Crystal structure of auracyanin, a “blue” copper protein from the green thermophilic...

doi:10.1006/jmbi.2000.4201 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 306, 47±67

Crystal Structure of Auracyanin, a ``Blue'' CopperProtein from the Green Thermophilic PhotosyntheticBacterium Chloroflexus aurantiacus

Charles S. Bond1, Robert E. Blankenship2, Hans C. Freeman1,3*J. Mitchell Guss1*, Megan J. Maher1, Fabiyola M. Selvaraj2

Matthew C. J. Wilce1 and Katrina M. Willingham1

1Department of BiochemistryUniversity of Sydney, NewSouth Wales, 2006, Australia2Department of Chemistry andBiochemistry, Arizona StateUniversity, Tempe, Arizona85287-1604, USA3School of Chemistry,University of Sydney, NewSouth Wales 2006, Australia

Present addresses: C. S. Bond, DeBiochemistry, University of DundeeScotland; M. C. J. Wilce, DepartmenUniversity of Western Australia, NeAustralia.

Abbreviations used: AdAz, Alcalazurin; BCB, blue Cu-binding protethaliana; CaAc-A, Chloro¯exus auranCaAc-B, Chloro¯exus aurantiacus aurcucumber basic protein (cucumbercucumber peeling cupredoxin; DPI,precision indicator; ESU, estimateduncertainty; Mc, mavicyanin; PBP,protein from pea pods; PoPc, poplaspinach basic protein (spinach planumecyanin.

E-mail addresses of the [email protected];[email protected]

0022-2836/01/010047±21 $35.00/0

Auracyanin B, one of two similar blue copper proteins produced by thethermophilic green non-sulfur photosynthetic bacterium Chloro¯exus aur-antiacus, crystallizes in space group P6422 (a � b � 115.7 AÊ , c � 54.6 AÊ ).The structure was solved using multiple wavelength anomalous dis-persion data recorded about the CuK absorption edge, and was re®nedat 1.55 AÊ resolution. The molecular model comprises 139 amino acid resi-dues, one Cu, 247 H2O molecules, one Clÿ and two SO4

2ÿ. The ®nalresidual and estimated standard uncertainties are R � 0.198,ESU � 0.076 AÊ for atomic coordinates and ESU � 0.05 AÊ for CuÐligandbond lengths, respectively. The auracyanin B molecule has a standardcupredoxin fold. With the exception of an additional N-terminal strand,the molecule is very similar to that of the bacterial cupredoxin, azurin.As in other cupredoxins, one of the Cu ligands lies on strand 4 of thepolypeptide, and the other three lie along a large loop between strands 7and 8. The Cu site geometry is discussed with reference to the aminoacid spacing between the latter three ligands. The crystallographicallycharacterized Cu-binding domain of auracyanin B is probably tethered tothe periplasmic side of the cytoplasmic membrane by an N-terminal tailthat exhibits signi®cant sequence identity with known tethers in severalother membrane-associated electron-transfer proteins.

# 2001 Academic Press

Keywords: auracyanin; copper protein; electron transfer; Chloro¯exus;photosynthesis
*Corresponding authors
partment of, Dundee DD1 5EH,t of Pharmacology,dlands, WA 6907,

igenes denitri®cansin from Arabidopsistiacus auracyanin A;acyanin B; CBP,plantacyanin); CPC,dffraction datastandard

putative blue Cur plastocyanin; SBP,tacyanin); Uc,

ding authors:

Introduction

Auracyanin is a ``blue'' single-copper protein(cupredoxin) produced by the green, ®lamentous,thermophilic, phototropic bacterium Chloro¯exusaurantiacus (Trost et al., 1988; McManus et al.,1992). Chloro¯exus is a member of the Chloro¯exa-ceae (Pierson & Castenholz, 1992), a family of bac-teria identi®ed by 16 S RNA analysis as one of theearliest branches of the evolutionary tree in thebacterial domain (Woese, 1987). Chloro¯exusoccupies an important position in current hypoth-eses concerning the evolution/origin of photosyn-thesis (Blankenship, 1992; Nitschke et al., 1998). Allother anoxygenic photosynthetic bacteria contain asoluble iron protein as an electron-transfer agent;Chloro¯exus lacks such an iron protein. Instead,electron transfer is mediated by a small blue cop-per protein, which is thought to ful®l the electron

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48 Auracyanin from Chloro¯exus aurantiacus

transfer role in much the same way as plastocyanindoes in oxygen-evolving photosynthetic organisms.The fact that at least one current survivor of theChloro¯exus family contains a copper protein butno iron protein suggests that the electron-transfersystem developed at a time when the release ofoxygen into a previously anoxic atmosphere hadmade iron less bio-available and copper more bio-available (due to the oxidation of soluble Fe(II)salts to insoluble Fe(OH)3, and insoluble Cu(I) sul-®des to soluble Cu(II) salts).

The photosynthetic apparatus of Chloro¯exus isby no means fully characterized. Many aspects ofthe current description of the electron-transportmechanism are based on comparisons with purplephotosynthetic bacteria, where extensive functionaland structural studies are available (Feick et al.,1995). In Chloro¯exus, photosynthesis appears to bea cyclic process (Figure 1). (i) Photons are absorbedin peripheral antenna complexes known as chloro-somes and the energy is transferred to the reactioncenter where electron transfer takes place(Blankenship et al., 1995). (ii) An electron in a``special pair'' of bacteriochlorophyll molecules,P870, is promoted to an excited state, and is thentransferred successively to a metal-free bacterio-chlorophyll (bacteriopheophytin) (Becker et al.,1991), a membrane-diffusible quinone (menaqui-none) (Vasmel & Amesz, 1983; Mulkidjanian et al.,1998), and a putative cytochrome bc1 complex. Allthese steps take place within the cytoplasmic mem-brane that separates the cytoplasm from the peri-plasm. (iii) In the ®nal step, the electron is returnedfrom the membrane-bound cytochrome bc1 complexvia the periplasm to the reaction center. The ulti-mate recipient of the electron in the reaction centeris the P870� special pair of bacteriochlorophyll mol-ecules, but the primary electron acceptor is thoughtto be a tetraheme cytochrome c554 molecule(Freeman & Blankenship, 1990). The most likelyagent for this ®nal electron-transfer step through

Figure 1. The photosynthetic electron-transfer cycle inChloro¯exus aurantiacus (adapted from Blankenship,1994).

the periplasm is a small metalloprotein. In purplebacteria, such an agent has been identi®ed as a sol-uble c-type cytochrome (Meyer & Donohue, 1995)or a high-potential iron protein, HiPIP (Menin et al.,1998). Neither a soluble cytochrome nor a HiPIPhas been found in Chloro¯exus, and the ®nal elec-tron-transfer step has been ascribed to auracyanin(McManus et al., 1992). In cyanobacteria, algae andhigher plants, an analogous electron-transfer step isperformed by the small, soluble, blue copper pro-tein, plastocyanin, which accepts an electron fromphotosystem II via a membrane-bound cytochromebf complex, and transports it to a P700� chloro-phyll-protein complex in photosystem I.

There is strong biochemical evidence that aura-cyanin is a peripheral membrane protein attachedto the periplasmic side of the cytoplasmic mem-brane. Cell rupture and ultracentrifugation yieldno detectable auracyanin in the mother liquorunless the membranes are washed with a salt or asalt-detergent mixture (McManus et al., 1992). Theattachment of auracyanin to the membrane mustbe suf®ciently ¯exible to permit electron accep-tance from a membrane-bound cytochrome bc com-plex, followed by electron donation to amembrane-bound reaction center. The distanceover which electrons have to be transferred is stilluncertain. We adduce below circumstantial evi-dence that the soluble part of the auracyanin mol-ecule is tethered to the membrane by a substantialN-terminal polypeptide tail. The tail is present inthe gene sequence, but is lost during the isolationof the soluble electron-transfer domain.

The need for such a tether arises because thephotosynthetic apparatus in Chloro¯exus is orga-nized in islands on the cytoplasmic membrane.The reaction center complexes are almost certainlylocated in a part of the membrane underneath ordirectly adjacent to the chlorosome from whoseantenna system they receive their energy(Blankenship et al., 1995). Thus, the need for elec-tron carriers that interact with the reaction center isrestricted to small (roughly 30 nm � 100 nm)islands of membrane. Large areas of membranehave no requirement for photosynthetic electrontransfer at all. In such a ``patchy'' type of electron-transport organization, electron carriers are notrequired uniformly throughout the periplasmicspace. Free diffusion of auracyanin within the peri-plasmic space would be uneconomical.

Similar logic favoring a non-uniform distributionof tethered electron carriers has previously beenapplied in the case of the green sulfur photosyn-thetic bacteria (Oh-oka et al., 1998). These organ-isms have a chlorosome antenna system similar tothat in Chloro¯exus (Blankenship et al., 1995).Electrons are transferred from a cytochrome bccomplex to the reaction center by a tethered, yetmobile, carrier, cytochrome cz.

A surprising complication is that Chloro¯exusproduces two distinct but related forms of auracya-nin. The functional signi®cance of the presence oftwo auracyanins is still unclear. The two proteins

Auracyanin from Chloro¯exus aurantiacus 49

have been labelled auracyanin A and auracyaninB (McManus et al., 1992). In the present work weuse the abbreviations CaAc-A and CaAc-B forthese proteins. CaAc-B is further sub-divided intoforms B1 and B2 as a result of differential proces-sing of the N-terminal segment mentioned above.CaAc-A (13.9 kDa, 139 residues), CaAc-B1(15.6 kDa, 155 residues) and CaAc-B2 (14.8 kDa,147 residues) have been sequenced chemically(McManus et al., 1992; Van Driessche et al., 1999).The complete gene sequences for CaAc-A (R. E. B.,M. Lince & R. Hiller, unpublished results) andCaAc-B (Lopez et al., 1996) correspond to 162 and235 residues, respectively. It follows that the formsof CaAc-A, CaAc-B1 and CaAc-B2 that are nor-mally isolated have lost 23, 80 and 88 N-terminalresidues, respectively. CaAc-B samples are in factcharacteristically heterogeneous.

We here report the crystal structure of an evenshorter CaAc-B protein comprising 140 amino acidresidues. Evidently, the 15 N-terminal residues inthe DNA-derived sequence of CaAc-B1 (or sevenresidues in the case of B2) were cleaved during theisolation and/or puri®cation of the protein. Thecleaved residues include Thr (ÿ2), which has beenidenti®ed as the attachment site of a polysaccaridecomponent (McManus et al., 1992). The sequence ofthis 140-residue form of CaAc-B, as determined bychemical amino acid sequence analysis (McManuset al., 1992; Van Driessche et al., 1999), is 38 % iden-tical with that of CaAc-A (Figure 2).

Figure 2. Alignment of the sequences of auracyanins A andue numbers correspond to the CaAc-B gene product whoseright of residue 1, the level of sequence identity between Aing residues in CaAc-B are denoted by ~. The sequence lathat the N-terminal sequence of B2 begins at ^. In the geneby 95 additional residues, i.e. residue 1 in the crystal structur

Some properties of CaAc-A and CaAc-B arecompared with the properties of three other wellcharacterized cupredoxins in Table 1. Thereduction potential E0 of both auracyanins A andB, 240 mV, is relatively low. Among the cupre-doxins listed by Sykes (1991), only Rhus verniciferastellacyanin has a lower E0 value of 184 mV. BothCaAc-A and CaAc-B have typical ``blue-Cu''electronic spectra with an intense charge-transferband at �600 nm and additional bands at�450 nm and �730 nm. The ratio e1/e2 betweenthe intensities of the 450 nm and 600 nm bands ishigher for CaAc-A than for CaAc-B. The values ofe1/e2 lead to the prediction of a rhombic X-bandEPR spectrum for the former and an axial X-bandEPR spectrum for the latter (Andrew, 1994), inagreement with observation (Table 1).

Results

General description, accuracy and precision ofthe crystallographic model

The molecular structure of CaAc-B is shown inFigure 3. As indicated above, the form of CaAc-Breported here comprises 140 residues, correspond-ing to residues 96-235 of the published genesequence of CaAc-B (Lopez et al., 1996), and resi-dues 16-155 of the protein sequence of CaAc-B1(McManus et al., 1992; Van Driessche et al., 1999).The residue labels have been changed to 1-140 in

d B (adapted from Van Driessche et al., 1999). The resi-crystal structure is reported here. For the residues to the

(139 residues) and B (140 residues) is 38 %. The Cu-bind-belled B represents both CaAC-Bl and CaAc-B2, exceptsequence of CaAc-B, the residue labelled 1 is preceded

e is residue 96 in the gene sequence.

Table 2. Re®nement details

Final residual R 0.198 (0.267)a

Final free residual Rfree 0.233 (0.285)a

Number (%) of reflections in Rfree test set 927 (3 %)Number of independent reflections 28,390Resolution range (AÊ ) 8.0-1.55Completeness for range (%) 98Number of non-H atoms, protein 1,037Number of non-H atoms, solvent 247Number of non-H atoms, Clÿ, SO4

2ÿ 11r.m.s. deviations from ideal valuesb

Bond lengths (AÊ ) 0.017Bond-angle distances (AÊ ) 0.018Intra-planar 1-distances (AÊ ) 0.025

Estimated standard uncertainties b, c

Coordinates, based on residual R (AÊ ) 0.077Coordinates, based on free residual Rfree (AÊ ) 0.079Coordinates, based on maximum likelihood (AÊ ) 0.035B-factors, based on maximum likelihood (AÊ 2) 1.039Mean temperature factors (AÊ 2)All atoms 20.6Protein atoms 16.5Protein main-chain atoms 15.2Cu atom 13.9All atoms of Cu-binding residues 12.5Atoms coordinated to Cu 12.1Clÿ 18.5Solvent atoms 37.3SO4

2ÿ on special position 39.6SO4

2ÿ in general position 49.1

a The value in parenthesis is the residual in the highest-reso-lution bin.

b Calculated using REFMAC (Collaborative ComputationalProject, No. 4, 1994).

c The estimated standard uncertainties (ESUs) were calcu-lated as the Cruickshank diffraction precision indicator(Cruickshank, 1999).

Table 1. Some properties of auracyanin compared with other cupredoxins

Property Auracyanin Aa Auracyanin Ba Plastocyaninb Azurinc CBPd

E0F mV (pH) 240 (8.0) 240 (8.0) 379 (7.0) 276 (7.0) 306(7.0)

l1 (nm) 453 457 460 460 448e1 (Mÿ1cmÿ1) 920 520 590 580 1240l2 (nm) 596 600 597 619 593e2 (Mÿ1cmÿ1) 3000 4500 4900 5100 2900e1/e2 0.31 0.12 0.12 0.11 0.43X-Band EPR Rhombic Axial Axial Axial Rhombicgz (�gjj) 2.21 - 2.23 2.26 2.21gx 2.02 - 2.05 2.06 2.02gy 2.06 - 2.05 2.06 2.08Ajj(10ÿ4 cmÿ1) 47 - 63 60 55

a Em (cited as E0F), l values and e values for CaAc-A and CaAc-B from McManus et al. (1992). Qualitative descriptions of EPR

derived from spectra in the same reference. EPR parameters for CaAc-A from Trost et al. (1988).b E0

F for poplar plastocyanin from McLeod et al. (1996). l and e values from Han et al. (1993). EPR data from Fee (1975).c E0

F and spectroscopic data for Alcaligenes denitri®cans azurin from Ainscough et al. (1987)d E0

F for cucumber basic protein from Battistuzzi et al. (1997). Other literature values: Em � 317 at pH 6.8 (Murata et al., 1982),E0

F � 340(�10) mV at 4.0 < pH < 8.5 (Nersissian et al., 1985). l and e values from Sakurai et al. (1986). Other literature values:l � 443, 597 nm, e � 2030, 3400 Mÿ1 cmÿ1 (Murata et al, 1982), l � 440, 595 nm, e � 1600, 2800 Mÿ1 cmÿ1 (Nersissian et al., 1998).EPR data from Colman et al. (1977). Similar values reported by Nersissian et al. (1998).


keeping with software and data-deposition require-ments. The N-terminal residue Ala1 was notlocated in electron density, so that only residues2-140 are included in the ®nal model. Nine side-chains were modelled with dual conformations. Inaddition to the protein atoms, 247 solventmolecules, one Clÿ and two SO4

2ÿ were identi®ed.Criteria for accuracy and precision are listed in

Table 2, which also includes values of the disorderparameter (temperature factor) B averaged overvarious groups of atoms. The estimated standarduncertainty (ESU), evaluated as the Cruickshankdiffraction data precision indicator (Cruickshank,1999), is sw(x) � 0.076 AÊ for an average atom(mean B � 20.6 AÊ 2), and 0.046 AÊ for the donoratoms at the Cu site (mean B � 12.1 AÊ 2).

The polypeptide fold

The core of the molecule is a sandwich of twob-sheets, formed by eight polypeptide strands in atypical cupredoxin fold (Figure 4). We shall refer tothe segments of polypeptide that contribute to theb-sandwich as ``strand 1'', ``strand 2``, etc., and tothe loops between them as ``loop 1-2``, ``loop 2-3``,etc. Details of the secondary structure elements aresummarized in Table 3. In comparison with othercupredoxins, the polypeptide fold has two distinc-tive features:

(i) Strand 1 is preceded by an N-terminal tail of14 residues. We shall call this tail strand 0. InFigure 3, strand 0 starts in a niche between strand6 and the non-helical part of strand 5, and mean-ders across the surface of the b-sandwich near its``southern'' end until it joins strand 1. It formspeptide-peptide H bonds both to strand 5[(Gly6)N � � �O(Trp96), (Gly6)O � � �N(Trp96), (Ser8)N � � �O(Ala93), (Asn9)N � � �O(Leu94)] and to strand6 [(Asn9)O � � �N(R111), (Val11)N � � �O(Thr109)].

The segment of strand 0 between residues 2 and 5is in an extended conformation, but is not shownas b in Figure 3, since it does not comply fullywith the inter-strand hydrogen-bonding criteriadescribed by Kabsch & Sander (1983).

Figure 3. Three views of the molecular structure ofChloro¯exus aurantiacus CaAc-B including the Cu atomand ligands (His57, Cys 122, His 127, Met132). Parts ofall eight polypeptide strands are in an extended confor-mation. Arrows indicate those segments that are Hbonded to an adjacent strand, as required by the Kabsch& Sander (1983) de®nition of b character. The represen-tation of the polypeptide backbone is color-rampedfrom blue at the N terminus to red at the C terminus.Residues Ala1 (not located by the structure analysis)and A1a2 (disordered) are omitted. Figure drawn withMOLSCRIPT (Kraulis, 1991). Top and Center, Ribbon-diagrams, related by a rotation of l80 � about the verticalaxis. Bottom, Ca diagram with every tenth residuemarked by a black sphere.


(ii) Strand 5, which is the region of greatestvariability among cupredoxins, is preceded by fourturns of a-helix, a single turn of 310 helix and anirregular segment. The helical and irregular seg-ments together form a large ¯ap, which lies outsidethe b-sandwich and in contact with it.

The Cu site

The coordination of the Cu atom in CaAc-B istypical of blue Cu protein sites. The Cu-bindinggroups are the side-chains of His57, Cys122,His127 and Met132. The `ùpstream'' ligand, His57,lies at the beginning (the ``northern'' end) of strand4. The remaining three ligands are located on loop7-8, the large loop between strands 7 and 8. TheCu atom is buried about 6 AÊ below the surface ofthe molecule, the shortest distance to the surfacebeing via the coordinated imidazole ring of thenorthern histidine residue, His127. The Cd2-Ne2

edge of this imidazole ring remote from the Cuatom is the only part of the Cu site that is exposedto the solvent. Details of the distorted trigonal-pyramidal Cu site geometry are shown in Table 4.

Both His57 and His127 are coordinated to theCu atom via their Nd1(imidazole) atom. In eachcase the Ne2(imidazole) atom is involved in aninteresting interaction. The Ne2 atom of His57 is H-bonded to an internal H2O molecule, Wat157(2.8 AÊ ), which in turn is H-bonded to the O(pep-tide) atoms of Ala24 (2.6 AÊ ) and Ala27 (2.7 AÊ ).These residues lie at the end of strand 1 and thebeginning of strand 2, respectively. Neither theimidazole ring of His57 nor the H-bonded solventatom Wat157 is accessible from the solvent. In thecase of His127, the Ne2 atom forms a 3.3-AÊ H bondto a Clÿ located on a crystallographic 2-fold axis(see below).

Other important H bonds at the Cu site includetwo N-H � � �S interactions, which link the Sg atomof Cys122 to the N(peptide) atoms of Asn58 (3.4 AÊ )and Phe124 (3.4 AÊ ), respectively. These H bondsare part of a network that stabilizes the Cu site(Table 5). Three of the interactions in this networklink Asn58, the residue adjacent to the upstreamhistidine ligand His57, to the cysteine ligandCys122 and its adjacent residue Thr123. The occur-rence of an asparagine residue immediately afterthe upstream histidine ligand, and the formation ofH bonds from that asparagine to the cysteineligand and to its neighboring residue, are charac-teristic of blue Cu sites (Adman, 1991). A furtherproperty that CaAc-B shares with other blue Cuproteins is that the Cu atom and the atoms of theside-chain of Cys122, Cu-Sg-Cb-Ca-N, are approxi-mately co-planar (Han et al., 1991).

A hydrophobic patch surrounding thenorthern histidine

As in other cupredoxins (Adman, 1991), theexposed edge of the imidazole ring of the down-stream (northern) histidine ligand His127 is sur-

Figure 4. Schematic of the topology (polypeptide fold)of CaAc-B, adapted from a similar diagram for azurin(Guss et al., 1988). The numerals identify the strands ofthe polypeptide backbone. The broken line represents alarge loop at the southern end of the auracyanin mol-ecule, culminating in the additional N-terminal strand(strand 0). The solid circles represent the locations of thefour Cu-binding residues.


rounded by predominantly hydrophobic residues(Figure 5(a)). Eight of these residues are located onloops at the northern end of the molecule (Leu28on loop 0-1, Leu53 and Val55 on loop 3-4, andPhe124, Pro125, Gly126, Leu129 and Alal30 onloop 7-8). Three more residues with side-chainscontributing to the hydrophobic patch lie on thehelical segment of the irregular strand 5 (Ala80,Leu83 and Phe84).

In the crystal, the hydrophobic region of eachCaAc-B molecule faces the corresponding region ofa second molecule. The molecules are related by a

2-fold symmetry axis, so that the contacts betweentheir hydrophobic patches occur in pairs. There areno direct H bonds, but solvent molecules provide abridge in a pair of N(Leu28)-H � � �O-H � � �O(Leu53)interactions. In addition, the exposed His127 imi-dazole rings of the two CaAc-B molecules arelinked by a pair of Ne2-H � � �Cl- H-bonds (�3.3 AÊ )via a Cl- on the 2-fold axis. The northern histidineresidue of each molecule lies in a depression of thehydrophobic surface, so that the (His127)Ne2-H � � �Cl- � � �H-Ne2(His 127) bridge is surrounded bya ring of non-bonded intermolecular contactsbetween non-polar side-chains. The annulus wherethe contacts occur represents a solvent-inaccessiblearea of 581 AÊ 2 in each molecule.

A polar region on the molecular surface

An unusual feature of the CaAc-B molecule is aregion of the molecular surface where the side-chains of two serine and ®ve threonine residuesare exposed to the solvent (Figure 5(b)). Such aconcentration of exposed -OH groups does notappear to occur in any other cupredoxin. A facileexplanation for the apparent uniqueness of thepolar patch in CaAc-B is that it is a recognition sitefor a biological redox partner. The crystal structureprovides no evidence for, or against, this hypoth-esis. Only three of the seven polar patch residues(Ser36, Thr117, Thr139) make direct or solvent-bridged H bonded contacts with neighbouringmolecules. Further, CaAc-A cannot have a similarpolar patch, since only three of the residues (Thr33,Ser36, Ser105) are conserved or conservatively sub-stituted in that protein (Figure 2).

Figure 5. (a) The hydrophobicsurface at the northern end ofChloro¯exus aurantiacus CaAc-B. (b)The polar patch on one side of theCaAc-B molecule (Thr33, Ser34,Ser36, Thr117, Thr135, Tbr137,Thr139). The orientation of the aur-acyanin molecule is related to thatin Figure 4 (bottom) by a rotationof �45 � about the vertical axis. (c)The hydrophobic surface at thenorthern end of Alcaligenes denitri®-cans azurin (AdAz). (d) The AdAzmolecule viewed from the sameangle as CaAc-B in (b). The yellowpolar patch in CaAc is absent inAdAz and other azurins. Colourcode: Yellow, Ser/Thr; red, Asp/Glu; blue, Lys/Arg/His; pink, Asn,Gln; green Tyr; gray, non-polar.

Table 3. Secondary structure assignments in auracyanin B

Strand/helix/loop Residues Location Peptide-peptide H bonds

Strand 0 1-14 N-terminal extensionof b-sandwich

Residues 6, 8, 9 to strand 5;residues 9, 11 to strand 6;

Strand 1 15-25 Residues 22-24 to strand 2a;residues 16-23, 25 to strand 3

Loop 1-2 25-28 North endStrand 2a 28-31 Residues 29, 31 to strand 1Kink 2a-2b 31-33 Dog-leg characteristic of

strand 2 in cupredoxinsStrand 2b 33-39 Residues 33-39 to strand 8Loop 2-3 39-41 South endStrand 3 41-52 Even-numbered residues 42-48

and 52 to strand 1; odd-numbered residues 41 and 43-49to strand 3

Loop 3-4 52-54 North endStrand 4 54-62 Odd-numbered residues 57-63 to

strand 5; even-numberedresidues 60-64 to strand 7

Loop 4-5 62-65 South enda-helix 66-77310-helix 80-82Irregular segment 83-94Strand 5 94-101 Residues 93-96 to strand 0;

residues 94, 95, 97, 100 to strand4; residue 101 to strand 6 (solepeptide-peptide interactionbetween strands 5 and 6)

Loop 5-6 101-103 North endStrand 6 103-112 Residue 103 and even-numbered

residues 104-112 to strand 3;residue 112 also to loop 2-3;residues 109, 111 to strand 0

Loop 6-7 112-115 South endStrand 7 115-122 Residues 119, 121 to strand 4;

even-numbered residues 116-122to strand 8

Loop 7-8 122-132 North end. Residues 122, 127,132 bind Cu.

Helix 126-130 Part of loop 7-8Strand 8 132-140 Odd-numbered residues 135-139

to strand 2b; even-numberedresidues 132-138 to strand 7

See Supplementary Material for tables of intramolecular hydrogen bonds.


Discussion

The polypeptide fold of auracyanin B closelyresembles that of azurin

A remarkable feature of the CaAC-B molecule isits close structural homology with azurin. Thepolypeptide backbones have identical topologies,with the exception that CaAc-B has an additionalstrand 0 (Figure 4). A superposition of the struc-tures of Chloro¯exus aurantiacus CaAc-B and Alcali-genes denitri®cans azurin (Baker, 1988) is shown inFigure 6 (the 1.8 AÊ structure of Alcaligenes denitri®-cans azurin is currently the azurin structure re®nedat the highest resolution). The diagram wasobtained by an iterative least-squares minimizationof the differences between the positions ofcorresponding Ca atoms, regions of obviousdifferences being progressively excluded. TheN-terminal strand 0 of CaAc-B, being absent inazurin, was omitted from the calculation. When Ca

atoms whose positions differ by >2.0 AÊ wereexcluded, the r.m.s. difference between the pos-itions of 89 corresponding Ca atoms was found tobe 0.795 AÊ ; if the cut-off distance was reduced to1.0 AÊ , the r.m.s. difference for 65 corresponding Ca

atoms (almost 50 % of the total) was 0.524 AÊ

(Table 6).Contrary to expectations based on sequence

homology alone (Van Driessche et al., 1999), CaAc-B has a much lower level of structural homologywith plastocyanin than with azurin. When theiterative superposition calculations describedabove were repeated for CaAc-B and PoPc (Gusset al., 1992), only 36 pairs of Ca atoms survivedwith a cut-off of 2.0 AÊ , and only 11 pairs of Ca

atoms survived with a cut-off of 1.0 AÊ (Table 6).Not only do fewer pairs of corresponding Ca

atoms have closely similar positions than in thesuperposition with azurin, but even for theseatoms the respective r.m.s. differences, 1.110 AÊ and0.587 AÊ , are signi®cantly larger.

Table 4. The dimensions of the Cu site in auracyanin B and other cupredoxins

Atomsa Plastocyaninb Auracyanin B Azurinc CBPd

A. Bond Lengths (AÊ )Cu-N(His57) 1.91 2.02 2.09 1.93Cu-S(Cys122) 2.07 2.19 2.15 2.16Cu-N(His127) 2.06 2.03 2.00 1.95Cu-S(Metl32) 2.82 2.84 3.11 2.61Cu � � �O(peptide56) 3.89 3.52 3.13 3.85

B. Bond Angles (deg.)N(His57)-Cu -S(Cys122) 132 132 135 138

-N(His127) 97 102 105 99-S(Met132) 88 80 77 83

S(Cys122)-Cu-N(His127) 121 117 119 110-S(Met132) 110 106 107 111

N(His127)-Cu-S(Met132) 101 105 96 112

C. Distance from NNS plane (AÊ )Cu 0.36 0.23 0.13 0.39S(Met132) 3.12 2.91 3.11 2.84

D. Angle between planes (deg.)e

N-Cu-N to S-Cu-S' 81.6 73.2 78.9 69.6`Tetragonal distortion' 0 8.4 2.7 12.0

a Residue numbers refer to auracyanin. The equivalent residues in the other proteins are: plastocyanin, His37, Cys84, His87,Met92; azurin, His46, Cys112, His117, Met121; CBP, His39, Cys79, His84 Met89. The O(peptide) atom is from the residue precedingthe ®rst (upstream) His residue

b Poplar (Populus nigra) plastocyanin (Guss et al., 1992), Protein Data Bank Reference 1PLC.c Alcaligenes denitri®cans azurin (Baker, 1988), Protein Data Bank reference 2AZA. Values averaged over independent molecules.d Cucumber basic protein (plantacyanin from Cucumis sativus) (Guss et al., 1996), Protein Data Bank reference 2CBP.e The angle between the N-Cu-N and S-Cu-S' planes has been proposed as a measure of the ``tetragonal distortions'' of blue Cu

sites (LaCroix et al., 1998). The tetragonal distortion in PoPc is assigned as zero.


Detailed comparisons between auracyanin Band azurin

Inspection of Figure 6 reveals that polypeptidestrands 1-8 (that is, the strands contributing to theb-sandwich) in CaAc-B superpose closely on thecorresponding strands in AdAz. Among the loopsconnecting these strands, only loop 5-6 is effec-tively identical in the two structures. Substantialstructural differences occur at loops 1-2, 2-3, 3-4,6-7 and 7-8. In each case, the structural differencere¯ects a difference between the numbers of resi-dues in the loop.

Table 5. Hydrogen bonds involving Cu-site residues in aurac

Atom X Atom Y

Asn a � 1 N SG Cys bAsn a � 1 ND2 OG Thr b�1Cys b N O Met dThr b � 1 N OD1 Asn a�1Phe b � 2 N SG Cys bHis c N O Phe c-3Tyr c � 1 OH OD1 Asp66Ala d-2 N O Gly c-1Gly d-1 N O Tyr c� 1Met d N O His cLys d� 1 NZ OD1 Asp66

Hydrogen bonds are listed as X-H � � �Y, i.e., X and Y are the pde®ned with respect to the Cu-binding residues His a, Cys b, His c ap

denotes an interaction that is described by the same residue numCaAc-B. * denotes an interaction in which the acceptor O atom belon

There are further signi®cant differences betweenCaAc-B and AdAz at the ¯ap which precedesstrand 5 in both structures (Figure 6). The mostpronounced differences occur at the ®rst turn ofhelix (residues 62-68 in CaAc-B), the northern loopfollowing the last turn of helix (residues 78-86),and the southern end of the irregular segment(residues 90-93, where residue 91 has no equivalentin AdAz). On the other hand, three of the fourturns of helix in the superposed molecules areeffectively identical, the r.m.s. difference being thesame as for the entire ensemble of Ca atomsincluded in the superposition. The fact that the

yanin B

d(X � � �Y) Equivalent interactions inAÊ Popc AdAz CBP

3.4p p p

3.0p p p

3.2p p p

2.9p p p

3.4 -p p

3.0p p p

2.6 - - -2.9

p*

p*

p*

2.9p

-p

2.9p p p

3.2 - - -

roton donor and acceptor, respectively. Residue numbers arend Met d. In CaAc-B, a � 57, b � 122, c � 127 and d � 132.

bers and atom types (peptide-N, peptide-O or side-chain) as ings to His c, instead of Gly c-1 as in CaAc-B

Figure 6. Stereo Ca diagram showing the structure of Chloro¯exus aurantiacus auracyanin B, red, superposed uponthat of Alcaligenes denitri®cans azurin, blue (PDB entry 2AZA). The superposition was optimized iteratively usingLSQMAN (Kleywegt & Jones, 1994). Only corresponding Ca atoms separated by less than 2 AÊ were included in eachminimization cycle.


helix is structurally conserved in CaAc-B andAdAz, while the segments that connect it to therest of the polypeptide are different, suggests thatit has a functional signi®cance. At this stage it isnot clear whether the helix contributes to the stab-ility of the molecule, the charge distribution, orsome other property.

The N-terminal tail (``strand 0``) of CaAc-B,which does not occur in azurin, hides a region ofthe molecular surface which in AdAz is exposed tothe solvent. The surface against which strand 0 ispacked in CaAc-B (at least in the crystalline state)comprises the middle and southern parts of strand5, and the southern end of strand 6. This region isone of those where the level of structural hom-ology with AdAz is particularly high (see above)

In Figure 7, the amino acid sequences of CaAc-Band AdAz are aligned according to the superposi-tion of the structures in Figure 6. The parts of thesequences where there is close structural homologycomprise 64 % of the CaAc-B molecule, and areenclosed in boxes. The overall level of sequence

Table 6. Results of pair-wise superpositions of auracyanin Bof Ca atoms whose positions differ by less than the cut-off dbetween these positions of these Ca atoms. Upper triangle ofCut-off distance 1.0 AÊ

CaAc-B

CaAc-B -AdAz 65 (0.524 AÊ )PoPc 11 (0.587 AÊ )

a Minimization failed. Insuf®cient atoms within 1.0 AÊ cut-off dista

identity is only 24 %, slightly lower than the value26 % obtained (Van Driessche et al., 1999) if thesequences are aligned in the absence of structuralevidence. Nevertheless, there are three segments ofthe structurally aligned molecules where the twosequences are identical or nearly so. These occur atpositions 57-61, 103-110 and 118-136 in CaAc. The®rst of these segments includes the upstream Cu-binding residue His57 and its structurally import-ant neighbour, Asn58; the second segment com-prises strand 6; and the third segment includes theremaining three Cu-binding residues, Cys122,His127 and Met132. The sequence of the strand 6segment is highly conserved in all known azurins,but the reason for sequence conservation is notclear.

The molecular surfaces surrounding the exposedimidazole ring edge of the northern histidineresidue in CaAc-B and AdAz are compared inFigure 5(a) and (c). As in other cupredoxins, thesurfaces are predominantly hydrophobic. The jux-taposition of the hydrophobic surfaces of adjacent

, azurin and plastocyanin. Each entry shows the numberistance. The value in parenthesis is the r.m.s. differencevalues: Cut-off distance 2.0 AÊ . Lower triangle of values:

AdAz PoPc

89 (0.795 AÊ ) 36 (1.110 AÊ )- 40 (1.293 AÊ )

0a -

nce.

Table 7. The fractions (%) of the solvent-accessible sur-face occupied by apolar, polar and charged residues inazurin (AdAz) and auracyanin B (CaAc-B). The calcu-lations were made with the SHOACC option in WHA-TIF (Vriend, 1990)

Fraction (%) of solvent-accessible surfacein

Residue type AdAz CaAc-BApolar 35 44Polar 27 37Charged 39 19

Figure 7. A structure-based alignment of the amino acid sequences of Chloro¯exus aurantiacus auracyanin B (CaAc)and Alcaligenes denitri®cans azurin (AdAz). ~ denotes the Cu-binding residues. Part of the pre-sequence of AdAz isincluded in fainter print. Figure drawn with ALSCRIPT (Barton, 1993). The sequence alignment is based on an opti-mized superposition of the three-dimensional structures (see Figure 6). Boxes enclose residues whose Ca atoms areseparated by less than 2 AÊ in the superposed structures. Arrows and cylinders denote b-strands and helices, respect-ively. Apparent differences in the assignment of residues to the b-strands and helices in the two structures, in regionswhere the boxes indicate that corresponding Ca positions are closely similar, are not signi®cant. They re¯ect differ-ences in inter-strand hydrogen bonding, which is one of the criteria used for the assignment of secondary structureby the Kabsch & Sander algorithm (Kabsch & Sander, 1983).


molecules in the crystals of CaAc-B, and even thepresence of a 2-fold axis between these surfaces,are similar to features in AdAz (see Figure 7 byNorris et al., 1983). In fact, analogous contactsoccur in 25 of the 29 structures of azurin, in theProtein Data Bank, though they do not necessarilyinvolve crystallographic symmetry or an exogen-ous bridging atom.

In other respects, the molecular surfaces ofCaAc-B and AdAz are strikingly different. Thepolar patch along one side of the CaAc-B molecule(Figure 5(b)) is absent in AdAz (Figure 5(d)). Thereare large differences between the fractions of themolecular surface of CaAc-B and AdAz occupiedby apolar, polar and charged residues, respectively(Table 7). In particular, the molecular surface ofCaAc-B is dominated by apolar and polar residues.Charged residues contribute only 19 % to the mol-ecular surface, compared with 39 % in AdAz. Inthe long run, these differences will no doubt befound to be related to the interactions between theproteins and their respective redox partners.

The Cu site in CaAc compared withother cupredoxins

In Table 4, the Cu-site dimensions in CaAc-B arecompared with those in poplar plastocyanin (PoPc),

Alcaligenes denitri®cans azurin (AdAz), and cucum-ber basic protein (CBP). Some physical properties ofthe same three representative cupredoxins areincluded in Table 1. Within the limits of precision,there is little to distinguish the Cu site dimensionsin the four proteins. There are few signi®cant differ-ences among corresponding bond lengths andangles, and most of these involve the axial Metligand. The Cu-S(Met) bond lengths increase inthe order CBP (2.61 AÊ ) < CaAc-B (2.82 AÊ ) � PoPc(2.84 AÊ ) < AdAz (3.11 AÊ ), and some bond anglesinvolving S(Met) are different at the 2s level. Theconformation of the Met side-chain in CaAc-B isall-trans, as in CBP and PoPc; as noted elsewhere,

Figure 8. The Cu site of auracyanin B in the context of other proteins with a blue Cu site. The letters C, H and Mrepresent the Cu-binding Cys, His and Met residues, which lie on the loop between the two C-terminal polypeptidestrands of all blue Cu proteins. The upstream His ligand located on polypeptide strand 4 is omitted. In stellacyanins,M is replaced by Q (Gln). In some laccases and at one blue Cu site of ceruloplasmin, M is replaced by L (a non-ligand Leu). The Cu sites are classi®ed according to the numbers of residues between the C, H and M/Q ligands. Inthis classi®cation, the Cu site of CaAc-B falls into the same category as the Cu sites of phytocyanins, rusticyanin andsulfocyanin. The dashed lines represent N-H � � � S hydrogen bonds from a backbone amide group to the thiolate Satom of the Cys ligand, as found in representative molecules for which crystal structure analyses are available. Theamide groups involved in the N-H � � � S bonds belong to (i) the residue following the upstream His ligand, and (ii)the residue two positions downstream (i.e. towards the C terminus) from the Cys ligand. In the schematics for ami-cyanin, plastocyanin, pseudoazurin and halocyanin, the letter P denotes that the second residue downstream fromthe Cys ligand is a Pro. All the schematics include an aromatic residue upstream (i.e. towards the N terminus) fromthe ligating Cys. If the position of the Cys is denoted by n, there is a Tyr at position (n ÿ 2) in halocyanin andCaAc-B, at position (n ÿ 3) in phytocyanins, at position (n ÿ 4) in sulfocyanin, and at positions (n ÿ 1), (n ÿ 2) and(n ÿ 3) in rusticyanin. Most of the sequences reported for the multi-copper proteins laccase, ascorbate oxidaseand ceruloplasmin have a Trp, Tyr or Phe at position (n ÿ 4). The Figure is based on at least one published crystalstructure for each type of molecule, with the exception of sulfocyanin, uclacyanin and laccase. Sequence data that arenot referenced elsewhere in this paper were obtained from SwissProt.


the Met side-chain in azurin and amicyanin isgauche at Ca-Cb and Cg-Sd (Guss et al., 1996). Azurinremains the only cupredoxin where there is a shortcontact between the Cu atom and an O(peptide)atom.

Attempts to establish correlations between thedimensions in Table 4 and the physical propertiesin Table 1 require caution. First, the estimateduncertainties of the Cu-ligand bond lengths rangefrom 0.05 AÊ to 0.08 AÊ . Second, while there isreasonable agreement among leading workers con-cerning the assignment of blue-Cu electronic struc-tures and spectra, there are unresolved con¯icts

between alternative hypotheses concerning the fac-tors that determine blue-Cu reduction potentials(Randall et al., 2000; Gray et al., 2000; Ryde et al.,2000).

At an empirical level, CaAc-B is one of the threeproteins in Tables 1 and 4 for which a low value ofthe spectroscopic ratio e1/e2 is associated with anaxial EPR spectrum, as expected (Andrew et al.,1994). Blue Cu sites with a low value of e1/e2 arealso predicted to have a small tetragonal distortion,i.e., the angle between the N-Cu-N and S-Cu-S'planes should be close the corresponding angle inplastocyanin (LaCroix et al., 1998). The tetragonal

Figure 9. Sequence-based classi®cation of blue single-Cu proteins. Proteins are grouped according to sequence iden-tity. Horizontal distances are proportional to the number of base-pair changes implied by sequence differences. Nocorrelation with evolutionary processes or time differences is implied. The diagram was compiled using PILEUP(Genetics Computer Group, 1994). The diagram shows that CaAc-A and CaAc-B are distinct from the azurins, butresemble them more closely than any other family of Cu protein. The separation of the auracyanins and azurins fromthe plastocyanins, rusticyanins, halocyanin, sulfocyanin, pseudoazurins and amicyanins occurs at the second branchfrom the origin on the left-hand side of the diagram. Only the phytocyanin sequences diverge sooner from the otherfamilies.


distortion of the Cu site in CaAc-B appears to belarger than this correlation leads one to expect.

The relatively small variations among the corre-sponding Cu site dimensions of different cupre-doxins are remarkable, since there are signi®cantdifferences among the polypeptide segments thatsupport the Cu site. Three of the four Cu ligandsin all cupredoxins lie along a large loop joining thetwo C-terminal polypeptide strands (loop 7-8). Ifcupredoxins are arranged in groups according tothe numbers of residues separating the three Cu-binding residues (Figure 8), it becomes apparentthat the conformation of loop 7-8 must be variablein order to accommodate differences in loop size.In terms of the loop-size criterion (which has beennoted by others, e.g. Sykes (1991)) the Cu site inCaAc-B (C-x-x-x-x-H-x-x-x-x-M) resembles azurin(C-x-x-x-x-H-x-x-x-M) between the ligating Cysand His, and plastocyanin (C-x-x-H-x-x-x-x-M)between the ligating His and Met. At ®rst sight,the inter-ligand intervals are re¯ected in the bondangles at the Cu atom as listed in Table 4(angles S(Cys122)-Cu-N(His127) and S(Cys122)-Cu-S(Met132) in CaAc-B are closer to thecorresponding angles in azurin, whereas angleN(His127)-Cu-S(Met132) is closer to that in plasto-cyanin), but most of the differences betweencorresponding angles are below the 2s level ofsigni®cance.

One reason why the differences among theinter-ligand intervals along loop 7-8 do not lead tolarger differences among the Cu site geometries isthat the cupredoxin Cu site is stabilized by ahighly conserved network of H bonds (Table 5).There is a particularly high level of similaritybetween the H bond networks in CaAc-B and CBP,consistent with the two proteins being grouped

together in Figure 8. It is to be noted that the over-all polypeptide fold of CaAc-B, being identicalwith that of azurin, is substantially different fromthat of CBP.

In the preceding discussion, we have said littleabout the upstream histidine ligand, i.e. His57 inCaAc-B. There is variety among the interactionsinvolving the side-chain of this residue. In CaAc-B,the Ne2 atom of His57 is H bonded to an internalsolvent molecule, Wat157, which in turn isH bonded to the O(peptide) atoms of Ala24 andAla27 on loop 1-2. In AdAz, the Ne2 atom of His47is H bonded to O Asn10 at the north end of strand1 (Baker, 1988). In PoPc, the Ne2 atom of His37 isH bonded to O Ala33 on loop 3-4 (Guss et al.,1992). In CBP and stellacyanin, the Ne2 atoms ofHis39 and His47, respectively, are solvent accessi-ble (Guss et al., 1996; Nersissian et al., 1998) and,regardless of what happens in the crystalline state,must interact with solvent molecules in solution.

Despite the variety of H bond acceptors at theupstream histidine residue, the orientations of theimidazole ring are very similar. In pair-wise super-positions of CaAc-B, AdAz, PoPc and CBP usingthe two Nd1(His) atoms and the S(Cys) atom ofeach Cu site, there is no signi®cant differencebetween the imidazole ring orientations at eitherthe upstream or the northern histidine, as indicatedby the angle of rotation of each imidazole ringabout its Cu-Nd1 bond. This suggests that theH bonds are a consequence, rather than a cause, ofthe imidazole ring orientation: an H bond isformed because an acceptor happens to be in anappropriate position with respect to the histidineNd2 atom, and not because the H bond is a meansof locking the imidazole ring into a favorableorientation. On the other hand, three H bonds


formed by the residue adjacent to the upstreamhistidine residue, an invariant asparagine residue,are highly conserved at cupredoxin Cu sites(Table 5). It is likely that this invariant asparaginestabilizes the position and orientation of theupstream histidine side-chain, and thereby makesan essential contribution to determining the Cu sitegeometry. In other words, the Cu site geometry isin¯uenced more by the interactions of the adjacentasparagine residue than by H bonding at the imi-dazole ring of the upstream histidine residue.

Yet another important feature of H bonding atthe Cu site in CaAc-B is that the cysteine ligand isthe acceptor of two N(peptide)-H � � �Sg(Cys)H bonds. It has been suggested that cupredoxinscan be classi®ed according to the number ofN-H � � �Sg(Cys) H bonds (Guss et al., 1996). Cupre-doxins with one N-H � � �Sg(Cys) H bond have a Cusite whose reduced (CuI) form is pH dependent,since the northern histidine dissociates from theCuI atom at low pH conditions. Below a thresholdpH which is characteristic of each protein, thereduction potential of such cupredoxins increases�60 mV/pH unit, resulting in a reduction or cut-off of redox activity. In cupredoxins with twoN-H � � �Sg(Cys) H bonds, the reduced form of theCu site is stabilized suf®ciently to prevent thedissociation of the northern histidine residue, sothat there is no reason for a reduction in redoxactivity at low pH values. Whether a cupredoxinbelongs to the `òne N-H � � �S'' or ``two N-H � � �S''class depends on the residue two positions down-stream (i.e. towards the C terminus) from the Cysresidue: if that residue is Pro, it cannot form one ofthe two N(peptide)-H � � �S bonds found in cupre-doxins where the residue is non-Pro.{ Accordingto this hypothesis, the redox behavior of auracya-nins should resemble that of azurin and CBP (pHdependent) rather than that of plastocyanin(pH independent). For CaAc-B, this predictionremains to be tested experimentally. However, theelectrochemical behavior of CaAc-B as a functionof pH is likely to be qualitatively similar to that ofCaAc-A, since neither protein has a Pro two resi-dues downstream from the Cys ligand (Figure 2),and both proteins have been reported to have amidpoint potential of 240 mV (Trost et al., 1988;McManus et al., 1992). The midpoint potential ofCaAc-A varies only in the range 240-210 mVbetween pH 4 and pH 9 (Selvaraj, 1999).

The generalization stated above may requiremodi®cation in the light of two exceptions thathave been reported recently. (i) The reduced form

{The blue Cu sites in the multi-Cu proteins laccaseand ascorbate oxidase also have only one N-H � � � Shydrogen bond to the thiolate S atom. In these proteins,the N-H � � � S hydrogen bond contributed by thepeptide group of the residue two positions downstreamfrom the Cys ligand remains intact. The missinghydrogen bond is the one from the N(peptide) atom ofthe residue following the upstream His ligand. Again,the relevant residue is Pro.

of CBP appears to undergo a previously unde-tected protonation at very low pH (Battistuzzi et al.,1997), so that it may be more correct to say thatthe presence of two N-H � � �Sg(Cys) H bonds makesa change in CuI coordination dif®cult but notimpossible (it has still to be determined whetherthe protonation in CBP occurs at the solvent-exposed upstream histidine or the downstreamnorthern histidine residue). (ii) The reduced formof the plastocyanin from a fern, Dryopteris crassirhi-zoma, fails to undergo a detectable change in CuI

coordination in the pH range 4-9. This has beenattributed to a p-p interaction between the sidechains of the ``northern'' histidine and an adjacentphenylalanine (Kohzuma et al., 1999). Thus, theprotonation and dissociation of the northern histi-dine imidazole ring may be inhibited by abnormalfactors, even in proteins where the Sg(Cys) atomforms only one N-H � � �S bond.

Speculations concerning the structure ofauracyanin A

In CaAc-A, the sequence of residues along loop7-8 is C-x-x-x-x-H-x-x-x-M-Q. This makes the pos-ition of CaAc-A in relation to Figure 8 equivocal. Ifthe Cu has the same Cys, His and Met ligands asin CaAc-B, then the interval between the His andMet ligands is different from that in CaAc-B. Inthis case, the polypeptide backbone at the Cu sitemust have different conformations in CaAc-A andCaAc-B, at least between the His and Met ligands.On the other hand, if the intervals between theligands in CaAc-A are the same as in CaAc-B, thenCaAc-A has different Cu ligands, Cys, His andGln. In this case, the Cu site is analogous to that instellacyanin, and CaAc-A is the ®rst cupredoxinoutside the phytocyanin sub-family to have Glninstead of Met at the Cu site.

In summary, the Cu sites in CaAc-A and CaAc-Beither have different backbone conformations ordifferent Cu-binding residues. The available physi-cal data do not enable us to distinguish betweenthese alternatives, but leave no doubt that there aresigni®cant differences between the Cu sites. Thereare substantial qualitative differences between boththe X-band EPR spectra and the resonance Ramanspectra of the two proteins (McManus et al., 1992).The values of the ratio e453/e596 calculated from thepublished UV visible spectra are 0.32 for CaAc-Aand 0.13 for CaAc-B (Table 1), indicating a rhombicCu site for the former and an axial Cu site for thelatter (Andrew et al., 1994). The values of e453/e596

also lead to an expectation that CaAc-A has alower nCu-S stretching frequency, a weaker Cu-S(Cys) bond and a stronger axial interaction thanCaAc-B (Andrew et al., 1994). It remains to be seenwhether the stronger axial interaction in CaAc-A isassociated with a shorter Cu-S(Met) bond or thepresence of O(Gln) instead of S(Met). At the timeof writing, the available evidence favors thehypothesis of a shorter Cu-S(Met) bond: while theX-band EPR spectrum of CaAc-A is qualitatively

Figure 10. Sequence identities between CaAc-B (Lopez et al., 1996), the azurin-like H.8-antigen from Neisseriagonorrhoea (Gotschlich & Seiff, 1987), and cytochrome cy from Rhodobacter capsulatus (Myllykallio et al., 1997).Bold numerals indicate the sequence number of the residue at the end of each segment. Vertical lines indicateidentity, broken lines indicate similarity (A, P; A, G; D, E, N, Q; S, T). The levels of sequence identity (similarity)between these segments are: CaAc-B/H.8-antigen, 55 % (86 %); CaAc-B/cytochrome cy, 45 % (62 %); cytochromecy/H.8-antigen, 52 % (62 %). The gene sequence numbers for CaAc-B have been adjusted to be consistent with thelabels used in the crystal structure (residue 96 in the gene sequence is shown as residue 1 above and in Figure 2).


similar to the spectra of CBP, rusticyanin and stel-lacyanin, the g- and A-values for CaAc-A agreemost closely with those for CBP, and least closelywith those for stellacyanin (Trost et al., 1988). Aclose similarity to the EPR spectrum of the Q99Mmutant of cucumber stellacyanin (Nersissian et al.,1998) is also to be noted. The crystallization andcrystal structure analysis of CaAc-A would be ofinterest.

Structure of auracyanin B in relationto function

The properties of CaAc-B are those of a typicalsoluble, blue Cu, electron-transfer protein: themolecular mass is relatively low (�14 kDa), thereduction potential (240 mV) is somewhat higherthan that of the CuII/CuI couple in aqueoussolution, the Cu site has the appropriate spectro-scopic signature, and the solvent-inaccessible Cuatom has N,N,S,S' ligands and a distorted trigonal-pyramidal geometry.

The coordinated imidazole ring of the northernhistidine residue provides at least one plausibleelectron-transfer pathway between the Cu atomand the molecular surface. The predominanthydrophobicity of the molecular surface sur-rounding the exposed edge of the northern histi-dine imidazole ring is characteristic ofcupredoxin structures. In the case of azurin ithas been inferred that the northern pathway isused for electron transfer, both in electronic self-exchange reactions (Groenewald et al., 1988) andin reactions with physiological redox partners(van de Kamp et al., 1990). A similar pathway isnow favored for electron transfer to plastocyaninfrom cytochrome f (Ubbink et al., 1998; Ejdebacket al., 2000) and from plastocyanin to P700�

(Sigfridsson et al., 1996).The coplanarity of the Cu atom and the side-

chain of Cys122 (see above) would be consistentwith the existence of a second electron-transferpathway (Han et al., 1991), but it is not clear wheresuch a pathway would lead. The residues adjacentto Cys122 are Ile121 and Thr123. Neither is likelyto engage in productive contacts and orbital over-lap with redox partners.

The N-terminal tail: a tether to theperiplasmic membrane?

In the gene sequence, the molecule studied inthe present work is preceded by 95 residues i.e.,the inferred starting Met is at position ÿ95 withrespect to the present structure (Lopez et al., 1996).It is not known whether the 95-residue polypeptidesegment remains intact and is present in the mem-brane in vivo, but we assume that this is the case.A plausible reason for the existence of these 95residues is that they form a protein-membranetether in vivo. Both the organization of the photo-synthetic apparatus in Chloro¯exus, and biochemi-cal evidence that auracyanin is attached to theperiplasmic side of the cytoplasmic membrane (seethe Introduction), make it probable that such atether exists. A hydropathy plot (Kyte & Doolittle,1982) (not shown) for the 95 residues is featureless,except for a signi®cantly hydrophobic segmentbetween residues ÿ65 and ÿ35. Noting that thehydrophobic segment is more than adequate for atrans-membrane helix, we suggest that the tetherthat links CaAc-B to the membrane comprises the35-residue polypeptide between the hydrophobicsegment and the soluble domain. A 35-residuetether would permit the redox-active body of themolecule to move within a 90-120 AÊ hemispherecentered about the point of attachment to themembrane. Both the putative electron donor, acytochrome bc1 complex, and the electron acceptor,cytochrome c554, would have to lie within thishemisphere. However, the tether may be longerthan 35 residues. At the upstream end, it mayinclude some of the residues in the hydrophobicsegment. At the downstream end, it may includepart of strand 0, if that strand becomes detachedfrom the rest of the soluble domain. The estimatedradius of 90-120 AÊ is obtained by a back-of-the-envelope calculation: The Ca-Ca distance betweenadjacent residues is �4.3 AÊ . The fully extendedlength of 35 residues would be �150 AÊ . The strandis unlikely to be fully extended. 60-80 % of max-imum � 90-120 AÊ .

The hypothesis that the pre-sequence of CaAc-Brepresents a tethering domain is supported by acomparison with two other proteins that areknown to be membrane-tethered. One is cyto-chrome cY, an electron carrier to the photosynthetic

Figure 11. A putative scheme for the evolution of photosynthetic organisms.


reaction center in Rhodobacter capsulatus(Myllykallio et al., 1997). In cyt cY, the N-terminaltether is a 98-residue uncleaved sequence, in whichthe ®rst 28 residues comprise a putative mem-brane-anchoring domain. The next 70 residueshave a high proline and alanine content, and areproposed to act as a ¯exible linker between themembrane anchor and the C-terminal cytochromedomain. A similar proline and alanine-rich seg-ment occurs in a membrane-linked, azurin-likeH.8-antigen (Gotschlich & Seiff, 1987).

There is a strong resemblance between the tetherin cytochrome cY, the tether in the H.8-antigen, andthe segment preceding the B-1 protein in the aminoacid sequence of CaAc-B. All three are rich in pro-line and alanine. Parts of the sequences are com-pared in Figure 10. A 33-residue segment of CaAc-B (between residues ÿ26 and �7 with respect tothe crystal structure) has 55 % sequence identityand 86 % sequence similarity with a 28-residuesegment of the H.8-antigen tether, and 45 % iden-tity and 62 % similarity with a 28-residue segmentof the cytochrome cY tether. The similarity of thesequences is consistent with a common (i.e. tether-ing) function. An evolutionary relationship is notnecessarily implied.

CaAc-A cannot have the same membrane anchoras CaAc-B. The gene sequence of CaAc-A (R. E. B.,M. Lince & R. Hiller, unpublished results) has ashorter N-terminal extension of only 23 residues,including a putative transit sequence. AnN-terminal blocking group is thought to anchorthe molecule to the membrane. Although the pre-cise chemical identi®cation of this group has not

yet been made, its mass and the fact that the genesequence includes a Cys residue in this positionstrongly suggest that it is an acetyl-N-cysteine-S-glycerol, which may then be esteri®ed to fattyacids that serve as the membrane anchor (vanDriessche et al., 1999).

Evolutionary relationship of auracyanin toother cupredoxins

What is the evolutionary connection, if any,between auracyanin with a likely function in cyclicelectron transfer in a photosynthetic bacterium,and plastocyanin with a similar function ofelectron transfer between the two photosystemsrequired for oxygenic photosynthesis?

Figure 11 shows a phylogenetic tree of the bluecopper proteins, including the auracyanins. Severalsimilar phylogenetic analyses of blue Cu proteinshave been published, some using sequence infor-mation (Van Driessche et al., 1999; RydeÂn & Hunt,1993; Albomaali et al., 1998) and at least one usingstructural data (Murphy et al., 1997). The overallconclusions are generally in agreement: an ances-tral blue Cu protein diversi®ed into a number offamilies; the azurins, plastocyanins, phytocyaninsand auracyanins form distinct clusters; the pseu-doazurins and amicyanins tend to be groupedclose to the plastocyanins. The grouping of halo-cyanin (from an archaeon (Scharf & Engelhard,1993)) with the plastocyanins (from cyanobacteria,algae and higher plants), and the grouping of sul-focyanin (from another archaeon (Castrasena et al.,1995)) with rusticyanin (from a true bacterium), are


surprising and may be the results of lateral genetransfers. However, the differences between thevarious analyses are suf®ciently large to indicatethat the connection between phylogeny and evol-utionary history is, at best, still tenuous. Figure 11places the auracyanins close to the azurins, inagreement with the structural evidence from thepresent work.

In the light of the above remarks, any discussionof the evolution of blue copper proteins is necess-arily speculative. We here explore the hypothesisthat there are parallels between the evolutionaryhistory of the blue copper proteins and the devel-opment of photosynthesis. We start from the pre-mise, stated by RydeÂn (1984), that the earliest bluecopper proteins functioned in electron-transfer pro-cesses before photosynthesis was invented.

A putative scheme for the evolution of photo-synthetic organisms is shown in Figure 11. Suchlinear schemes are now considered to be an over-simpli®cation, since they make no allowance forthe frequent occurrence of lateral gene transfers(Doolittle, 1999). Subject to this caveat, schemessuch as Figure 11 are useful for providing a frame-work for the discussion of evolutionary processes.

In Figure 11, an ancestral blue copper proteincan plausibly be placed after, but near, the junctionlabeled ``Last Common Ancestor''. The earliestazurins probably evolved in the branch labeled``Bacteria''. In contemporary biota, azurin-contain-ing genera are found in three of the four branchesof the proteobacteria, which include the purplephotosynthetic bacteria (RydeÂn 1984). The ®rstbranch to be associated with an authentic blue Cuprotein other than azurin is the one that leads toChloro¯exus. Thus, the structural similarities anddifferences between CaAc-B and azurin makeintuitive sense. A common ancestral protein orazurin itself evolved to produce a different patternof surface charges (presumably related to therecognition of redox partners) and an additionalN-terminal polypeptide strand (to act as amembrane tether). In the case of CaAc-A, thepolypeptide loops at the Cu site of azurinremained unchanged. In the case of CaAc-B, theloop between the His and Met ligands becameenlarged by one residue. It is possible that that thismutation created an operational advantage, suchas improved interaction with a redox partner.

The next dramatic development in the hypotheti-cal scenario of Figure 11 was the `Ùnknown fusionevent'', which produced organisms having both``Type 1`` and ``Type 2`` reaction centers (not to beconfused with ``Type 1'' and ``Type 2`` Cu sites inCu proteins). We assume that the ``Type 2`` reac-tion center involved in the fusion included an aura-cyanin-type electron-transfer protein. However, therequirement for electron transfer underwentchange. With two types of reaction centers operat-ing in series, there was a need for electron transferfrom one to the other. This function may initiallyhave been performed by a cytochrome, since somecyanobacteria still carry the genetic information to

produce a soluble cytochrome, but invoke it onlyunder conditions of copper deprivation where theproduction of plastocyanin is inhibited (Merchant,1998). In time, possibly as a result of a decreasingbio-availability of Fe and an increasing bio-avail-ability of Cu, plastocyanin emerged as the pre-ferred electron carrier between the two types ofreaction center.

The evolutionary relationship between plasto-cyanin and auracyanin is uncertain. In a purelyfunctional sense, there was a change from an aura-cyanin-like protein to a new electron carrier, plas-tocyanin, which did not need a membrane tether,lacked the N-terminal strand 0, lacked the helix-containing ¯ap preceding strand 5, had the Cysand His ligands at the Cu site separated by a two-residue loop instead of a four-residue loop, and,thanks to a change in H bonding at the Cu siteshad the ability to control electron transfer as afunction of pH. This had the advantage that thereduced form of the electron carrier, CuI-plastocya-nin, was redox-inactive in a low-pH environment.Assuming that the pH in the interior of a cyano-bacterium was low, the electron carried by plasto-cyanin was ``safe'' until the molecule docked withan electron acceptor that was suf®ciently basic todeprotonate the northern histidine residue andthus restore the Cu center to its redox-active form(Guss et al., 1986). In due course, endosymbiosisresulted in the appearance of cyanobacteria as thephotosynthetic apparatus of eukaryotic algae andplants. Plastocyanin continued to evolve as theelectron carrier between photosystems II and I,electrostatic recognition of its electron donor andelectron acceptor being improved by the develop-ment of features such as an acidic patch on themolecular surface. In this speculative odyssey, aur-acyanin represents an important link.

Materials and Methods

Cell growth, protein purificationand characterization.

Chloro¯exus aurantiacus strain J10-¯ was obtained fromthe American Type Culture Collection (ATCC) and wasgrown in modi®ed medium D as described by Pierson &Castenholz (1974). Three-day old cultures were har-vested by centrifugation. The purity of the culture wasroutinely checked by light microscopy.

The puri®cation of auracyanin followed the procedureof McManus et al. (1992), modi®ed to improve the yieldas described by Selvaraj (1999). Cells were disruptedusing sonication, the auracyanin released from the mem-branes by 1 M KCl salt treatment, and the auracyaninsprecipitated using ammonium sulfate fractionation. Thepellet was dialyzed, concentrated by ultra®ltration andapplied to a Sephadex G-100 gel ®ltration column. Thefractions containing auracyanins were chromatographedon DEAE-Sephacel ion-exchange media and eluted with50 mM NaCl. The auracyanin-containing fractions weresubjected to iso-electric focusing (IEF) using a BioRadRotofor IEF apparatus. Two blue-colored fractions withpI values 4.5 and 3.8, respectively, were recovered. Thelatter fraction contained CaAc-B. Final puri®cation was

Table 8. Quality of the diffraction dataa

Data setWavelength

(AÊ )Resolution

(AÊ ) Measurements UniqueCompletenessb

(%) Rsym

Wilson B(AÊ 2)

l1 1.3799 2.40 60149 7527 84.0 (97.2) 0.035 (0.057) 23.9l2 1.3779 2.40 60652 7222 80.6 (92.9) 0.038 (0.056) 24.4l3 1.3876 2.40 60415 7613 85.0 (96.4) 0.031 (0.052) 24.1l4 1.3050 2.40 59569 7389 82.4 (99.0) 0.037 (0.056) 24.0l5 0.7817 1.55 352714 21893 68.5 (92.3) 0.047 (0.227) 15.0l0 1.5418 2.00 186155 14333 94.2 (92.5) 0.077 (0.247) 22.5l5 � l0 Combined 1.55 538,869 29,884 93.5 (98.3) 0.093 (0.225) 15.0

a Figures in brackets apply to the highest-resolution shell.b The overall completeness was reduced by strong ice rings at �3.5 AÊ and �2.6 AÊ . Most other bins were >95 % complete.


carried out by chromatography on an FPLC Superose 12gel-®ltration column.

Compared with CaAc-B2, the CaAc-B sample used forcrystallization lacked seven amino acid residues from theN terminal end. The molecular mass, determined bymatrix-assisted laser desorption ionization time-of-¯ight(MALDI-TOF) spectrometry using sinapinic acid asthe matrix and horse heart cytochrome c as an internalstandard, was 14,405 Da. The identity of the N-terminalresidues was con®rmed by chemical sequencing.

Crystallization

A single crystal of CaAc-B grew in a hanging dropconsisting of 1 ml of protein solution (�6.5 mg/ml pro-tein, 20 mM Tris buffer (pH 8.6), 10 mM NaCl) and 1 mlof reservoir solution (100 mM Hepes buffer (pH 7.5),2 M Li2SO4). When a cat's whisker dipped into themother liquor was used to streak-seed fresh drops,microcrystals were produced. These microcrystals wereused as seeds in further drops, yielding dark bluehexagonal prisms with dimensions up to 0.3 mm� 0.3 mm � 0.6 mm.

Single-wavelength data collection

A crystal was washed quickly in a solution of 20 %MPD in arti®cial mother liquor, and was placed directlyin a stream of pre-cooled (�115 K) nitrogen gas. Data to2.0 AÊ resolution were recorded on an in-house R-AXIS IIimage-plate diffractometer mounted on a Rigaku RU-200rotating-anode generator with a Cu target (l � 1.5418 AÊ )and focussing mirror optics. The data were indexed witha primitive hexagonal unit cell (a � b � 115.7 AÊ ,c � 54.6 AÊ ). Scaling in both P6 and P622 yielded similar

Table 9. (Upper) Anomalous and (lower) dispersive differenc

nref hDano

l1 5936 0.3l2 5982 0.3l3 6024 0.3l4 5795 0.2

nref R(I)is

l1 - l3 7463 0.030l2 - l3 7492 0.038l4 - l3 7281 0.039

a nresf, no. of re¯ections; hDanoi, average anomalous differencmaximum anomalous difference; R(I)iso, isomorphous residual; hjDiso

phous difference.

values of Rsym, suggesting that the higher-symmetryLaue group was to be preferred. Systematic absencesindicated that the space group was P6222 or itsenantiomer P6422. We shall refer to this set of in-housedata as l0.

Molecular replacement calculations

Attempts to solve the structure by molecular replace-ment using the known structures of other cupredoxins(azurin, plastocyanin, rusticyanin, cucumber basicprotein, and a consensus model of the cupredoxinGreek-key fold (E. T. Adman, personal communicationbased on Murphy et al., 1997) as search models wereunsuccessful.

Multiple-wavelength data collection

As in the structure analysis of cucumber basic protein,CBP (Guss et al., 1988, 1996), multiple wavelength anom-alous dispersion (MAD) data were recorded about the Kedge (�1.38 AÊ ) of the single Cu atom in the molecule.The data were recorded on BioCARS beamline 14D atthe Advanced Photon Source, Argonne National Labora-tory. In a ¯uorescence scan the Cu K edge had an in¯ec-tion point at 8985 eV, and a peak at 8998 eV. Theequivalent in¯ection points for a Cu standard and forCBP were at 8979 eV and 8990 eV, respectively (Gusset al., 1988). In the light of this observation, data wererecorded at 4 wavelengths: l1, 1.3799 AÊ , 8985 eV (thein¯ection point); l2, 1.3779 AÊ , 8998 eV (the f'' peak); l3,1.3876 AÊ , 8935 eV (a low energy remote from absorptionedge); and l4, 1.3050 AÊ , 9500 eV (a high energy remotefrom absorption edge). The data recorded at each wave-length comprised a complete data set (0-25 �) and a com-plete anomalous-scattering data set (180-205 �), where

esa

i hjDanoji jDanoj max

7.7 11410.0 1597.5 958.4 114

o hjDisoij jDisoj max

5.3 1575.8 1366.1 124

e; hjDanoji, average absolute anomalous difference; jDanojmax,ji, average isomorphous difference; jDisojmax, maximum isomor-


the rotation angles took into account the space groupand orientation of the crystal specimen. Finally a high-resolution data set was recorded at l5, 0.7817 AÊ (range0-60 �). All data were recorded at �115 K.

In principle, the data at l1 provided suf®cient infor-mation to determine the position of the Cu atom; thedata at l2 were expected to supplement the data at l1;the data at l3, which contained no anomalous signal,provided duplicate observations from which the self-consistency of the data could be assessed; the data at l4

provided a further set of reference intensities; and thedata at l5 were for use in the subsequent re®nement.The data were processed and scaled using theHKL package (Otwinowski & Minor, 1997), and aresummarized in Table 8.

MAD structure analysis

The structure analysis was performed at 2.4 AÊ resol-ution, using the CCP4 suite (Collaborative Compu-tational Project, 1994). A consistent solution for theCu site was obtained (i) from anomalous difference Pat-terson syntheses calculated using the data sets a l1,l2 and l4, and (ii) from six isomorphous differencePatterson syntheses calculated using all possible pairs ofthe data sets recored at l1, l2, l3 and l4. The anomalousand dispersive intensity differences upon which the anal-ysis depended are listed in Table 9. The calculationswere performed with RSPS (Collaborative Compu-tational Project, 1994). Both the positive and negativevalues of the approximate Cu coordinates, (0.444, 0.283,0.139), were then re®ned with MLPHARE (CollaborativeComputational Project, 1994) in the enantiomeric spacegroups P6422 and P6222. The data at l3 were used as``native'', and the data at l1, l2 and l4 as ``derivatives''(in the manner of Ramakrishnan & Biou, who call thereference data ``native'', and the data from which anom-alous and dispersive differences are derived ``deriva-tives'' (Ramakrishnan & Biou, 1997)). When theanomalous scattering contributions were included,re®nement in spacegroup P6422 yielded phases which,after solvent ¯attening with dm0 (Cowtan, 1994), pro-duced a readily interpretable map. No additional metalsites were found in any Fourier maps calculated withphases based on the re®ned Cu position.

The wARP package (Perrakis et al., 1999) was used toextend the MAD phases to the resolution limit (1.55 AÊ )of the l5 data. The relatively poor precision of the low-resolution re¯ections in the l5 data was remedied bymerging the l0 data. The warpNtrace method then pro-duced a trace of 135 of the 140 main-chain residues,as well as 50 % of the side-chains. Model building ofthe entire polypeptide with the exception of theN-terminal residue was completed manually.

Refinement

The model was re®ned against a 115 K dataset com-prising the in-house data originally recorded at l0,1.5418 AÊ , and the synchrotron dataset later recorded atl5, 0.7817 AÊ . The re®nement was made by means ofREFMAC (Collaborative Computational Project, 1994),using a maximum-likelihood target. The cross-validationresidual Rfree (BruÈ nger, 1992a) was based on 3 % of there¯ections. The bulk solvent model from X-PLOR(BruÈ nger, 1992b) was included at a late stage of there®nement by taking the model from REFMAC, applyingthe correction in X-PLOR, and returning the corrected

structure amplitudes to REFMAC. This improved themaps and led to an immediate drop of 3 % in R andRfree. Spherical residual density features that were higherthan three times the average electron density for the unitcell were identi®ed as solvent (water) atoms, providedthat they were appropriately located with respect to atleast one plausible hydrogen-bonding partner. Theywere added to the model with ARP (CollaborativeComputational Project, 1994). Model re-building withO (Jones et al., 1991) and the addition of solvent atomswere guided by changes in Rfree and by other criteriaprovided in the structure-validation software WHATCH-ECK (Hooft et al., 1996) and PROCHECK (Laskowskiet al., 1993).

The N-terminal residue, Ala1 was not located in den-sity, and was omitted from the model. Thus, the asym-metric unit ®nally submitted to re®nement comprisedone auracyanin polypeptide (139 amino acid residues),one Cu atom, 247 water molecules two sulfate ions anda chloride ion. The atoms of one SO4

2ÿ and the Clÿ werere®ned with occupancies 0.25 and 0.5, respectively,re¯ecting the location of the S(sulfate) atom on a 3-foldspecial position with point symmetry 222, and thelocation of the C1ÿ on a 2-fold axis. Some 11 watermolecules lie so close to 2-fold axes that the pair ofsymmetry-related positions cannot be occupied simul-taneously, and these disordered water molecules werealso re®ned with occupancy 0.5. Nine residues weremodelled with dual conformations suggested by omitelectron density maps and con®rmed by improvementsin the electron density and Rfree. At the end of the re®ne-ment, residual electron density equivalent to about two-thirds of a solvent peak persisted at the Cu site.Attempts to model this electron density by introducingparameters representing anisotropy, partial reduction orpartial loss of metal were unsuccessful. The ®nalresiduals were R � 0.198 and Rfree � 0.233, respectively.In a Ramachandran plot (not shown), the onlynon-glycine residue lying slightly outside the core areaswas Ala2, the N-terminal residue of the model.

Protein Data Bank accession number

Crystallographic coordinates for auracyanin B fromChlorofexus aurantiacus have been deposited with theRCSB Protein Data Bank as entry 1QHQ.

Acknowledgments

Helpful discussions with Dr E. T. Adman and Dr A.Nersissian are gratefully acknowledged. H.C.F. andJ.M.G. thank the Australian Research Council for grantsA29230677 and A29601726, and R.E.B. acknowledgessupport from the Arizona State University AstrobiologyInstitute, part of the NASA Astrobiology Institute pro-gram. Access to BioCARS Sector 14 at the AdvancedPhoton Source at Argonne, Illinois, was provided by theAustralian Synchrotron Research Program, which isfunded by the Commonwealth of Australia as a MajorNational Research Facility. M.J.M. was a Research Fellowof the Australian Synchrotron Research Program. Bio-CARS Sector 14 is supported by grant RR07707 from theU.S. National Institutes of Health, National Center forResearch Resources. The Advanced Photon Source issupported by the U.S. Department of Energy, Basic


Energy Sciences, Of®ce of Energy Research, under Con-tract No. W-31-109-Eng-38.

References

Adman, E. T. (1991). Copper protein structures. Advan.Protein Chem. 42, 145-197.

Albomali, B., Taylor, H. V. & Weser, U. (1998). Evol-utionary aspects of copper binding centers incopper proteins. Struct. Bond. 9I, 91-190.

Ainscough, E. W., Bingham, A. G., Brodie, A. M., Ellis,W. R., Gray, H. B., Loehr, T. M., Plowman, J. E.,Norris, G. E. & Baker, E. N. (1987). Spectrochemicalstudies on the blue copper protein azurin fromAlcaligenes denitri®cans. Biochemistry, 26, 71-82.

Andrew, C. R., Yeom, H., Valentine, J. S., Karlsson,B. G., Bonander, N., van Pouderoyen, G., Canters,G. W., Loehr, T. M. & Sanders-Loehr, J. (1994).Raman spectroscopy as an indicator of Cu-S bond-length in Type-1 and Type-2 copper cysteinteproteins. J. Am. Chem. Soc. 116, 11489-11498.

Baker, E. N. (1988). Structure of azurin from Alcaligenesdenitri®cans. Re®nement at 1.8 AÊ resolution andcomparison of the two crystallographically indepen-dent molecules. J. Mol. Biol. 203, 1071-1095.

Barton, G. J. (1993). ALSCRIPT - a tool to format mul-tiple sequence alignments. Protein Eng. 6, 37-40.

Battistuzzi, G., Borsari, M., Loschi, L. & Sola, M. (1997).Redox thermodynamics, acid-base equilibria andsalt-induced effects for the cucumber basic protein.General implications for blue-copper proteins. J. Biol.Inorg. Chem. 2, 350-359.

Becker, M., Nagarajan, V., Middendorf, D., Parson,W. W., Martin, J. E. & Blankenship, R. E. (1991).Temperature dependence of the initial electron-transfer kinetics in photosynthetic reaction centersof Chloro¯exus aurantiacus. Biochim. Biophys. Acta.1057, 299-312.

Blankenship, R. E. (1992). Origin and early evolution ofphotosynthesis. Photosynth. Res. 33, 91-111.

Blankenship, R. E. (1994). Protein structure, electrontransfer and evolution of prokaryotic photosyntheticreaction centers. Antonie van Leeuwenhoek, 65, 311-329.

Blankenship, R. E., Olson, J. M. & Miller, M. (1995).Antenna complexes from green photosyntheticbacteria. In Anoxygenic Photosynthetic Bacteria(Blankenship, R. E., Madigan, M. T. & Bauer, C. E.,eds), pp. 399-435, Kluwer Academic Publishers,Dordrecht, The Netherlands.

BruÈ nger, A. T. (1992a). Free R-value - a novel statisticalquantity for assessing the accuracy of crystal struc-tures. Nature, 355, 472-474.

BruÈ nger, A. T. (1992b). X-PLOR, Version 3.1. A Systemfor Crystallography and NMR, Yale University Press,New Haven, CT, USA.

Castrasena, J., Luebben, M. & Samste, M. (1995). Newarchaebacterial genes coding for redox proteins:implications for the evolution of aerobic metab-olism. J. Mol. Biol. 250, 202-210.

Collaborative Computational Project No. 4 (1994). TheCCP4 Suite: programs for protein crystallography.Acta Crystallog. sect. D, 150, 760-763.

Colman, P. M., Freeman, H. C., Guss, J. M., Murata, M.,Norris, V. A., Ramshaw, J. A. M., Venkatappa, M. P.& Vickery, L. E. (1977). Preliminary crystallographicdata for a basic copper-containing protein fromcucumber seedlings. J. Mol. Biol. 112, 649-650.

Cowtan, K. (1994). DM: an automated procedure forphase improvement by density modi®cation. JointCCP4 and ESF-EACBM Newsletter on Protein Crystal-lography, 31, 34-38; see also Collaborative Compu-tational Project.

Cruickshank, D. W. J. (1999). Remarks about proteinstructure precision. Acta Crystallog. sect D, 55, 583-601.

Doolittle, W. F. (1999). Phylogenetic classi®cation andthe universal tree. Science, 284, 2124-2128.

Ejdeback, M., Bergkvist, A., Karlsson, B. G. & Ubbink,M. (2000). Side-chain interactions in the plastocya-nin-cytochrome f complex. Biochemistry, 39, 5022-5027.

Fee, J. A. (1975). Copper proteins. Systems containingthe ``blue'' copper center. Struct. Bond. 23, 1-60.

Feick, R., Shiozawa, J. A. & Ertlmaier, A. (1995). Bio-chemical and spectroscopic properties of the reac-tion center of the green ®lamentous bacterium,Chloro¯exus aurantiacus. In Anoxygenic PhotosyntheticBacteria (Blankenship, R. E., Madigan, M. T. &Bauer, C. E., eds), pp. 699-708, Kluwer AcademicPublishers, Dordrecht, The Netherlands.

Freeman, J. C. & Blankenship, R. E. (1990). Isolation andcharacterization of the membrane-bound cyto-chrome c-554 from the thermophilic green photo-synthetic bacterium Chloro¯exus aurantiacus.Photosynth. Res. 23, 29-38.

Genetics Computer Group (1994). PILEUP ProgramManual, Wisconsin Package, Version 8, Genetics Com-puter Group, 575 Science Drive, Madison, WI53711, USA.

Gotschlich, E. C. & Seiff, M. E. (1987). Identi®cation andgene structure of an azurin-like protein with a lipo-protein signal peptide in Neisseria gonorrhoeae.FEMS Microbiol. Letters, 43, 253-255.

Gray, H. B., MalmstroÈm, B. G. & Williams, R. J. P.(2000). Copper coordination in blue proteins. J. Biol.Inorg. Chem. 5, 551-559.

Groenewald, C. M., Ouwerling, M. C., Erkelens, C. &Canters, G. W. (1988). H-1 nuclear magnetic reson-ance study of the protonation behavior of the histi-dine residues and the electron self-exchangereaction of azurin from Alcaligenes denitri®cans.J. Mol. Biol. 200, 189-199.

Guss, J. M., Harrowell, P. R., Murata, M., Norris, V. A.& Freeman, H. C. (1986). Crystal structure analysesof reduced (Cul) poplar plastocyanin at six pHvalues. J Mol. Biol. 192, 361 387..

Guss, J. M., Merritt, E. A., Phizackerley, R. P., Hedman,B., Murata, M., Hodgson, K. O. & Freeman, H. C.(1988). Phase determination by multiple-wavelengthX-ray diffraction: crystal structure of a basic ``blue''copper protein from cucumbers. Science, 241, 806-811.

Guss, J. M., Bartunik, H. D. & Freeman, H. C. (1992).Accuracy and precision in protein structure anal-ysis: restrained least-squares re®nement of thestructure of poplar plastocyanin at 1.33 AÊ resol-ution. Acta Crystallog. sect. B, 48, 790-811.

Guss, J. M., Merritt, E. A., Phizackerley, R. P. &Freeman, H. C. (1996). The structure of a phytocya-nin, the basic blue protein from cucumber, re®nedat 1.8 AÊ resolution. J Mol. Biol. 262, 686-705.

Han, J., Adman, E. T., Beppu, T., Codd, R., Freeman,H. C., Huq, L., Loehr, T. M. & Sanders-Loehr, J.(1991). Resonance Raman spectra of plastocyaninand pseudoazurin - evidence for conserved cysteine


ligand conformations in cupredoxins (blue copperproteins). Biochemistry, 30, 10904-10913.

Han, J., Loehr, T. M., Lu, Y., Valentine, J. S., Averill,B. A. & Sanders-Loehr, J. (1993). Resonance Ramanexcitation pro®les indicate multiple Cys! Cucharge transfer transitions in type 1 copper proteins.J Amer. Chem. Soc. 115, 4256-4263.

Hooft, R. W. W., Vriend, G., Sander, C. & Abola, E. E.(1996). Errors in protein structures. Nature, 381, 272.

Jones, T. A., Zou, J.-Y., Cowan, S. W. & Kjeldgaard, M.(1991). Improved methods for building proteinmodels in electron density maps and the location oferrors in these models. Acta Crystallog. sect A, 47,110-119.

Kabsch, W. & Sander, C. (1983). Dictionary of proteinsecondary structure: pattern recognition of hydro-gen-bonded and geometrical features. Biopolymers,22, 2577-2637.

Kleywegt, G. J. & Jones, T. A. (1994). A super position.ESF/CCP4 Newsletter, 31, 9-14.

Kohzuma, T., Inoue, T., Yoshizaki, F., Sasakawa, Y.,Onodera, K., Nagatomo, S., Kitagawa, T., Uzawa,S., Isobe, Y., Sugimura, Y., Gotowda, M. & Kai, Y.(1999). The structure and unusual pH dependenceof plastocyanin from the fern Dryopteris crassirhi-zoma. The protonation of an active-site histidine ishindered by p-p interactions. J Biol. Chem. 274,11817-11823.

Kraulis, P. J. (1991). MOLSCRIPT: a program to produceboth detailed and schematic plots of protein struc-tures. J. Appl Crystallog. 24, 946-950.

Kyte, J. & Doolittle, R. F. (1982). A simple method fordisplaying the hydropathic character of a protein.J. Mol. Biol. 157, 105-132.

LaCroix, L. B., Randall, D. W., Nersissian, A. M.,Hoitink, C. W. G., Canters, G. W., Valentine, J. S. &Solomon, E. W. (1998). Spectroscopic and geometricvariations in perturbed blue copper centers: elec-tronic structures of stellacyanin and cucumber basicprotein. J. Am. Chem. Soc. 120, 9621-9631.

Laskowski, R. A., MacArthur, M. W., Moss, D. S. &Thornton, J. M. (1993). PROCHECK: a program tocheck the stereochemical quality of protein struc-tures. J. Appl. Crystallog. 26, 283-291.

Lopez, J. C., Dracheva, S. & Blankenship, R. E. (1996).Gene sequence for auracyanin B from Chloro¯exusaurantiacus. GenBank, Accession No. AAB38318.

McLeod, D. D. N., Freeman, H. C., Harvey, I., Lay, P. A.& Bond, A. M. (1996). Voltammetry of plastocyaninat a graphite electrode: effects of structure, chargeand electrolyte. Inorg. Chem. 35, 7156-7165.

McManus, J. D., Brune, D. C., Han, J., Sanders-Loehr, J.,Meyer, T. E., Cusanovich, M. A., Tollin, G. &Blankenship, R. E. (1992). Isolation, characterization,and amino acid sequences of auracyanins, blue cop-per proteins from the green photosynthetic bacter-ium Chloro¯exus aurantiacus. J Biol. Chem. 267, 6531-6540.

Menin, L., Gaillard, J., Parot, P., Schoepp, B., Nitschke,W. & Vermeglio, A. (1998). Role of HiPIP aselectron donor to the RC-bound cytochrome inphotosynthetic purple bacteria. Photosynth. Res. 55,343-348.

Merchant, S. (1998). Synthesis of metalloproteinsinvolved in photosynthesis: plastocyanin and cyto-chromes. In The Molecular Biology of Chloroplasts andMitochondria in Chlamydomonas (Rochaix, J.-D.,Goldschmidt-Clermont, M. & Merchant, S., eds),

pp. 597-611, Kluwer Academic Publishers,Dordrecht, The Netherlands.

Meyer, T. E. & Donohue, T. J. (1995). Cytochromes, ironsulfur, and copper proteins mediating electrontransfer from the Cyt bc1, complex to photosyn-thetic reaction center complexes. In AnoxygenicPhotosynthetic Bacteria (Blankenship, R. E., Madigan,M. T. & Bauer, C. E., eds), pp. 725-745, KluwerAcademic Publishers, Dordrecht, The Netherlands.

Mulkidjanian, A. Y., Hochkoeppler, A., Zannoni, D.,Drachev, L. A., Melandri, B. A. & Venturoli, G.(1998). Photosynthetic electrogenic events in nativemembranes of Chloro¯exus aurantiacus. Flash-induced charge displacements in the acceptorquinone complex of the photosynthetic reactioncenter. Photosynth. Res. 56, 75-82.

Murata, M., Begg, G. S., Lambrou, F., Leslie, B.,Simpson, R. J., Freeman, H. C. & Morgan, F. J.(1982). Amino acid sequence of a basic blue proteinfrom cucumber seedlings. Proc. Natl Acad. Sci. USA,79, 6434-6437.

Murphy, M. E. P., Lindley, P. F. & Adman, E. T. (1997).Structural comparison of cupredoxin domains:domain recycling to construct proteins with novelfunctions. Protein Sci. 6, 761-770.

Myllykallio, H., Jenney, F. E., Moomaw, C. R.,Slaughter, C. A. & Daldal, F. (1997). Cytochrome cY

of Rhodobacter capsulatus is attached to the cyto-plasmic membrane by an uncleaved signalsequence-like anchor. J Bacteriol. 179, 2623-2631.

Nersissian, A. M., Babayan, M. A., Sarkissian, L. K.,Sarukhanian, E. G. & Nalbandyan, R. M. (1985).Studies on plantacyanin. II. NMR data, redox prop-erties, reaction with nitrite, and the formation ofcomplex with plastocyanin. Biochim. Biophys. Acta,830, 195-205.

Nersissian, A. M., Immoos, C., Hill, M. G., Hart, P. J.,Williams, G., Herrmann, R. G. & Valentine, J. S.(1998). Uclacyanins, stellacyanins, and planta-cyanins are distinct subfamilies of phytocyanins:plant-speci®c mononuclear blue copper proteins.Protein Sci. 7, 1915-1929.

Nitschke, W., Muhlenhof, U. & Liebl, U. (1998). Evol-ution. In Photosynthesis, a Comprehensive Treatise(Raghavendra, A. S., ed.), pp. 285-304, CambridgeUniversity Press, Cambridge, UK.

Norris, G. E., Anderson, B. F. & Baker, E. N. (1983).Structure of azurin from Alcaligenes denitri®cans at2.5 AÊ resolution. J. Mol. Biol. 165, 501-521.

Oh-oka, H., Iwaki, M. & Rob, S. (1998). Membrane-bound cytochrome cz couples quinol oxidoreductaseto the P840 reaction center complex in isolatedmembranes of the green sulfur bacterium Chloro-bium tepidum. Biochemistry, 37, 12293-12300.

Otwinowski, Z. & Minor, W. (1997). Processing of X-raydiffraction data collected in oscillation mode.Methods Enzymol. 276, 307-326.

Perrakis, A., Morris, R. & Lamzin, V. S. (1999). Auto-mated protein model building combined withiterative structure re®nement. Nature Struct. Biol. 6,458-463.

Pierson, B. K. & Castenholz, R. W. (1974). Phototrophicgliding ®lamentous bacterium of hot springs, Chlor-o¯exus aurantiacus. Arch. Microbiol. 100, 5-24.

Pierson, B. K. & Castenholz, R. W. (1992). The familyChloro¯exaceae. In The Prokaryotes (Balows, A.,Truper, H. G., Dworkin, M., Schliefer, K. H. &Harder, W., eds), pp. 3754-3774, Springer-Verlag,Berlin.


Ramakrishnan, V. & Biou, V. (1997). Treatment of multi-wavelength anomalous diffraction data as a specialcase of multiple isomorphous replacement. MethodsEnzymol. 276, 538-557.

Randall, D. W., Gamelin, D. R., LaCroix, L. B. &Solomon, E. I. (2000). Electronic structure contri-butions to electron transfer in blue Cu and CuA.J. Biol. Inorg. Chem, 5, 16-19.

Ryde, U., Olsson, M. H. M., Roos, B. O., DeKerpel,J. O. A. & Pierloot, K. (2000). On the role of strainin blue copper proteins. J. Biol. Inorg. Chem. 5, 565-574.

RydeÂn, L. (1984). Structure and evolution of the smallblue proteins. In Copper Proteins and Copper Enzymes(Lontie, R., ed.), vol. 1, pp. 157-182, CRC Press,Boca Raton, FL, USA.

RydeÂn, L. & Hunt, L. T. (1993). Evolution of proteincomplexity - the blue copper-containing oxidasesand related proteins. J. Mol. Evol. 36, 41-66.

Sakurai, T., Sawada, S. & Nakahara, A. (1986). Reson-ance Raman spectra of cucumber basic blue copperprotein ``plantacyanin''. Inorg. Chim. Acta, 123, L21-L22.

Scharf, B. & Engelhard, M. (1993). Halocyanin, anarchaebacterial blue copper protein (Type I) fromNatronobacterium pharaonis. Biochemistry, 32, 12894-12900.

Selvaraj, F. M. (1999). Studies on the metalloproteinsfrom the photosynthetic bacteria Chloro¯exus auran-tiacus and Chlorobium tepidum. PhD Dissertation,Arizona State University, Tempe, AZ, USA.

Sigfridsson, K., He, S. P., Modi, S., Bendall, D. S., Gray,J. & Hansson, O. (1996). A comparative ¯ash-photolysis study of electron transfer from pea andspinach plastocyanins to spinach Photosystem 1. Areaction involving a rate-limiting conformationalchange. Photosynth. Res. 50, 11-21.

Sykes, A. G. (1991). Active-site properties of the bluecopper proteins. Advan. Inorg. Chem. 36, 377-408.

Trost, J. T., McManus, J. D., Freeman, J. C.,Ramakrishna, B. L. & Blankenship, R. E. (1988).Auracyanin: a blue copper protein from the greenphotosynthetic bacterium Chloro¯exus aurantiacus.Biochemistry, 27, 7858-7863.

Ubbink, M., Ejdeback, M., Karlsson, B. G. & Bendall,D. S. (1998). The structure of the complex of plasto-cyanin and cytochrome f, determined by paramag-

netic NMR and restrained rigid-body moleculardynamics. Structure, 6, 323-335.

Van de Kamp, M., Silvestrini, M. C., Brunori, M., VanBeeumen, J., Hali, F. C. & Canters, G. W. (1990).Involvement of the hydrophobic patch of azurin inthe electron transfer reactions with cytochrome c551,and nitrite reductase. Eur. J. Biochem. 194, 109-118.

Van Driessche, G., Hu, W., Van de Werken, G., Selvaraj,F., McManus, J. D., Blankenship, R. E. & VanBeeumen, J. J. (1999). Auracyanin A from the ther-mophilic green gliding photosynthetic bacteriumChloro¯exus aurantiacus represents an unusual classof small blue copper proteins. Protein Sci. 8, 947-957.

Vasmel, H. & Amesz, J. (1983). Photoreduction of mena-quinone in the reaction center of the green photo-synthetic bacterium Chloro¯exus aurantiacus. Biochim.Biophys Acta, 724, 118-122.

Vriend, G. (1990). WHAT IF - A molecular modelingand drug design program. J. Mol. Graph. 8, 52-56.

Woese, C. R. (1987). Bacterial evolution. Microbiol. Rev.51, 221-271.

Edited by R Huber

(Received 18 May 2000; received in revised form 27September 2000; accepted 27 September 2000)

http://www.academicpress.com/jmb

Supplementary material comprising six tables(intramolecular hydrogen bonds, intermolecularcontacts via a solvent bridge, polypeptide b andg-turns, and contributions of charged, polar andapolar residues to the solvent-accessible surfacesof CaAc-B and Alcaligenes denitri®cans azurin) isavailable on IDEAL

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Crystal structure of auracyanin, a “blue” copper protein from the green thermophilic...

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