Amino acid sequence of [2Fe-2S] ferredoxin from Clostridium pasteurianum

The complete amino acid sequence of the [2Fe-2S] ferredoxin from the saccharolytic anaerobe Clostridium pasteurianum has been determined by automated Edman degradation of the whole protein and of peptides obtained by tryptic and by staphylococcal protease digestion. The polypeptide chain consists of 102 amino acids, including 5 cysteine residues in positions 11, 14, 24, 56, and 60. The sequence has been analyzed for hydrophilicity and for secondary structure predictions. In its native state the protein is a dimer, each subunit containing one [2Fe-2S] cluster, and it has a molecular weight of 23,174, including the four iron and inorganic sulfur atoms. The extinction coefficient of the native protein is 19,400 M-1 cm-1 at 463 nm. The positions of the cysteine residues, four of which are most probably the ligands of the [2Fe-2S] cluster, on the polypeptide chain of this protein are very different from those found in other [2Fe-2S] proteins, and in other ferredoxins in general. In addition, whole sequence comparisons of the [2Fe-2S] ferredoxin from C. pasteurianum with a number of other ferredoxins did not reveal any significant homologies. The likely occurrence of several phylogenetically unrelated ferredoxin families is discussed in the light of these observations.     

Solution structure of HndAc: a thioredoxin-like domain involved in the NADP-reducing hydrogenase complex

The NADP-reducing hydrogenase complex from Desulfovibrio fructosovorans is a heterotetramer encoded by the hndABCD operon. Sequence analysis indicates that the HndC subunit (52 kDa) corresponds to the NADP-reducing unit, and the HndD subunit (63.5 kDa) is homologous to Clostridium pasteurianum hydrogenase. The role of HndA and HndB subunits (18.8 kDa and 13.8 kDa, respectively) in the complex remains unknown. The HndA subunit belongs to the [2Fe-2S] ferredoxin family typified by C. pasteurianum ferredoxin. HndA is organized into two independent structural domains, and we report in the present work the NMR structure of its C-terminal domain, HndAc. HndAc has a thioredoxin-like fold consisting in four beta-strands and two relatively long helices. The [2Fe-2S] cluster is located near the surface of the protein and bound to four cysteine residues particularly well conserved in this class of proteins. Electron exchange between the HndD N-terminal [2Fe-2S] domain (HndDN) and HndAc has been previously evidenced, and in the present studies we have mapped the binding site of the HndDN domain on HndAc. A structural analysis of HndB indicates that it is a FeS subunit with 41% similarity with HndAc and it contains a possible thioredoxin-like fold. Our data let us propose that HndAc and HndB can form a heterodimeric intermediate in the electron transfer between the hydrogenase (HndD) active site and the NADP reduction site in HndC.     

Structural differences between [2Fe-2S] clusters in spinach ferredoxin and in the "red paramagnetic protein" from Clostridium pasteurianum. A resonance Raman study

The [2Fe-2S] ferredoxin ("Red paramagnetic protein", RPP) from C. pasteurianum has been found to be composed of two identical subunits of 10,000 +/- 2 000 daltons, each containing a [2Fe-2S] cluster. Resonance Raman (RR) spectra of RPP have been obtained at 23 degrees K, and compared to those of spinach ferredoxin (Sp Fd). Ten modes of the [2Fe-2S] chromophore were observed in the 100-450 cm-1 range. Assignments of non fundamental modes in the 500-900 cm-1 range allowed correlations between fundamental stretching modes of RPP and Sp Fd. Although assuming a [2Fe-2S] structure, the chromophore of RPP differs from that of Sp Fd by its conformation and by a slight weakening of Fe-S bonds, involving both the inorganic core and the cysteine ligands.     

Engineering a three-cysteine, one-histidine ligand environment into a new hyperthermophilic archaeal Rieske-type [2Fe-2S] ferredoxin from Sulfolobus solfataricus

We heterologously overproduced a hyperthermostable archaeal low potential (E(m) = -62 mV) Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus strain P-1 and its variants in Escherichia coli to examine the influence of ligand substitutions on the properties of the [2Fe-2S] cluster. While two cysteine ligand residues (Cys(42) and Cys(61)) are essential for the cluster assembly and/or stability, the contributions of the two histidine ligands to the cluster assembly in the archaeal Rieske-type ferredoxin appear to be inequivalent as indicated by much higher stability of the His(64) --> Cys variant (H64C) than the His(44) --> Cys variant (H44C). The x-ray absorption and resonance Raman spectra of the H64C variant firmly established the formation of a novel, oxidized [2Fe-2S] cluster with one histidine and three cysteine ligands in the archaeal Rieske-type protein moiety. Comparative resonance Raman features of the wild-type, natural abundance and uniformly (15)N-labeled ARF and its H64C variant showed significant mixing of the Fe-S and Fe-N stretching characters for an oxidized biological [2Fe-2S] cluster with partial histidine ligation.     

Repair of nitric oxide-modified ferredoxin [2Fe-2S] cluster by cysteine desulfurase (IscS)

Iron-sulfur proteins are among the sensitive targets of the nitric oxide cytotoxicity. When Escherichia coli cells are exposed to nitric oxide, iron-sulfur clusters are modified forming protein-bound dinitrosyl iron complexes. Such modified protein dinitrosyl iron complexes are stable in vitro but are efficiently repaired in aerobically growing E. coli cells even without any new protein synthesis. Here we show that cysteine desulfurase encoded by the gene iscS of E. coli can directly convert the ferredoxin dinitrosyl iron complex to the ferredoxin [2Fe-2S] cluster in the presence of L-cysteine in vitro. A reassembly of the [2Fe-2S] cluster in the ferredoxin dinitrosyl iron complex does not require any addition of iron or other protein components. Furthermore, a complete removal of the dinitrosyl iron complex from ferredoxin prevents reassembly of the [2Fe-2S] cluster in the protein. The results suggest that cysteine desulfurase (IscS) together with L-cysteine can efficiently repair the nitric oxide-modified ferredoxin [2Fe-2S] cluster and that the iron center in the dinitrosyl iron complex may be recycled for the reassembly of iron-sulfur clusters in proteins.     

NMR structure of the [2Fe-2S] ferredoxin domain from soluble methane monooxygenase reductase and interaction with its hydroxylase.

The soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath) is a multicomponent enzyme system required for the conversion of methane to methanol. It comprises a hydroxylase, a regulatory protein, and a reductase. The reductase contains two domains: an NADH-binding and FAD-containing flavin domain and a ferredoxin (Fd) domain carrying a [2Fe-2S] cofactor. Here, we report the solution structure of the reduced form of the 98-amino acid Fd domain (Blazyk, J. L., and Lippard, S. J. Unpublished results) determined by nuclear magnetic resonance (NMR) spectroscopy and restrained molecular dynamics calculations. The structure consists of six beta strands arranged into two beta sheets as well as three alpha helices. Two of these helices form a helix-proline-helix motif, unprecedented among [2Fe-2S] proteins. The [2Fe-2S] cluster is coordinated by the sulfur atoms of cysteine residues 42, 47, 50, and 82. The 10.9 kDa ferredoxin domain of the reductase protein transfers electrons to carboxylate-bridged diiron centers in the 251 kDa hydroxylase component of sMMO. The binding of the Fd domain with the hydroxylase was investigated by NMR spectroscopy. The hydroxylase binding surface on the ferredoxin protein has a polar center surrounded by patches of hydrophobic residues. This arrangement of amino acids differs from that by which previously studied [2Fe-2S] proteins interact with their electron-transfer partners. The critical residues on the Fd domain involved in this binding interaction map well onto the universally conserved residues of sMMO enzymes from different species. We propose that the [2Fe-2S] domains in these other sMMO systems have a fold very similar to the one found here for M. capsulatus (Bath) MMOR-Fd.     

The [2Fe-2S] protein I (Shetna protein I) from Azotobacter vinelandii is homologous to the [2Fe-2S] ferredoxin from Clostridium pasteurianum

 The [2Fe-2S] protein from Azotobacter vinelandii that was previously known as iron-sulfur protein I, or Shethna protein I, has been shown to be encoded by a gene belonging to the major nif gene cluster. Overexpression of this gene in Escherichia coli yielded a dimeric protein of which each subunit comprises 106 residues and contains one [2Fe-2S] cluster. The sequence of this protein is very similar to that of the [2Fe-2S] ferredoxin from Clostridium pasteurianum (2FeCpFd), and the four cysteine ligands of the [2Fe-2S] cluster occur in the same positions. The A. vinelandii protein differs from the C. pasteurianum one by the absence of the N-terminal methionine, the presence of a five-residue C-terminal extension, and a lesser number of acidic and polar residues. The UV-visible absorption and EPR spectra, as well as the redox potentials of the two proteins, are nearly identical. These data show that the A. vinelandii FeS protein I, which is therefore proposed to be designated 2FeAvFdI, is the counterpart of the [2Fe-2S] ferredoxin from C. pasteurianum. The occurrence of the 2FeAvFdI-encoding gene in the nif gene cluster, together with the previous demonstration of a specific interaction between the 2FeCpFd and the nitrogenase MoFe protein, suggest that both proteins might be involved in nitrogen fixation, with possibly similar roles. Received: 21 December 1998 / Accepted: 1 March 1999     

Vertebrate-type and plant-type ferredoxins: crystal structure comparison and electron transfer pathway modelling

Crystallographic analysis of a fully functional, truncated bovine adrenodoxin, Adx(4-108), has revealed the structure of a vertebrate-type [2Fe-2S] ferredoxin at high resolution. Adrenodoxin is involved in steroid hormone biosythesis in adrenal gland mitochondria by transferring electrons from adrenodoxin reductase to different cytochromes P450. Plant-type [2Fe-2S] ferredoxins interact with photosystem I and a diverse set of reductases.A systematic structural comparison of Adx(4-108) with plant-type ferredoxins which share about 20 % sequence identity yields these results. (1) The ferredoxins of both types are partitioned into a large, strictly conserved core domain bearing the [2Fe-2S] cluster and a smaller interaction domain which is structurally different for both subfamilies. (2) In both types, residues involved in interactions with reductase are located at similar positions on the molecular surface and coupled to the [2Fe-2S] cluster via structurally equivalent hydrogen bonds. (3) The accessibility of the [2Fe-2S] cluster differs between Adx(4-108) and the plant-type ferredoxins where a solvent funnel leads from the surface to the cluster. (4) All ferredoxins are negative monopoles with a clear charge separation into two compartments, and all resulting dipoles but one point into a narrow cone located in between the interaction domain and the [2Fe-2S] cluster, possibly controlling predocking movements during interactions with redox partners. (5) Model calculations suggest that FE1 is the origin of electron transfer pathways to the surface in all analyzed [2Fe-2S] ferredoxins and that additional transfer probability for electrons tunneling from the more buried FE2 to the cysteine residue in position 92 of Adx is present in some.     

Spectroscopic characterization of the novel iron-sulfur cluster in Pyrococcus furiosus ferredoxin

Pyrococcus furiosus ferredoxin is the only known example of a ferredoxin containing a single [4Fe-4S] cluster that has non-cysteinyl ligation of one iron atom, as evidenced by the replacement of a ligating cysteine residue by an aspartic acid residue in the amino acid sequence. The properties of the iron-sulfur cluster in both the aerobically and anaerobically isolated ferredoxin have been characterized by EPR, magnetic circular dichroism, and resonance Raman spectroscopies. The anaerobically isolated ferrodoxin contains a [4Fe-4S]+,2+ cluster with anomalous properties in both the oxidized and reduced states which are attributed to aspartate and/or hydroxide coordination of a specific iron atom. In the reduced form, the cluster exists with a spin mixture of S = 1/2 (20%) and S = 3/2 (80%) ground states. The dominant S = 3/2 form has a unique EPR spectrum that can be rationalized by an S = 3/2 spin Hamiltonian with E/D = 0.22 and D = +3.3 +/- 0.2 cm-1. The oxidized cluster has an S = 0 ground state, and the resonance Raman spectrum is characteristic of a [4Fe-4S]2+ cluster except for the unusually high frequency for the totally symmetric breathing mode of the [4Fe-4S] core, 342 cm-1. Comparison with Raman spectra of other [4Fe-4S]2+ centers suggests that this behavior is diagnostic of anomalous coordination of a specific iron atom. The iron-sulfur cluster is shown to undergo facile and quantitative [4Fe-4S] in equilibrium [3Fe-4S] interconversion, and the oxidized and reduced forms of the [3Fe-4S] cluster have S = 1/2 and S = 2 ground states, respectively. In both redox states the [3Fe-4S]0,+ cluster exhibits spectroscopic properties analogous to those of similar clusters in other bacterial ferredoxins, suggesting non-cysteinyl coordination for the iron atom that is removed by ferricyanide oxidation. Aerobic isolation induces partial degradation of the [4Fe-4S] cluster to yield [3Fe-4S] and possibly [2Fe-2S] centers. Evidence is presented to show that only the [4Fe-4S] form of this ferredoxin exists in vivo.     

Tertiary structure of Bacillus thermoproteolyticus [4Fe-4S] ferredoxin. Evolutionary implications for bacterial ferredoxins

The structure of a low-potential [4Fe-4S] ferredoxin from Bacillus thermoproteolyticus has been solved using anomalous scattering data from iron atoms in the diffraction data of native crystals and refined partially to a crystallographic R-factor of 0.33, with 2.3 A (1 A = 0.1 nm) resolution data. The least-squares refinement based on the Bijvoet differences has determined that the four iron atoms in the cluster are an equal distance, approximately 2.8 A, apart. The NH ... S hydrogen bonds between polypeptide nitrogen atoms, and both cysteine and inorganic sulfur atoms, are present, as in ferrodoxin from Peptococcus aerogenes. The polypeptide chain of the B. thermoproteolyticus ferredoxin has a fold closely similar to that of 2[4Fe-4S] ferredoxin from P. aerogenes. The structural correspondence indicates strongly that both types of ferredoxin evolved from a common ancestor. The second cluster-binding region in P. aerogenes ferredoxin corresponds to the alpha-helix in B. thermoproteolyticus ferredoxin. The secondary-structure predictions strongly suggest that the alpha-helix is generally present in the monocluster-type ferredoxins. The conformational change to alpha-helix, insertions of a loop and a protrusion, as well as the absence of the second cluster in B. thermoproteolyticus ferredoxin, result in the lack of 2-fold symmetry present in P. aerogenes ferredoxin. So, the track of gene duplication is no longer detectable in the tertiary structure alone. The evolutionary events that may have occurred in the ferredoxins with the [4Fe-4S] cluster are discussed.     

Amino acid sequence of a four-iron-four-sulphur ferredoxin isolated from Bacillus stearothermophilus.

1. The primary structure of a 4Fe-4S ferredoxin from Bacillus stearothermophilus was determined and shown to consist of a single polypeptide chain of 81 amino acid residues. The molecular weight of the holoprotein is about 9120. 2. There are only four cysteine residues in the molecule; three of these are located near the N-terminus as a Cys-X-X-Cys-X-X-Cys segment, and the fourth cysteine residue is followed by a proline and located in the C-terminal half. 3. The Fe-S chromophore in B. stearothermophilus ferredoxin was previously well characterized and was shown to consist of a single 4Fe-4S cluster. This ferredoxin sequence establishes for the first time the relative location of the four cysteine residues necessary to bind the 4Fe-4S cluster of a 4Fe ferredoxin, and is in agreement with the criteria for the relative positions of the cysteines proposed from X-ray-crystallographic studies on an 8Fe (two 4Fe-4S clusters) ferredoxin. 4. The sequence of B. stearothermophilus ferredoxin is homologous in many segments to that of other bacterial ferredoxins, the degree of homology being greater towards ferredoxins from Desulfovibrio gigas and photosynthetic bacteria than to Clostridial ferredoxins. 5. The presence of a relatively higher number of glutamic acid and lower number of cysteine residues in the molecule may explain the greater thermal stability and oxygen-insenstivity of this ferredoxin.     

Electrochemical and spectroscopic characterization of the conversion of the 7Fe into the 8Fe form of ferredoxin III from Desulfovibrio africanus. Identification of a [4Fe-4S] cluster with one non-cysteine ligand.

Desulfovibrio africanus ferredoxin III is a protein (Mr 6585) containing one [3Fe-4S]1+,0 and one [4Fe-4S]2+,1+ core cluster when aerobically isolated. The amino acid sequence contains only seven cysteine residues, the minimum required to ligand these two clusters. Cyclic voltammery by means of direct electrochemistry at a pyrolytic-graphite-'edge' electrode promoted by neomycin shows that, when reduced, the [3Fe-4S]0 centre reacts rapidly with Fe(II) ion to form a [4Fe-4S]2+ cluster. The latter, which can be reduced at a redox potential similar to that of the other [4Fe-4S] cluster, must include non-thiolate ligation. We propose that the carboxylate side chain of aspartic acid-14 is the most likely candidate, since this amino acid occupies the position of a cysteine residue in the sequence typical of an 8Fe ferredoxin. The magnetic properties at liquid-He temperature of this novel cluster, studied by low-temperature magnetic-c.d. and e.p.r. spectroscopy, are diamagnetic in the oxidized state and S = 3/2 in the one-electron-reduced state. This cluster provides a plausible model for the ligation states of the [4Fe-4S]1+ core in the S = 3/2 cluster of the iron protein of nitrogenase and in Bacillus subtilis glutamine:phosphoribosyl pyrophosphate amidotransferase.     

A new class of [2Fe-2S]-cluster-containing protoporphyrin (IX) ferrochelatases

Protoporphyrin (IX) ferrochelatase catalyses the insertion of ferrous iron into protoporphyrin IX to form haem. These ferrochelatases exist as monomers and dimers, both with and without [2Fe-2S] clusters. The motifs for [2Fe-2S] cluster co-ordination are varied, but in all cases previously reported, three of the four cysteine ligands are present in the 30 C-terminal residues and the fourth ligand is internal. In the present study, we demonstrate that a group of micro-organisms exist which possess protoporphyrin (IX) ferrochelatases containing [2Fe-2S] clusters that are co-ordinated by a group of four cysteine residues contained in an internal amino acid segment of approx. 20 residues in length. This suggests that these ferrochelatases have evolved along a different lineage than other bacterial protoporphyrin (IX) ferrochelatases. For example, Myxococcus xanthus protoporphyrin (IX) ferrochelatase ligates a [2Fe-2S] cluster via cysteine residues present in an internal segment. Site-directed mutagenesis of this ferrochelatase demonstrates that changing one cysteine ligand into serine results in loss of the cluster, but unlike eukaryotic protoporphyrin (IX) ferrochelatases, this enzyme retains its activity. These data support a role for the [2Fe-2S] cluster in iron affinity, and strongly suggest convergent evolution of this feature in prokaryotes.     

The crystal structure of Trichomonas vaginalis ferredoxin provides insight into metronidazole activation

Crystallographic studies revealing the three-dimensional structure of the oxidized form of the [2Fe-2S] ferredoxin from Trichomonas vaginalis (TvFd) are presented. TvFd, a member of the hydrogenosomal class of ferredoxins, possesses a unique combination of redox and spectroscopic properties, and is believed to be the biological molecule that activates the drug metronidazole reductively in the treatment of trichomoniasis. It is the first hydrogenosomal ferredoxin to have its structure determined. The structure of TvFd reveals a monomeric, 93 residue protein with a fold similar to that of other known [2Fe-2S] ferredoxins. It contains nine hydrogen bonds to the sulfur atoms of the cluster, which is more than the number predicted on the basis of the spectroscopic data. The TvFd structure contains a large dipole moment like adrenodoxin, and appears to have a similar interaction domain. Our analysis demonstrates that TvFd has a unique cavity near the iron-sulfur cluster that exposes one of the inorganic sulfur atoms of the cluster to solvent. This cavity is not seen in any other [2Fe-2S] ferredoxin with known structure, and is hypothesized to be responsible for the high rate of metronidazole reduction by TvFd.     

Characterization and cloning of an extremely thermostable, Pyrococcus furiosus-type 4Fe ferredoxin from Thermococcus profundus.

An extremely thermostable [4Fe-4S] ferredoxin was isolated under anaerobic conditions from a hyperthermophilic archaeon Thermococcus profundus, and the ferredoxin gene was cloned and sequenced. The nucleotide sequence of the ferredoxin gene shows the ferredoxin to comprise 62 amino acid residues with a sequence similar to those of many bacterial and archaeal 4Fe (3Fe) ferredoxins. The unusual Fe-S cluster type, which was identified in the resonance Raman and EPR spectra, has three cysteines and one aspartate as the cluster ligands, as in the Pyrococcus furiosus 4Fe ferredoxin. Under aerobic conditions, a ferredoxin was purified that contains a [3Fe-4S] cluster as the major Fe-S cluster and a small amount of the [4Fe-4S] cluster. Its N-terminal amino acid sequence is the same as that of the anaerobically-purified ferredoxin up to the 26th residue. These results indicate that the 4Fe ferredoxin was degraded to 3Fe ferredoxin during aerobic purification. The aerobically-purified ferredoxin was reversibly converted back to the [4Fe-4S] ferredoxin by the addition of ferrous ions under reducing conditions. The anaerobically-purified [4Fe-4S] ferredoxin is quite stable; little degradtion was observed over 20 h at 100 degrees C, while the half-life of the aerobically-purified ferredoxin is 10 h at 100 degrees C. Both the anaerobically- and aerobically-purified ferredoxins were found to function as electron acceptors for the pyruvate-ferredoxin oxidoreductase purified from the same archaeon.     

A [2Fe-2S] protein from the hyperthermophilic bacterium Aquifex aeolicus.

Overexpression in Escherichia coli of the fdx4 gene from Aquifex aeolicus has allowed isolation and characterization of the first hyperthermophilic [2Fe-2S](Scys)(4) protein, a homodimer of M = 2 x 12.4 kDa with one [2Fe-2S] cluster per subunit. This protein is undamaged by heating to 100 degrees C for at least three hours. The primary structure, in particular the characteristic distribution of the four cysteine ligands of the metal site, and the spectroscopic properties of the A. aeolicus protein relate it to well characterized [2Fe-2S] proteins from Clostridium pasteurianum and Azotobacter vinelandii. These proteins are also homologous to subunits or domains of hydrogenases and NADH-ubiquinone oxidoreductase (Complex I) of respiratory chains. The A. aeolicus [2Fe-2S] protein is thus representative of a presumably novel protein fold involved in a variety of functions in very diverse cellular backgrounds.     

An Isc-type extremely thermostable [2Fe-2S] ferredoxin from Aquifex aeolicus. Biochemical,spectroscopic, and unfolding studies

Analysis of the genome of the hyperthermophilic bacterium Aquifex aeolicus has revealed the presence of a previously undetected gene potentially encoding a plant- and mammalian-type [2Fe-2S] ferredoxin. Expression of that gene in Escherichia coli has yielded a novel thermostable [2Fe-2S] ferredoxin (designated ferredoxin 5) whose sequence is most similar to those of ferredoxins involved in the assembly of iron-sulfur clusters (Isc-Fd). It nevertheless differs from the latter proteins by having deletions near its N- and C-termini, and no cysteine residues other than those involved in [2Fe-2S] cluster coordination. Resonance Raman, low-temperature MCD and EPR studies show close spectral similarities between ferredoxin 5 and the Isc-Fd from Azotobacter vinelandii. M?ssbauer spectra of the reduced protein were analyzed with an S = 1/2 spin Hamiltonian and interpreted in the framework of the ligand field model proposed by Bertrand and Gayda. The redox potential of A. aeolicus ferredoxin 5 (-390 mV) is in keeping with its relatedness to Isc-Fd. Unfolding experiments showed that A. aeolicus ferredoxin 5 is highly thermostable (T(m) = 106 degrees C at pH 7), despite being devoid of features (e.g., high content of charged residues) usually associated with extreme thermal stability. Searches for genes potentially encoding plant-type [2Fe-2S] ferredoxins have been performed on the sequenced genomes of hyperthermophilic organisms. None other than the two proteins from A. aeolicus were retrieved, indicating that this otherwise widely distributed group of proteins is barely represented among hyperthermophiles.     

Crystal structure of the 2[4Fe-4S] ferredoxin from Chromatium vinosum: evolutionary and mechanistic inferences for [3/4Fe-4S] ferredoxins.

The crystal structure of the 2[4Fe-4S] ferredoxin from Chromatium vinosum has been solved by molecular replacement using data recorded with synchrotron radiation. The crystals were hexagonal prisms that showed a strong tendency to develop into long tubes. The hexagonal prisms diffracted to 2.1 A resolution at best, and a structural model for C. vinosum ferredoxin has been built with a final R of 19.2%. The N-terminal domain coordinates the two [4Fe-4S] clusters in a fold that is almost identical to that of other known ferredoxins. However, the structure has two unique features. One is a six-residue insertion between two ligands of one cluster forming a two-turn external loop; this short loop changes the conformation of the Cys 40 ligand compared to other ferredoxins and hampers the building of one NH...S H-bond to one of the inorganic sulfurs. The other remarkable structural element is a 3.5-turn alpha-helix at the C-terminus that covers one side of the same cluster and is linked to the cluster-binding domain by a six-residue external chain segment. The charge distribution is highly asymmetric over the molecule. The structure of C. vinosum ferredoxin strongly suggests divergent evolution for bacterial [3/4Fe-4S] ferredoxins from a common ancestral cluster-binding core. The unexpected slow intramolecular electron transfer rate between the clusters in C. vinosum ferredoxin, compared to other similar proteins, may be attributed to the unusual electronic properties of one of the clusters arising from localized changes in its vicinity rather than to a global structural rearrangement.