Hybrid cluster proteins (HCPs) from Desulfovibrio desulfuricans ATCC 27774 and Desulfovibrio vulgaris (Hildenborough): X-ray structures at 1.25 Å resolution using synchrotron radiation

The structures of the hybrid cluster proteins (HCPs) from the sulfate-reducing bacteria Desulfovibrio desulfuricans (ATCC 27774) and Desulfovibrio vulgaris (Hildenborough) have been elucidated at a resolution of 1.25 A using X-ray synchrotron radiation techniques. In the case of the D. desulfuricans protein, protein isolation, purification, crystallization and X-ray data collection were carried out under strict anaerobic conditions, whereas for the D. vulgaris protein the conditions were aerobic. However, both structures are essentially the same, comprising three domains and two iron-sulfur centres. One of these centres situated near the exterior of the molecules in domain 1 is a cubane [4Fe-4S] cluster, whereas the other, located at the interface of the three domains, contains the unusual four-iron cluster initially found in the D. vulgaris protein. Details of the structures and the associated EPR spectroscopy of the D. desulfuricans protein are reported herein. These structures show that the nature of the hybrid cluster, containing both oxygen and sulfur bridges, is independent of the presence of oxygen in the isolation and crystallization procedure and also does not vary significantly with changes in the oxidation state. The structures and amino acid sequences of the HCP are compared with the recently elucidated structure of the catalytic subunit of a carbon monoxide dehydrogenase from Carboxydothermus hydrogenoformans and related dehydrogenases. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-001-0326-y.     

Gaseous hydrocarbons associated with black layer induced by the interaction of cyanobacteria and Desulfovibrio desulfuricans†

Black layer is a condition of high-sand-content golf greens that results in a subsurface blackened layer in the sand produced by sulfate-reducing bacteria. Black layer can be the product of an interaction of cyanobacteria and sulfate-reducing bacteria and may or may not be toxic to the grass growing on the sand. The organic byproducts of the cyanobacteria coat and plug the sand thereby creating an anoxic environment for development of the sulfate- reducing bacteria. The present study was initiated to determine the range of gaseous hydrocarbons evolved from black layered sand produced by the interaction of two genera of cyanobacteria, Nostoc and Oscillatoria, and Desulfovibrio desulfuricans. The gaseous hydrocarbons measured included methane, ethane, ethylene, and propylene. In nonblackened sand, Nostoc evolved the highest levels of these gases, Oscillatoria evolved relatively low levels except for propylene, and D. desulfuricans evolved the smallest quantities of the gases. When the cyanobacteria and D. desulfuricans were combined to develop black layered sand some changes occurred in the evolution of the gases. Evolution of the gases from Nostoc + D. desulfuricans decreased or remained the same relative to Nostoc alone, and increased relative to D. desulfuricans alone. Except for propylene evolution, gases from Oscillatoria + D. desulfuricans increased relative to Oscillatoria or D. desulfuricans alone. Propylene evolution from Oscillatoria + D. desulfuricans remained unchanged relative to Oscillatoria alone, but increased relative to D. desulfuricans alone. The gases measured are discussed relative to the organisms observed and the conditions of the study.     

M?ssbauer study of the native, reduced and substrate-reacted Desulfovibrio gigas aldehyde oxido-reductase.

The Desulfovibrio gigas aldehyde-oxido-reductase contains molybdenum and iron-sulfur clusters. M?ssbauer spectroscopy was used to characterize the iron-sulfur clusters. Spectra of the enzyme in its oxidized, partially reduced and benzaldehyde-reacted states were recorded at different temperatures and applied magnetic fields. All the iron atoms in D. gigas aldehyde oxido-reductase are organized as [2Fe-2S] clusters. In the oxidized enzyme, the clusters are diamagnetic and exhibit a single quadrupole doublet with parameters (delta EQ = 0.62 +/- 0.02 mm/s and delta = 0.27 +/- 0.01 mm/s) typical for the [2Fe-2S]2+ state. M?ssbauer spectra of the reduced clusters also show the characteristics of a [2Fe-2S]1+ cluster and can be explained by a spin-coupling model proposed for the [2Fe-2S] cluster where a high-spin ferrous ion (S = 2) is antiferromagnetically coupled to a high-spin ferric ion (S = 5/2) to form a S = 1/2 system. Two ferrous sites with different delta EQ values (3.42 mm/s and 2.93 mm/s at 85 K) are observed for the reduced enzyme, indicating the presence of two types of [2Fe-2S] clusters in the D. gigas enzyme. Taking this observation together with the re-evaluated value of iron content (3.5 +/- 0.1 Fe/molecule), it is concluded that, similar to other Mo-hydroxylases, the D. gigas aldehyde oxido-reductase also contains two spectroscopically distinguishable [2Fe-2S] clusters.     

Mo–Cu metal cluster formation and binding in an orange protein isolated from Desulfovibrio gigas

The orange protein (ORP) isolated from the sulfate-reducing bacterium Desulfovibrio gigas (11.8 kDa) contains a mixed-metal sulfide cluster of the type [S₂MoS₂CuS₂MoS₂]^3- noncovalently bound to the polypeptide chain. The D. gigas ORP was heterologously produced in Escherichia coli in the apo form. Different strategies were used to reconstitute the metal cluster into apo-ORP and obtain insights into the metal cluster synthesis: (1) incorporation of a synthesized inorganic analogue of the native metal cluster and (2) the in situ synthesis of the metal cluster on the addition to apo-ORP of copper chloride and tetrathiomolybdate or tetrathiotungstate. This latter procedure was successful, and the visible spectrum of the Mo–Cu reconstituted ORP is identical to the one reported for the native protein with absorption maxima at 340 and 480 nm. The ¹H–¹⁵N heteronuclear single quantum coherence spectra of the reconstituted ORP obtained by strategy 2, in contrast to strategy 1, exhibited large changes, which required sequential assignment in order to identify, by chemical shift differences, the residues affected by the incorporation of the cluster, which is stabilized inside the protein by both electrostatic and hydrophobic interactions.     

Hexaheme nitrite reductase from Desulfovibrio desulfuricans. M?ssbauer and EPR characterization of the heme groups

M?ssbauer and EPR spectroscopy were used to characterize the heme prosthetic groups of the nitrite reductase isolated from Desulfovibrio desulfuricans (ATCC 27774), which is a membrane-bound multiheme cytochrome capable of catalyzing the 6-electron reduction of nitrite to ammonia. At pH 7.6, the as-isolated enzyme exhibited a complex EPR spectrum consisting of a low-spin ferric heme signal at g = 2.96, 2.28, and 1.50 plus several broad resonances indicative of spin-spin interactions among the heme groups. EPR redox titration studies revealed yet another low-spin ferric heme signal at g = 3.2 and 2.14 (the third g value was undetected) and the presence of a high-spin ferric heme. M?ssbauer measurements demonstrated further that this enzyme contained six distinct heme groups: one high-spin (S = 5/2) and five low-spin (S = 1/2) ferric hemes. Characteristic hyperfine parameters for all six hemes were obtained through a detailed analysis of the M?ssbauer spectra. D. desulfuricans nitrite reductase can be reduced by chemical reductants, such as dithionite or reduced methyl viologen, or by hydrogenase under hydrogen atmosphere. Addition of nitrite to the fully reduced enzyme reoxidized all five low-spin hemes to their ferric states. The high-spin heme, however, was found to complex NO, suggesting that the high-spin heme could be the substrate binding site and that NO could be an intermediate present in an enzyme-bound form.     

DsrC is involved in fermentative growth and interacts directly with the FlxABCD–HdrABC complex in Desulfovibrio vulgaris Hildenborough

DsrC is a key protein in dissimilatory sulfur metabolism, where it works as co-substrate of the dissimilatory sulfite reductase DsrAB. DsrC has two conserved cysteines in a C-terminal arm that are converted to a trisulfide upon reduction of sulfite. In sulfate-reducing bacteria, DsrC is essential and previous works suggested additional functions beyond sulfite reduction. Here, we studied whether DsrC also plays a role during fermentative growth of Desulfovibrio vulgaris Hildenborough, by studying two strains where the functionality of DsrC is impaired by a lower level of expression (IPFG07) and additionally by the absence of one conserved Cys (IPFG09). Growth studies coupled with metabolite and proteomic analyses reveal that fermentation leads to lower levels of DsrC, but impairment of its function results in reduced growth by fermentation and a shift towards more fermentative metabolism during sulfate respiration. In both respiratory and fermentative conditions, there is increased abundance of the FlxABCD–HdrABC complex and Adh alcohol dehydrogenase in IPFG09 versus the wild type, which is reflected in higher production of ethanol. Pull-down experiments confirmed a direct interaction between DsrC and the FlxABCD–HdrABC complex, through the HdrB subunit. Dissimilatory sulfur metabolism, where sulfur compounds are used for energy generation, is a key process in the ecology of anoxic environments, and is more widespread among bacteria than previously believed. Two central proteins for this type of metabolism are DsrAB dissimilatory sulfite reductase and its co-substrate DsrC. Using physiological, proteomic and biochemical studies of Desulfovibrio vulgaris Hildenborough and mutants affected in DsrC functionality, we show that DsrC is also relevant for fermentative growth of this model organism and that it interacts directly with the soluble FlxABCD-HdrABC complex that links the NAD(H) pool with dissimilatory sulfite reduction.     

Interaction of the active site of the Ni–Fe–Se hydrogenase from Desulfovibrio vulgaris Hildenborough with carbon monoxide and oxygen inhibitors

The study of Ni–Fe–Se hydrogenases is interesting from the basic research point of view because their active site is a clear example of how nature regulates the catalytic function of an enzyme by the change of a single residue, in this case a cysteine, which is replaced by a selenocysteine. Most hydrogenases are inhibited by CO and O₂. In this work we studied these inhibition processes for the Ni–Fe–Se hydrogenase from Desulfovibrio vulgaris Hildenborough by combining catalytic activity measurements, followed by mass spectrometry or chronoamperometry, with Fourier transform IR spectroscopy experiments. The results show that the CO inhibitor binds to Ni in both conformations of the active site of this hydrogenase in a way similar to that in standard Ni–Fe hydrogenases, although in one of the CO-inhibited conformations the active site of the Ni–Fe–Se hydrogenase is more protected against the attack by O₂. The inhibition of the Ni–Fe–Se hydrogenase activity by O₂ could be explained by oxidation of the terminal cysteine ligand of the active-site Ni, instead of the direct attack of O₂ on the bridging site between Ni and Fe.     

Can cofactor-binding sites in proteins be flexible? Desulfovibrio desulfuricans flavodoxin binds FMN dimer

Flavodoxins catalyze redox reactions using the isoalloxazine moiety of the flavin mononucleotide (FMN) cofactor stacked between two aromatic residues located in two peptide loops. At high FMN concentrations that favor stacked FMN dimers in solution, isothermal titration calorimetric studies show that these dimers bind strongly to apo-flavodoxin from Desulfovibrio desulfuricans (30 degrees C, 20 mM Hepes, pH 7, K(D) = 5.8 microM). Upon increasing the temperature so the FMN dimers dissociate (as shown by (1)H NMR), only one-to-one (FMN-to-protein) binding is observed. Calorimetric titrations result in one-to-one binding also in the presence of phosphate or sulfate (30 degrees C, 13 mM anion, pH 7, K(D) = 0.4 microM). FMN remains dimeric in the presence of phosphate and sulfate, suggesting that specific binding of a divalent anion to the phosphate-binding site triggers ordering of the peptide loops so only one isoalloxazine can fit. Although the physiological relevance of FMN and other nucleotides as dimers has not been explored, our study shows that high-affinity binding to proteins of such dimers can occur in vitro. This emphasizes that the cofactor-binding site in flavodoxin is more flexible than previously expected.     

Redox intermediates of Desulfovibrio gigas [NiFe] hydrogenase generated under hydrogen. M?ssbauer and EPR characterization of the metal centers

The hydrogenase (EC 1.2.2.1) of Desulfovibrio gigas is a complex enzyme containing one nickel center, one [3Fe-4S] and two [4Fe-4S] clusters. Redox intermediates of this enzyme were generated under hydrogen (the natural substrate) using a redox-titration technique and were studied by EPR and M?ssbauer spectroscopy. In the oxidized states, the two [4Fe-4S]2+ clusters exhibit a broad quadrupole doublet with parameters (apparent delta EQ = 1.10 mm/s and delta = 0.35 mm/s) typical for this type of cluster. Upon reduction, the two [4Fe-4S]1+ clusters are spectroscopically distinguishable, allowing the determination of their midpoint redox potentials. The cluster with higher midpoint potential (-290 +/- 20 mV) was labeled Fe-S center I and the other with lower potential (-340 +/- 20 mV), Fe-S center II. Both reduced clusters show atypical magnetic hyperfine coupling constants, suggesting structural differences from the clusters of bacterial ferredoxins. Also, an unusually broad EPR signal, labeled Fe-S signal B', extending from approximately 150 to approximately 450 mT was observed concomitantly with the reduction of the [4Fe-4S] clusters. The following two EPR signals observed at the weak-field region were tentatively attributed to the reduced [3Fe-4S] cluster: (i) a signal with crossover point at g approximately 12, labeled the g = 12 signal, and (ii) a broad signal at the very weak-field region (approximately 3 mT), labeled the Fe-S signal B. The midpoint redox potential associated with the appearance of the g = 12 signal was determined to be -70 +/- 10 mV. At potentials below -250 mV, the g = 12 signal began to decrease in intensity, and simultaneously, the Fe-S signal B appeared. The transformation of the g = 12 signal into the Fe-S signal B was found to parallel the reduction of the two [4Fe-4S] clusters indicating that the [3Fe-4S]o cluster is sensitive to the redox state of the [4Fe-4S] clusters. Detailed redox profiles for the previously reported Ni-signal C and the g = 2.21 signal were obtained in this study, and evidence was found to indicate that these two signals represent two different oxidation states of the enzyme. Finally, the mechanistic implications of our results are discussed.     

Structure refinement of the aldehyde oxidoreductase from Desulfovibrio gigas (MOP) at 1.28 Å

The sulfate-reducing bacterium aldehyde oxidoreductase from Desulfovibrio gigas (MOP) is a member of the xanthine oxidase family of enzymes. It has 907 residues on a single polypeptide chain, a molybdopterin cytosine dinucleotide (MCD) cofactor and two [2Fe-2S] iron-sulfur clusters. Synchrotron data to almost atomic resolution were collected for improved cryo-cooled crystals of this enzyme in the oxidized form. The cell constants of a=b=141.78 A and c=160.87 A are about 2% shorter than those of room temperature data, yielding 233,755 unique reflections in space group P6(1)22, at 1.28 A resolution. Throughout the entire refinement the full gradient least-squares method was used, leading to a final R factor of 14.5 and Rfree factor of 19.3 (4sigma cut-off) with "riding" H-atoms at their calculated positions. The model contains 8146 non-hydrogen atoms described by anisotropic displacement parameters with an observations/parameters ratio of 4.4. It includes alternate conformations for 17 amino acid residues. At 1.28 A resolution, three Cl- and two Mg2+ ions from the crystallization solution were clearly identified. With the exception of one Cl- which is buried and 8 A distant from the Mo atom, the other ions are close to the molecular surface and may contribute to crystal packing. The overall structure has not changed in comparison to the lower resolution model apart from local corrections that included some loop adjustments and alternate side-chain conformations. Based on the estimated errors of bond distances obtained by blocked least-squares matrix inversion, a more detailed analysis of the three redox centres was possible. For the MCD cofactor, the resulting geometric parameters confirmed its reduction state as a tetrahydropterin. At the Mo centre, estimated corrections calculated for the Fourier ripples artefact are very small when compared to the experimental associated errors, supporting the suggestion that the fifth ligand is a water molecule rather than a hydroxide. Concerning the two iron-sulfur centres, asymmetry in the Fe-S distances as well as differences in the pattern of NH.S hydrogen-bonding interactions was observed, which influences the electron distribution upon reduction and causes non-equivalence of the individual Fe atoms in each cluster.     

Spin distribution of the H-cluster in the Hox–CO state of the [FeFe] hydrogenase from Desulfovibrio desulfuricans: HYSCORE and ENDOR study of 14N and 13C nuclear interactions

Hydrogenases are enzymes which catalyze the reversible cleavage of molecular hydrogen into protons and electrons. In [FeFe] hydrogenases the active center is a 6Fe6S cluster, referred to as the “H-cluster.” It consists of the redox-active binuclear subcluster ([2Fe]_H) coordinated by CN⁻ and CO ligands and the cubane-like [4Fe–4S]_H subcluster which is connected to the protein via Cys ligands. One of these Cys ligands bridges to the [2Fe]_H subcluster. The CO-inhibited form of [FeFe] hydrogenase isolated from Desulfovibrio desulfuricans was studied using advanced EPR methods. In the H_ox–CO state the open coordination site at the [2Fe]_H subcluster is blocked by extrinsic CO, giving rise to an EPR-active S = 1/2 species. The CO inhibited state was prepared with ¹³CO and illuminated under white light at 273 K. In this case scrambling of the CO ligands occurs. Three ¹³C hyperfine couplings of 17.1, 7.4, and 3.8 MHz (isotropic part) were observed and assigned to ¹³CO at the extrinsic, the bridging, and the terminal CO-ligand positions of the distal iron, respectively. No ¹³CO exchange of the CO ligand to the proximal iron was observed. The hyperfine interactions detected indicate a rather large distribution of the spin density over the terminal and bridging CO ligands attached to the distal iron. Furthermore, ¹⁴N nuclear spin interactions were measured. On the basis of the observed ¹⁴N hyperfine couplings, which result from the CN⁻ ligands of the [2Fe]_H subcluster, it has been concluded that there is very little unpaired spin density on the cyanides of the binuclear subcluster.

Ab initio structure solution of a dimeric cytochrome c 3 from Desulfovibrio gigas containing disulfide bridges

The 1.2?Å resolution crystal structure of the 29?kDa di-tetrahaem cytochrome c ₃ from the sulfate reducing bacterium Desulfovibrio gigas was solved by ab initio methods, making this the largest molecule to be solved by this procedure. The actual refined model of the cysteine-linked dimeric molecule reveals that this molecule is very similar to the non-covalently linked symmetrical dimer of the di-tetrahaem cytochrome c ₃ from Desulfomicrobium norvegicum. Each monomer has the typical polypeptide fold, haem arrangement and iron coordination found for the tetrahaem cytochrome c ₃ molecules. The interface between the covalently linked monomers in the asymmetric unit of the crystal shows a pseudo two-fold arrangement, disturbed from symmetry by crystal packing forces. The fact that D. gigas contains a dimeric tetrahaem cytochrome with solvent accessible disulfide bridges and that this cytochrome specifically couples hydrogen oxidation to thiosulfate reduction in bacterial extracts provides an interesting aspect related to disulfide exchange reactions in this microorganism.     

Multi-frequency EPR and high-resolution M?ssbauer spectroscopy of a putative [6Fe-6S] prismane-cluster-containing protein from Desulfovibrio vulgaris (Hildenborough). Characterization of a supercluster and superspin model protein.

The putative [6Fe-6S] prismane cluster in the 6-Fe/S-containing protein from Desulfovibrio vulgaris, strain Hildenborough, has been enriched to 80% in 57Fe, and has been characterized in detail by S-, X-, P- and Q-band EPR spectroscopy, parallel-mode EPR spectroscopy and high-resolution 57Fe M?ssbauer spectroscopy. In EPR-monitored redox-equilibrium titrations, the cluster is found to be capable of three one-electron transitions with midpoint potentials at pH 7.5 of +285, +5 and -165 mV. As the fully reduced protein is assumed to carry the [6Fe-6S]3+ cluster, by spectroscopic analogy to prismane model compounds, four valency states are identified in the titration experiments: [6Fe-6S]3+, [6Fe-6S]4+, [6Fe-6S]5+, [6Fe-6S]6+. The fully oxidized 6+ state appears to be diamagnetic at low temperature. The prismane protein is aerobically isolated predominantly in the one-electron-reduced 5+ state. In this intermediate state, the cluster exists in two magnetic forms: 10% is low-spin S = 1/2; the remainder has an unusually high spin S = 9/2. The S = 1/2 EPR spectrum is significantly broadened by ligand (2.3 mT) and 57Fe (3.0 mT) hyperfine interaction, consistent with a delocalization of the unpaired electron over 6Fe and indicative of at least some nitrogen ligation. At 35 GHz, the g tensor is determined as 1.971, 1.951 and 1.898. EPR signals from the S = 9/2 multiplet have their maximal amplitude at a temperature of 12 K due to the axial zero-field splitting being negative, D approximately -0.86 cm-1. Effective g = 15.3, 5.75, 5.65 and 5.23 are observed, consistent with a rhombicity of [E/D] = 0.061. A second component has g = 9.7, 8.1 and 6.65 and [E/D] = 0.108. When the protein is reduced to the 4+ intermediate state, the cluster is silent in normal-mode EPR. An asymmetric feature with effective g approximately 16 is observed in parallel-mode EPR from an integer spin system with, presumably, S = 4. The fully reduced 3+ state consists of a mixture of two S = 1/2 ground state. The g tensor of the major component is 2.010, 1.825 and 1.32; the minor component has g = 1.941 and 1.79, with the third value undetermined. The sharp line at g = 2.010 exhibits significant convoluted hyperfine broadening from ligands (2.1 mT) and from 57Fe (4.6 mT). Zero-field high-temperature M?ssbauer spectra of the protein, isolated in the 5+ state, quantitatively account for the 0.8 fractional enrichment in 57Fe, as determined with inductively coupled plasma mass spectrometry.(ABSTRACT TRUNCATED AT 400 WORDS)     

NMR assignment of the apo-form of a Desulfovibrio gigas protein containing a novel Mo–Cu cluster

We report the 98% assignment of the apo-form of an orange protein, containing a novel Mo–Cu cluster isolated from Desulfovibrio gigas. This protein presents a region where backbone amide protons exchange fast with bulk solvent becoming undetectable. These residues were assigned using ¹³C-detection experiments.     

[NiFe] hydrogenase from Desulfovibrio desulfuricans ATCC 27774: gene sequencing, three-dimensional structure determination and refinement at 1.8 Å and modelling studies of its interaction with the tetrahaem cytochrome c 3

The primary and three-dimensional structures of a [NiFe] hydrogenase isolated from D. desulfitricans ATCC 27774 were determined, by nucleotide analysis and single-crystal X-ray crystallography. The three-dimensional structural model was refined to R=0.167 and Rfree=0.223 using data to 1.8 A resolution. Two unique structural features are observed: the [4Fe-4S] cluster nearest the [NiFe] centre has been modified [4Fe-3S-3O] by loss of one sulfur atom and inclusion of three oxygen atoms; a three-fold disorder was observed for Cys536 which binds to the nickel atom in the [NiFe] centre. Also, the bridging sulfur atom that caps the active site was found to have partial occupancy, thus corresponding to a partly activated enzyme. These structural features may have biological relevance. In particular, the two less-populated rotamers of Cys536 may be involved in the activation process of the enzyme, as well as in the catalytic cycle. Molecular modelling studies were carried out on the interaction between this [NiFe] hydrogenase and its physiological partner, the tetrahaem cytochrome c3 from the same organism. The lowest energy docking solutions were found to correspond to an interaction between the haem IV region in tetrahaem cytochrome c3 with the distal [4Fe-4S] cluster in [NiFe] hydrogenase. This interaction should correspond to efficient electron transfer and be physiologically relevant, given the proximity of the two redox centres and the fact that electron transfer decay coupling calculations show high coupling values and a short electron transfer pathway. On the other hand, other docking solutions have been found that, despite showing low electron transfer efficiency, may give clues on possible proton transfer mechanisms between the two molecules.     

Nine-haem cytochrome c from Desulfovibrio desulfuricans ATCC 27774 : primary sequence determination, crystallographic refinement at 1.8 and modelling studies of its interaction with the tetrahaem cytochrome c 3

A monomeric nine-haem cytochrome c (9Hcc) with 292 amino acid residues was isolated from cells of the sulfate- and nitrate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 grown under both nitrate- and sulfate-respiring conditions. The nucleotide sequence encoding the 292 residues was determined, allowing the correction of about 10% of the previous primary structure, determined from 1.8?Å electron density maps. The refinement at 1.8?Å resolution of the structural model was completed, giving an R-value of 16.5%. The nine haem groups are arranged into two tetrahaem clusters, located at both ends of the molecule, with Fe-Fe distances and local protein fold very similar to tetrahaem cytochromes c ₃, and the extra haem is located asymmetrically between the two regions. The new primary sequence determination confirmed the 39% sequence homology found between this cytochrome and the C-terminal region (residues 229–514) of the high-molecular-weight cytochrome c (Hmc) from D. vulgaris Hildenborough, providing strong evidence of structural similarity between 9Hcc and the C-terminal region of Hmc. The interaction between 9Hcc and the tetrahaem cytochrome c ₃ from the same organism was studied by modelling methods, and the results suggest that a specific interaction is possible between haem 4 of tetrahaem cytochrome c ₃ and haem 1 or haem 2 of 9Hcc, in agreement with previous kinetic experiments which showed the catalytic effect of the tetrahaem cytochrome c ₃ upon the reduction of 9Hcc by the [NiFe] hydrogenase from D. desulfuricans ATCC 27774. These studies suggest a role for 9Hcc as part of the assembly of redox proteins involved in recycling the molecular hydrogen released by the cell as a result of substrate oxidation.     

Cr(VI) detoxification by Desulfovibrio vulgaris strain Hildenborough: microbe–metal interactions studies

Toxic heavy metals constitute a worldwide environmental pollution problem. Bioremediation technologies represent efficient alternatives to the classic cleaning-up of contaminated soil and ground water. Most toxic heavy metals such as chromium are less soluble and toxic when reduced than when oxidized. Sulfate-reducing bacteria (SRB) are able to reduce heavy metals by a chemical reduction via the production of H₂S and by a direct enzymatic process involving hydrogenases and c₃ cytochromes. We have previously reported the effects of chromate [Cr(VI)] on SRB bioenergetic metabolism and the molecular mechanism of the metal reduction by polyhemic cytochromes. In the current work, we pinpoint the bacteria–metal interactions using Desulfovibrio vulgaris strain Hildenborough as a model. The bacteria were grown in the presence of high Cr(VI) concentration, where they accumulated precipitates of a reduced form of chromium, trivalent chromium [Cr(III)], on their cell surfaces. Moreover, the inner and outer membranes exhibited precipitates that shared the spectroscopic signature of trivalent chromium. This subcellular localization is consistent with enzymatic metal reduction by cytochromes and hydrogenases. Regarding environmental significance, our findings point out the Cr(VI) immobilization mechanisms of SRB; suggesting that SRB are highly important in metal biogeochemistry.