Aldehyde oxidoreductase activity in Desulfovibrio alaskensis NCIMB 13491 EPR assignment of the proximal [2Fe-2S] cluster to the Mo site.

A novel molybdenum iron-sulfur-containing aldehyde oxidoreductase (AOR) belonging to the xanthine oxidase family was isolated and characterized from the sulfate-reducing bacterium Desulfovibrio alaskensis NCIMB 13491, a strain isolated from a soured oil reservoir in Purdu Bay, Alaska. D. alaskensis AOR is closely related to other AORs isolated from the Desulfovibrio genus. The protein is a 97-kDa homodimer, with 0.6 +/- 0.1 Mo, 3.6 +/- 0.1 Fe and 0.9 +/- 0.1 pterin cytosine dinucleotides per monomer. The enzyme catalyses the oxidation of aldehydes to their carboxylic acid form, following simple Michaelis-Menten kinetics, with the following parameters (for benzaldehyde): K(app/m)= 6.65 microM; V app = 13.12 microM.min(-1); k(app/cat) = 0.96 s(-1). Three different EPR signals were recorded upon long reduction of the protein with excess dithionite: an almost axial signal split by hyperfine interaction with one proton associated with Mo(V) species and two rhombic signals with EPR parameters and relaxation behavior typical of [2Fe-2S] clusters termed Fe/S I and Fe/S II, respectively. EPR results reveal the existence of magnetic interactions between Mo(V) and one of the Fe/S clusters, as well as between the two Fe/S clusters. Redox titration monitored by EPR yielded midpoint redox potentials of -275 and -325 mV for the Fe/S I and Fe/S II, respectively. The redox potential gap between the two clusters is large enough to obtain differentiated populations of these paramagnetic centers. This fact, together with the observed interactions among paramagnetic centers, was used to assign the EPR-distinguishable Fe/S I and Fe/S II to those seen in the reported crystal structures of homologous enzymes.     

NADPH-dependent reductases involved in the detoxification of reactive carbonyls in plants

Reactive carbonyls, especially α,β-unsaturated carbonyls produced through lipid peroxidation, damage biomolecules such as proteins and nucleotides; elimination of these carbonyls is therefore essential for maintaining cellular homeostasis. In this study, we focused on an NADPH-dependent detoxification of reactive carbonyls in plants and explored the enzyme system involved in this detoxification process. Using acrolein (CH(2) = CHCHO) as a model α,β-unsaturated carbonyl, we purified a predominant NADPH-dependent acrolein-reducing enzyme from cucumber leaves, and we identified the enzyme as an alkenal/one oxidoreductase (AOR) catalyzing reduction of an α,β-unsaturated bond. Cloning of cDNA encoding AORs revealed that cucumber contains two distinct AORs, chloroplastic AOR and cytosolic AOR. Homologs of cucumber AORs were found among various plant species, including Arabidopsis, and we confirmed that a homolog of Arabidopsis (At1g23740) also had AOR activity. Phylogenetic analysis showed that these AORs belong to a novel class of AORs. They preferentially reduced α,β-unsaturated ketones rather than α,β-unsaturated aldehydes. Furthermore, we selected candidates of other classes of enzymes involved in NADPH-dependent reduction of carbonyls based on the bioinformatic information, and we found that an aldo-keto reductase (At2g37770) and aldehyde reductases (At1g54870 and At3g04000) were implicated in the reduction of an aldehyde group of saturated aldehydes and methylglyoxal as well as α,β-unsaturated aldehydes in chloroplasts. These results suggest that different classes of NADPH-dependent reductases cooperatively contribute to the detoxification of reactive carbonyls.     

Kinetic behavior of Desulfovibrio gigas aldehyde oxidoreductase encapsulated in reverse micelles

We report the kinetic behavior of the enzyme aldehyde oxidoreductase (AOR) from the sulfate reducing bacterium Desulfovibrio gigas (Dg) encapsulated in reverse micelles of sodium bis-(2-ethylhexyl) sulfosuccinate in isooctane using benzaldehyde, octaldehyde, and decylaldehyde as substrates. Dg AOR is a 200-kDa homodimeric protein that catalyzes the conversion of aldehydes to carboxylic acids. Ultrasedimentation analysis of Dg AOR-containing micelles showed the presence of 100-kDa molecular weight species, confirming that the Dg AOR subunits can be dissociated. UV-visible spectra of encapsulated Dg AOR are indistinguishable from the enzyme spectrum in solution, suggesting that both protein fold and metal cofactor are kept intact upon encapsulation. The catalytic constant (k(cat)) profile as a function of the micelle size W(0) (W(0)=[H(2)O]/[AOT]) using benzaldehyde as substrate showed two bell-shaped activity peaks at W(0)=20 and 26. Furthermore, enzymatic activity for octaldehyde and decylaldehyde was detected only in reverse micelles. Like for the benzaldehyde kinetics, two peaks with both similar k(cat) values and W(0) positions were obtained. EPR studies using spin-labeled reverse micelles indicated that octaldehyde and benzaldehyde are intercalated in the micelle membrane. This suggests that, though Dg AOR is found in the cytoplasm of bacterial cells, the enzyme may catalyze the reaction of substrates incorporated into a cell membrane.     

Nitrite reduction by xanthine oxidase family enzymes: a new class of nitrite reductases

Mammalian xanthine oxidase (XO) and Desulfovibrio gigas aldehyde oxidoreductase (AOR) are members of the XO family of mononuclear molybdoenzymes that catalyse the oxidative hydroxylation of a wide range of aldehydes and heterocyclic compounds. Much less known is the XO ability to catalyse the nitrite reduction to nitric oxide radical (NO). To assess the competence of other XO family enzymes to catalyse the nitrite reduction and to shed some light onto the molecular mechanism of this reaction, we characterised the anaerobic XO- and AOR-catalysed nitrite reduction. The identification of NO as the reaction product was done with a NO-selective electrode and by electron paramagnetic resonance (EPR) spectroscopy. The steady-state kinetic characterisation corroborated the XO-catalysed nitrite reduction and demonstrated, for the first time, that the prokaryotic AOR does catalyse the nitrite reduction to NO, in the presence of any electron donor to the enzyme, substrate (aldehyde) or not (dithionite). Nitrite binding and reduction was shown by EPR spectroscopy to occur on a reduced molybdenum centre. A molecular mechanism of AOR- and XO-catalysed nitrite reduction is discussed, in which the higher oxidation states of molybdenum seem to be involved in oxygen-atom insertion, whereas the lower oxidation states would favour oxygen-atom abstraction. Our results define a new catalytic performance for AOR—the nitrite reduction—and propose a new class of molybdenum-containing nitrite reductases.     

Kinetic and Structural Studies of Aldehyde Oxidoreductase from Desulfovibrio gigas Reveal a Dithiolene-Based Chemistry for Enzyme Activation and Inhibition by H2O2

Mononuclear Mo-containing enzymes of the xanthine oxidase (XO) family catalyze the oxidative hydroxylation of aldehydes and heterocyclic compounds. The molybdenum active site shows a distorted square-pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. The XO family member aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is an exception as presents in its catalytically competent form an equatorial oxo ligand instead of the sulfido ligand. Despite this structural difference, inactive samples of DgAOR can be activated upon incubation with dithionite plus sulfide, a procedure similar to that used for activation of desulfo-XO. The fact that DgAOR does not need a sulfido ligand for catalysis indicates that the process leading to the activation of inactive DgAOR samples is different to that of desulfo-XO. We now report a combined kinetic and X-ray crystallographic study to unveil the enzyme modification responsible for the inactivation and the chemistry that occurs at the Mo site when DgAOR is activated. In contrast to XO, which is activated by resulfuration of the Mo site, DgAOR activation/inactivation is governed by the oxidation state of the dithiolene moiety of the pyranopterin cofactor, which demonstrates the non-innocent behavior of the pyranopterin in enzyme activity. We also showed that DgAOR incubation with dithionite plus sulfide in the presence of dioxygen produces hydrogen peroxide not associated with the enzyme activation. The peroxide molecule coordinates to molybdenum in a η² fashion inhibiting the enzyme activity.     

Biochemical/spectroscopic characterization and preliminary X-ray analysis of a new aldehyde oxidoreductase isolated from Desulfovibrio desulfuricans ATCC 27774

Aldehyde oxidoreductase (AOR) activity has been found in different sulfate reducing organisms (Moura, J. J. G., and Barata, B. A. S. (1994) in Methods in Enzymology (Peck, H. D., Jr., and LeGall, J., Eds.), Vol. 243, Chap. 4. Academic Press; Rom?o, M. J., Kn?blein, J., Huber, R., and Moura, J. J. G. (1997) Prog. Biophys. Mol. Biol. 68, 121-144). The enzyme was purified to homogeneity from extracts of Desulfovibrio desulfuricans (Dd) ATCC 27774, a sulfate reducer that can use sulfate or nitrate as terminal respiratory substrates. The protein (AORDd) is described as a homodimer (monomer, circa 100 kDa), contains a Mo-MCD pterin, 2 x [2Fe-2S] clusters, and lacks a flavin group. Visible and EPR spectroscopies indicate a close similarity with the AOR purified from Desulfovibrio gigas (Dg) (Barata, B. A. S., LeGall, J., and Moura, J. J. G. (1993) Biochemistry 32, 11559-11568). Activity and substrate specificity for different aldehydes were determined. EPR studies were performed in native and reduced states of the enzyme and after treatment with ethylene glycol and dithiothreitol. The AORDd was crystallized using ammonium sulfate as precipitant and the crystals belong to the space group P6(1)22, with unit cell dimensions a = b = 156.4 and c = 177.1 A. These crystals diffract to beyond 2.5 A resolution and a full data set was measured on a rotating anode generator. The data were used to solve the structure by Patterson Search methods, using the model of AORDg.     

Spectroscopic studies on APS reductase isolated from the hyperthermophilic sulfate-reducing archaebacterium Archaeglobus fulgidus.

Adenylyl sulfate (APS) reductase, the key enzyme of the dissimilatory sulfate respiration, catalyzes the reduction of APS (the activated form of sulfate) to sulfite with release of AMP. A spectroscopic study was carried out with the APS reductase purified from the extremely thermophilic sulfate-reducing archaebacterium Archaeoglobus fulgidus DSM 4304. Combined ultraviolet/visible spectroscopy and low temperature electron paramagnetic resonance (EPR) studies were used in order to characterize the active centers and the reactivity towards AMP and sulfite of this enzyme. The A. fulgidus APS reductase is an iron-sulfur flavoprotein containing two distinct [4Fe-4S] clusters (Centers I and II) very similar to the homologous enzyme from Desulfovibrio gigas. Center I, which has a high redox potential, is reduced by AMP and sulfite, and Center II has a very negative redox potential.     

The novel tungsten-iron-sulfur protein of the hyperthermophilic archaebacterium, Pyrococcus furiosus, is an aldehyde ferredoxin oxidoreductase. Evidence for its participation in a unique glycolytic pathway

The anaerobic archaebacterium, Pyrococcus furiosus, grows optimally at 100 degrees C by a fermentative-type metabolism in which H2, CO2, and organic acids are end products. The growth of this organism is stimulated by tungsten, and, from it, a novel, red-colored, tungsten-iron-sulfur protein, abbreviated RTP, has been purified (Mukund, S., and Adams, M. W. W. (1990) J. Biol. Chem. 265, 11508-11516). RTP (Mr approximately 85,000) contained approximately 1W, 7Fe, and 5 acid-labile sulfide atoms/molecule and exhibited unique EPR properties. The physiological function of the protein, however, was unknown. We show here that RTP is an inactive form of an aldehyde ferredoxin oxidoreductase (AOR). The active enzyme was obtained by rapid purification under anaerobic conditions using buffers containing dithiothreitol and glycerol. AOR catalyzed the oxidation of a range of aliphatic aldehydes with an optimum temperature for activity above 90 degrees C, but it did not oxidize glucose or glyceraldehyde 3-phosphate, nor reduce NAD(P), and its activity was independent of CoA. The active (AOR) and inactive (RTP) forms of the enzyme were indistinguishable in their contents of metals and acid-labile sulfide and in their EPR properties. The latter are though to originate from two nonidentical and spin-coupled iron-sulfur clusters, whereas the tungsten in this enzyme, which was not detectable by EPR, appears to be present as a novel pterin cofactor. Inhibition and activation studies indicated that AOR contains a catalytically essential W-SH group that is not present in RTP, the inactive form. AOR is a new type of aldehyde-oxidizing enzyme and is the first aldehyde oxidoreductase to be purified from an archaebacterium or a nonactogenic anaerobic bacterium. Its physiological role in P. furiosus is proposed as the oxidation of glyceraldehyde to glycerate in a unique, partially nonphosphorylated, glycolytic pathway that generates acetyl-CoA from glucose without the participation of nicotinamide nucleotides.     

Purification and characterization of the tungsten enzyme aldehyde:ferredoxin oxidoreductase from the hyperthermophilic denitrifier Pyrobaculum aerophilum

A tungsten-containing aldehyde:ferredoxin oxidoreductase (AOR) has been purified to homogeneity from Pyrobaculum aerophilum. The N-terminal sequence of the isolated enzyme matches a single open reading frame in the genome. Metal analysis and electron paramagnetic resonance (EPR) spectroscopy indicate that the P. aerophilum AOR contains one tungsten center and one [4Fe-4S]^2+/1+ cluster per 68-kDa monomer. Native AOR is a homodimer. EPR spectroscopy of the purified enzyme that has been reduced with the substrate crotonaldehyde revealed a W(V) species with g_zyx values of 1.952, 1.918, 1.872. The substrate-reduced AOR also contains a [4Fe-4S]¹⁺ cluster with S=3/2 and zero field splitting parameters D=7.5 cm^–1 and E/D=0.22. Molybdenum was absent from the enzyme preparation. The P. aerophilum AOR lacks the amino acid sequence motif indicative for binding of mononuclear iron that is typically found in other AORs. Furthermore, the P. aerophilum AOR utilizes a 7Fe ferredoxin as the putative physiological redox partner, instead of a 4Fe ferredoxin as in Pyrococcus furiosus. This 7Fe ferredoxin has been purified from P. aerophilum, and the amino acid sequence has been identified using mass spectrometry. Direct electrochemistry of the ferredoxin showed two one-electron transitions, at –306 and –445 mV. In the presence of 55 M ferredoxin the AOR activity is 17% of the activity obtained with 1 mM benzyl viologen as an electron acceptor.     

Correlating EPR and X-ray structural analysis of arsenite-inhibited forms of aldehyde oxidoreductase

Two arsenite-inhibited forms of each of the aldehyde oxidoreductases from Desulfovibrio gigas and Desulfovibrio desulfuricans have been studied by X-ray crystallography and electron paramagnetic resonance (EPR) spectroscopy. The molybdenum site of these enzymes shows a distorted square-pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. Arsenite addition to active as-prepared enzyme or to a reduced desulfo form yields two different species called A and B, respectively, which show different Mo(V) EPR signals. Both EPR signals show strong hyperfine and quadrupolar couplings with an arsenic nucleus, which suggests that arsenic interacts with molybdenum through an equatorial ligand. X-ray data of single crystals prepared from EPR-active samples show in both inhibited forms that the arsenic atom interacts with the molybdenum ion through an oxygen atom at the catalytic labile site and that the sulfido ligand is no longer present. EPR and X-ray data indicate that the main difference between both species is an equatorial ligand to molybdenum which was determined to be an oxo ligand in species A and a hydroxyl/water ligand in species B. The conclusion that the sulfido ligand is not essential to determine the EPR properties in both Mo–As complexes is achieved through EPR measurements on a substantial number of randomly oriented chemically reduced crystals immediately followed by X-ray studies on one of those crystals. EPR saturation studies show that the electron transfer pathway, which is essential for catalysis, is not modified upon inhibition. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

A cytoplasmic nickel-iron hydrogenase with high specific activity from Desulfovibrio multispirans sp. N., a new species of sulfate reducing bacterium

A hydrogenase from a new species of sulfate reducing bacterium has been isolated and characterized. In contrast to other hydrogenases isolated from Desulfovibrio, this enzyme is found in the cytoplasmic fraction rather than in the periplasm. The specific activity of the enzyme, as measured in the hydrogen evolution assay, is twice as high as the specific activity of the hydrogenase from D. gigas. It also differentiates itself from the periplasmic Desulfovibrio hydrogenases by being more active in the hydrogen evolution rather than in the hydrogen uptake assay. The enzyme was shown to contain 0.9 atoms of nickel, 11 atoms of iron and 10 atoms of labile sulfide per mole of enzyme. It exhibits an unusually low intensity of the g = 2.31 nickel EPR signal in the isolated enzyme but shows a normal intensity for the g = 2.19 nickel EPR signal when reduced under hydrogen.     

Isolation of fumarate reductase from Desulfovibrio multispirans, a sulfate reducing bacterium

Fumarate reductase was isolated and purified 100-fold to homogeneity from Desulfovibrio multispirans, a new species of sulfate-reducing bacteria. The enzyme contained 1 mol of non-covalently bound FAD and four subunits with Mr 45,000, 32,000, 30,000 and 27,000. EPR spectroscopy showed the existence of two iron-sulfur clusters. The absorption spectrum showed a broad region of high absorbance from 450 nm to 300 nm with a protein peak at 278 nm. The ratio of A278:A400 was 2.60. The specific activity was 110 mumoles H2/mg of protein. The Km for fumarate was 2.5 mM. The activation energy was 8.7 kcal/mol. Electron transport from H2 to fumarate in intact cells was inhibited by 2-heptyl-4-hydroxy-quinoline-N-oxide, a quinone inhibitor, indicating the participation of quinone (probably menaquinone) in fumarate reduction.     

Carbon monoxide inhibits superoxide dismutase and stimulates reactive oxygen species production by Desulfovibrio desulfuricans 1388

The hypothesis that oxidative stress characterized by enhanced superoxide generation underlies the toxicity of some factors to living organisms has been investigated. It is shown that CO (5-6% in gas phase) changed some growth parameters (mu, t(d)) of the sulfate-reducing bacterium Desulfovibrio desulfuricans 1388. Enhanced O(2)(-) generation registered by EPR spectroscopy and adrenochrome method was observed when cells were incubated under CO. The SOD activity in cells from the exponential growth phase growing under CO was decreased 1.5-fold compared with the control cells growing under Ar. SOD activities in cells from the stationary growth phase growing with or without CO were comparable. The results support the concept that CO toxicity for sulfate-reducing bacteria is an oxidative stress that arises in cells oxidizing CO to CO(2).     

Characterization of the cytochrome system of a nitrogen-fixing strain of a sulfate-reducing bacterium: Desulfovibrio desulfuricans strain Berre-Eau

Two c-type cytochromes were purified and characterized by electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopic techniques, from the sulfate-reducer nitrogen-fixing organism, Desulfovibrio desulfuricans strain Berre-Eau (NCIB 8387). The purification procedures included several chromatographic steps on alumina, carboxymethylcellulose and gel filtration. A tetrahaem and a monohaem cytochrome were identified. The multihaem cytochrome has visible, EPR and NMR spectra with general properties similar to other low-potential bis-histidinyl axially bound haem proteins, belonging to the class of tetrahaem cytochrome c3 isolated from other Desulfovibrio species. The monohaem cytochrome c553 is ascorbate-reducible and its EPR and NMR data are characteristic of a cytochrome with methionine-histidine ligation. Their properties are compared with other homologous proteins isolated from sulfate-reducing bacteria.     

Purification and characterization of the formate dehydrogenase from Desulfovibrio vulgaris Hildenborough

Abstract Formate dehydrogenase from Desulfovibrio vulgaris Hildenborough, a sulfate-reducing bacterium, has been isolated and characterized. The enzyme is composed of three subunits. A high molecular mass subunit (83 500 Da) is proposed to contain a molybdenum cofactor, a 27 000 Da subunit is found to be similar to the Fe-S subunit of the formate dehydrogenase from Escherichia coli and a low molecular mass subunit (14000 Da) holds a c -type heme. The presence of heme c in formate dehydrogenase is reported for the first time and is correlated to the peculiar low oxidoreduction potential of the metabolism of these strictly anaerobic bacteria. In vitro measurements have shown that a monoheme cytochrome probably acts as a physiological partner of the enzyme in the periplasm.     

Purification and characterization of an iron superoxide dismutase and a catalase from the sulfate-reducing bacterium Desulfovibrio gigas

The iron-containing superoxide dismutase (FeSOD; EC 1.15.1.1) and catalase (EC 1.11.1.6) enzymes constitutively expressed by the strictly anaerobic bacterium Desulfovibrio gigas were purified and characterized. The FeSOD, isolated as a homodimer of 22-kDa subunits, has a specific activity of 1,900 U/mg and exhibits an electron paramagnetic resonance (EPR) spectrum characteristic of high-spin ferric iron in a rhombically distorted ligand field. Like other FeSODs from different organisms, D. gigas FeSOD is sensitive to H(2)O(2) and azide but not to cyanide. The N-terminal amino acid sequence shows a high degree of homology with other SODs from different sources. On the other hand, D. gigas catalase has an estimated molecular mass of 186 +/- 8 kDa, consisting of three subunits of 61 kDa, and shows no peroxidase activity. This enzyme is very sensitive to H(2)O(2) and cyanide and only slightly sensitive to sulfide. The native enzyme contains one heme per molecule and exhibits a characteristic high-spin ferric-heme EPR spectrum (g(y,x) = 6.4, 5.4); it has a specific activity of 4,200 U/mg, which is unusually low for this class of enzyme. The importance of these two enzymes in the context of oxygen utilization by this anaerobic organism is discussed.     

EPR characterization of the molybdenum(V) forms of formate dehydrogenase from Desulfovibrio desulfuricans ATCC 27774 upon formate reduction

The EPR characterization of the molybdenum(V) forms obtained on formate reduction of both as-prepared and inhibited formate dehydrogenase from Desulfovibrio desulfuricans ATCC 27774, an enzyme that catalyzes the oxidation of formate to CO(2), is reported. The Mo(V) EPR signal of the as-prepared formate-reduced enzyme is rhombic (g(max)=2.012, g(mid)=1.996, g(min)=1.985) and shows hyperfine coupling with two nuclear species with I=1/2. One of them gives an anisotropic splitting and is not solvent exchangeable (A(max)=11.7, A(mid)=A(min)=non-detectable, A-values in cm(-1)x10(-4)). The second species is exchangeable with solvent and produces a splitting at the three principal g-values (A(max)=7.7, A(mid)=10.0, A(min)=9.3). The hyperfine couplings of the non-solvent and solvent exchangeable nuclei are assigned to the hydrogen atoms of the beta-methylene carbon of a selenocysteine and to a Mo ligand whose nature, sulfydryl or hydroxyl, is still in debate. The Mo(V) species obtained in the presence of inhibitors (azide or cyanide) yields a nearly axial EPR signal showing only one detectable splitting given by nuclear species with I=1/2 (g(max)=2.092, g(mid)=2.000, g(min)=1.989, A(max)=non-detectable, A(mid)=A(min)=7.0), which is originated from the alpha-proton donated by the formate to a proximal ligand of the molybdenum. The possible structures of both paramagnetic molybdenum species (observed upon formate reduction in presence and absence of inhibitors) are discussed in comparison with the available structural information of this enzyme and the structural and EPR properties of the closely related formate dehydrogenase-H from Escherichia coli.     

Purification and characterization of bisulfite reductase (desulfofuscidin) from Desulfovibrio thermophilus and its complexes with exogenous ligands

A dissimilatory bisulfite reductase has been purified from a thermophilic sulfate-reducing bacterium Desulfovibrio thermophilus (DSM 1276) and studied by EPR and optical spectroscopic techniques. The visible spectrum of the purified bisulfite reductase exhibits absorption maxima at 578.5, 392.5 and 281 nm with a weak band around 700 nm. Photoreduction of the native enzyme causes a decrease in absorption at 578.5 nm and a concomitant increase in absorption at 607 nm. When reduced, the enzyme reacts with cyanide, sulfite, sulfide and carbon monoxide to give stable complexes. The EPR spectrum of the native D. thermophilus bisulfite reductase shows the presence of a high-spin ferric signal with g values at 7.26, 4.78 and 1.92. Upon photoreduction the high-spin ferric heme signal disappeared and a typical 'g = 1.94' signal of [4Fe-4S] type cluster appeared. Chemical analyses show that the enzyme contains four sirohemes and eight [4Fe-4S] centers per mol of protein. The molecular mass determined by gel filtration was found to be 175 kDa. On SDS-gel electrophoresis the enzyme presents a main band of 44 to 48 kDa. These results suggest that the bisulfite reductase contains probably one siroheme and two [4Fe-4S] centers per monomer. The dissimilatory bisulfite reductase from D. thermophilus presents some homologous properties with desulfofuscidin, the bisulfite reductase isolated from Thermodesulfobacterium commune (Hatchikian, E.C. and Zeikus, J.G. (1983) J. Bacteriol. 153, 1211-1220).     

EPR and redox properties of Desulfovibrio vulgaris Miyazaki hydrogenase: comparison with the Ni-Fe enzyme from Desulfovibrio gigas.

We have carried out a detailed redox titration monitored by EPR on the hydrogenase from Desulfovibrio vulgaris Miyazaki. Typical 3Fe and nickel signals have been observed, which are very similar to those given by Desulfovibrio gigas hydrogenase in all the characteristic redox states of the enzyme. This confirms that D. vulgaris Miyazaki hydrogenase is a Ni-Fe enzyme closely related to that from D. gigas, as was recently proposed on the basis of sequence comparisons (Deckers, H.M., Wilson, F.R. and Voordouw, G. (1990) J. Gen. Microb. 136, 2021-2028).