Desulfovibrio Gigas Research Articles

The membrane-bound high-molecular-mass cytochromes c from Desulfovibrio gigas and Desulfovibrio vulgaris Hildenborough; EPR and Mössbauer studies

The high-molecular-mass cytochromes c (Hmcs) from the sulfate-reducing bacteria Desulfovibrio gigas and Desulfovibrio vulgaris (Hildenborough) were found to be strongly bound to the cytoplasmic membrane. After detergent solubilization they were shown to be water soluble and to be similar to those previously isolated from the soluble fractions in terms of N-terminal sequence, molecular mass, UV-visible and EPR spectroscopies. In D. gigas, higher amounts of Hmc can be obtained from the membranes than from the soluble fraction. This enabled further characterization of both cytochromes. The apparent heme reduction potentials of both Hmcs, determined at pH 7.5 through visible and EPR redox titrations, span a large range of redox potentials, approximately between 0 and –280 mV, and can be roughly divided into three groups: four to five hemes have E0s of –30 mV to –100 mV, three to four hemes have E0s around –170 mV, and seven to eight hemes have a lower E0 of –250 to –280 mV. Several of these redox potentials are strongly pH dependent. Mossbauer studies of oxidized and reduced D. vulgaris Hmc show that this protein contains two high-spin hemes in both oxidation states. The rate of reduction of both Hmcs with the periplasmic hydrogenases from the corresponding organisms is extremely slow.

The cytochrome c3 superfamily: amino acid sequence of a dimeric octahaem cytochrome c3 (M(r) 26,000) isolated from Desulfovibrio gigas.

Cytochrome c3 (M(r) 26000) isolated from Desulfovibrio gigas is a dimeric cytochrome consisting of two identical subunits of 109 amino acids, each of which contains four haem groups. On the basis of its amino acid sequence, this cytochrome clearly belongs to the cytochrome c3 superfamily, and will be classified in class III of the c-type cytochromes as defined by Ambler [(1980) in From Cyclotrons to Cytochromes (Robinson, A. B. and Kaplan, N. O., eds.), pp. 263-279, Academic Press, London]. It contains ten cysteine and nine histidine residues in each subunit, and eight cysteines and eight histidines linked to the four haem groups were found to be invariant on alignment of all known cytochrome c3 sequences. Two intermolecular disulphide bridges have been determined between cysteine residues 5 and 46 of the two monomers. Cytochrome c3 (M(r) 26,000) from D gigas is clearly different from cytochrome c3 (M(r) 13,000) from the same strain, with which it shows only 27% sequence identity. Compared with cytochrome c3 (M(r) 26,000) from D. desulfuricans Norway, the three-dimensional structure of which has been determined, 26.95% of the residues have been conserved. In the enzyme from D. desulfuricans Norway, hydrophobic interactions have been described across the dimer interface. Residues involved in similar interactions seem to be well conserved in the equivalent D. gigas cytochrome. This sequence provides structural data to allow specification of this new subclass of polyhaem cytochromes. Furthermore, D. gigas cytochrome c3 (M(r) 26,000) is the first polyhaem cytochrome shown to contain two disulphide bridges linking two identical subunits, which could induce more rigid folding. The folding and the evolution of this family of polyhaem cytochromes are discussed.

Electron-dense granules in Desulfovibrio gigas do not consist of inorganic triphosphate but of a glucose pentakis(diphosphate).

Under certain growth conditions the sulfate-reducing bacterium Desulfovibrio gigas forms electron-dense granules in the cells which had been claimed to consist of a magnesium triphosphate). We observed granules after cultivation in media with a low Fe2+ or NH4+ concentration and reinvestigated the nature of the electron-dense bodies. Energy-dispersive X-ray analysis of the granules in the cells showed that they contain large amounts of P, Mg, and K. Gel electrophoresis and chromatographic analyses of isolated granules which had been dissolved in 20 mM EDTA, however, revealed discrepancies with commercially available polyphosphates. 31P-NMR spectra also lacked the peaks in the -22-ppm region which are characteristic for inner phosphates of polyphosphates confirming that the phosphocompound as isolated from the electron-dense bodies of D. gigas did not consist of polyphosphates. Using multinuclear NMR spectroscopy we showed that the electron-dense bodies of D. gigas contained a novel metabolite which was identified as alpha-glucose 1,2,3,4,6-pentakis(diphosphate).

Overproduction of the rbo gene product from Desulfovibrio species suppresses all deleterious effects of lack of superoxide dismutase in Escherichia coli.

In an attempt to isolate the superoxide dismutase (SOD) gene from the anaerobic sulfate-reducing bacterium Desulfoarculus baarsii, a DNA fragment was isolated which functionally complemented an Escherichia coli mutant (sodA sodB) deficient in cytoplasmic SODs. This region carries two open reading frames with sequences which are very similar to that of the rbo-rub operon from Desulfovibrio vulgaris. Independent expression of the rbo and rub genes from ptac showed that expression of rbo was responsible for the observed phenotype. rbo overexpression suppressed all deleterious effects of SOD deficiency in E. coli, including inactivation by superoxide of enzymes containing 4Fe-4S clusters and DNA damage produced via the superoxide-enhanced Fenton reaction. Thus, rbo restored to the sodA sodB mutant the ability to grow on minimal medium without the addition of branched amino acids, and growth on gluconate and succinate carbon sources was no longer impaired. The spontaneous mutation rate, which is elevated in SOD-deficient mutants, returned to the wild-type level in the presence of Rbo, which also restored aerobic viability of sodA sodB recA mutants. Rbo from Desulfovibrio vulgaris, but not Desulfovibrio gigas desulforedoxin, which corresponds to the NH2-terminal domain of Rbo, complemented sod mutants. The physiological role of Rbo in sulfate-reducing bacteria is unknown. In E. coli, Rbo may permit the bacterium to avoid superoxide stress by maintaining functional (reduced) superoxide sensitive 4Fe-4S clusters. It would thereby restore enzyme activities and prevent the release of iron that occurs after cluster degradation and presumably leads to DNA damage.

Structure of the Ni Sites in Hydrogenases by X-ray Absorption Spectroscopy. Species Variation and the Effects of Redox Poise

Structural information obtained from the analysis of nickel K-edge X-ray absorption spectroscopic data of [NiFe]hydrogenases from Desulfovibrio gigas, Thiocapsa roseopersicina, Desulfovibrio desulfuricans (ATCC 27774), Escherichia coli (hydrogenase-1), Chromatium vinosum, and Alcaligenes eutrophus H16 (NAD+-reducing, soluble hydrogenase), poised in different redox states, is reported. The data allow the active-site structures of enzymes from several species to be compared, and allow the effects of redox poise on the structure of the nickel sites to be examined. In addition, the structure of the nickel site obtained from recent crystallographic studies of the D. gigas enzyme (Volbeda, A.; Charon, M.-H.; Piras, C.; Hatchikian, E. C.; Frey, M.; Fontecilla-Camps, J. C. Nature 1995, 373, 580−587) is compared with the structural features obtained from the analysis of XAS data from the same enzyme. The nickel sites of all but the oxidized (as isolated) sample of A. eutrophus hydrogenase are quite similar. The nickel K-edge energies shift 0.9−1.5 eV to lower energy upon reduction from oxidized (forms A and B) to fully reduced forms. This value is comparable with no more than a one-electron metal-centered oxidation state change. With the exception of T. roseopersicina hydrogenase, most of the edge energy shift (∼0.8 eV) occurs upon reduction of the oxidized enzymes to the EPR-silent intermediate redox level (SI). Analysis of the XANES features assigned to 1s → 3d electronic transitions indicates that the shift in energy that occurs for reduction of the enzymes to the SI level may be attributed at least in part to an increase in the coordination number from five to six. The smallest edge energy shift is observed for the T. roseopersicina enzyme, where the XANES data indicate that the nickel center is always six-coordinate. With the exception of the oxidized sample of A. eutrophus hydrogenase, the EXAFS data are dominated by scattering from S-donor ligands at ∼2.2 Å. The enzyme obtained from T. roseopersicina also shows evidence for the presence of O,N-donor ligands. The data from A. eutrophus hydrogenase are unique in that they indicate that a significant structural change occurs upon reduction of the enzyme. EXAFS data obtained from the oxidized (as isolated) A. eutrophus enzyme indicate that the EXAFS is dominated by scattering from 3−4 N,O-donor atoms at 2.06(2) Å, with contributions from 2−3 S-donor ligands at 2.35(2) Å. This changes upon reduction to a more typical nickel site composed of ∼4 S-donor ligands at a Ni−S distance of 2.19(2) Å. Evidence for the presence of atoms in the 2.4−2.9 Å distance range is found in most samples, particularly the reduced enzymes (SI, form C, and R). The analysis of these data is complicated by the fact that it is difficult to distinguish between S and Fe scattering atoms at this distance, and by the potential presence of both S and another metal atom at similar distances. The results of EXAFS analysis are shown to be in general agreement with the published crystal structure of the D. gigas enzyme.

Structure and function of mononuclear molybdenum enzymes

An account of recent advances in our understanding of enzymes possessing mononuclear molybdenum active sites is presented. On the basis of a variety of structural studies, and in conjunction with comparisons among the growing number of these enzymes for which the amino acid sequences are known, it is evident that these enzymes fall into three principal families as represented by xanthine oxidase, sulfite oxidase and DMSO reductase. Structural features of the active sites of these three families are compared and contrasted, and specific mechanistic implications of the recently reported crystal structure for the aldehyde oxidoreductase from Desulfovibrio gigas are discussed.

Protein crystallography with non-detergent sulfobetaines

The use in protein crystal growth of the newly described non-detergent solubilizers/stabilizers sulfobetaines (NDSB) has been investigated with crystallographic methods. In the classic model of egg white lysozyme crystallization by NaCl, NDSB increased the protein solubility and the crystal growth rate without influencing the quality of crystals as shown by Wilson plots. Sulphur anomalous X-ray scattering was used to show the absence of any bound NDSB in the crystals. A practical example of the use of NDSB is given for Desulfovibrio Gigas type II ferrodoxin, where NDSB contributed to reduction in crystal twinning and resulted in the growth of a new crystal form. With their unique combination of solubilizing and stabilizing properties, the NDSB molecules might prove to be valuable additives for protein crystal growth.

Cloning, Expression, and Sequence Analysis of the Three Genes Encoding Quinoline 2-Oxidoreductase, a Molybdenum-containing Hydroxylase from Pseudomonas putida 86

The three genes coding for quinoline 2-oxidoreductase (Qor) of Pseudomonas putida 86 were cloned and sequenced. The qor genes are clustered in the transcriptional order medium (M) small (S), large (L) and code for three subunits of 288 (QorM), 168 (QorS), and 788 (QorL) amino acids, respectively. Formation of active quinoline 2-oxidoreductase and degradation of quinoline occurred in a recombinant P. putida KT2440 clone. The amino acid sequences of Qor show significant homology to various prokaryotic molybdenum containing hydroxylases and to eukaryotic xanthine dehydrogenases. QorS contains two conserved motifs for [2Fe-2S] clusters. The binding motif for the N-terminal [2Fe-2S] cluster corresponds to the binding site of bacterial and chloroplast-type [2Fe-2S] ferredoxins, whereas the amino acid pattern of the internal [2Fe-2S] center apparently is a distinct feature of molybdenum-containing hydroxylases, showing no homology to any other described [2Fe-2S] binding motif. The medium subunit QorM presumably contains the FAD, but no conserved sequence areas or described motifs of FAD, NAD, NADP, or ATP binding were detected. Putative binding sites of the molybdopterin cytosine dinucleotide cofactor were detected in QorL by comparison with "contacting segments" recently described in aldehyde oxidoreductase from Desulfovibrio gigas (Romão, M. J., Archer, M., Moura, I., Moura, J. J. G., LeGall, J., Engh, R., Schneider, M., Hof, P., and Huber, R. (1995) Science 270, 1170-1176).

Interactions of 77Se and 13CO with Nickel in the Active Site of Active F420-nonreducing Hydrogenase from Methanococcus voltae

The selenium-containing F420-nonreducing hydrogenase from Methanococcus voltae was prepared in the Nia(I) middle dotCO state. The effect of illumination on this light-sensitive species was studied. EPR studies were carried out with enzyme containing natural selenium or with enzyme enriched in 77Se. Samples were prepared with either CO or 13CO. In the Nia(I) middle dotCO state, the nuclear spins of both 77Se (I = 1/2) and 13C (I = 1/2) interacted with the nickel-based unpaired electron, suggesting that they are positioned on opposite sites of the nickel ion. In the light-induced signal, the interaction with 13CO was lost. The 77Se nuclear spin introduced an anisotropic hyperfine splitting in both the dark and light-induced EPR signals. The data on the active enzyme of M. voltae are difficult to reconcile with the crystal structure of the inactive hydrogenase of Desulfovibrio gigas (Volbeda, A., Charon, M. H., Piras, C., Hatchikian, E. C., Frey, M., and Fontecilla Camps, J. C. (1995) Nature 373, 580-587) and suggest a structural change in the active site upon activation of the enzyme.

Diversity of Nitrogen Fixation Genes in the Symbiotic Intestinal Microflora of the Termite Reticulitermes speratus

The diversity of nitrogen-fixing organisms in the symbiotic intestinal microflora of a lower termite, Reticulitermes speratus, was investigated without culturing the resident microorganisms. Fragments of the nifH gene, which encodes the dinitrogenase reductase, were directly amplified from the DNA of the mixed microbial population in the termite gut and were clonally isolated. The phylogenetic analysis of the nifH product amino acid sequences showed that there was a remarkable diversity of nitrogenase genes in the termite gut. A large number of the termite nifH sequences were most closely related to those of a firmicute, Clostridium pasteurianum, with a few being most closely related to either the (gamma) subclass of the proteobacteria or a sequence of Desulfovibrio gigas. Some of the others were distantly related to those of the bacteria and were seemingly derived from the domain Archaea. The phylogenetic positions of these nifH sequences corresponded to those of genera found during a previous determination of rRNA-based phylogeny of the termite intestinal microbial community, of which a majority consisted of new, yet-uncultivated species. The results revealed that we have little knowledge of the organisms responsible for nitrogen fixation in termites.

Cytochrome c3 from Desulfovibrio gigas: crystal structure at 1.8 A resolution and evidence for a specific calcium-binding site.

Crystals of the tetraheme cytochrome c3 from sulfate-reducing bacteria Desulfovibrio gigas (Dg) (MW 13 kDa, 111 residues, four heme groups) were obtained and X-ray diffraction data collected to 1.8 A resolution. The structure was solved by the method of molecular replacement and the resulting model refined to a conventional R-factor of 14.9%. The three-dimensional structure shows many similarities to other known crystal structures of tetraheme c3 cytochromes, but it also shows some remarkable differences. In particular, the location of the aromatic residues around the heme groups, which may play a fundamental role in the electron transfer processes of the molecule, are well conserved in the cases of hemes I, III, and IV. However, heme II has an aromatic environment that is completely different to that found in other related cytochromes c3. Another unusual feature is the presence of a Ca2+ ion coordinated by oxygen atoms supplied by the protein within a loop near the N-terminus. It is speculated that this loop may be stabilized by the presence of this Ca2+ ion, may contribute to heme-redox perturbation, and might even be involved in the specificity of recognition with its redox partner.

Studies of light-induced nickel EPR signals in hydrogenase: comparison of enzymes with and without selenium

Upon reduction under hydrogen-argon atmosphere, the nickel-hydrogenases generally show a characteristic rhombic EPR spectrum which is known as NiC. Illumination of this state at temperatures below 60 K has previously been shown to cause the disappearance of the Ni-C signal and the simultaneous appearance of two overlapping signals, here referred to as NiL1 and NiL2, which revert to the Ni-C state at higher temperatures. These phenomena have been compared in three nickel-containing hydrogenases, the [NiFe]-hydrogenases from Desulfovibrio gigas and Desulfovibrio fructosouorans, and the selenium-containing soluble [NiFeSe]-hydrogenase from Desulfomicrobium baculatum. Significant differences were observed between these enzymes. (1) The NiC, NiL1 and NiL2 EPR spectra were almost identical for D. gigas and D. fructosouorans hydrogenases, but the rates of photoconversion of NiC to NiL1 were different, being about 5 times slower for D. fructosouorans than for D. gigas in H 2O. (2) The kinetic isotope effect in 2H 20/H 20 was a factor of thirty in D. gigas, but only two in D. fructosouorans. Dm. baculatum hydrogenase showed almost no kinetic isotope effect on the NiC to NiL1 conversion, but an effect on the conversion of NiL1 to NiL2. The kinetic isotope effects indicate that the processes involve the movement of a hydrogen nucleus. (3) The NiL1 species converted to into NiL2 in the dark at a rate that was virtually temperature-independent below 30 K, indicative of a proton tunnelling process. (4) The conversion of NiL1 to NiL2 was partly reversed by light in Dm. baculatum hydrogenase, but not in the [NiFe]-hydrogenases. (5) Prolonged illumination of the three enzymes induced the appearance of a third light-induced signal, NiL3. The new signal was rhombic, with features at g = 2.41, 2.16 (the third component being unresolved) in the [NiFe]-hydrogenases and g = 2.48, 2.16, 2.03 for the [NiFeSe]-enzyme. (6) Splittings caused by by spin-spin interactions with [4Fe-4S] clusters were detected for all the illuminated signals, NiL1, NiL2 and NiL3. These were quantitatively different for the three enzymes. (7) Broadening of the NiC signals in H 2O compared with 2H 20 was observed in the g 1 and g 2 components of D. gigas and D. fructosovorans hydrogenases, but not for Dm. baculatum. This broadening effect was not seen with any of the NiL species. These comparative effects are discussed in terms of subtle differences in the structure and protein environment of the nickel site, and access to exchangeable hydrons.

An NMR-Derived Model for the Solution Structure of Oxidized Thermotoga maritima 1[Fe4-S4] Ferredoxin

The solution structure of the 60-residue 1[Fe4-S4] ferredoxin from the hyperthermophilic bacterium Thermotoga maritima was determined based on 683 distance and 35 dihedral angle restraints that were obtained from NMR data. In addition, data known from crystallographic studies of ferredoxins was used for modeling of the iron-sulfur cluster and its environment. The protein shows a globular fold very similar to the fold of the related 1[Fe4-S4] ferredoxins from Desulfovibrio gigas and Desulfovibrio africanus, and elements of regular secondary structure similar to those in other ferredoxins were found in the T. maritima protein. In particular, the T. maritima protein displayed a beta-sheet structure made up of strands located at the very NH(2) and COOH termini of the protein, and an internal alpha-helix. The internal beta-sheet observed in the D. gigas and D. africanus ferredoxins could not be confirmed in T. maritima ferredoxin and is thus suggested to be only weakly present or even absent in this protein. This result suggests that thermostability in ferredoxins is not necessarily correlated with the content of stable elements of regular secondary structure.

Spin-spin interactions between the Ni site and the [4Fe-4S] centers as a probe of light-induced structural changes in active Desulfovibrio gigas hydrogenase.

In typical NiFe hydrogenases like that from Desulfovibrio gigas, the active state of the enzyme which is obtained by incubation under hydrogen gas gives a characteristic Ni-C electron paramagnetic resonance (EPR) signal at g = 2.19, 2.14, and 2.01. The Ni-C species is light-sensitive, being converted upon illumination at temperatures below 100 K in a mixture of different Ni-L species, the most important giving an EPR signal at g = 2.30, 2.12, and 2.05. This photoprocess is considered to correspond to the dissociation of a hydrogen species initially coordinated to the Ni ion in the Ni-C state. When the [4Fe-4S] centers of the enzyme are reduced, the proximal [4Fe-4S]1+ cluster interacts magnetically with the Ni center, which leads to complex split Ni-C or split Ni-L EPR spectra only detectable below 10 K. In order to probe the structural changes induced in the Ni center environment by the photoprocess, these spin-spin interactions were analyzed in D. gigas hydrogenase by simulating the split Ni-L spectra recorded at different microwave frequencies. We shown that, upon illumination, the relative arrangement of the Ni and [4Fe-4S] centers is not modified but that the exchange interaction between them is completely canceled. Moreover, the rotations undergone by the Ni center magnetic axes in the photoconversion were determined. Taken together, our results support a Ni-C structure in which the hydrogen species is not in the first coordination sphere of the Ni ion but is more likely bound to a sulfur atom of a terminal cysteine ligand of the Ni center.

Influence of Sulfur Metalation on the Accessibility of the Ni(II/I) Couple in [N,N'-Bis(2-mercaptoethyl)-1,5-diazacyclooctanato]nickel(II): Insight into the Redox Properties of [NiFe]-Hydrogenase.

A redox model study of [NiFe] hydrogenase has examined a series of five polymetallics based on the metalation of the dithiolate complex [1,5-bis(mercaptoethyl)-1,5-diazacyclooctane]Ni(II), Ni-1. Crystal structures of three polymetallics of the series have been reported earlier: [(Ni-1)(2)()Ni]Cl(2)(), [(Ni-1)(2)()FeCl(2)()](2)(), and [(Ni-1)(3)()(ZnCl)(2)()]Cl(2)(). Two are described here: [(Ni-1)(2)()Pd]Cl(2)().2H(2)()Ocrystallizes in the monoclinic system, space group P2(1)/c with cell constants a = 12.212(4) Å, b = 7.642(2) Å, c = 16.625(3) Å, beta = 107.69(2) degrees, V = 1443.230(0) Å(3), Z = 2, R = 0.051, and R(w) = 0.056. [(Ni-1)(2)()CoCl]PF(6)() crystallizes in the triclinic system, space group P&onemacr;, with cell constants a = 8.14(2) Å, b = 13.85(2) Å, c = 15.67(2) Å, alpha = 113.59(10) degrees, beta = 101.84(14) degrees, gamma = 94.0(2) degrees, V = 1561.620(0)Å(3), Z = 2, R = 0.072, and R(w) = 0.077. In all Ni-1 serves as a bidentate metallothiolate ligand with a "hinge" angle in the range 105-118 degrees and Ni-M distances of 2.7- 3.7 Å. The most accessible redox event is shown by EPR and electrochemistry to reside in the N(2)S(2)Ni unit and is the Ni(II/I) couple. Charge neutralization of the thiolate sulfurs by metalation can (dependent on the interacting metal) stabilize the Ni(I) state as efficiently as methylation forming a thioether. The implication of these results for the heterometallic active site of [NiFe]-hydrogenase as structured from Desulfovibrio gigas (Volbeda, A., et al. Nature, 1995, 373, 580), the generality of the Ni(&mgr;-SR)(2)M hinge structure, and a possible explanation for the unusual redox potentials are discussed.

Structure of the [NiFe] Hydrogenase Active Site: Evidence for Biologically Uncommon Fe Ligands

Crystallographic data on the [NiFe] hydrogenase from Desulfovibrio gigas are presented that provide new information on the structure and mode of action of its dihydrogen activating metal center. Recently we found this center to contain, besides Ni, a second metal ion which was tentatively assigned to Fe (Volbeda, A.; Charon, M. H.; Piras, C.; Hatchikian, E. C.; Frey, M.; Fontecilla-Camps, J. C. Nature 1995, 373, 580−587). This assignment is now unambiguously confirmed by a crystallographic analysis using 3 Å resolution X-ray data collected at wavelengths close to either side of the Fe absorption edge. Moreover, we report the structure of another crystal form of the as-purified D. gigas hydrogenase refined at 2.54 Å resolution, showing that the active site Fe binds three diatomic ligands. The electron density map shows an additional small peak at a position bridging the two active site metal ions, which may be assigned to some form of oxygen. This bridging oxygen species is proposed to be the signature of the inactive form of the enzyme. An infrared analysis similar to the one reported for Chromatium vinosum hydrogenase (Bagley, K. A.; Duin, E. C.; Roseboom, W.; Albracht, S. P. J.; Woodruff, W. H. Biochemistry 1995, 34, 5527−5535) shows the existence of three bands at exceptionally high frequencies, that shift their position in a concerted fashion depending on the redox state of the enzyme. Based on these high frequencies, the diatomic Fe ligands may be assigned to nonexchangeable triply bonded molecules, possible candidates being CO, CN- and NO. The frequency shifts of the infrared bands suggest a redox role for the Fe center during catalysis. Based on the new crystal structure and a number of spectroscopic results, possible modes of hydrogen binding to the active site are discussed.

A Local Spin Model To Describe the Magnetic Interactions in Biological Molecules Containing [4Fe−4S]+Clusters. Application to Ni−Fe Hydrogenases

Structural information on the relative arrangement of paramagnetic centers can be obtained through the quantitative study of their magnetic interactions based on the numerical simulation of EPR spectra recorded at different microwave frequencies. In a recent work, we have shown that such studies must explicitly take into account the delocalization of the magnetic moments over polynuclear clusters, and we have presented a local spin model suited for valence-localized clusters (Bertrand, P.; More, C.; Guigliarelli, B.; Fournel, A.; Bennet, B.; Howes, B. J. Am. Chem. Soc. 1994, 116, 3078−3086). In this paper, we examine the more general situation in which the cluster contains partially or completely delocalized mixed-valence pairs. This situation is encountered in particular in the case of [4Fe−4S] clusters which are ubiquitous in biological systems. A model is presented and developed for a system containing a [4Fe−4S]+ cluster magnetically coupled to a mononuclear center. This model is then applied to the study of the magnetic interactions between the nickel center and the proximal [4Fe−4S]+ cluster in the active form of Desulfovibrio gigas hydrogenase. The arrangement at atomic resolution deduced from this study is compared to that given by a recent X-ray crystal study carried out on the inactive form of the same enzyme (Volbeda, A.; Charon, M. H.; Piras, C.; Hatchikian, E. C.; Frey, M.; Fontecilla-Camps, J. C. Nature 1995, 373, 580−587).

Crystal structure of the xanthine oxidase-related aldehyde oxido-reductase from D. gigas.

The crystal structure of the aldehyde oxido-reductase (Mop) from the sulfate reducing anaerobic Gram-negative bacterium Desulfovibrio gigas has been determined at 2.25 A resolution by multiple isomorphous replacement and refined. The protein, a homodimer of 907 amino acid residues subunits, is a member of the xanthine oxidase family. The protein contains a molybdopterin cofactor (Mo-co) and two different [2Fe-2S] centers. It is folded into four domains of which the first two bind the iron sulfur centers and the last two are involved in Mo-co binding. Mo-co is a molybdenum molybdopterin cytosine dinucleotide. Molybdopterin forms a tricyclic system with the pterin bicycle annealed to a pyran ring. The molybdopterin dinucleotide is deeply buried in the protein. The cis-dithiolene group of the pyran ring binds the molybdenum, which is coordinated by three more (oxygen) ligands.

Purification and characterization of a benzylviologen-linked, tungsten-containing aldehyde oxidoreductase from Desulfovibrio gigas.

Desulfovibrio gigas NCIMB 9332 cells grown in ethanol-containing medium with 0.1 microM tungstate contained a benzylviologen-linked aldehyde oxidoreductase. The enzyme was purified to electrophoretic homogeneity and found to be a homodimer with a subunit M(r) of 62,000. It contained 0.68 +/- 0.08 W, 4.8 Fe, and 3.2 +/- 0.2 labile S per subunit. After acid iodine oxidation of the purified enzyme, a fluorescence spectrum typical for form A of molybdopterin was obtained. Acetaldehyde, propionaldehyde, and benzaldehyde were excellent substrates, with apparent Km values of 12.5, 10.8, and 20 microM, respectively. The natural electron acceptor is not yet known; benzylviologen was used as an artificial electron acceptor (apparent Km, 0.55 mM). The enzyme was activated by potassium ions and strongly inhibited by cyanide, arsenite, and iodoacetate. In the as-isolated enzyme, electron paramagnetic resonance studies readily detected W(V) as a complex signal with g values in the range of 1.84 to 1.97. The dithionite-reduced enzyme exhibited a broad signal at low temperature with g = 2.04 and 1.92; this is indicative of a [4Fe-4S]1+ cluster interacting with a second paramagnet, possibly the S = 1 system of W(IV). Until now W-containing aldehyde oxidoreductases had only been found in two Clostridium strains and two hyperthermophilic archaea. The D. gigas enzyme is the first example of such an enzyme in a gram-negative bacterium.

The Role of Nickel in Hydrogenases: Implications for a Heterodinuclear Active Site

Abstract Hydrogenases (H2ases) are enzymes that catalyze the reversible two-electron redox chemistry of H2. Most (but not all) H2ases are now known to contain Ni, which is intimately associated with unusual epr signals that are characteristic of the enzymes. A recent crystal structure of the H2ase from Desulfovibrio gigas reveals that the Ni in these enzymes is not an isolated metal center, but part of a Ni, Fe dinuclear cluster. The structure of the biological Ni, Fe cluster is compared with those of model complexes, and the redox chemistry of the model systems is examined for mechanistic insights into the role played by the active site cluster. Coupled with biophysical studies designed to probe the role of the Ni center, these studies fail to provide support for schemes that involve Ni-centered redox chemistry or utilize Ni as a binding site for the substrate (H2) or for inhibitors like CO. Instead, they point to the involvement of the cysteinate ligands and/or the Fe center in the catalysis of H2 redox chemistry.