Sulfate-reducing Bacterium Desulfovibrio Vulgaris Research Articles

Localization of Cytochromes in the Outer Membrane of Desulfovibrio vulgaris (Hildenborough) and their Role in Anaerobic Biocorrosion

A protocol was developed whereby the outer and cytoplasmic membranes of the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) were isolated and partially characterized. The isolated outer membrane fractions from cultures grown under high (100 ppm) and low (5 ppm) Fe2+ conditions were compared by SDS-PAGE electrophoresis, and showed that several protein bands were derepressed under the low iron conditions, most notably at 50kDa, and 77.5kDa. Outer membrane isolated from low iron cultured cells was found to contain two proteins, 77.5 kDa and 62.5 kDa in size, that reacted with a heme-specific stain and were referred to as high molecular weight cytochromes. Studies conducted on the low iron isolated outer membrane by a phosphate/mild steel hydrogen evolution system showed that addition of the membrane fraction caused an immediate acceleration in H2 production. A new model for the anaerobic biocorrosion of mild steel is proposed.

Guanylcobamide and hypoxanthylcobamide-Corrinoids formed by Desulfovibrio vulgaris

The corrinoids synthesized by the sulfate-reducing bacterium Desulfovibrio vulgaris were analyzed. The compounds found were guanylcobamide and hypoxanthylcobamide; structures were determined by mass spectrometry, 1H-NMR, and ultraviolet/visible spectroscopy. D. vulgaris used externally added guanine to form guanylcobamide, as demonstrated with 8-14C-guanine. Addition of adenine did not lead to the formation of adenylcobamide (pseudovitamin B12), whereas 5,6-dimethylbenzimidazole was transformed into vitamin B12.

Desulfoviridin, a multimeric-dissimilatory sulfite reductase from Desulfovibrio vulgaris (Hildenborough). Purification, characterization, kinetics and EPR studies.

Conditions for the rigorous purification of desulfoviridin, the dissimilatory sulfite reductase from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) have been established. A final purification by fast protein liquid chromatography yields at least three distinct bands that each exhibit the characteristic absorption spectrum of desulfoviridin. Two of these have been extensively characterized by amino acid analysis, isoelectric focusing, polyacrylamide gel electrophoresis, and formulation of the prosthetic centers. Each contains two pairs of [Fe4S4] and siroheme units. These results stand in marked contrast to recent work claiming significant demetallation of siroheme, excess iron content, and the presence of Fe6S6 clusters. These proposals are critically assessed in light of our results and other published work. Steady-state kinetic parameters have been determined: kcat(SO3(2-) = 0.31 mol SO3(2-).s-1.mol heme-1, Km = 0.06 mM; kcat(NO2-) = 0.038 mol NO2-.s-1.mol heme-1, Km = 0.028 mM; kcat(NH2OH) = 29 mol NH2OH.s-1.mol heme-1, Km = 48 mM. A detailed comparison is made with the Escherichia coli and spinach assimilatory sulfite reductase enzymes and spinach nitrite reductase. Highly purified samples of dissimilatory sulfite reductase display an electron paramagnetic resonance spectrum characteristic of rhombic high spin ferric heme centers, while the fully reduced enzyme shows EPR features typical of [Fe4S4] clusters. The magnetic properties of the prosthetic centers are further characterized by variable temperature experiments and spin quantitation.

Structure Analysis of Cytochrome c3 From Desulfovibrio vulgaris Hildenborough at 1·9 Å Resolution

The three-dimensional X-ray structure of cytochrome c3 from sulfate-reducing bacteria Desulfovibrio vulgaris Hildenborough (DvH) (Mr 13 kDa, 107 residues, 4 heme groups) has been determined at 1·9 Å resolution, by the method of molecular replacement, using the homologous part of the refined structure of cytochrome c3 from D. vulgaris Miyazaki F (DvMF). Crystals of c3 DvH were obtained with space group P61, a = 77·0 Å, c = 77·2 Å, Z = 12, corresponding to two independent molecules in the asymmetric unit. The structure was refined to an R-factor of 19·6%.The structures of the two molecules are analyzed, compared with each other and also with that of c3 DvMF. The main-chain atoms are, for the three structures, generally within 1·0 Å. The intramolecular heme edge to edge distances and interplanar angles indicate two groups of values. Shorter distances are associated with near-normal angles, while longer distances with acute angles. Moreover, two of the four hemes, II and IV, are close to only one other heme, while the remaining two hemes, I and III, have two close neighbors each. The two histidine residues that co-ordinate the heme irons on the fifth and sixth positions are nearly parallel, except in the case of heme II. The only substitution from DvMF which is inside the molecule, A68V, occurs in the vicinity of that same heme. However, the non-paralellism between the two flanking histidine residues was also observed in DvMF. Heme II has a conserved higher exposure to solvent and one of the lowest redox potentials in the fully oxidized forms of the two cytochromes. A comparison between data obtained by spectroscopic techniques, nuclear magnetic resonance and electron paramagnetic resonance, and the structural results presented here, indicates two types of interactions, between hemes I and II and between hemes III and IV.

Regulation of the Periplasmic [Fe] Hydrogenase by Ferrous Iron in Desulfovibrio vulgaris (Hildenborough)

The periplasmic [Fe] hydrogenase from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) DSM 8303 was found to be regulated by ferrous iron availability. During growth with 5 ppm of iron, the enzyme derepressed and the specific activity increased approximately fourfold, whereas the presence of 100 ppm of ferrous iron repressed the enzyme. The repression-derepression phenomenon with ferrous iron was found to be operative when the cells were cultured under either hydrogen or nitrogen gas. This is the first reported case showing that the hydrogenase enzyme is regulated by iron, and the implications of this finding relative to the corrosion industry are discussed.

Purification and biochemical characterization of a putative [6Fe-6S] prismane-cluster-containing protein from Desulfovibrio vulgaris (Hildenborough).

A novel iron-sulfur protein has been isolated from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough). It is a stable monomeric protein, which has a molecular mass of 52 kDa, as determined by sedimentation-equilibrium centrifugation. Analysis of the metal and acid-labile sulfur content of the protein revealed the presence of 6.3 +/- 0.4 Fe/polypeptide and 6.2 +/- 0.7 S2-/polypeptide. Non-iron transition metals, heme, flavin and selenium were absent. Combining these data with the observation of a very anisotropic S = 1/2 [6Fe-6S]3+ prismane-like EPR signal in the dithionite-reduced protein, we believe that we have encountered the first example of a prismane-cluster-containing protein. The prismane protein has a slightly acidic amino acid composition and isoelectric point (pI = 4.9). The ultraviolet/visible spectrum is relatively featureless (epsilon 280 = 81 mM-1.cm-1, epsilon 400 = 25 mM-1.cm-1, epsilon 400,red = 14 mM-1.cm-1). The shape of the protein is approximately globular (S20.w = 4.18 S). The N-terminal amino acid sequence is MFS/CFQS/C QETAKNTG. Polyclonal antibodies against the protein were raised. Cytoplasmic localization was inferred from subcellular fractionation studies. Cross-reactivity of antibodies against this protein indicated the occurrence of a similar protein in D. vulgaris (Monticello) and Desulfovibrio desulfuricans (ATCC 27774). We have not yet identified a physiological function for the prismane protein despite trials for some relevant enzyme activities.

Biochemical and spectroscopic characterization of the high molecular weight cytochrome c from Desulfovibrio vulgaris Hildenborough expressed in Desulfovibrio desulfuricans G200.

The gene of high molecular weight, multiheme cytochrome c (Hmc) from the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough has been overexpressed in Desulfovibrio desulfuricans G200. The recombinant protein has been purified. Its molecular weight (65,600), amino acid composition, and NH2-terminal sequence were found to be identical to those of the wild-type protein. The recombinant protein has been spectroscopically characterized (optical spectrum, EPR, circular dichroism) and compared to the wild-type protein. We have found 16 hemes per molecule by iron analysis and the pyridine hemochrome test. Both high- and low-spin features were observed in the EPR spectrum. A detailed spin quantitation analysis indicates 1 or 2 high-spin hemes and 14 or 15 low-spin hemes per molecule. The redox potentials of the hemes determined by voltammetric techniques gave an average of three different values, 0, -100, and -250 mV (versus NHE), for the wild-type and the recombinant cytochrome. The low potential values are similar to the values observed for the bis(histidinyl) coordinated hemes of cytochrome c3. A comparison of the arrangement of heme binding sites and coordinated histidines in the amino acid sequences of cytochrome c3 and Hmc has shown that the latter contains four domains, three of which are complete c3-like domains, while the fourth represents an incomplete c3-like domain which may contain His-Met coordinated hemes. These data are in agreement with the detailed study of the number and types of hemes reported in this paper.

The primary structure of rubrerythrin, a protein with inorganic pyrophosphatase activity from Desulfovibrio vulgaris. Comparison with hemerythrin and rubredoxin.

The complete polypeptide chain of rubrerythrin from the sulfate reducing bacterium Desulfovibrio vulgaris, strain Hildenborough NCIB 8303, was found by protein chemical techniques to consist of 191 residues and to have the amino acid sequence [sequence: see text] The C-terminal part of the protein (position 153----191) shows the typical sequence features of rubredoxin, a protein with a nonheme iron center also present in the same and other Desulfovibrio species. Based on the known three-dimensional structure of D. desulfuricans rubredoxin, we propose that the C-terminal part of rubrerythrin is folded in a similar way and suggest that the deletion of the extra 10 residues is compatible with the same basic rubredoxin-fold. After characterization of the C-terminal region, and in contrast to what could be expected from previously published spectroscopic analyses, the N-terminal region 1-152 of rubrerythrin appears to have no sequence similarity with the eukaryotic protein hemerythrin which is known to contain a binuclear iron center bound by 5 histidine ligands. However, the N-terminal region of rubrerythrin does contain 5 histidine residues but they are differently spaced along the peptide chain. We suggest that at least one of the 3 histidine residues located in the rubredoxin-like center of rubrerythrin may be liganded to one iron atom of the hemerythrin-like center. This paper is the first sequence report of a protein with pyrophosphatase activity although the physiological substrate for the rubrerythrin may be not inorganic pyrophosphate.

Cloning and sequencing of the gene for rubrerythrin from Desulfovibrio vulgaris (Hildenborough)

The gene coding for rubrerythrin from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) has been cloned and sequenced. Rubrerythrin is known to contain two types of iron sites: one rubredoxin-like FeS4 center in each of the two identical subunits and one hemerythrin-like diiron site per dimer [LeGall, J., et al. (1988) Biochemistry 27, 1636-1642]. The gene encodes a polypeptide of 191 amino acids, and a normal ribosome binding site is located 11-6 base pairs upstream from the translational start of the gene. There is no evidence for the presence of a leader sequence, suggesting a cytoplasmic location for the protein. The rubrerythrin gene is not part of any other known transcriptional unit in the D. vulgaris genome. The nucleotide sequence encodes four Cys residues, the minimum required for ligation to iron in rubredoxin. The pairs of Cys residues occur in Cys-X-X-Cys sequences as they do in rubredoxin, but the 12-residue spacing between the Cys pairs in rubrerythrin is less than half that in rubredoxins. A pair of Arg residues flanking one Cys residue may contribute to the much more positive reduction potential of the rubredoxin-like site in rubrerythrin compared to that of rubredoxin. While the amino acid sequence of rubrerythrin shows no significant overall homology with that of any known protein, the C-terminal region does share some homology with rubredoxin sequences. If folding of the rubredoxin-like amino acid sequence domain in rubrerythrin is similar to that in rubredoxins, then three His residues are brought into proximity.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular dynamics simulations of the cytochrome c3-rubredoxin complex from Desulfovibrio vulgaris.

Molecular dynamics simulations have been carried out on the complex formed between the tetraheme cytochrome c3 and the iron protein rubredoxin from the sulfate-reducing bacterium Desulfovibrio vulgaris. These simulations were performed both with explicit solvent water molecules included, and without solvent molecules using a distance-dependent dielectric constant to approximate the screening effects of solvent. The results of both simulations are strikingly different, indicating that the representation of environmental effects is important in such simulations. For example, a striking adaptation of the two proteins seen in the nonsolvated simulation is not seen when explicit solvent water is included; in fact, the complex appears to become weaker in the solvated simulation. Nonetheless, the iron-iron distance decreases more significantly in the solvated simulation than in the nonsolvated simulation. It was found that in both cases molecular dynamics optimized the structures further than energy minimization alone.

Primary structure of the assimilatory-type sulfite reductase from Desulfovibrio vulgaris (Hildenborough): cloning and nucleotide sequence of the reductase gene

The nucleotide sequence encoding the structural gene (651 bp) and flanking regions for the assimilatory-type sulfite reductase from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) was determined after cloning a 1.4 kb HindIII/SalI genomic fragment possessing the gene into Bluescript pBS(+)KS. The primary structure of the protein was deduced, and the molecular mass of the apoprotein was estimated as 24 kDa. The amino acid sequence of the polypeptide shows some similarities at putative [Fe4S4] cluster binding sites in comparison with the heme protein subunit of the larger Escherichia coli and Salmonella typhimurium sulfite reductases and spinach nitrite reductase. This is the first reported sequence of a member of a new class of low molecular weight assimilatory sulfite-reducing enzymes recently identified in a number of anaerobic bacteria [Moura, I., Lina, A. R., Moura, J. J. G., Xavier, A. V., Fauque, G., Peck, H. D., & Le Gall, J. (1986) Biochem. Biophys. Res. Commun. 141, 1032-1041].

Expression of the gene encoding cytochrome c3 from the sulfate-reducing bacterium Desulfovibrio vulgaris in the purple photosynthetic bacterium Rhodobacter sphaeroides

The gene encoding cytochrome c 3 ( cyc-gene) from Desulfovibrio vulgaris (Hildenborough) was cloned by G. Voordouw and S. Brenner (1986, Eur. J. Biochem. 159, 347–351) . The gene was expressed in Escherichia coli but only the apoprotein was observed (W. Pollock, P. Chemerika, M. Forrest, J. Beatty, and G. Voordouw, 1989, J. Gen. Microbiol. 135, 2319–2328) . In this study, the cyc-gene was cloned into the broad host range vector pRK404 and then introduced into the purple photosynthetic bacterium Rhodobacter sphaeroides. Cells grown anaerobically produced a significant amount of recombinant cytochrome c 3. The purified protein contains four hemes and the N-terminal protein sequence is identical to the published sequence of the native cytochrome c 3. Thus, R. sphaeroides was able to produce the mature cytochrome c 3 by combining the four steps of protein synthesis, exporting the protein across the membrane, cleaving the signal peptide, and inserting four hemes. It appears that the D. vulgaris promoter is not very efficiently used by R. sphaeroides. However, replacement of the promoter with a R. sphaeroides promoter should result in cytochrome c 3 overproduction.

Coordination and redox properties of a novel triheme cytochrome from Desulfovibrio vulgaris (Hildenborough)

A high molecular weight multiheme c-type cytochrome from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) has been spectroscopically characterized and compared with the tetraheme cytochrome c3. The protein contains a pentacoordinate high-spin heme (gz 6.0) and two hexacoordinate low-spin hemes (gz 2.95, gy 2.27, gx 1.48). From analysis of the g values for the low-spin hemes by the procedure of Blumberg and Peisach (Palmer, 1983) and comparison with with the optical spectra from a variety of c-type cytochromes, it is likely that these low-spin hemes are bound by two histidine residues. The NO derivative displayed typical rhombic EPR features (gx 2.07, gz 2.02, gy 1.99). Addition of azide does not lead to coupling between heme chromophores, but the ligand is accessible to the high-spin heme. The use of a glassy-carbon electrode to perform direct (no promoter) electrochemistry on the cytochrome is illustrated. Differential pulse polarography of the native protein gave two waves with reduction potentials of -59 (5) and -400 (8) mV (versus NHE). The cyanide adduct gave two waves with reduction potentials of -263 (8) and -401 (8) mV. The cytochrome was found to catalyze the reduction of nitrite and hydroxylamine.

Time-resolved fluorescence studies of flavodoxin. Fluorescence decay and fluorescence anisotropy decay of tryptophan in Desulfovibrio flavodoxins

The time-resolved fluorescence characteristics of tryptophan in flavodoxin isolated from the sulfate-reducing bacteria Desulfovibrio vulgaris and Desulfovibrio gigas have been examined. By comparing the results of protein preparations of normal and FMN-depleted flavodoxin, radiationless energy transfer from tryptophan to FMN has been demonstrated. Since the crystal structure of the D. vulgaris flavodoxin is known, transfer rate constants from the two excited states 1La and 1Lb can be calculated for both tryptophan residues (Trp 60 and Trp 140). Residue Trp 60, which is very close to the flavin, transfers energy very rapidly to FMN, whereas the rate of energy transfer from the remote Trp 140 to FMN is much smaller. Both tryptophan residues have the indole rings oriented in such a way that transfer will preferentially take place from the 1La excited state. The fluorescence decay of all protein preparations turned out to be complex, the parameter values being dependent on the emission wavelength. Several decay curves were analyzed globally using a model in which tryptophan is involved in some nanosecond relaxation process. A relaxation time of about 2 ns was found for both D. gigas apo- and holo-flavodoxin. The fluorescence anisotropy decay of both Desulfovibrio FMN-depleted flavodoxins is exponential, whereas that of the two holoproteins is clearly non-exponential. The anisotropy decay was analyzed using the same model as that applied for fluorescence decay. The tryptophan residues turned out to be immobilized in the protein. A time constant of a few nanoseconds results from energy transfer from tryptophan to flavin, at least for D. gigas flavodoxin. The single tryptophan residue in D. gigas flavodoxin occupies a position in the polypeptide chain remote from the flavin prosthetic group. Because of the close resemblance of steady-state and time-resolved fluorescence properties of tryptophan in both flavodoxins, the center to center distance between tryptophan and FMN in D. gigas flavodoxin is probably very similar to the distance between Trp 140 and FMN in D. vulgaris flavodoxin (i.e. 20 A).

Electron transport in sulfate‐reducing bacteria

A hypothetical model of the complex formed between the iron-sulfur protein rubredoxin and the tetraheme cytochrome c3 from the sulfate-reducing bacteria Desulfovibrio vulgaris (Hildenborough) has been proposed utilizing computer graphic modeling, computational methods and NMR spectroscopy. The proposed complex appears feasible on the basis of complementary electrostatic interaction and steric factors and is consistent with the data from NMR experiments. In this model, the non-heme iron atom of rubredoxin is in close proximity to heme 1 of cytochrome c3. The complex is stabilized by charge-pair interactions and hydrogen bonds. This complex is compared to the flavodoxin-cytochrome c3 complex previously proposed [Stewart, D. E., LeGall, J., Moura, I., Moura, J. J. G., Peck, H. D. Jr, Xavier, A. V., Weiner, P. K. & Wampler, J. E. (1988) Biochemistry 27, 2444-2450] and new NMR data shows that both proteins interact with the same heme group of the cytochrome as postulated.

Formation of dithionite by sulfite reduction with Desulfovibrio vulgaris hydrogenase and cytochrome c3.

Sulfite is reduced to dithionite under hydrogen atmosphere with periplasmic hydrogenase and cytochrome c3 from a sulfate-reducing bacterium Desulfovibrio vulgaris Miyazaki K.

A hypothetical model of the flavodoxin-tetraheme cytochrome c3 complex of sulfate-reducing bacteria.

A hypothetical model of the flavodoxin-tetraheme cytochrome c3 electron-transfer complex from the sulfate-reducing bacterium Desulfovibrio vulgaris has been constructed by using interactive computer graphics based on electrostatic potential field calculations and previous NMR experiments. Features of the proposed complex are (1) van der Waals contact between the flavin mononucleotide prosthetic group of flavodoxin and one heme of the cytochrome, (2) unique complementarity of electrostatic fields between the region surrounding this heme and the region surrounding the exposed portion of the flavin mononucleotide group of flavodoxin, and (3) no steric interferences between the two polypeptide chains in the complex. This complex is consistent with all structural and spectroscopic data available.

Putative signal peptide on the small subunit of the periplasmic hydrogenase from Desulfovibrio vulgaris.

We sequenced the NH2 terminus of the large and small subunits of the periplasmic hydrogenase from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) and found that the small subunit lacks a region of 34 NH4-terminal amino acids coded by the gene for the small subunit (G. Voordouw and S. Brenner, Eur. J. Biochem. 148:515-520, 1985). We suggest that this region constitutes a signal peptide based on comparison with known procaryotic signal peptides.

Refined structure of cytochrome c3 at 1.8 Å resolution

The structure of cytochrome c 3 from the sulfate-reducing bacterium Desulfovibrio vulgaris Miyazaki has been successfully refined at 1.8 Å resolution. The crystallographic R factor is 0.176 for 9907 significant reflections. The isotropic temperature factors of individual atoms were refined and a total of 47 water molecules located on the difference map were incorporated in the refinement. The four heme groups are closely packed, with adjacent pairs of heme planes being nearly perpendicular to each other. The fifth and the sixth ligands of the heme iron atoms are histidine residues with N ε2-Fe distances ranging from 1.88 Å to 2.12 Å. The histidine co-ordination to the heme iron is different for each heme group. The heme groups are all highly exposed to solvent, although the actual regions exposed differ among the hemes. The four heme groups are located in different environments, and the heme planes are deformed from planarity. The differences in the heme structures and their environments indicate that the four heme groups are non-equivalent. The chemical as well as the physical properties of cytochrome c 3 should be interpreted in terms of the structural non-equivalence of the heme groups. The characteristic secondary structures of the polypeptide chain of this molecule are three short α-helices, two short β-strands and ten reverse turns.

Flavodoxin from the sulfate reducing bacterium Desulfovibrio vulgaris. Its structure at 2.5 A resolution.

Crystals of oxidized Desulfovibrio vulgaris flavodoxin have been studied by X-ray diffraction techniques. The structure has been determined at 2.5 A resolution. The molecule consists of a pleated sheet core with two sections of helix on each side of the sheet. The FMN group is distinguishable: it is mostly burried but 7- and 8-Methyl groups appear to be at the surface of the protein.