Biomedical subjects

J LeGall

Publications and source records attributed to J LeGall.

At least 73 records · Page 4Linked to original sources

PubMed1993

Structural studies on Desulfovibrio gigas cytochrome c3 by two-dimensional 1H-nuclear-magnetic-resonance spectroscopy.

Several aromatic amino acid residues and haem resonances in the fully reduced form of Desulfovibrio gigas cytochrome c3 are assigned, using two-dimensional 1H n.m.r., on the basis of the interactions between the protons of the aromatic amino acids and the haem protons as well as the intrahaem distances known from the X-ray structure [Kissinger (1989) Ph.D. Thesis, Washington State University]. The interhaem interactions observed in the n.m.r. spectra are in full agreement with the D. gigas X-ray structure and also with the n.m.r. data from Desulfovibrio vulgaris (Hildenborough) [Turner, Salgueiro, LeGall and Xavier (1992) Eur. J. Biochem. 210, 931-936]. The good correlation between the calculated ring-current shifts and the observed chemical shifts strongly supports the present assignments. Observation of the two-dimensional nuclear-Overhauser-enhancement spectra of the protein in the reduced, intermediate and fully oxidized stages led to the ordering of the haems in terms of their midpoint redox potentials and their identification in the X-ray structure. The first haem to oxidize is haem I, followed by haems II, III and IV, numbered according to the Cys ligand positions in the amino acid sequences [Mathews (1985) Prog. Biophys. Mol. Biol. 54, 1-56]. Although the haem core architecture is the same for the different Desulfovibrio cytochromes c3, the order of redox potentials is different.

Bacterial Proteins↗

PubMed1993

Subunit composition, crystallization and preliminary crystallographic studies of the Desulfovibrio gigas aldehyde oxidoreductase containing molybdenum and [2Fe-2S] centers.

The Desulfovibrio gigas aldehyde oxidoreductase contains molybdenum bound to a pterin cofactor and [2Fe-2S] centers. The enzyme was characterized by SDS/PAGE, gel-filtration and analytical ultracentrifugation experiments. It was crystallized at 4 degrees C, pH 7.2, using isopropanol and MgCl2 as precipitants. The crystals diffract beyond 0.3-nm (3.0-A) resolution and belong to space group P6(1)22 or its enantiomorph, with cell dimensions a = b = 14.45 nm and c = 16.32 nm. There is one subunit/asymmetric unit which gives a packing density of 2.5 x 10(-3) nm3/Da (2.5 A3/Da), consistent with the experimental crystal density, rho = 1.14 g/cm3. One dimer (approximately 2 x 100 kDa) is located on a crystallographic twofold axis.

Aldehyde Oxidoreductases↗

PubMed1993

Rubredoxin oxidase, a new flavo-hemo-protein, is the site of oxygen reduction to water by the "strict anaerobe" Desulfovibrio gigas.

A rubredoxin-oxygen oxidoreductase, a homodimer with a molecular weight of 43 kDa per monomer, was found to be a component of an electron transfer chain that couples the reduction of oxygen to water with NADH oxidation. This FAD-containing protein appears to contain a new type of heme group. The electron transfer chain is not inhibited by cyanide and azide. In contrast, CO decreases NADH oxidation rate and also induces release of the prosthetic groups from the native terminal reductase.

Chromatography, Gel↗

PubMed1993

Isolation and characterization of flavoredoxin, a new flavoprotein that permits in vitro reconstitution of an electron transfer chain from molecular hydrogen to sulfite reduction in the bacterium Desulfovibrio gigas.

A new FMN-containing flavoprotein isolated from Desulfovibrio gigas provided maximum coupling efficiency for the reduction of bisulfite from molecular H2. This protein, which is distinct from flavodoxin and for which the name flavoredoxin is proposed, is required for reconstitution of an electron transfer chain between hydrogenase and bisulfite reductase. A Ca(2+)-binding protein functions as a modulator in the presence of Ca2+ in the process. The finding of a membrane-bound cytochrome c with a molecular weight of 104,000 Da that is also active in this electron transfer chain provides an explanation for the energetic linkage between periplasmic and cytoplasmic proteins in this sulfate-reducing bacterium.

Amino Acid Sequence↗

PubMed1993

Resonance Raman studies of iron-only hydrogenases.

The nature of the iron-sulfur clusters in oxidized and reduced forms of Fe-only hydrogenases from Desulfovibrio vulgaris, Thermotoga maritima, and Clostridium pasteurianum has been investigated by resonance Raman spectroscopy. The results indicate the presence of ferredoxin-like [4Fe-4S]2+,+ and [2Fe-2S]2+,+ clusters in both T. maritima hydrogenase and C. pasteurianum hydrogenase I, but only [4Fe-4S]2+,+ clusters in D. vulgaris hydrogenase. This necessitates a reevaluation of the iron-sulfur cluster composition of C. pasteurianum hydrogenase I and indicates that the resonance Raman bands in the oxidized hydrogenase that were previously attributed to the hydrogen activating center [Macor, K. A., Czernuszewicz, R. S., Adams, M. W. W., & Spiro, T. G. (1987) J. Biol. Chem. 262, 9945-9947] arise from an indigenous [2Fe-2S]2+ cluster. No resonance Raman bands that could be uniquely attributed to the oxidized or reduced hydrogen activating center were observed. This suggests that the hydrogen activating center is a novel Fe center that is unrelated to any known type of Fe-S cluster.

Clostridium↗

PubMed1992

Structural studies of Desulfovibrio vulgaris ferrocytochrome c3 by two-dimensional NMR.

Two-dimensional NMR has been used to make specific assignments for the four haems in Desulfovibrio vulgaris (Hildenborough) ferrocytochrome c3 and to determine their haem core architecture. The NMR signals from the haem protons were assigned according to type using two-dimensional NMR experiments which led to four sets of signals, one for each of the haems. Specific assignments were obtained by calculating the ring current shifts which arise from other haems and aromatic residues. Observation of interhaem NOEs confirmed the assignments and established that the relative orientation of the haems is identical to that found in the crystal structure of D. vulgaris (Miyazaki F.) ferricytochrome c3. Assignments were also made for all the aromatic residues except for the haem ligands and F20, which is shifted under the main envelope of signals. The NOEs observed between these aromatic protons and haem protons confirm the similarity between the structures in solution and in the crystal. The assignments reported here are the basis for the cross-assignments of the four microscopic haem redox potentials to specific haems in the protein structure [Salgueiro, C. A., Turner, D. L., Santos, H., LeGall, J. and Xavier, A. V. (1992) FEBS Lett., in the press]

Amino Acid Sequence↗

PubMed1992

Assignment of the redox potentials to the four haems in Desulfovibrio vulgaris cytochrome c3 by 2D-NMR.

Using 2D-NMR the four haems of Desulfovibrio vulgaris (Hildenborough) cytochrome c3 within the X-ray structure were fully cross assigned according to their redox potential. The strategy used was based on a complete network of chemical exchange connectivities between the NMR signals obtained for all oxidation levels to the corresponding ones in the fully reduced spectrum [1992, Eur. J. Biochem., in press]. This unequivocal cross-assignment disagrees with earlier results obtained for the similar protein from Desulfovibrio vulgaris (Miyazaki F.).

Cytochrome c Group↗

PubMed1992

Structure determination of a novel cyclic phosphocompound isolated from Desulfovibrio desulfuricans.

The structure of a novel diphosphodiester compound recently detected in Desulfovibrio desulfuricans cells [Santos, Fareleira, Pedregal, LeGall & Xavier (1991) Eur. J. Biochem. 201, 283-287] was fully elucidated using a combination of n.m.r. techniques in aqueous and in methanolic solutions. The novel metabolite was identified as 3-methyl-1,2,3,4-tetrahydroxybutane-1,3-cyclic bisphosphate, and the minimum energy conformation is presented. The two chiral centres have the relative configuration RS.

Butanols↗

PubMed1992

Partial purification and characterization of the first hydrogenase isolated from a thermophilic sulfate-reducing bacterium.

A soluble [NiFe] hydrogenase has been partially purified from the obligate thermophilic sulfate-reducing bacterium Thermodesulfobacterium mobile. A 17% purification yield was obtained after four chromatographic steps and the hydrogenase presents a purity index (A398 nm/A277 nm) equal to 0.21. This protein appears to be 75% pure on SDS-gel electrophoresis showing two major bands of molecular mass around 55 and 15 kDa. This hydrogenase contains 0.6-0.7 nickel atom and 7-8 iron atoms per mole of enzyme and has a specific activity of 783 in the hydrogen uptake reaction, of 231 in the hydrogen production assay and of 84 in the deuterium-proton exchange reaction. The H2/HD ratio is lower than one in the D2-H+ exchange reaction. The enzyme is very sensitive to NO, relatively little inhibited by CO but unaffected by NO2-. The EPR spectrum of the native hydrogenase shows the presence of a [3Fe-4S] oxidized cluster and of a Ni(III) species.

Chromatography, Gel↗

PubMed1992

Direct spectroscopic evidence for the presence of a 6Fe cluster in an iron-sulfur protein isolated from Desulfovibrio desulfuricans (ATCC 27774)

A novel iron-sulfur protein was purified from the extract of Desulfovibrio desulfuricans (ATCC 27774) to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a monomer of 57 kDa molecular mass. It contains comparable amounts of iron and inorganic labile sulfur atoms and exhibits an optical spectrum typical of iron-sulfur proteins with maxima at 400, 305, and 280 nm. Mössbauer data of the as-isolated protein show two spectral components, a paramagnetic and a diamagnetic, of equal intensity. Detailed analysis of the paramagnetic component reveals six distinct antiferromagnetically coupled iron sites, providing direct spectroscopic evidence for the presence of a 6Fe cluster in this newly purified protein. One of the iron sites exhibits parameters (delta EQ = 2.67 +/- 0.03 mm/s and delta = 1.09 +/- 0.02 mm/s at 140 K) typical for high spin ferrous ion; the observed large isomer shift indicates an iron environment that is distinct from the tetrahedral sulfur coordination commonly observed for the iron atoms in iron-sulfur clusters and is consistent with a penta- or hexacoordination containing N and/or O ligands. The other five iron sites are most probably high spin ferric. Three of them show parameters characteristic for tetrahedral sulfur coordination. In correlation with the EPR spectrum of the as-purified protein which shows a resonance signal at g = 15.3 and a group of signals between g = 9.8 and 5.4, this 6Fe cluster is assigned to an unusual spin state of 9/2 with zero field splitting parameters D = -1.3 cm-1 and E/D = 0.062. Other EPR signals attributable to minor impurities are also observed at the g = 4.3 and 2.0 regions. The diamagnetic Mössbauer component represents a second iron cluster, which, upon reduction with dithionite, displays an intense S = 1/2 EPR signal with g values at 2.00, 1.83, and 1.31. In addition, an EPR signal of the S = 3/2 type is also observed for the dithionite-reduced protein.

Amino Acid Sequence↗

PubMed1992

Mössbauer characterization of the tetraheme cytochrome c3 from Desulfovibrio baculatus (DSM 1743). Spectral deconvolution of the heme components.

Mössbauer spectroscopy was used to study the tetraheme cytochrome c3 from Desulfovibrio baculatus (DSM 1743). Samples with different degrees of reduction were prepared using a redoxtitration technique. In the reduced cytochrome c3, all four hemes are reduced and exhibit diamagnetic Mössbauer spectra typical for low-spin ferrous hemes (S = 0). In the oxidized protein, the hemes are low-spin ferric (S = 1/2) and exhibit overlapping magnetic Mössbauer spectra. A method of differential spectroscopy was applied to deconvolute the four overlapping heme spectra and a crystal-field model was used for data analysis. Characteristic Mössbauer spectral components for each heme group are obtained. Hyperfine and crystal-field parameters for all four hemes are determined from these deconvoluted spectra.

Cytochrome c Group↗

PubMed1991

A thermodynamic model for the cooperative functional properties of the tetraheme cytochrome c3 from Desulfovibrio gigas.

A thermodynamic model is presented to describe the redox behaviour of the tetraheme cytochrome c3 from Desulfovibrio gigas. This molecule displays different intrinsic redox potentials for the four hemes and during the redox titration process, interactions among different hemes occur, thus altering the values of redox potentials according to which of the hemes are oxidized [Santos, H., Moura, J.J.G., Moura, I., LeGall, J. & Xavier, A.V. (1984) Eur. J. Biochem. 141, 283-296]. This complex cooperative behaviour [Xavier, A.V. (1986) J. Inorg. Biochem. 28, 239-243] has been analyzed here using an I2H4-interaction network [Cornish-Bowden, A. & Koshland, D.E. Jr (1970) J. Biol. Chem. 245, 6241-6250] coupled to a proton-linked equilibrium between two tertiary structures. Such a formalism, which requires a reduced number of parameters, is able to fully account quantitatively for the pH dependence of the NMR redox-titration curves. The 'redox-Bohr' effect is discussed in terms of the available structure and thermodynamic data and a functional mechanism is proposed.

Binding Sites↗

PubMed1991

Kinetic study of the reduction mechanism for Desulfovibrio gigas cytochrome c3.

The kinetic aspects of the reduction process in cytochrome c3 from Desulfovibrio gigas have been investigated over a wide range of pH values ranging between pH 5.8 and pH 9.8. The data have been analyzed in the framework of an I2H4 interaction network coupled to a proton-linked equilibrium between two tertiary structures (Cornish-Bowden, A. & Koshland, D.E. Jr (1970) J. Biol. Chem. 245, 6241-6250). The kinetic rate constants for the reduction of the four hemes for the two tertiary conformations have been characterized in the framework of the thermodynamic network obtained from the equilibrium analysis (Coletta, M., Catarino, T., LeGall, J.J. & Xavier, A.V. (1991) Eur. J. Biochem. 202, 1101-1106). The intrinsic reduction rate constants determined by reaction with sodium dithionite for two hemes (namely heme 4 and heme 1) are significantly faster than those for the other two heme residues. In view of the equilibrium redox properties, heme 4 (with the fastest reduction rate) may then work as the kinetic electron-capturing site for the electrons from sodium dithionite. The transfer to hemes 2 and 3 then occurs by virtue of their free-energy levels at equilibrium. At our experimental conditions, there is also transfer of electrons to hemes 2 and 3 from heme 1, which is reduced at a slower rate than heme 4, thus contributing to the biphasic kinetics observed for the overall process. The kinetic parameters obtained are discussed in terms of the mechanism proposed for the coupling between the electron and proton transfer, as induced by the heme/heme cooperativity network.

Cytochrome c Group↗

PubMed1991

Simulation of the electrochemical behavior of multi-redox systems. Current potential studies on multiheme cytochromes.

The direct unmediated electrochemical response of the tetrahemic cytochrome c3 isolated from sulfate reducers Desulfovibrio baculatus (DSM 1743) and D. vulgaris (strain Hildenborough), was evaluated using different electrode systems [graphite (edge cut), gold, semiconductor (InO2) and mercury)] and different electrochemical methods (cyclic voltammetry and differential pulse voltammetry). A computer program was developed for the theoretical simulation of a complete cyclic voltammetry curve, based on the method proposed by Nicholson and Shain [Nicholson, R.S. & Shain, I. (1964) Anal. Chem. 36, 706-723], using the Gauss-Legendre method for calculation of the integral equations. The experimental data obtained for this multi-redox center protein was deconvoluted in to the four redox components using theoretically generated cyclic voltammetry curves and the four mid-point reduction potentials determined. The pH dependence of the four reduction potentials was evaluated using the deconvolution method described.

Carbon↗

PubMed1991

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)

Amino Acid Sequence↗

PubMed1991

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.

Amino Acid Sequence↗

PubMed1991

In vivo 31P-NMR studies of Desulfovibrio species. Detection of a novel phosphorus-containing compound.

The phosphorus metabolism of sulfate-reducing bacteria was, for the first time, probed by in vivo 31P NMR. A novel phosphoric anhydride diester compound was detected in Desulfovibrio desulfuricans ATCC 27774 at intracellular concentrations up to 5 mM. The compound has been extracted and partially purified by anion-exchange chromatography and analysed by 31P, 13C and 1H NMR. These studies show that the novel phosphorus-containing compound is formed by five carbon atoms and is probably cyclic, with a Mr of approximately 300. Various Desulfovibrio strains were examined in vivo for the presence of this phosphorus-containing compound. Detectable amounts of the novel metabolite were found in D. desulfuricans ATCC 27774 when grown on lactate/sulfate, lactate/thiosulfate or pyruvate/sulfate. The phosphorus-containing compound was not detected when this strain of D. desulfuricans was grown on lactate/nitrate or pyruvate; neither was it detected in two other strains which, like D. desulfuricans ATCC 27774, have the capability of utilizing nitrate as a terminal electron acceptor.

Desulfovibrio↗

PubMed1991

Direct evidence of the metal-free nature of sirohydrochlorin in desulfoviridin.

We have obtained direct evidence that the majority of the sirohydrochlorin chromophore in the dissimilatory sulfite reductase desulfoviridin from Desulfovibrio gigas, is not associated with any metal. The evidence comes from resonance Raman measurements of native and deuterated samples of desulfoviridin. The breathing mode v4 (or v4*) at 1336 cm-1 in the native enzyme is downshifted to 1326 cm-1 upon deuteration. This mode is not sensitive to deuteration if a metal is present at the center of the chromophore inside protein or in solution. The results also establish the existence of exchangeable core hydrogen(s) at the pyrrolic nitrogen(s).

Desulfovibrio↗