ORIGINAL PAPER

Structure at 1.0 A resolution of a high-potential iron–sulfurprotein involved in the aerobic respiratory chainof Rhodothermus marinus

Meike Stelter • Ana M. P. Melo • Gudmundur O. Hreggvidsson •

Sigridur Hjorleifsdottir • Lıgia M. Saraiva • Miguel Teixeira •

Margarida Archer

Received: 7 May 2009 / Accepted: 19 October 2009 / Published online: 18 November 2009

� SBIC 2009

Abstract The aerobic respiratory chain of the thermo-

halophilic bacterium Rhodothermus marinus, a nonphoto-

synthetic organism from the Bacteroidetes/Chlorobi group,

contains a high-potential iron–sulfur protein (HiPIP) that

transfers electrons from a bc1 analog complex to a caa3

oxygen reductase. Here, we describe the crystal structure of

the reduced form of R. marinus HiPIP, solved by the sin-

gle-wavelength anomalous diffraction method, based on

the anomalous scattering of the iron atoms from the [4Fe–

4S]3?/2? cluster and refined to 1.0 A resolution. This is the

first structure of a HiPIP isolated from a nonphotosynthetic

bacterium involved in an aerobic respiratory chain. The

structure shows a similar environment around the cluster as

the other HiPIPs from phototrophic bacteria, but reveals

several features distinct from those of the other HiPIPs

of phototrophic bacteria, such as a different fold of the

N-terminal region of the polypeptide due to a disulfide

bridge and a ten-residue-long insertion.

Keywords High-potential iron–sulfur protein �Crystal structure � Rhodothermus marinus �Electron transfer chain

Abbreviations

HiPIP High-potential iron–sulfur protein

RmHip Rhodothermus marinus high-potential

iron–sulfur protein

SLS Swiss Light Source

Tris Tris(hydroxymethyl)aminomethane

Rms Root mean square

M. Stelter and A. M. P. Melo contributed equally to this work.

The Rhodothermus marinus high-potential iron–sulfur protein

coordinates and structure factors have been deposited in the Protein

Data Bank with accession code 3H31.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00775-009-0603-8) contains supplementarymaterial, which is available to authorized users.

M. Stelter � L. M. Saraiva � M. Teixeira � M. Archer (&)

Instituto de Tecnologia Quımica e Biologica,

Universidade Nova de Lisboa,

Av. da Republica (EAN),

2780-157 Oeiras, Portugal

e-mail: archer@itqb.unl.pt

Present Address:M. Stelter

The European Synchrotron Radiation Facility,

BP 220, 38043 Grenoble Cedex 9, France

A. M. P. Melo

Eco-Bio, Instituto de Investigacao Cientıfica Tropical,

Av. da Republica (EAN),

2784-505 Oeiras, Portugal

A. M. P. Melo

Faculdade de Engenharia e Ciencias Naturais,

Universidade Lusofona de Humanidades e Tecnologias,

Av. do Campo Grande, 376,

1749-024 Lisbon, Portugal

G. O. Hreggvidsson � S. Hjorleifsdottir

Matis ohf, Gylfaflot 5,

112 Reykjavık, Iceland

G. O. Hreggvidsson � S. Hjorleifsdottir

University of Iceland,

Askja, Sturlugata 7,

101 Reykjavık, Iceland

123

J Biol Inorg Chem (2010) 15:303–313

DOI 10.1007/s00775-009-0603-8

Introduction

Iron–sulfur clusters were probably among the first cata-

lysts that Earth prebiotic chemistry used [1]. Iron–sulfur

proteins are widespread in nature, from the most primitive

organisms to eukaryotes, where they assume several

biological roles, namely, in electron transfer, substrate

recognition, catalysis, and transcriptional regulation [2].

Ferredoxins and some oxidoreductases, such as the

NADH:quinone oxidoreductase (complex I) [3], have

[4Fe–4S] centers that alternate between the 2?/1? redox

states. However, in high-potential iron–sulfur proteins

(HiPIPs), small (8–10 kDa) soluble proteins containing a

cubane [4Fe–4S] cluster bound to the protein backbone by

four iron–sulfur cysteine bonds, the [4Fe–4S] centers

switch between the 3?/2? states, at a higher redox

potential.

For a long time, HiPIPs were thought to be confined to

phototrophs (e.g., Allochromatium vinosum [4]), until a

HiPIP was found in the membranes of Rhodothermus

marinus [5], a chemoorganotrophic, thermohalophilic

Gram-negative bacterium from the Bacteriodetes/Chlorobi

group. This R. marinus HiPIP (RmHip) was detected in

whole cells and membrane extracts by the characteristic

EPR signal of its oxidized form, and was shown to be

able to reduce the caa3 oxygen reductase [6], one of the

heme-copper oxygen reductases from the R. marinus

aerobic respiratory chain. Later, a HiPIP was also shown

to be involved in both the aerobic and the photosynthetic

electron transfer chains of the facultative phototroph

Rhodoferax fermentans [7]. This type of iron–sulfur

center, initially considered only as an electron carrier, has

recently been shown to be endowed with catalytic activ-

ities, such as in heterooligomeric proteins, as the human

and Sacharomyces cerevisiae primases [8, 9], the hete-

rodisulfide reductases (from, e.g., Methanothermobacter

marburgensis [10]), the Tmc membrane-bound complex

from Desulfovibrio vulgaris [11], and the base excision

repair glycosylases, where it is involved in DNA binding

and repair [12].

The oxidized HiPIP centers have an S = � ground state,

which produces a quasi-axial EPR spectrum with values of

gmax * 2.1 and gmed,min * 2.03. The reduced form of

HiPIP has an S = 0 ground state and is EPR-silent. HiPIPs

are characterized by a high reduction potential that ranges

from ?50 to ?500 mV [13, 14], in contrast to ferredoxins,

which have lower reduction potentials ranging, in general,

from -450 to -100 mV [2].

In the pursuit of a full characterization of the

R. marinus respiratory chain, we report here the high-

resolution structure of one of its electron carriers, the

HiPIP.

Materials and methods

Bacterial growth and nucleic acid manipulation

Rhodothermus marinus strains PRQ62B and ITI378 were

grown essentially according to a previously described

procedure [15]. Unless otherwise indicated, the Esche-

richia coli strains XL2Blue (Stratagene) and BL21-Gold

(DE3) (Stratagene) were grown in Luria–Bertani medium,

with the appropriate antibiotics.

DNA manipulation procedures were carried out

according to standard methods [16]. A DNA fragment

encoding the processed form of the RmHip was amplified

from the genomic DNA of R. marinus ITI378, in a PCR

catalyzed by the DNA polymerase PFUturbo (Stratagene),

using 50-gtggaacatcgcatatggccgagctg and 50-gtagccaatcg-

gatcctttcaggtcg as forward and reverse oligonucleotides,

respectively. The obtained blunt end product was digested

with NdeI and BamHI (New England Biolabs), purified and

cloned in pET12a (Novagen), previously digested with the

same enzymes, and transformed in E. coli XL2Blue. DNA

was isolated and sequenced, and an errorless rmhip gene

containing plasmid was transformed in E. coli BL21-Gold

(DE3). RmHip expression was induced at an optical den-

sity at 600 nm of 0.6, with 0.5 mM isopropyl-b-D-thioga-

lactopyranoside, in M9B medium [17] supplemented with

0.8 mg mL-1 FeSO4�4H2O, at 310 K, overnight in a 10 L

reactor, yielding 3.5 g of cells (wet weight) per liter of

culture.

Purification procedures

All purification procedures were performed at 277 K.

E. coli cells resuspended in 20 mM Tris-HCl pH 7.5

(buffer A) were broken in a French press at 6,000 psi and

the cell debris was removed by centrifugation (20,000g,

15 min). Soluble and membrane fractions were separated

by ultracentrifugation at 138,000g for 2 h. Since the

recombinant HiPIP was present in both fractions, the solu-

ble extract was used for purification. Its ionic strength was

lowered by consecutive dilution and concentration steps in a

Diaflo with a 3 kDa cutoff membrane and buffer A, and it

was applied onto a Q-Sepharose column, previously equil-

ibrated in buffer A, and a linear gradient of 0–250 mM

NaCl in buffer A was performed. The fraction containing

RmHip, which was eluted between 80 and 150 mM NaCl,

was concentrated and applied onto a 115 mL G 50 gel fil-

tration column that had previously been equilibrated with

buffer A plus 150 mM NaCl with a flux of 0.25 mL min-1.

The protein sample was then loaded onto a MonoQ column

and, according to the sodium dodecyl sulfate polyacryl-

amide gel electrophoresis, pure HiPIP was eluted at

304 J Biol Inorg Chem (2010) 15:303–313

123

approximately 150 mM NaCl in buffer A in two pools

corresponding to the oxidized and reduced forms of the

protein, exhibiting absorbance ratios (280 nm/380 nm) of

1.6 and 2.1, respectively.

EPR spectra were recorded using a Bruker EMX spec-

trometer equipped with an Oxford Instruments continuous-

flow helium cryostat at 9.39 MHz microwave frequency,

2.0 mW microwave power, 1 mT modulation amplitude, and

10 K. UV–vis absorption spectra were acquired with a Shi-

madzu UV-1603 spectrophotometer at room temperature.

Crystallization and X-ray diffraction data collection

Crystals of the reduced recombinant RmHip were grown

using the hanging-drop vapor-diffusion method. One

microliter of a 10 mg mL-1 protein solution in 20 mM

Tris-HCl buffer pH 7.5 and 0.1 M NaCl was mixed with

0.5 lL of a precipitant solution (2 M ammonium sulfate,

8% dioxane, and 50 mM sodium citrate pH 4) and equil-

ibrated against 1 mL precipitant solution at 293 K. Light-

brown rod-shaped crystals grew in 2 days to a final size of

0.5 mm 9 0.07 mm 9 0.07 mm. For in-house data col-

lection, a crystal was cryoprotected in 30% glycerol,

1.5 M ammonium sulfate, 8% dioxane, and 50 mM

sodium citrate pH 4 and flash-frozen using a nylon loop in

a cold nitrogen stream. The crystal diffracted to 1.78 A

resolution using an Enraf–Nonius Cu Ka rotating anode

generator and a complete data set was collected at 100 K

on a 300 mm Marresearch image-plate detector. For syn-

chrotron data collection, crystals were flash-frozen in

liquid nitrogen using as cryoprotectant 35% sucrose in

1.3 M ammonium sulfate, 8% dioxane, and 50 mM

sodium citrate pH 4. X-ray diffraction data were collected

at 0.918 A on the X06SA beamline at the Swiss Light

Source (SLS; Villigen, Switzerland) with a Mar225

mosaic CCD detector. A complete X-ray diffraction data

set was collected from a single crystal that diffracted to

1.0 A resolution. These data were recorded in two steps at

different exposure times to accurately measure both the

strongest low-resolution and the weakest high-resolution

reflections. The diffraction data were indexed and inte-

grated using MOSFLM [18] and scaled with SCALA from

the CCP4 program suite [19]. The crystals belong to the

orthorhombic space group P212121 with unit-cell param-

eters a = 27.27, b = 43.34, and c = 46.79 A for SLS

data. The asymmetric unit contains one molecule, result-

ing in an apparent Vm of 1.71 A3 Da-1 and a low solvent

content of about 28%.

Structure determination and refinement

Intensity data were transformed to structure factor ampli-

tudes with the program TRUNCATE [19]. The structure of

RmHip was solved by the single wavelength anomalous

dispersion method based on the anomalous signal of the

iron atoms from the [4Fe–4S] cluster and using the in-

house diffraction data. The positions of the iron atoms were

determined with SHELXD [20] after the structure factors

had been analyzed using SHELXC [21] and the solution

was characterized by a correlation coefficient of CCAll/

Weak of 51.4/34.2 and a Patterson figure of merit of 33.4.

Phase calculation and density modification were performed

using the program SHELXE [22]. The assumption of a

solvent content of 28% and the application of 20 cycles of

density modification resulted in the phase refinement con-

verging with a pseudo-free correlation coefficient of

around 61% and led to a well-defined experimental elec-

tron density map that enabled automated model building

with ARP/wARP [23] of 31 (residues 7–24 and 48–60) of

74 residues. The partial model was completed manually

using COOT [24], alternated with structure refinement

using REFMAC [19] and automated water-molecule

searching using ARP [25]. The model refinement was

continued against the synchrotron diffraction data using

SHELXL [26] in a first step to a resolution of 1.2 A. After

the initial 20 cycles of isotropic refinement, anisotropic

atomic displacement parameters were refined. The resolu-

tion was then carefully increased to 1.0 A in several steps,

alternated with manual protein and solvent model correc-

tion. Before each step of including higher-resolution data,

ten cycles of anisotropic refinement and full-matrix least-

squares refinement were carried out, fixing the atomic

displacement parameters and using zero damping and a

zero shift multiplier. One percent of randomly selected

reflections (521) were used for cross-validation. Inspection

of the sigmaA electron density maps [27] allowed addition

of water molecules and building of alternative conforma-

tions for several amino acid residues. The final ten

refinement cycles included hydrogen atoms on riding

positions. Standard restraints were applied to the geomet-

rical [28] and isotropic or anisotropic displacement

parameters [26], except for the [4Fe–4S] group, which was

not restrained. The final values of R and Rfree converged to

10.4 and 14.4%, respectively. In a final step, the full least-

squares matrix for the unrestrained coordinates was

inverted to obtain reliable estimates of the standard

uncertainties of the atom positions.

Structure analysis and comparison

Secondary structure assignments were made with the pro-

gram DSSP [29]. The exploration of protein–protein

interfaces in the crystal packing for prediction of probable

quaternary structures was carried out with the PISA service

at EBI [30]. Amino acid sequence databases were searched

using NCBI BLAST [31]. Pairwise and multiple structure

J Biol Inorg Chem (2010) 15:303–313 305

123

based sequence alignments were done using the ALIGN3D

and SALIGN tools in Modeller [32] and corrected manu-

ally upon visual comparison of the superposed protein

models. For HiPIPs of unknown structure, the sequence

alignment was based on the BLAST alignment and was

corrected manually taking into account the conserved or

conservatively substituted aromatic residues in the struc-

ture-based alignment. All figures of protein structures were

generated with PyMOL [33].

Results and discussion

Protein production

The sequence of the first 35 amino acid residues of the

processed form of the HiPIP previously obtained from

the R. marinus PRQ-62B strain [6] was used to screen

a partial genome library of R. marinus ITI 378 (G.O.

Hreggvidsson, S. Hjorleifsdottir, O.H. Fridjonsson and S.

Skirnisdottir, unpublished results), retrieving the total

sequence of the gene encoding RmHip, deposited at

GenBank with accession number FJ873797. The N-ter-

minal sequences of the mature form of the HiPIPs from

both strains display more than 94% identity. The com-

parison of the amino acid sequence of the purified HiPIP

with the sequence deduced from the rmhip gene showed

that the latter encodes 53 additional amino acid residues

in the N-terminal side of the protein, probably repre-

senting a periplasmic signal peptide of the twin arginine

translocation pathway [34], as is typically observed in

HiPIPs.

With the purpose of obtaining the crystallographic

structure of RmHip, the DNA region encoding the pro-

cessed form of RmHip (74 amino acid residues) was pro-

duced in E. coli as an 8.1 kDa protein. EPR and UV–vis

spectra of the recombinant HiPIP (Fig. 1) attested that it is

spectroscopically identical to the wild-type protein isolated

from R. marinus PRQ-62B [5].

X-ray data and model quality

Crystals of reduced recombinant RmHip were used to

collect X-ray diffraction data in-house and at SLS to a

resolution of 1.8 and 1.0 A, respectively. Data collection

and processing statistics are summarized in Table 1.

The X-ray structure of RmHip was determined by sin-

gle-wavelength anomalous dispersion and the anisotropic

structure refinement to 1.0 A resolution converged to val-

ues of R = 10.4 and Rfree = 14.4%. Statistics of refine-

ment and model quality are presented in Table 2. All non-

glycine and non-proline residues lie in the most favored

(88.5%) or additionally allowed (11.5%) regions of the

Ramachandran plot. The final model contains 74 protein

residues, the [4Fe–4S] cluster, and 140 water molecules, of

which 103 are fully occupied and 37 are modeled with half

Fig. 1 Spectroscopic properties

of the recombinant

Rhodothermus marinus high-

potential iron–sulfur protein

(RmHip). a EPR spectrum of

the oxidized high-potential

iron–sulfur protein (HiPIP), at

10 K; b UV–vis absorption

spectra of the oxidized and

reduced HiPIP at room

temperature

Table 1 Data collection and processing statistics

X-ray source In-house X-ray

generator

X06SA beamline,

SLS

Wavelength (A) 1.541 0.918

Space group P212121 P212121

Cell parameters (A) 27.6, 43.8, 45.9 27.27, 43.34, 46.79

Resolution range (A) 17.6–1.78

(1.88–1.78)

46.8–1.00

(1.05–1.00)

Rmerge (%) 5.2 (16.6) 12.8 (39.6)

Rpim (%) 2.0 (5.2) 5.6 (22.7)

Ranom (%) 5.0 (8.3)

Number of observed

reflections

71,993 (8,992) 140,359 (17,004)

Number of unique

reflections

5,565 (735) 30,715 (4,410)

Mean [I/r(I)] 31.7 (12.7) 9.2 (2.9)

Completeness (%) 97.8 (91.9) 100 (100)

Multiplicity 12.9 (12.2) 4.6 (3.9)

SLS Swiss Light Source

Values in parentheses refer to the highest resolution bin

306 J Biol Inorg Chem (2010) 15:303–313

123

occupancy. Side chains of ten residues are modeled with

discrete alternative conformations. Despite some disconti-

nuities in their electron density, the N- and C-termini were

modeled into two distinct conformations comprising the

first two and last four residues, respectively. The refined

electron density map is generally very well defined, except

for Pro29 to Pro31 (which form a protruding turn), Pro47,

and the side chains of surface residues Gln49 and Gln54.

Structure analysis and comparison with other HiPIPs

The RmHip structure comprises one a-helix (residues

13–21), one 310-helix (35–37), a short b-sheet formed by

two antiparallel b-strands (41–42, 52–53), and seven

hydrogen-bonded turns and five bends, which wrap around

the [4Fe–4S] cluster (Fig. 2). A disulfide bridge between

Cys5 and Cys50 links the N-terminus to the core of the

protein. The [4Fe–4S] cluster is covalently attached to the

polypeptide chain through Cys35, Cys38, Cys53, and Cys66

and the intracluster bond distances (Table S1) are within the

range observed for other HiPIP structures [35–41]. The

[4Fe–4S] cluster is completely shielded from the solvent

and the binding pocket is formed by hydrophobic and aro-

matic residues. Analysis of the RmHip structure with PISA

suggests a monomer as the most probable assembly, in

accordance with previous studies indicating a monomer in

solution [6]. Although the wild-type HiPIP has been iso-

lated from R. marinus membranes, and is not detached even

by chaotropic agents, the structure does not reveal any

transmembrane or amphipathic helices that could attach it

to the membranes; therefore, its attachment has to occur

either through electrostatic interactions or possibly via

association with other membrane complexes.

Superposition of the RmHip structure with the other

available HiPIP structures yields rms distance values

around 1.4–1.5 A for the aligned CA atoms (Fig. 3,

Table 3). All HiPIP structures reported to date show a

similar fold, particularly in the protein core surrounding the

[4Fe–4S] cluster, whereas the largest differences are

observed in external loops owing to insertions and dele-

tions in the primary structure. Most HiPIP structures form a

salient loop of variable size (‘‘left loop’’, Fig. 3, residues

25–39, A. vinosum numbering, Fig. 4), which is absent in

RmHip and Rhodocyclus tenuis HiPIP. In contrast, the

structure of RmHip is the only known structure that shows

a large protruding extension on the opposite side of the

peptide (‘‘right side extension’’, Fig. 3) formed by residues

7–15 and comprising the N-terminus of the a-helix. In fact,

the N-terminal a-helix of RmHip is significantly longer

(nine residues) than the one present in its counterparts (five

to six residues). This structural element is conserved in

most HiPIPs for which the structure is available, with the

exception of that of Halorhodospira halophila (Fig. 3).

Table 2 Summary of refinement statistics and model quality

Resolution range (A) 10.0–1.0

Number of unique reflections

in refinement [Fo [ 4r(Fo)]

56,286 (43,085)

Working set (no. of reflections) 55,765 (42,735)

Test set (no. of reflections) 521 (350)

Completeness (%) 100

R factor (%) [Fo [ 4r(Fo)] 10.4 (8.5)

Free R factor (%) 14.4 (10.3)

Number of non-hydrogen atoms 808

Number of protein atomsa 660

Number of hetero atomsb 8

Number of solvent atomsa 140

Weighted rms deviation

from ideal values

Bond length (A) 0.017

Angle distances (A) 0.042

Average B factor (A2)

Main chain 8.2

Side chain 11.7

All protein atoms 10.0

Water molecules 23.0

[4Fe–4S] cluster 5.3

a Non-hydrogen atomsb [4Fe-4S]2? cluster

Fig. 2 The RmHip structure.

The [4Fe–4S] cluster is

shown in sphere and stick

representation (Fe in orange,

S in yellow), and the side chains

of the cysteines that are attached

to the cluster and of those

forming the disulfide bridge are

shown in stick representation. a‘‘Front view’’ and b ‘‘side view’’

J Biol Inorg Chem (2010) 15:303–313 307

123

Another remarkable feature revealed by the RmHip struc-

ture is the existence of a disulfide bridge between Cys5 and

Cys50 that links the N-terminus to the core of the protein

and should contribute to the stabilization of the afore-

mentioned N-terminal protrusion.

Although these two unique structural elements of

RmHip (‘‘right side’’ N-terminal protrusion and disulfide

bridge) are not present in any of the 3D structures of

HiPIPs already described (Fig. 4), the primary structures of

the HiPIPs from the gammaproteobacteria NOR5-3 and

Congregibacter litoralis KT71, and from Salinibacter ru-

ber and Pedobacter heparinus, which, like R. marinus, also

belong to the Bacteroidetes/Chlorobi phylum, contain

regions that may harbor these features (Figs. 4, 5). It is

noteworthy that the presence of a disulfide bridge has also

been suggested for the HiPIP of the alphaproteobacterium

Fig. 3 Stereoview of the superposed CA traces of the different HiPIP

structures in ribbon representation. Molecules are colored as follows:

Rhodothermus marinus in red, Rhodocyclus tenuis in purple,

Allochromatium vinosum in green, Ectothiorhodospira vacuolata in

yellow, Rhodoferax fermentans in blue, and Halorhodospira halo-phila in cyan. The structures of the Thermochromatium tepidum and

Marichromatium purpuratum HiPIPs are not shown because their

fold is almost identical to the fold of the A. vinosum molecule. The

dashed pink ellipse indicates the region where RmHip has a singular

protrusion (‘‘right side extension’’). The dashed green ellipseindicates the region where all molecules except Rhodocyclus tenuisHiPiP and RmHip have an extension (‘‘left loop’’)

Table 3 Summary of data for known high-potential iron–sulfur protein structures

Organism PDB accession code Resolution (A) NPDB E0 (mV) Nalgn Rmsd (A) Nid Nsim

Rhodothermus marinus 3H31 1.0 74 260

Allochromatium vinosum 1HIPa 2.0 85 346h 53 1.37 19 22

Thermochromatium tepidum 1IUAb 0.8 83 323i 55 1.43 19 21

Marichromatium purpuratum 3HIPc 2.8 83 390h 54 1.39 17 20

Ectothiorhodospira vacuolata iso-II 1HPId 1.8 71 150h 52 1.47 18 22

Halorhodospira halophila iso-I 2HIPe 2.5 71 120h 52 1.44 10 14

Rhodoferax fermentans 1HLQf 1.5 75 351j 54 1.46 16 18

Rhodocyclus tenuis 1ISUg 1.5 62 310h 58 1.45 22 24

Salinibacter ruber 33 43

Congregibacter litoralis 26 39

Pedobacter heparinus 1 29 39

Pedobacter heparinus 2 27 40

Gammaproteobacterium NOR5-3 27 45

Acidithiobacillus ferrooxidans 11 15

For structures with several chains in the asymmetric unit, comparison is done using chain A.

PDB Protein Data Bank, NPDB number of protein residues in the pdb file, E0 redox potential, Nalgn number of aligned equivalent positions using

Modeller, Rmsd root mean square distance between CA positions after superposition using Modeller, Nid number of identical residues in

alignment with RmHip, Nsim number of similar residues in alignment with RmHipa [35], b[36], c[37], d[38], e[39], f[40], g[41], h[37], i[36], j[53]

308 J Biol Inorg Chem (2010) 15:303–313

123

Acidithiobacillus ferrooxidans [42]. A structure-based

amino acid sequence alignment with RmHip (Fig. 4) fur-

ther suggests the absence of the ‘‘left’’ loop in the HiPIPs

of S. ruber, P. heparinus, NOR5-3, and C. litoralis as well

as in the shorter HiPIP of A. ferroxidans, which does not

contain the ‘‘right side’’ extension. A dendrogram produced

from a wider set of selected amino acid sequences showed

that the HiPIPs whose primary structure may harbor the N-

terminal protrusion and the disulfide bridge affiliate inde-

pendently (Fig. 5), and irrespective of their phylogenetic

nature. Furthermore, it indicates that the RmHip is the first

example of a subtype of HiPIPs. Intriguingly, so far HiPIPs

have been detected only in Proteobacteria, a more recent

phylum, and in a more ancient one, Bacteroidetes/

Chlorobi.

The anchoring of the N-terminal protrusion to the core

of the protein, through the disulfide bond, is reminiscent of

what is observed in seven-iron ferredoxins from hyper-

thermophilic archaea, in which the iron–sulfur-containing

domain is stabilized by the presence of an N-terminal

domain, in many cases containing a Zn2? structural site

[43], and can thus contribute to the thermostability of

HiPIPs of this subtype. Regarding the role of the disulfide

bridge, the latter have been correlated with protein stabil-

ization in thermophiles [44], which is consistent with the

thermophilicity of R. marinus and the high Tm of about

353 K determined for RmHip ([6] and data not shown).

However, the former is also present in the mesophilic

HiPIPs of P. heparinus.

The iron–sulfur cluster geometry and its immediate

environment are highly conserved among the HiPIP

structures reported to date. In particular, five hydrogen

bonds are established between the amide nitrogen atoms

of the peptide backbone and the [4Fe–4S] cluster: four to

the SG atoms of the cluster-binding cysteines, and one to

the cluster S1 atom. However, in RmHip, two of these

bonds are shorter than their counterparts in the available

3D structures (Table S2), and there is an additional

amide nitrogen (of Ile56) in hydrogen-bonding distance

to the SG of Cys53 (3.40 A). The reduction potential of

RmHip is within the range of the known HiPIPs, cor-

roborating the idea that other parameters determine its

redox potential beyond the expected influence of protein-

cluster N–H���S hydrogen bonding [45, 46]. Actually the

way in which different parameters influence the redox

potential of [4Fe–4S] ferredoxins is still a matter of

debate. Although there is consensus that the cluster

environment and solvent accessibility of the cluster

dominate the redox potential (the latter is inconsiderable

in HiPIPs, the clusters being shielded from solvent),

some studies suggest that the hydrogen bonding is not a

determinant factor but rather the dipoles from backbone

amides in proximity to the [4Fe–4S] cluster, whether

hydrogen-bonded or not [41, 47]. The positions and

orientations of the eight amide groups claimed to have

the largest influence on the reduction potential [47] are

quite conserved in all the HiPIP structures (data not

shown). Only in the R. tenuis and H. halophila HiPIPs

are two amides in slightly different positions, and in the

latter case this coincides with the relatively low reduc-

tion potential of ?120 mV. In the HiPIPs of R. marinus

and R. tenuis, amide 66 (RmHip numbering) corresponds

1 10 20 30 40 50 60 70 . . . . . . . .

R. marinus ----------AELTCTDVSGLTAEEIQMRE----SLQYTDHSP-------------YPDKTCANCQLYVPA--ESPDQCGGCQL--I-KG-PIHPNGYCTSWVQKATA. vinosum SAPANAVAAD---------------NATAI----ALKYNQDATKSERVAAARPGLPPEEQHCADCQFMQADAAGATDEWKGCQL--FPGK-LINVNGWCASWTLKAGT. tepidum AAPANAVTAD---------------DPTAI----ALKYNQDATKSERVAAARPGLPPEEQHCANCQFMQANV--GEGDWKGCQL--FPGK-LINVNGWCASWTLKAGM. purpuratum -VPANAVTES---------------DPAAV----ALKYHRDAASSERVAAARPGLPPEEQHCENCQFMNPDS--AAADWKGCQL--FPGK-LINLSGWCASWTLRAGE. vacuolata ---MERLSED---------------DPAAQ----ALEYRHDASSVQ---HPA---YEEGQTCLNCLLYTDA---SAQDWGPCSV--FPGK-LVSANGWCTAWVAR--H. halophila ---EPRAEDG-----------------HA------HDYVNEAADASG--HPR---YQEGQLCENCAFWGEA---VQDGWGRCTHPDFDEV-LVKAEGWCSVYAPAS-R. fermentans --AAPLVAET---------------DANAK----SLGYVADTTKADKTKYPK---HTKDQSCSTCALYQGK---TA-PQGACPL--FAGK-EVVAKGWCSAWAKKA-R. tenuis -------------------GTN---AAMRK----AFNYQDTAK-------------NG-KKCSGCAQFVPG--ASPTAAGGCKV--IPGDNQIAPGGYCDAFIVKK-S. ruber ----------ASADCSDLSRLSDAQKQRRKQQVNALNYVKASP-------------KPNKNCANCQLYQQE--KYGSGCGGCQL--FPG--PVAGEGYCSSWAKQS-C. litoralis ----------AALVCADPGKMTSAQESVRK----TLRYTEQSS-------------DSTKTCAGCAFYSGA----QGACGSCSI--FDGN-AVNPAGHCDSWSAAS-NOR5-3 ----------DSLVCADPASMSSAQESVRR----TLKYTEISS-------------DPAKTCAACEFFHVA-KDGTDGCGTCEM--F-SGEPVNPQGHCDSWSVDS-P. heparinus1 ----------KIDPCDDMTGVSPAELAKRK----KLAYVNKSP-------------IEDSHCSNCALYLPP--GKGKSCGGCVL--F-KG-PVRPTGYCAYWAPINNP. heparinus2 ----------DDFKCGDYSNVSPEELAKRK----KLGYVEKSP-------------DPERECQKCNLFIPK--GAEKTCGGCIL--F-KG-PVNKEGSCTYWAEQVSA. ferrooxid -----------AGNCPGT-------TPKA-----EVQYQPHP--------------KGKDQCSVCANFIAP--------KCCKV--VAGP--VAPDGYCIAFTPMPA

Y C C C G C

Fig. 4 Structure-based sequence alignment of known HiPIP struc-

tures, whereby the primary sequences of the molecules from

Salinibacter ruber, Congregibacter litoralis, Acidithiobacillus ferro-oxidans, gammaproteobacterium NOR5-3, and Pedobacter heparinuswere manually aligned. The sequences are labeled with the names of

the organisms: Rhodothermus marinus (R. marinus), Rhodoferaxfermentans (R. fermentans), Rhodocyclus tenuis (R. tenuis), Salinib-acter ruber (S. ruber), Congregibacter litoralis (C. litoralis),

gammaproteobacterium NOR5-3 (NOR5-3), Pedobacter heparinus(ZP_04327229.1 and ZP_04327256.1) (P. heparinus1 and P. hepari-nus2), and Acidithiobacillus ferrooxidans (A. ferrooxid). The

following residues are highlighted: cysteines (red); aromatic residues

in the cluster environment in the HiPIPs of known structure (cyan);

conserved residues in Rhodothermus marinus, Salinibacter ruber,

Congregibacter litoralis, NOR5-3 and Pedobacter heparinus (R19

and K33 are also conserved in Rhodocyclus tenuis) (gray). The

residues for which the backbone amide nitrogens form hydrogen

bonds with the sulfur atoms of the cluster (S1 and SG) are underlined.

The pink box and the green boxes evidence residues forming the

‘‘right side’’ extension (N-terminal protrusion) in RmHip and the

‘‘left’’ extension absent in RmHip, respectively

J Biol Inorg Chem (2010) 15:303–313 309

123

to a proline residue, and is the only dipole where

the oxygen points towards the cluster. The proline

should have a very different backbone amide dipole from

that of any other residue and should therefore contribute

largely to raise the reduction potential [48]. In summary,

the specific redox properties of HiPIPs are clearly

modulated by a series of subtle structural differences of

the cluster environment, including the adjustment of the

hydrogen-bond network, polar interactions of side chains

with cluster sulfur atoms, and various effects that

specifically stabilize one redox level of the active site

[48–50].

The [4Fe–4S] cluster binding pocket is, as expected,

formed by well conserved hydrophobic (Leu22, Leu40,

Leu55, and Ile60, RmHip numbering unless otherwise

mentioned) and aromatic (Tyr24, Tyr41, Ty65, and Trp69)

residues (Fig. 6). The role of the core aromatic residues is

to maintain a hydrophobic barrier for exclusion of solvent

from the cluster cavity to inhibit the oxidation of

the reduced cluster, thus raising the redox potential, and

preventing the hydrolytic degradation of the cluster [49]. In

RmHip, the hydroxyl group of Tyr24 forms the usual

hydrogen bond with a backbone amide nitrogen atom (NH

of His61) and is additionally hydrogen-bonded to the

Fig. 5 Dendrogram of representative HiPIP amino acid sequences

from organisms of the Bacteroidetes/Chlorobi phylum and of a, b, c,

and d subdivisions of Proteobacteria. Ac, Acidiphilium cryptum(YP_001235590.1); Ae, Alkalilimnicola ehrlichii (YP_742426.1); Af,Acidithiobacillus ferrooxidans (YP_002220762.1); Am, Alteromonasmacleodii (YP_002128217.1); Av, Allochromatium vinosum(ZP_04773031.1); Bb, Bdellovibrio bacteriovorus (NP_968669.1);

Bv, Burkholderia vietnamensis (YP_001109919.1); Cl1, Congregi-bacter litorali (ZP_01104040.1); Cl2, Congregibacter litorali(ZP_01104665.1); Ev, Ectothiorhodospira vacuolata (P38524.1);

Gpb, gammaproteobacterium NOR5 (YP_002660774); Hh, Halorho-dospira halophila (P04168.2); Ma, Marinobacter sp. (ZP_

01738296.1); Mgp, marine gammaproteobacteria (ZP_01616964.1);

Mp, Marichromatium purpuratum (P59860.1); Mpe, Methylobiumpetroleum (YP_001021387.1); Pa, Paracoccus sp. (P00264.1); Ph1,

Pedobacter heparinus (ZP_04327229.1); Ph2, Pedobacter heparinus

(ZP_04327256.1) Pn, Polynucleobacter necessarius (YP_

001155579.1); Pp, Photobacterium profundum (YP_130336.1); Rb,

Rhodobacterales bacterium (ZP_01450491.1); Rf, Rhodoferax fer-mentans (P80882.1); Rg, Rubrivivax gelatinosus (BAC55219.1); Rgl,Rhodopila globiformis (P38589.1); Rm, Rhodothermus marinus(FJ873797); Rp1, Rhodopseudomonas palustris (YP_532822.1);

Rp2, Rhodopseudomonas palustris (YP_570740.1); Rp3, Rhodo-pseudomonas palustris (YP_779769.1); Rpi, Ralstonia pickettii(YP_001900922.1); Rs1, Ralstonia solanacearum (NP_521207.1);

Rs2, Ralstonia solanacearum (YP_002254080.1); Rt, Rhodocyclustenuis (P33678.1); Sr, Salinibacter ruber (YP_446920.1); Th,

Thioalkalivibrio sp. (YP_002514019.1); Tp, Thiococcus pfennigii(P00263.1); Tr, Thiocapsa roseopersicina (P00261.1); Tt, Thermo-chromatium tepidum (P80176.1); Va, Vibrio angustum(ZP_01235462.1)

310 J Biol Inorg Chem (2010) 15:303–313

123

carboxylate group of Asp26 (Fig. 7), conferring rigidity to

the protein backbone. This tyrosine residue, conserved in

almost all amino acid sequences of known HiPIPs [14]

(Fig. 4), is proposed to play an important role in protecting

the cluster from solvent and stabilizing the HiPIP through

hydrogen bonds and electrostatic interactions [50]. Gly64,

in close proximity to Tyr24, is also highly conserved,

probably because the structural constraints imposed by the

interactions of Tyr24 would not permit space for any side

chains [14, 42]. Tyr41, Tyr65, and Trp69 correspond to

conservatively substituted aromatic residues, except in the

Chromatiaceae (i.e., A. vinosum, Marichromatium purpu-

ratum, Thermochromatium tepidum) structures, where

Tyr41 is replaced by a methionine, although the preceding

residue is a phenylalanine. A. vinosum Phe66 is conserved

in many structures, with the exceptions of RmHip and

R. tenuis HiPIP, where an isoleucine occupies the corre-

sponding position. However, in R. tenuis HiPIP there is a

phenylalanine (Phe10) at the front of the molecule that

occupies a similar position relative to the cluster as the

A. vinosum Phe66 (Fig. 6). Therefore, RmHip is so far the

only structure showing four aromatic residues in the cluster

environment, instead of the usual five (six in H. halophila).

The electrostatic surface potential map of RmHip pre-

sents a hydrophobic patch on the face adjacent to the [4Fe–

4S] cluster (Fig. 8a). This patch is conserved among

HiPIPs [14] and is formed by Met18, Leu22, Leu40,

Leu55, Ile56, and Val70 in RmHip. The periphery of the

front face also contains patches of preponderant negative

charge, in particular on the lower right side, formed by

Glu14 and Glu15. The remaining part of the surface con-

tains charged and polar regions (Fig. 8b), though overall

more negative charges. RmHiP is able to donate electrons

to the caa3 oxygen reductase [6] with a significantly higher

turnover than cytochrome c (208 vs. 10 min-1) [6, 51]. The

cytochrome c domain of the caa3 oxidase is the likely entry

point for electrons in the oxidase [15]. A hydrophobic

patch is present on the surface of this c domain as well

(Fig. 8c), adjacent to the exposed heme edge where

docking for electron transfer is suggested to occur [52],

with patches of positive electrostatic potential on the

periphery. The molecular recognition between electron

transfer partners could thus involve charge complementary

and hydrophobic interactions for the initial orientation and

docking of the soluble electron carrier.

In summary, the RmHip structure at atomic resolution

gives a detailed view of the overall architecture and the

metal cluster environment for the first HiPIP from a non-

phototrophic bacterium. Although, in the cluster environ-

ment, the structure presents a fold similar to the folds of the

HiPIPs from phototrophic bacteria, it reveals a unique

disulfide bridge anchoring the singular extended N-termi-

nus to the core of the protein and thus possibly contributing

to the high thermostability of the R. marinus protein.

Fig. 6 Closeup view of the cluster environment in RmHip. The

[4Fe–4S] cluster is shown in a ball-and-stick representation and the

side chains of the aromatic residues in RmHip and the other HiPIP

structures are drawn in stick representation. Molecules are colored as

follows: Rhodothermus marinus in red, Rhodocyclus tenuis in purple,

Allochromatium vinosum in green, and Halorhodospira halophila in

cyan. For sake of clarity, the structures of Marichromatium purpu-ratum, Thermochromatium tepidum, Rhodoferax fermentans, and

Ectothiorhodospira vacuolata are omitted owing to the identical

positions displayed by their aromatic residues compared with

Allochromatium vinosum

Fig. 7 Closeup of the region around Tyr24. The hydrogen bonds

between its hydroxyl group and the backbone amide nitrogen atom of

His61 and one side-chain oxygen of Asp26 are shown as dashed redlines and are labeled with the distances. The refined sigmaA-weighted

2Fo - Fc electron density map is contoured at 1.0r

J Biol Inorg Chem (2010) 15:303–313 311

123

Acknowledgments We are grateful to Nuno A.M. Felix for excel-

lent technical assistance, to Ana Coelho, from the Mass Spectrometry

Service of Instituto de Tecnologia Quımica e Biologica, and to Joao

Carita for cell growth. We thank Carlos Frazao for advice on high-

resolution refinement and the European Synchrotron Radiation

Facility for provision of synchrotron radiation facilities. X-ray data

collection at SLS was supported by the European Commission under

the Sixth Framework Programme through the Key Action: Strength-

ening the European Research Area, Research Infrastructures, contract

no. RII3-CT-2004-506008. This work was supported by Fundacao

para a Ciencia e a Tecnologia (PTDC/BIA-PRO/66833/2006 to M.A.,

POCTI/BIA-PRO/58608/2004 to M.T., REEQ/336/BIO/05, PTDC/

BIA-PRO/67105/2006 to A.M.P.M.). M.S. received a grant from

Fundacao para a Ciencia e a Tecnologia (BPD/24193/2005).

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