R Shastry and H Roder ldquoParallel pathways incytochrome c551 foldingrdquo Journal of Molecular Biology vol 330no 5 pp 1145ndash1152 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Scientifica 15

chaperonerdquo Journal of the American Chemical Society vol 127no 11 pp 3716ndash3717 2005

[69] A D Goddard J M Stevens F Rao et al ldquoc-Type cytochromebiogenesis can occur via a natural Ccm system lacking CcmHCcmG and the heme-binding histidine of CcmErdquo Journal ofBiological Chemistry vol 285 no 30 pp 22882ndash22889 2010

[70] J M Aramini K Hamilton P Rossi et al ldquoSolution NMRstructure backbone dynamics and heme-binding propertiesof a novel cytochrome c maturation protein CcmE fromDesulfovibrio vulgarisrdquo Biochemistry vol 51 no 18 pp 3705ndash3707 2012

[71] T Uchida J M Stevens O Daltrop et al ldquoThe interactionof covalently bound heme with the cytochrome c maturationprotein CcmErdquo Journal of Biological Chemistry vol 279 no 50pp 51981ndash51988 2004

[72] I Garcıa-Rubio M Braun I Gromov L Thony-Meyer andA Schweiger ldquoAxial coordination of heme in ferric CcmEchaperone characterized by EPR spectroscopyrdquo BiophysicalJournal vol 92 no 4 pp 1361ndash1373 2007

[73] L Thony-Meyer ldquoHaem-polypeptide interactions duringcytochrome c maturationrdquo Biochimica et Biophysica Acta vol1459 no 2-3 pp 316ndash324 2000

[74] J A Keightley D Sanders T R Todaro A Pastuszyn and J AFee ldquoCloning expression in Escherichia coli of the cytochromec552 gene from Thermus thermophilus HB8 Evidence forgenetic linkage to an ATP- binding cassette protein and initialcharacterization of the cycA gene productsrdquo Journal of Biologi-cal Chemistry vol 273 no 20 pp 12006ndash12016 1998

[75] E M Monika B S Goldman D L Beckman and R GKranz ldquoA thioreduction pathway tethered to the membranefor periplasmic cytochromes c biogenesis In vitro and in vivostudiesrdquo Journal of Molecular Biology vol 271 no 5 pp 679ndash692 1997

[76] M Throne-Holst L Thony-Meyer and L HederstedtldquoEscherichia coli ccm in-frame deletion mutants can produceperiplasmic cytochrome b but not cytochrome crdquo FEBS Lettersvol 410 no 2-3 pp 351ndash355 1997

[77] U Ahuja and L Thony-Meyer ldquoDynamic features of a hemedelivery system for cytochrome c maturationrdquo Journal of Bio-logical Chemistry vol 278 no 52 pp 52061ndash52070 2003

[78] J W A Allen P D Barker O Daltrop et al ldquoWhy isnrsquot ldquostan-dardrdquo heme good enough for c-type and di-type cytochromesrdquoDalton Transactions no 21 pp 3410ndash3418 2005

[79] K Inaba and K Ito ldquoStructure and mechanisms of the DsbB-DsbA disulfide bond generation machinerdquo Biochimica et Bio-physica Acta vol 1783 no 4 pp 520ndash529 2008

[80] H Kadokura F Katzen and J Beckwith ldquoProtein disulfidebond formation in prokaryotesrdquoAnnual Review of Biochemistryvol 72 pp 111ndash135 2003

[81] S R Shouldice B Heras P M Walden M Totsika M ASchembri and J L Martin ldquoStructure and function of DsbA akey bacterial oxidative folding catalystrdquoAntioxidants and RedoxSignaling vol 14 no 9 pp 1729ndash1760 2011

[82] R Metheringham L Griffiths H Crooke S Forsythe and JCole ldquoAn essential role for DsbA in cytochrome c synthesis andformate-dependent nitrite reduction by Escherichia coli K-12rdquoArchives of Microbiology vol 164 no 4 pp 301ndash307 1995

[83] Y Sambongi and S J Ferguson ldquoMutants of Escherichia colilacking disulphide oxidoreductases DsbA and DsbB cannotsynthesise an exogenous monohaem c-type cytochrome exceptin the presence of disulphide compoundsrdquo FEBS Letters vol398 no 2-3 pp 265ndash268 1996

[84] D AMavridou S J Ferguson and JM Stevens ldquoThe interplaybetween the disulfide bond formation pathway and cytochromec maturation in Escherichia colirdquo FEBS Letters vol 586 no 12pp 1702ndash1707 2012

[85] C U Stirnimann A Rozhkova U Grauschopf M G GrutterR Glockshuber and G Capitani ldquoStructural basis and kineticsof DsbD-dependent cytochrome c maturationrdquo Structure vol13 no 7 pp 985ndash993 2005

[86] N Ouyang Y-G Gao H-Y Hu and Z-X Xia ldquoCrystalstructures of E coli CcmGand itsmutants reveal key roles of theN-terminal120573-sheet and the fingerprint regionrdquoProteins vol 65no 4 pp 1021ndash1031 2006

[87] M A Edeling L W Guddat R A Fabianek et al ldquoCrys-tallization and preliminary diffraction studies of native andselenomethionine CcmG (CycY DsbE)rdquoActa CrystallographicaD vol 57 no 9 pp 1293ndash1295 2001

[88] R A Fabianek M Huber-Wunderlich R Glockshuber PKunzler H Hennecke and L Thony-Meyer ldquoCharacterizationof the Bradyrhizobium japonicum CycY protein a membrane-anchored periplasmic thioredoxin that may play a role as areductant in the biogenesis of c-type cytochromesrdquo Journal ofBiological Chemistry vol 272 no 7 pp 4467ndash4473 1997

[89] G B Kallis and A Holmgren ldquoDifferential reactivity of thefunctional sulfhydryl groups of cysteine-32 and cysteine-32present in the reduced form of thioredoxin from Escherichiacolirdquo Journal of Biological Chemistry vol 255 no 21 pp 10261ndash10265 1980

[90] P T Chivers andR T Raines ldquoGeneral acidbase catalysis in theactive site of Escherichia coli thioredoxinrdquoBiochemistry vol 36no 50 pp 15810ndash15816 1997

[91] X M Zheng J Hong H Y Li D H Lin and H Y HuldquoBiochemical properties and catalytic domain structure of theCcmH protein from Escherichia colirdquo Biochim Biophys Actavol 1824 no 12 pp 1394ndash1400 2012

[92] UAhujaA Rozhkova RGlockshuber LThony-Meyer andOEinsle ldquoHelix swapping leads to dimerization of the N-terminaldomain of the c-type cytochrome maturation protein CcmHfrom Escherichia colirdquo FEBS Letters vol 582 no 18 pp 2779ndash2786 2008

[93] R A Fabianek T Hofer and L Thony-Meyer ldquoCharacteriza-tion of the Escherichia coli CcmH protein reveals new insightsinto the redox pathway required for cytochrome c maturationrdquoArchives of Microbiology vol 171 no 2 pp 92ndash100 1999

[94] E Reid J Cole and D J Eaves ldquoThe Escherichia coli CcmGprotein fulfils a specific role in cytochrome c assemblyrdquo Bio-chemical Journal vol 355 no 1 pp 51ndash58 2001

[95] Q Ren U Ahuja and LThony-Meyer ldquoA bacterial cytochromec heme lyase CcmF forms a complex with the heme chaperoneCcmE and CcmH but not with apocytochrome crdquo Journal ofBiological Chemistry vol 277 no 10 pp 7657ndash7663 2002

[96] E H Meyer P Giege E Gelhaye et al ldquoAtCCMH an essentialcomponent of the c-type cytochrome maturation pathway inArabidopsis mitochondria interacts with apocytochrome crdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 44 pp 16113ndash16118 2005

[97] C Rios-Velazquez R Coller and T J Donohue ldquoFeatures ofRhodobacter sphaeroides CcmFHrdquo Journal of Bacteriology vol185 no 2 pp 422ndash431 2003

[98] C Sanders M Deshmukh D Astor R G Kranz and F DaldalldquoOverproduction of CcmG and CcmFHRc fully suppresses thec-type cytochrome biogenesis defect of Rhodobacter capsulatus

16 Scientifica

CcmI-null mutantsrdquo Journal of Bacteriology vol 187 no 12 pp4245ndash4256 2005

[99] A F Verissimo H Yang X Wu C Sanders and F DaldalldquoCcmI subunit of CcmFHI heme ligation complex functionsas an apocytochrome c chaperone during c-type cytochromematurationrdquo Journal of Biological Chemistry vol 286 no 47 pp40452ndash40463 2011

[100] K Bryson L J McGuffin R L Marsden J J Ward J SSodhi and D T Jones ldquoProtein structure prediction servers atUniversity College Londonrdquo Nucleic Acids Research vol 33 no2 pp W36ndashW38 2005

[101] D T Jones ldquoProtein secondary structure prediction basedon position-specific scoring matricesrdquo Journal of MolecularBiology vol 292 no 2 pp 195ndash202 1999

[102] C Sanders C Boulay and F Daldal ldquoMembrane-spanning andperiplasmic segments of CcmI have distinct functions duringcytochrome c biogenesis in Rhodobacter capsulatusrdquo Journal ofBacteriology vol 189 no 3 pp 789ndash800 2007

[103] D Han K Kim J Oh J Park and Y Kim ldquoTPR domainof NrfG mediates complex formation between heme lyaseand formate-dependent nitrite reductase in Escherichia coliO157H7rdquo Proteins vol 70 no 3 pp 900ndash914 2008

[104] D J Eaves J Grove W Staudenmann et al ldquoInvolvement ofproducts of the nrfEFG genes in the covalent attachment ofhaem c to a novel cysteine-lysine motif in the cytochrome c552nitrite reductase from Escherichia colirdquo Molecular Microbiol-ogy vol 28 no 1 pp 205ndash216 1998

[105] J Nilsson B Persson and G Von Heijne ldquoConsensus predic-tions of membrane protein topologyrdquo FEBS Letters vol 486 no3 pp 267ndash269 2000

[106] E di Silvio A di Matteo F Malatesta and C Travaglini-Allocatelli ldquoRecognition and binding of apocytochrome c toP aeruginosa CcmI a component of cytochrome c maturationmachineryrdquo Biochim Biophys Acta vol 1834 no 8 pp 1554ndash1561 2013

[107] N Zeytuni and R Zarivach ldquoStructural and functional dis-cussion of the tetra-trico-peptide repeat a protein interactionmodulerdquo Structure vol 20 no 3 pp 397ndash405 2012

[108] C Sanders S Turkarslan D-W Lee O Onder R G Kranz andF Daldal ldquoThe cytochrome c maturation components CcmFCcmH and CcmI form a membrane-integral multisubunitheme ligation complexrdquo Journal of Biological Chemistry vol283 no 44 pp 29715ndash29722 2008

[109] A F Verissimo M A Mohtar and F Daldal ldquoThe hemechaperone ApoCcmE forms a ternary complex with CcmI andapocytochrome crdquoThe Journal of Biological Chemistry vol 288no 9 pp 6272ndash6283 2013

[110] P D Barker J C Ferrer M Mylrajan et al ldquoTransmutation of aheme proteinrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 90 no 14 pp 6542ndash65461993

[111] J Simon and L Hederstedt ldquoComposition and function ofcytochrome c biogenesis system IIrdquoThe FEBS Journal vol 278no 22 pp 4179ndash4188 2011

[112] T R H M Kouwen and J M van Dijl ldquoInterchangeablemodules in bacterial thiol-disulfide exchange pathwaysrdquo Trendsin Microbiology vol 17 no 1 pp 6ndash12 2009

[113] A Crow A Lewin O Hecht et al ldquoCrystal structure andbiophysical properties of Bacillus subtilis BdbD An oxidizingthioldisulfide oxidoreductase containing a novel metal siterdquoJournal of Biological Chemistry vol 284 no 35 pp 23719ndash23733 2009

[114] M C Moller and L Hederstedt ldquoExtracytoplasmic processesimpaired by inactivation of trxA (thioredoxin gene) in Bacillussubtilisrdquo Journal of Bacteriology vol 190 no 13 pp 4660ndash46652008

[115] A Lewin A Crow A Oubrie and N E Le Brun ldquoMolecularbasis for specificity of the extracytoplasmic thioredoxin ResArdquoJournal of Biological Chemistry vol 281 no 46 pp 35467ndash35477 2006

[116] C L Colbert QWu P J A Erbel K H Gardner and J Deisen-hofer ldquoMechanism of substrate specificity in Bacillus subtilisResA a thioredoxin-like protein involved in cytochrome cmaturationrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 103 no 12 pp 4410ndash44152006

[117] UAhuja P Kjelgaard B L Schulz LThony-Meyer andLHed-erstedt ldquoHaem-delivery proteins in cytochrome c maturationsystem IIrdquoMolecular Microbiology vol 73 no 6 pp 1058ndash10712009

[118] M L D Page P P Hamel S T Gabilly et al ldquoA homologof prokaryotic thiol disulfide transporter CcdA is required forthe assembly of the cytochrome b6f complex in Arabidopsischloroplastsrdquo Journal of Biological Chemistry vol 279 no 31pp 32474ndash32482 2004

[119] A Rozhkova C U Stirnimann P Frei et al ldquoStructural basisand kinetics of inter- and intramolecular disulfide exchange inthe redox catalyst DsbDrdquoThe EMBO Journal vol 23 no 8 pp1709ndash1719 2004

[120] T Schiott M Throne-Holst and L Hederstedt ldquoBacillussubtilis CcdA-defective mutants are blocked in a late step ofcytochrome C biogenesisrdquo Journal of Bacteriology vol 179 no14 pp 4523ndash4529 1997

[121] R E Feissner C S Beckett J A Loughman and R G KranzldquoMutations in cytochrome assembly and periplasmic redoxpathways in Bordetella pertussisrdquo Journal of Bacteriology vol187 no 12 pp 3941ndash3949 2005

[122] L S ErlendssonMMoller and L Hederstedt ldquoBacillus subtilisStoA is a thiol-disulfide oxidoreductase important for sporecortex synthesisrdquo Journal of Bacteriology vol 186 no 18 pp6230ndash6238 2004

[123] L S Erlendsson R M Acheson L Hederstedt and N E LeBrun ldquoBacillus subtilis ResA is a thiol-disulfide oxidoreductaseinvolved in cytochrome c synthesisrdquo Journal of BiologicalChemistry vol 278 no 20 pp 17852ndash17858 2003

[124] L S Erlendsson and L Hederstedt ldquoMutations in the thiol-disulfide oxidoreductases BdbC and BdbD can suppresscytochrome c deficiency of CcdA-defective Bacillus subtiliscellsrdquo Journal of Bacteriology vol 184 no 5 pp 1423ndash1429 2002

[125] M E Dumont J F Ernst D M Hampsey and F ShermanldquoIdentification and sequence of the gene encoding cytochromec heme lyase in the yeast Saccharomyces cerevisiaerdquoThe EMBOJournal vol 6 no 1 pp 235ndash241 1987

[126] M E Dumont J F Ernst and F Sherman ldquoCoupling of hemeattachment to import of cytochrome c into yeast mitochondriaStudies with heme lyase-deficient mitochondria and alteredapocytochromes crdquo Journal of Biological Chemistry vol 263 no31 pp 15928ndash15937 1988

[127] B San Francisco E C Bretsnyder and R G Kranz ldquoHumanmitochondrial holocytochrome c synthasersquos hemebindingmat-uration determinants and complex formationwith cytochromecrdquo Proceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 9 pp E788ndashE797 2012

Scientifica 17

[128] L Schaefer A Ballabio and H Y Zoghbi ldquoCloning andcharacterization of a putative human holocytochrome c-typesynthetase gene (HCCS) isolated from the critical region formicrophthalmia with linear skin defects (MLS)rdquoGenomics vol34 no 2 pp 166ndash172 1996

[129] S K Prakash T A Cormier A E McCall et al ldquoLoss ofholocytochrome c-type synthetase causes the male lethality ofX-linked dominant microphthalmia with linear skin defects(MLS) syndromerdquo Human Molecular Genetics vol 11 no 25pp 3237ndash3248 2002

[130] I Wimplinger M Morleo G Rosenberger et al ldquoMutations ofthe mitochondrial holocytochrome c-type synthase in X-linkeddominant microphthalmia with linear skin defects syndromerdquoTheAmerican Journal of HumanGenetics vol 79 no 5 pp 878ndash889 2006

[131] S Kiryu-Seo KGamo T Tachibana K Tanaka andHKiyamaldquoUnique anti-apoptotic activity of EAAC1 in injured motorneuronsrdquoTheEMBO Journal vol 25 no 14 pp 3411ndash3421 2006

[132] R Lill R A Stuart M E Drygas F E Nargang and W Neu-pert ldquoImport of cytochrome c heme lyase into mitochondriaa novel pathway into the intermembrane spacerdquo The EMBOJournal vol 11 no 2 pp 449ndash456 1992

[133] H Steiner A Zollner A Haid W Neupert and R LillldquoBiogenesis of mitochondrial heme lyases in yeast Importand folding in the intermembrane spacerdquo Journal of BiologicalChemistry vol 270 no 39 pp 22842ndash22849 1995

[134] A Zollner G Rodel and A Haid ldquoMolecular cloning andcharacterization of the Saccharomyces cerevisiae CYT2 geneencoding cytochrome-c1-heme lyaserdquo European Journal of Bio-chemistry vol 207 no 3 pp 1093ndash1100 1992

[135] V Corvest D A Murrey D G Bernard D B Knaff B Guiardand P PHamel ldquoc-Type cytochrome assembly in Saccharomycescerevisiae a key residue for apocytochrome c1lyase interactionrdquoGenetics vol 186 no 2 pp 561ndash571 2010

[136] V Corvest D A Murrey M Hirasawa D B Knaff B Guiardand P P Hamel ldquoThe flavoprotein Cyc2p a mitochondrialcytochrome c assembly factor is a NAD(P)H-dependent haemreductaserdquo Molecular Microbiology vol 83 no 5 pp 968ndash9802012

[137] D G Bernard S Quevillon-Cheruel S Merchant B Guiardand P P Hamel ldquoCyc2p a membrane-bound flavopro-tein involved in the maturation of mitochondrial c-typecytochromesrdquo Journal of Biological Chemistry vol 280 no 48pp 39852ndash39859 2005

[138] J G Kleingardner and K L Bren ldquoComparing substratespecificity between cytochrome cmaturation and cytochrome cheme lyase systems for cytochrome c biogenesisrdquo Metallomicsvol 3 no 4 pp 396ndash403 2011

[139] C Travaglini-Allocatelli S Gianni VMorea A Tramontano TSoulimane and M Brunori ldquoExploring the cytochrome c fold-ing mechanism cytochrome c552 fromThermus thermophilusfolds through an on-pathway intermediaterdquo Journal of BiologicalChemistry vol 278 no 42 pp 41136ndash41140 2003

[140] L Zhang and L Guarente ldquoHeme binds to a short sequencethat serves a regulatory function in diverse proteinsrdquoTheEMBOJournal vol 14 no 2 pp 313ndash320 1995

[141] S Hira T Tomita T Matsui K Igarashi and M Ikeda-SaitoldquoBach1 a heme-dependent transcription factor reveals pres-ence of multiple heme binding sites with distinct coordinationstructurerdquo IUBMB Life vol 59 no 8-9 pp 542ndash551 2007

[142] T K Kuhl A Wiszligbrock N Goradia et al ldquoAnalysis of Fe(III)heme binding to cysteine-containing heme-regulatory motifs

in proteinsrdquo ACS Chemical Biology vol 8 no 8 pp 1785ndash17932013

[143] R L Moore J M Stevens and S J Ferguson ldquoMitochondrialcytochrome c synthase CP motifs are not necessary for hemeattachment to apocytochrome crdquo FEBS Letters vol 585 no 21pp 3415ndash3419 2011

[144] S Gianni M Brunori and C Travaglini-Allocatelli ldquoRefoldingkinetics of cytochrome c551 reveals a mechanistic differencebetween urea and guanidinerdquo Protein Science vol 10 no 8 pp1685ndash1688 2001

[145] S Gianni C Travaglini-Allocatelli F Cutruzzola M BrunoriM C R Shastry and H Roder ldquoParallel pathways incytochrome c551 foldingrdquo Journal of Molecular Biology vol 330no 5 pp 1145ndash1152 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

16 Scientifica

CcmI-null mutantsrdquo Journal of Bacteriology vol 187 no 12 pp4245ndash4256 2005

[99] A F Verissimo H Yang X Wu C Sanders and F DaldalldquoCcmI subunit of CcmFHI heme ligation complex functionsas an apocytochrome c chaperone during c-type cytochromematurationrdquo Journal of Biological Chemistry vol 286 no 47 pp40452ndash40463 2011

[100] K Bryson L J McGuffin R L Marsden J J Ward J SSodhi and D T Jones ldquoProtein structure prediction servers atUniversity College Londonrdquo Nucleic Acids Research vol 33 no2 pp W36ndashW38 2005

[101] D T Jones ldquoProtein secondary structure prediction basedon position-specific scoring matricesrdquo Journal of MolecularBiology vol 292 no 2 pp 195ndash202 1999

[102] C Sanders C Boulay and F Daldal ldquoMembrane-spanning andperiplasmic segments of CcmI have distinct functions duringcytochrome c biogenesis in Rhodobacter capsulatusrdquo Journal ofBacteriology vol 189 no 3 pp 789ndash800 2007

[103] D Han K Kim J Oh J Park and Y Kim ldquoTPR domainof NrfG mediates complex formation between heme lyaseand formate-dependent nitrite reductase in Escherichia coliO157H7rdquo Proteins vol 70 no 3 pp 900ndash914 2008

[104] D J Eaves J Grove W Staudenmann et al ldquoInvolvement ofproducts of the nrfEFG genes in the covalent attachment ofhaem c to a novel cysteine-lysine motif in the cytochrome c552nitrite reductase from Escherichia colirdquo Molecular Microbiol-ogy vol 28 no 1 pp 205ndash216 1998

[105] J Nilsson B Persson and G Von Heijne ldquoConsensus predic-tions of membrane protein topologyrdquo FEBS Letters vol 486 no3 pp 267ndash269 2000

[106] E di Silvio A di Matteo F Malatesta and C Travaglini-Allocatelli ldquoRecognition and binding of apocytochrome c toP aeruginosa CcmI a component of cytochrome c maturationmachineryrdquo Biochim Biophys Acta vol 1834 no 8 pp 1554ndash1561 2013

[107] N Zeytuni and R Zarivach ldquoStructural and functional dis-cussion of the tetra-trico-peptide repeat a protein interactionmodulerdquo Structure vol 20 no 3 pp 397ndash405 2012

[108] C Sanders S Turkarslan D-W Lee O Onder R G Kranz andF Daldal ldquoThe cytochrome c maturation components CcmFCcmH and CcmI form a membrane-integral multisubunitheme ligation complexrdquo Journal of Biological Chemistry vol283 no 44 pp 29715ndash29722 2008

[109] A F Verissimo M A Mohtar and F Daldal ldquoThe hemechaperone ApoCcmE forms a ternary complex with CcmI andapocytochrome crdquoThe Journal of Biological Chemistry vol 288no 9 pp 6272ndash6283 2013

[110] P D Barker J C Ferrer M Mylrajan et al ldquoTransmutation of aheme proteinrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 90 no 14 pp 6542ndash65461993

[111] J Simon and L Hederstedt ldquoComposition and function ofcytochrome c biogenesis system IIrdquoThe FEBS Journal vol 278no 22 pp 4179ndash4188 2011

[112] T R H M Kouwen and J M van Dijl ldquoInterchangeablemodules in bacterial thiol-disulfide exchange pathwaysrdquo Trendsin Microbiology vol 17 no 1 pp 6ndash12 2009

[113] A Crow A Lewin O Hecht et al ldquoCrystal structure andbiophysical properties of Bacillus subtilis BdbD An oxidizingthioldisulfide oxidoreductase containing a novel metal siterdquoJournal of Biological Chemistry vol 284 no 35 pp 23719ndash23733 2009

[114] M C Moller and L Hederstedt ldquoExtracytoplasmic processesimpaired by inactivation of trxA (thioredoxin gene) in Bacillussubtilisrdquo Journal of Bacteriology vol 190 no 13 pp 4660ndash46652008

[115] A Lewin A Crow A Oubrie and N E Le Brun ldquoMolecularbasis for specificity of the extracytoplasmic thioredoxin ResArdquoJournal of Biological Chemistry vol 281 no 46 pp 35467ndash35477 2006

[116] C L Colbert QWu P J A Erbel K H Gardner and J Deisen-hofer ldquoMechanism of substrate specificity in Bacillus subtilisResA a thioredoxin-like protein involved in cytochrome cmaturationrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 103 no 12 pp 4410ndash44152006

[117] UAhuja P Kjelgaard B L Schulz LThony-Meyer andLHed-erstedt ldquoHaem-delivery proteins in cytochrome c maturationsystem IIrdquoMolecular Microbiology vol 73 no 6 pp 1058ndash10712009

[118] M L D Page P P Hamel S T Gabilly et al ldquoA homologof prokaryotic thiol disulfide transporter CcdA is required forthe assembly of the cytochrome b6f complex in Arabidopsischloroplastsrdquo Journal of Biological Chemistry vol 279 no 31pp 32474ndash32482 2004

[119] A Rozhkova C U Stirnimann P Frei et al ldquoStructural basisand kinetics of inter- and intramolecular disulfide exchange inthe redox catalyst DsbDrdquoThe EMBO Journal vol 23 no 8 pp1709ndash1719 2004

[120] T Schiott M Throne-Holst and L Hederstedt ldquoBacillussubtilis CcdA-defective mutants are blocked in a late step ofcytochrome C biogenesisrdquo Journal of Bacteriology vol 179 no14 pp 4523ndash4529 1997

[121] R E Feissner C S Beckett J A Loughman and R G KranzldquoMutations in cytochrome assembly and periplasmic redoxpathways in Bordetella pertussisrdquo Journal of Bacteriology vol187 no 12 pp 3941ndash3949 2005

[122] L S ErlendssonMMoller and L Hederstedt ldquoBacillus subtilisStoA is a thiol-disulfide oxidoreductase important for sporecortex synthesisrdquo Journal of Bacteriology vol 186 no 18 pp6230ndash6238 2004

[123] L S Erlendsson R M Acheson L Hederstedt and N E LeBrun ldquoBacillus subtilis ResA is a thiol-disulfide oxidoreductaseinvolved in cytochrome c synthesisrdquo Journal of BiologicalChemistry vol 278 no 20 pp 17852ndash17858 2003

[124] L S Erlendsson and L Hederstedt ldquoMutations in the thiol-disulfide oxidoreductases BdbC and BdbD can suppresscytochrome c deficiency of CcdA-defective Bacillus subtiliscellsrdquo Journal of Bacteriology vol 184 no 5 pp 1423ndash1429 2002

[125] M E Dumont J F Ernst D M Hampsey and F ShermanldquoIdentification and sequence of the gene encoding cytochromec heme lyase in the yeast Saccharomyces cerevisiaerdquoThe EMBOJournal vol 6 no 1 pp 235ndash241 1987

[126] M E Dumont J F Ernst and F Sherman ldquoCoupling of hemeattachment to import of cytochrome c into yeast mitochondriaStudies with heme lyase-deficient mitochondria and alteredapocytochromes crdquo Journal of Biological Chemistry vol 263 no31 pp 15928ndash15937 1988

[127] B San Francisco E C Bretsnyder and R G Kranz ldquoHumanmitochondrial holocytochrome c synthasersquos hemebindingmat-uration determinants and complex formationwith cytochromecrdquo Proceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 9 pp E788ndashE797 2012

Scientifica 17

[128] L Schaefer A Ballabio and H Y Zoghbi ldquoCloning andcharacterization of a putative human holocytochrome c-typesynthetase gene (HCCS) isolated from the critical region formicrophthalmia with linear skin defects (MLS)rdquoGenomics vol34 no 2 pp 166ndash172 1996

[129] S K Prakash T A Cormier A E McCall et al ldquoLoss ofholocytochrome c-type synthetase causes the male lethality ofX-linked dominant microphthalmia with linear skin defects(MLS) syndromerdquo Human Molecular Genetics vol 11 no 25pp 3237ndash3248 2002

[130] I Wimplinger M Morleo G Rosenberger et al ldquoMutations ofthe mitochondrial holocytochrome c-type synthase in X-linkeddominant microphthalmia with linear skin defects syndromerdquoTheAmerican Journal of HumanGenetics vol 79 no 5 pp 878ndash889 2006

[131] S Kiryu-Seo KGamo T Tachibana K Tanaka andHKiyamaldquoUnique anti-apoptotic activity of EAAC1 in injured motorneuronsrdquoTheEMBO Journal vol 25 no 14 pp 3411ndash3421 2006

[132] R Lill R A Stuart M E Drygas F E Nargang and W Neu-pert ldquoImport of cytochrome c heme lyase into mitochondriaa novel pathway into the intermembrane spacerdquo The EMBOJournal vol 11 no 2 pp 449ndash456 1992

[133] H Steiner A Zollner A Haid W Neupert and R LillldquoBiogenesis of mitochondrial heme lyases in yeast Importand folding in the intermembrane spacerdquo Journal of BiologicalChemistry vol 270 no 39 pp 22842ndash22849 1995

[134] A Zollner G Rodel and A Haid ldquoMolecular cloning andcharacterization of the Saccharomyces cerevisiae CYT2 geneencoding cytochrome-c1-heme lyaserdquo European Journal of Bio-chemistry vol 207 no 3 pp 1093ndash1100 1992

[135] V Corvest D A Murrey D G Bernard D B Knaff B Guiardand P PHamel ldquoc-Type cytochrome assembly in Saccharomycescerevisiae a key residue for apocytochrome c1lyase interactionrdquoGenetics vol 186 no 2 pp 561ndash571 2010

[136] V Corvest D A Murrey M Hirasawa D B Knaff B Guiardand P P Hamel ldquoThe flavoprotein Cyc2p a mitochondrialcytochrome c assembly factor is a NAD(P)H-dependent haemreductaserdquo Molecular Microbiology vol 83 no 5 pp 968ndash9802012

[137] D G Bernard S Quevillon-Cheruel S Merchant B Guiardand P P Hamel ldquoCyc2p a membrane-bound flavopro-tein involved in the maturation of mitochondrial c-typecytochromesrdquo Journal of Biological Chemistry vol 280 no 48pp 39852ndash39859 2005

[138] J G Kleingardner and K L Bren ldquoComparing substratespecificity between cytochrome cmaturation and cytochrome cheme lyase systems for cytochrome c biogenesisrdquo Metallomicsvol 3 no 4 pp 396ndash403 2011

[139] C Travaglini-Allocatelli S Gianni VMorea A Tramontano TSoulimane and M Brunori ldquoExploring the cytochrome c fold-ing mechanism cytochrome c552 fromThermus thermophilusfolds through an on-pathway intermediaterdquo Journal of BiologicalChemistry vol 278 no 42 pp 41136ndash41140 2003

[140] L Zhang and L Guarente ldquoHeme binds to a short sequencethat serves a regulatory function in diverse proteinsrdquoTheEMBOJournal vol 14 no 2 pp 313ndash320 1995

[141] S Hira T Tomita T Matsui K Igarashi and M Ikeda-SaitoldquoBach1 a heme-dependent transcription factor reveals pres-ence of multiple heme binding sites with distinct coordinationstructurerdquo IUBMB Life vol 59 no 8-9 pp 542ndash551 2007

[142] T K Kuhl A Wiszligbrock N Goradia et al ldquoAnalysis of Fe(III)heme binding to cysteine-containing heme-regulatory motifs

in proteinsrdquo ACS Chemical Biology vol 8 no 8 pp 1785ndash17932013

[143] R L Moore J M Stevens and S J Ferguson ldquoMitochondrialcytochrome c synthase CP motifs are not necessary for hemeattachment to apocytochrome crdquo FEBS Letters vol 585 no 21pp 3415ndash3419 2011

[144] S Gianni M Brunori and C Travaglini-Allocatelli ldquoRefoldingkinetics of cytochrome c551 reveals a mechanistic differencebetween urea and guanidinerdquo Protein Science vol 10 no 8 pp1685ndash1688 2001

[145] S Gianni C Travaglini-Allocatelli F Cutruzzola M BrunoriM C R Shastry and H Roder ldquoParallel pathways incytochrome c551 foldingrdquo Journal of Molecular Biology vol 330no 5 pp 1145ndash1152 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Scientifica 17

[128] L Schaefer A Ballabio and H Y Zoghbi ldquoCloning andcharacterization of a putative human holocytochrome c-typesynthetase gene (HCCS) isolated from the critical region formicrophthalmia with linear skin defects (MLS)rdquoGenomics vol34 no 2 pp 166ndash172 1996

[129] S K Prakash T A Cormier A E McCall et al ldquoLoss ofholocytochrome c-type synthetase causes the male lethality ofX-linked dominant microphthalmia with linear skin defects(MLS) syndromerdquo Human Molecular Genetics vol 11 no 25pp 3237ndash3248 2002

[130] I Wimplinger M Morleo G Rosenberger et al ldquoMutations ofthe mitochondrial holocytochrome c-type synthase in X-linkeddominant microphthalmia with linear skin defects syndromerdquoTheAmerican Journal of HumanGenetics vol 79 no 5 pp 878ndash889 2006

[131] S Kiryu-Seo KGamo T Tachibana K Tanaka andHKiyamaldquoUnique anti-apoptotic activity of EAAC1 in injured motorneuronsrdquoTheEMBO Journal vol 25 no 14 pp 3411ndash3421 2006

[132] R Lill R A Stuart M E Drygas F E Nargang and W Neu-pert ldquoImport of cytochrome c heme lyase into mitochondriaa novel pathway into the intermembrane spacerdquo The EMBOJournal vol 11 no 2 pp 449ndash456 1992

[133] H Steiner A Zollner A Haid W Neupert and R LillldquoBiogenesis of mitochondrial heme lyases in yeast Importand folding in the intermembrane spacerdquo Journal of BiologicalChemistry vol 270 no 39 pp 22842ndash22849 1995

[134] A Zollner G Rodel and A Haid ldquoMolecular cloning andcharacterization of the Saccharomyces cerevisiae CYT2 geneencoding cytochrome-c1-heme lyaserdquo European Journal of Bio-chemistry vol 207 no 3 pp 1093ndash1100 1992

[135] V Corvest D A Murrey D G Bernard D B Knaff B Guiardand P PHamel ldquoc-Type cytochrome assembly in Saccharomycescerevisiae a key residue for apocytochrome c1lyase interactionrdquoGenetics vol 186 no 2 pp 561ndash571 2010

[136] V Corvest D A Murrey M Hirasawa D B Knaff B Guiardand P P Hamel ldquoThe flavoprotein Cyc2p a mitochondrialcytochrome c assembly factor is a NAD(P)H-dependent haemreductaserdquo Molecular Microbiology vol 83 no 5 pp 968ndash9802012

[137] D G Bernard S Quevillon-Cheruel S Merchant B Guiardand P P Hamel ldquoCyc2p a membrane-bound flavopro-tein involved in the maturation of mitochondrial c-typecytochromesrdquo Journal of Biological Chemistry vol 280 no 48pp 39852ndash39859 2005

[138] J G Kleingardner and K L Bren ldquoComparing substratespecificity between cytochrome cmaturation and cytochrome cheme lyase systems for cytochrome c biogenesisrdquo Metallomicsvol 3 no 4 pp 396ndash403 2011

[139] C Travaglini-Allocatelli S Gianni VMorea A Tramontano TSoulimane and M Brunori ldquoExploring the cytochrome c fold-ing mechanism cytochrome c552 fromThermus thermophilusfolds through an on-pathway intermediaterdquo Journal of BiologicalChemistry vol 278 no 42 pp 41136ndash41140 2003

[140] L Zhang and L Guarente ldquoHeme binds to a short sequencethat serves a regulatory function in diverse proteinsrdquoTheEMBOJournal vol 14 no 2 pp 313ndash320 1995

[141] S Hira T Tomita T Matsui K Igarashi and M Ikeda-SaitoldquoBach1 a heme-dependent transcription factor reveals pres-ence of multiple heme binding sites with distinct coordinationstructurerdquo IUBMB Life vol 59 no 8-9 pp 542ndash551 2007

[142] T K Kuhl A Wiszligbrock N Goradia et al ldquoAnalysis of Fe(III)heme binding to cysteine-containing heme-regulatory motifs

in proteinsrdquo ACS Chemical Biology vol 8 no 8 pp 1785ndash17932013

[143] R L Moore J M Stevens and S J Ferguson ldquoMitochondrialcytochrome c synthase CP motifs are not necessary for hemeattachment to apocytochrome crdquo FEBS Letters vol 585 no 21pp 3415ndash3419 2011

[144] S Gianni M Brunori and C Travaglini-Allocatelli ldquoRefoldingkinetics of cytochrome c551 reveals a mechanistic differencebetween urea and guanidinerdquo Protein Science vol 10 no 8 pp1685ndash1688 2001

[145] S Gianni C Travaglini-Allocatelli F Cutruzzola M BrunoriM C R Shastry and H Roder ldquoParallel pathways incytochrome c551 foldingrdquo Journal of Molecular Biology vol 330no 5 pp 1145ndash1152 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology