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F Guerlesquin

Publications and source records attributed to F Guerlesquin.

At least 37 records · Page 2Linked to original sources
PubMed1995

Purification and characterization of the formate dehydrogenase from Desulfovibrio vulgaris Hildenborough.

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 (14,000 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.

Amino Acid Sequence↗
PubMed1995

Control of the redox potential in c-type cytochromes: importance of the entropic contribution.

The enthalpic and entropic components of the redox free energy variation of cytochrome c553 from Desulfovibrio vulgaris Hildenborough and its mutant Y64V, flavocytochrome b2 from Saccharomyces cerevisiae, and the different hemes of cytochromes c3 from Desulfovibrio vulgaris Miyazaki and Desulfovibrio desulfuricans Norway have been determined in 0.1 M Tris-HCl pH 7.0 (7.6 for cytochromes c3) at 25 degrees C by using nonisothermal potentiometric titrations. The set of available experimental data demonstrates that the entropic component plays an important role in the control of the redox potential in c-type and b-type cytochromes. The variation of the entropic component within the class of cytochromes characterized by a positive value of E degrees ' is proposed to be mainly determined by the variation of the exposure of the heme propionates to the solvent. In the case of tetraheme cytochromes c3, the thermodynamic characteristics vary largely among the hemes belonging to the same molecule, which reflects the environmental peculiarities of each heme and also the heme-heme redox interactions. This study substantiates the existence of compensatory effects between large and opposite contributions to E degree ' predicted by all the current theoretical models which are based on electrostatic free energy calculations.

Animals↗
PubMed1995

Structure and dynamics of ferrocytochrome c553 from Desulfovibrio vulgaris studied by NMR spectroscopy and restrained molecular dynamics.

The solution structure of Desulfovibrio vulgaris Hildenborough (DvH) ferrocytochrome c553 has been determined by nuclear magnetic resonance spectroscopy and combined simulated annealing/high temperature restrained molecular dynamics calculations. This three-stage protocol consists of an initial determination of overall fold from randomised co-ordinates, followed by a 20 picosecond exploratory stage, during which the non-bonded terms are simplified to facilitate as broad a sampling of conformational space as possible, and a 26 picosecond refinement stage, using the full AMBER force field. This latter stage systematically improved the energetic and convergence characteristics of the ensemble, while still satisfying the experimental restraints. Forty structures have been obtained from a total of 875 distance constraints for this protein of 79 amino acid residues. The root-mean-square deviation over all residues with respect to the mean is 0.70(+/- 0.12)A for the backbone (N, C alpha and C') atoms. Two conformations of the turn motif at the solvent/heme cleft interface have been identified, both fulfilling the experimental data and having equally viable energetic characteristics. The stability of the ensemble and the dynamic characteristics have been further investigated by subjecting ten of the structures to constraint-free molecular dynamics calculations (130 picoseconds) in vacuo. The structures were found to be stable to within 1.5 A of the initial backbone conformation. Comparison with the dynamic behaviour of the restrained molecular dynamics calculations has been used to identify regions of inherent flexibility in the molecule.

Cytochrome c Group↗
PubMed1995

Biochemical properties of a beta-xylosidase from Clostridium cellulolyticum.

A 43-kDa beta-xylosidase from Clostridium cellulolyticum was purified to homogeneity. The enzyme releases xylose from p-nitrophenylxylose and xylodextrins with a degree of polymerization ranging between 2 and 5. The N-terminal amino acid sequence of the enzyme showed homologies with three other bacterial beta-xylosidases. By proton nuclear magnetic resonance spectroscopy, the enzyme was found to act by inverting the beta-anomeric configuration.

Amino Acid Sequence↗
PubMed1994

Effects of the Tyr64 substitution on the stability of cytochrome c553, a low oxidoreduction-potential cytochrome from Desulfovibrio vulgaris Hildenborough.

Cytochrome c553 from sulfate-reducing bacteria is a low-oxidoreduction-potential cytochrome. The primary and tertiary structures show notable differences when compared to mitochondrial cytochromes. Tyr64 replacement in cytochrome c553 provides evidence that this residue is not directly involved in the potential modulation but is mostly implicated in the hydrogen-bond network around the heme. While the different variants obtained did not induce drastic structural modifications, they did affect the stability of the protein. This decrease of stability in acidic and alkaline environments was observed by variations in the optical spectra and by mass spectrometry. In addition, the mobility of aromatic side-chain was found to be increased in the mutant proteins as monitored by two-dimensional NMR spectroscopy.

Base Sequence↗
PubMed1994

Crystal structure of cytochrome c3 from Desulfovibrio desulfuricans Norway at 1.7 A resolution.

The crystal structure of cytochrome c3 (M(r) 13,000) from Desulfovibrio desulfuricans (118 residues, four heme groups) has been crystallographically refined to 1.7 A resolution using a simulated annealing method, based on the structure-model at 2.5 A resolution, already published. The final R-factor for 10,549 reflections was 0.198 covering the range from 5.5 to 1.7 A resolution. The individual temperature factors were refined for a total of 1059 protein atoms, together with 126 bound solvent molecules. The structure has been analyzed with respect to its detailed conformational properties, secondary structure features, temperature factor behaviour, bound solvent sites and heme geometry and ligation. The characteristic secondary structures of the polypeptide chain of this molecule are one extended alpha-helix, a short beta-strand and 13 reverse turns. The four heme groups are located in different structural environments, all highly exposed to solvent. The particular structural features of the heme environments are compared to the four hemes of the cytochrome c3 from Desulfovibrio vulgaris Miyazaki.

Amino Acid Sequence↗
PubMed1994

Active site geometry in the high oxido-reduction potential rusticyanin from Thiobacillus ferrooxidans.

Rusticyanin is a blue copper protein involved in the oxidation of iron catalyzed by Thiobacillus ferrooxidans. This protein is characterized by a high oxido-reduction potential and a high stability at low pH. The three dimensional structure of this protein is still unknown and in order to investigate the geometric properties of the copper center which could be correlated to the high oxido-reduction potential, we have studied rusticyanin by UV-Visible, EPR and NMR spectroscopies, at different pH values. Our results suggest that rusticyanin is stable between pH 2 and pH 9 and that the copper center does not undergo significant geometric modifications in this pH range. Moreover, the copper atom could be buried more deeply in the protein than in other type I copper proteins and the atomic distance Cu-S(Met), one of the four bonds involved in copper coordination, is probably shorter in rusticyanin than in other cupredoxins. These two properties of the copper site are expected to be responsible, in part, for the high oxido-reduction potential observed in rusticyanin.

Azurin↗
PubMed1994

Involvement of histidine residues in the catalytic mechanism of hydrogenases.

In spite of their structural and amino acid sequence differences, Fe-only and Ni-containing hydrogenases achieved the same catalytic reactions. A chemical modification of histidine residues using a highly specific reagent (pentaammineruthenium II) has been carried out on Desulfovibrio vulgaris Hildenborough Fe-hydrogenase and Desulfovibrio desulfuricans Norway Ni-Fe-Se-hydrogenase. The preliminary results obtained suggest the existence of a general mechanism involving histidine residues in the two groups of hydrogenases. These residues may be part of the histidine-containing motive shown to be present in both Fe- and Ni-Fe-hydrogenase sequences by Hydrophobic Cluster Analysis. This analysis also allows us to suggest a functional role for the small subunit of Desulfovibrio vulgaris Hildenborough Fe-hydrogenase.

Amino Acid Sequence↗
PubMed1994

Amino-acid sequence of the cytochrome c3 (M(r) 26,000) from Desulfovibrio desulfuricans Norway and a comparison with those of the other polyhemic cytochromes from Desulfovibrio.

The amino-acid sequence of an octaheme cytochrome c3 isolated from Desulfovibrio desulfuricans Norway is presented. The protein molecule (M(r) 26,000) comprises two identical subunits of 111 amino acids with the characteristics typical of tetrahemic cytochrome c3 class. Comparisons between the amino-acid sequences and physiological properties of cytochrome c3 (M(r) 26,000) and cytochromes c3 (M(r) 13,000) isolated from various species of Desulfovibrio showed the existence of considerable differences. In order to distinguish between the various subclasses in the cytochrome c3 superfamily, the amino-acid sequence of cytochrome c3 (M(r) 26,000) was compared with six known cytochrome c3 (M(r) 13,000) sequences as well as with the sequence of the four c3-like domains of a high molecular weight cytochrome c (Hmc) containing 16 hemes per molecule of 65,500 Da, isolated from Desulfovibrio vulgaris Hildenborough. The evolution and phylogenetic relationships of these various polyhemic cytochromes are discussed.

Amino Acid Sequence↗
PubMed1994

Characterization and oxidoreduction properties of cytochrome c3 after heme axial ligand replacements.

Cytochrome c3 (M(r) 13,000) is a tetrahemic cytochrome in which the four heme iron atoms are coordinated by 2 histidine residues at the axial positions. The presence of several oxidoreduction centers in the same molecule raises the question of their coupling. To investigate this mechanism, four single mutations were introduced in cytochrome c3 by site-directed mutagenesis, leading to the replacement of each histidine, the sixth axial ligand of the heme iron atom, by a methionine residue. Characterization of the new set of molecules using biochemical and biophysical techniques was carried out. The novel methionine was correctly coordinated to the iron atom of hemes 3 and 4 in H25M and H70M cytochromes c3, respectively, and this coordination induced a large increase in the oxidoreduction potential of the mutated heme. In contrast, in the case of H22M and H35M cytochromes c3, in which the corresponding methionine is in an oxidized form, only slight changes in redox potential values were observed. In H22M, H25M, and H35M cytochromes c3, two conformations of the molecule were possible, in which the methionine is either free or coordinated to the iron atom. The rate constants for the electron exchange reactions between the cytochrome mutants and the hydrogenase were measured using electrochemical techniques. Distinct behaviors were revealed depending on the mutation. The values of the rate constants for the electron exchange reactions are interpreted in terms of intramolecular electron exchange among the four hemes of the cytochrome.

Base Sequence↗
PubMed1994

The protein moiety modulates the redox potential in cytochromes c.

Cytochrome c is one of the most thoroughly documented oxidoreduction proteins. Its electron transfer activity, which involves an association between the heme group and the polypeptidic chain, is correlated with the redox potential value of the heme group. The redox potential covers a wide range up to 0.8 V, an extreme case being observed in the low-potential cytochromes c from sulfate reducing bacteria. On of the main roles of the polypeptidic moiety consists of modulating the redox potential value of the heme group. In this paper, some structural factors that seem likely to be involved in maintaining the redox potential value are described.

Cytochrome c Group↗
PubMed1994

Involvement of electrostatic interactions in cytochrome c complex formations.

Structural studies on various electron transfer complexes involving the tetrahemic cytochrome c3 provided evidence that one of the hemes (heme 4) is the interacting site on the molecule. The reactivity of this particular heme is allocated to the positive charges found around the heme group which are strongly involved in the electrostatic interaction processes. Electrostatic and hydrophobic effects in complex formation are considered on the basis of two electron transfer complex examples: the soluble cytochrome c-cytochrome c peroxidase and the membrane bound photosynthetic reaction center.

Amino Acid Sequence↗
PubMed1993

Overexpression of Desulfovibrio vulgaris Hildenborough cytochrome c553 in Desulfovibrio desulfuricans G200. Evidence of conformational heterogeneity in the oxidized protein by NMR.

Plasmid pRC41, containing the cyf gene encoding cytochrome c533 from Desulfovibrio vulgaris Hildenborough, was transferred by conjugation from Escherichia coli to Desulfovibrio desulfuricans G200. The structural properties of the purified protein were studied by one-dimensional and two-dimensional NMR. A heterogeneity in the folding of the cytochrome isolated from D. vulgaris Hildenborough and from D. desulfuricans G200 was observed for the oxidized from. Temperature, pH and salt-dependence studies indicated that the heterogeneity does not result from an intermediate in the protein unfolding process, but derives from two conformations which are not in dynamic equilibrium.

Amino Acid Sequence↗
PubMed1993

Extensive 1H NMR resonance assignment of proteins using natural abundance gradient-enhanced 13C-1H correlation spectroscopy.

The reliability and completeness of 1H NMR resonance assignment can be improved by the use of 13C-1H HSQC correlation spectra on unlabelled protein samples using pulsed field gradients. This technique is illustrated on a 5.2 mM sample of the 79 residue Desulfovibrio vulgaris ferrocytochrome c553. Protons attached to the same carbon can be unambiguously paired in a HSQC spectrum. Contrary to 1H, most amino acids exhibit characteristic 13C chemical shift ranges, which can be used for 13C assignment. This technique is especially useful for long side chain residues, such as Gln, Glu, Lys, Arg.

Amino Acid Sequence↗
PubMed1993

Purification and characterization of endoglucanase C from Clostridium cellulolyticum. Catalytic comparison with endoglucanase A.

An Escherichia coli clone was constructed to overproduce endoglucanase C (CelCCC) from Clostridium cellulolyticum. This construction made it easier to isolate the enzyme but, as observed in the case of endoglucanase A (CelCCA) from the same organism, the purification led to the isolation of two forms of the cellulase differing in their molecular masses, 48 kDa and 41 kDa. N-terminal sequence analysis of both purified enzymes showed that the shorter form was probably the result of partial proteolysis near the COOH-extremity. The difference in mass indicated that the shorter protein lacks the C-terminal reiterated domains (20-24-amino-acid twice-repeated sequences). These particular domains are characteristic of clostridial cellulases acting on cellulose by the mean of cellulosomal particles. Biochemical and enzymic studies were performed on each form of CelCCC, and revealed that their temperature and pH optima were identical, but their catalytic parameters were quite different. Furthermore, the differences of enzymic behavior observed between the two forms of CelCCC are almost identical to those already noted in the case of the two forms of CelCCA. The stereoselectivity of the reaction catalysed by CelCCC and CelCCA was determined using proton NMR spectroscopy; CelCCC acts by configuration inversion, whereas CelCCA acts by configuration retention. The degradation patterns on cellodextrins (ranging from cellotriose to cellohexaose) and chromophoric cellodextrins (from p-nitrophenyl-cellobiose to p-nitrophenyl-cellopentaose) were also investigated in both forms of CelCCC and CelCCA. It emerged that the natural cellodextrins degradation patterns of CelCCC and CelCCA were very similar but the utilization of p-nitrophenyl-cellodextrins showed the existence of considerable differences between these two endoglucanases in terms of cleavage-site position and catalytic parameters. CelCCC and CelCCA were found not to act synergistically on the tested substrates.

Base Sequence↗
PubMed1993

Intramolecular electron transfer in ferredoxin II from Desulfovibrio desulfuricans Norway.

In order to elucidate the role of the two (4Fe-4S) clusters in ferredoxins and to determine whether an electron-transfer mechanism may occur between the clusters, the in vitro reduction of cytochrome c3 and cytochrome c553 by Desulfovibrio desulfuricans Norway ferredoxin II was studied using spectrophotometric techniques. Ferredoxin II, covalently cross-linked with either cytochrome c3 or c553, is an obligate intermediate in cytochrome reduction by pyruvate dehydrogenase. Both titration of the complex formation under 1H-NMR spectroscopy and cross-linking experiments between ferredoxin II and either cytochrome c3 or cytochrome c553 gave a stoichiometric ratio of 1:1. Modelling the protein yielded differences between the charge distributions around the two (Fe-S) clusters. The fact that Cluster 2 is blocked in the electron-transfer domain facing the cytochrome interacting heme, indicates Cluster 1 receives electron from pyruvate dehydrogenase. Consecutively, cytochrome reduction occurs owing to an intramolecular electron exchange between the two clusters of the ferredoxin. The properties of two (Fe-S) cluster ferredoxins are compared to those of monocluster ferredoxins and discussed in evolutionary terms.

Amino Acid Sequence↗
PubMed1992

Reactivity of [Fe] and [Ni-Fe-Se] hydrogenases with their oxido-reduction partner: the tetraheme cytochrome c3.

In order to understand the electron transfer mechanisms for the [Fe] and [Ni-Fe] hydrogenases, a kinetic study of cytochrome c3 reduction has been undertaken. Cyclic voltammetry and controlled-potential amperometry techniques have been used to investigate the intermolecular electron-transfer reaction between cytochrome c3 and [Fe] hydrogenase from Desulfovibrio vulgaris Hildenborough. Electron-transfer cross-reactions between [Fe] or [Ni-Fe-Se] hydrogenase and cytochrome c3 from Desulfovibrio vulgaris Hildenborough or Desulfovibrio desulfuricans Norway have been studied. Some structural implications are considered from these experimental data.

Cytochrome c Group↗