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A V Xavier

Publications and source records attributed to A V Xavier.

At least 37 records · Page 2Linked to original sources
PubMed1999

Desulfovibrio gigas neelaredoxin. A novel superoxide dismutase integrated in a putative oxygen sensory operon of an anaerobe.

Neelaredoxin, a small non-heme blue iron protein from the sulfate-reducing bacterium Desulfovibrio gigas [Chen, L., Sharma, P., LeGall, J., Mariano, A.M., Teixeira M. and Xavier, A.V. (1994) Eur. J. Biochem. 226, 613-618] is shown to be encoded by a polycistronic unit which contains two additional open reading frames (ORF-1 and ORF-2) coding for chemotaxis-like proteins. ORF-1 has domains highly homologous with those structurally and functionally important in methyl-accepting chemotaxis proteins, including two putative transmembrane helices, potential methylation sites and the interaction domain with CheW proteins. Interestingly, ORF-2 encodes a protein having homologies with CheW proteins. Neelaredoxin is also shown to have significant superoxide dismutase activity (1200 U. mg-1), making it a novel type of iron superoxide dismutase. Analysis of genomic data shows that neelaredoxin-like putative polypeptides are present in strict anaerobic archaea, suggesting that this is a primordial superoxide dismutase. The three proteins encoded in this operon may be involved in the oxygen-sensing mechanisms of this anaerobic bacterium, indicating a possible transcriptional mechanism to sense and respond to potential stress agents.

Amino Acid Sequence↗
PubMed1999

The superoxide dismutase activity of desulfoferrodoxin from Desulfovibrio desulfuricans ATCC 27774.

Desulfoferrodoxin (Dfx), a small iron protein containing two mononuclear iron centres (designated centre I and II), was shown to complement superoxide dismutase (SOD) deficient mutants of Escherichia coli [Pianzzola, M.J., Soubes M. & Touati, D. (1996) J. Bacteriol. 178, 6736-6742]. Furthermore, neelaredoxin, a protein from Desulfovibrio gigas containing an iron site similar to centre II of Dfx, was recently shown to have a significant SOD activity [Silva, G., Oliveira, S., Gomes, C.M., Pacheco, I., Liu, M.Y., Xavier, A.V., Teixeira, M., Le Gall, J. & Rodrigues-Pousada, C. (1999) Eur. J. Biochem. 259, 235-243]. Thus, the SOD activity of Dfx isolated from the sulphate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 was studied. The protein exhibits a SOD activity of 70 U x mg-1, which increases approximately 2.5-fold upon incubation with cyanide. Cyanide binds specifically to Dfx centre II, yielding a low-spin iron species with g-values at 2.27 (g perpendicular) and 1.96 (g parallel). Upon reaction of fully oxidized Dfx with the superoxide generating system xanthine/xanthine oxidase, Dfx centres I and II become partially reduced, suggesting that Dfx operates by a redox cycling mechanism, similar to those proposed for other SODs. Evidence for another SOD in D. desulfuricans is also presented - this enzyme is inhibited by cyanide, and N-terminal sequence data strongly indicates that it is an analogue to Cu,Zn-SODs isolated from other sources. This is the first indication that a Cu-containing protein may be present in a sulphate-reducing bacterium.

Amino Acid Sequence↗
PubMed1998

Functional and mechanistic studies of cytochrome c3 from Desulfovibrio gigas: thermodynamics of a "proton thruster".

Nuclear magnetic resonance and visible spectroscopies were used to determine the thermodynamic parameters of the four hemes in cytochrome c3 from Desulfovibrio gigas at 298 and 277 K and to investigate the mechanism of electron/proton energy transduction. Data obtained in the pH range from 5 to 9 were analyzed according to a model in which the hemes interact with each other (redox cooperativities) and with an ionizable center (redox-Bohr cooperativities). The results obtained at the two temperatures allow the deconvolution of the entropic contribution to the free energy of the four hemes, to the acid-base equilibrium of the ionizable center, and to the network of cooperativities among the five centers. The redox potentials of the hemes are modulated by the enthalpic contribution to the free energy, and evidence for the participation of the propionates of heme I in the redox-Bohr effect is presented. The network of interactions between the centers in this protein facilitates the concerted transfer of electrons and protons, in agreement with the "proton thruster" mechanism proposed for electronic to protonic energy transduction by cytochromes c3.

Cytochrome c Group↗
PubMed1998

Replacement of lysine 45 by uncharged residues modulates the redox-Bohr effect in tetraheme cytochrome c3 of Desulfovibrio vulgaris (Hildenborough).

The structural basis for the pH dependence of the redox potential in the tetrahemic Desulfovibrio vulgaris (Hildenborough) cytochrome c3 was investigated by site-directed mutagenesis of charged residues in the vicinity of heme I. Mutation of lysine 45, located in the neighborhood of the propionates of heme I, by uncharged residues, namely threonine, glutamine and leucine, was performed. The replacement of a conserved charged residue, aspartate 7, present in the N-terminal region and near heme I was also attempted. The analysis of the redox interactions as well as the redox-Bohr behavior of the mutated cytochromes c3 allowed the conclusion that residue 45 has a functional role in the control of the pKa of the propionate groups of heme I and confirms the involvement of this residue in the redox-Bohr effect.

Amino Acid Substitution↗
PubMed1998

Solution structure of Desulfovibrio vulgaris (Hildenborough) ferrocytochrome c3: structural basis for functional cooperativity.

Desulfovibrio vulgaris cytochrome c3 is a 14 kDa tetrahaem cytochrome that plays a central role in energy transduction. The three-dimensional structure of the ferrocytochrome at pH 8.5 was solved through two-dimensional 1H-NMR. The structures were calculated using a large amount of experimental information, which includes upper and lower distance limits as well as dihedral angle restraints. The analysis allows for fast-flipping aromatic residues and flexibility in the haem plane. The structure was determined using 2289 upper and 2390 lower distance limits, 63 restricted ranges for the phi torsion angle, 88 stereospecific assignments out of the 118 stereopairs with non-degenerate chemical shifts (74.6%), and 115 out of the 184 nuclear Overhauser effects to fast-flipping aromatic residues (62.5%), which were pseudo-stereospecifically assigned to one or the other side of the ring. The calculated NMR structures are very well defined, with an average root-mean-square deviation value relative to the mean coordinates of 0.35 A for the backbone atoms and 0.70 A for all heavy-atoms. Comparison of the NMR structures of the ferrocytochrome at pH 8.5 with the available X-ray structure of the ferricytochrome at pH 5.5 reveals that the general fold of the molecule is very similar, but that there are some distinct differences. Calculation of ring current shifts for the residues with significantly different conformations confirms that the NMR structures represent better its solution structure in the reduced form. Some of the localised differences, such as a reorientation of Thr24, are thought to be state-dependent changes that involve alterations in hydrogen bond networks. An important rearrangement in the vicinity of the propionate groups of haem I and involving the covalent linkage of haem II suggests that this is the critical region for the functional cooperativities of this protein.

Amino Acid Sequence↗
PubMed1998

Characterisation of a new rubredoxin isolated from Desulfovibrio desulfuricans 27774: definition of a new family of rubredoxins.

A new rubredoxin from the sulphate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774, grown with nitrate as terminal electron acceptor, was isolated and characterised. The protein is an 8.5 kDa monomer containing one iron atom per molecule, with a reduction potential of 25 +/- 5 mV at pH 7.6. Like the recombinant Rdl protein from D. vulgaris, expressed in Escherichia coli [Lumpio, H.L., Shenvi, N.V., Garg, R.P., Summers, A.O. and Kurtz, D.M., J. Bacteriol. 179 (1997) 4607-4615], it contains an unusual spacing of four amino acids between the first two of the iron coordinating cysteinyl residues. This difference is reflected in the structure of the iron centre, as observed by visible and EPR spectroscopies. All together, these features make these proteins the first members of a new family of rubredoxins.

Amino Acid Sequence↗
PubMed1998

Determination of solution structures of paramagnetic proteins by NMR.

Standard procedures for using nuclear Overhauser enhancements (NOE) between protons to generate structures for diamagnetic proteins in solution from NMR data may be supplemented by using dipolar shifts if the protein is paramagnetic. This is advantageous since the electron -nuclear dipolar coupling provides relatively long-range geometric information with respect to the paramagnetic centre which complements the short-range distance constraints NOEs. Several different strategies have been developed to date, but none of these attempts to combine data from NOEs and dipolar shifts in the initial stages of structure calculation or to determine three dimensional protein structures together with their magnetic properties. This work shows that the magnetic and atomic structures are highly correlated and that it is important to have additional constraints both to provide starting parameters for the magnetic properties and to improve the definition of the best fit. Useful parameters can be obtained for haem proteins from Fermi contact shifts; this approach is compared with a new method based on the analysis of dipolar shifts in haem methyl groups with respect to data from horse and tuna ferricytochromes c. The methods developed for using data from NOEs and dipolar shifts have been incorporated in a new computer program, PARADYANA, which is demonstrated in application to a model data set for the sequence of the haem octapeptide known as microperoxidase-8.

Anisotropy↗
PubMed1997

Characterization of the [NiFe] hydrogenase from the sulfate reducer Desulfovibrio vulgaris Hildenborough.

The [NiFe] hydrogenase from Desulfovibrio vulgaris Hildenborough was isolated from the cytoplasmic membranes and characterized by EPR spectroscopy. It has a total molecular mass of 98.7 kDa (subunits of 66.4 and 32.3 kDa), and contains 1 nickel and 12 Fe atoms per heterodimer. The catalytic activities for hydrogen consumption and production were determined to be 174 and 89 mumol H2.min-1.mg-1, respectively. As isolated, under aerobic conditions, this hydrogenase exhibits EPR signals characteristic of the nickel centers in [NiFe] hydrogenases (Ni-A signal at gx,y,z = 2.32, 2.23 and approximately 2.0 and Ni-B signal at gx,y,z = 2.33, 2.16 and approximately 2.0) as well as an intense quasi-isotropic signal centered at g = 2.02 due to the oxidized [3Fe-4S] center. The redox profile under hydrogen atmosphere is remarkably similar to that of other [NiFe] hydrogenases. The signals observed for the oxidized state disappear, first being substituted by the Ni-C type signal (gx,y,z = 2.19, 2.14, approximately 2.01), which upon long incubation under hydrogen yields the split Ni-C signal due to interaction with the reduced [4Fe-4S] centers.

Amino Acid Sequence↗
PubMed1997

Studies on the redox centers of the terminal oxidase from Desulfovibrio gigas and evidence for its interaction with rubredoxin.

Rubredoxin-oxygen oxidoreductase (ROO) is the final component of a soluble electron transfer chain that couples NADH oxidation to oxygen consumption in the anaerobic sulfate reducer Desulfovibrio gigas. It is an 86-kDa homodimeric flavohemeprotein containing two FAD molecules, one mesoheme IX, and one Fe-uroporphyrin I per monomer, capable of fully reducing oxygen to water. EPR studies on the native enzyme reveal two components with g values at approximately 2.46, 2.29, and 1.89, which are assigned to low spin hemes and are similar to the EPR features of P-450 hemes, suggesting that ROO hemes have a cysteinyl axial ligation. At pH 7.6, the flavin redox transitions occur at 0 +/- 15 mV for the quinone/semiquinone couple and at -130 +/- 15 mV for the semiquinone/hydroquinone couple; the hemes reduction potential is -350 +/- 15 mV. Spectroscopic studies provided unequivocal evidence that the flavins are the electron acceptor centers from rubredoxin, and that their reduction proceed through an anionic semiquinone radical. The reaction with oxygen occurs in the flavin moiety. These data are strongly corroborated by the finding that rubredoxin and ROO are located in the same polycistronic unit of D. gigas genome. For the first time, a clear role for a rubredoxin in a sulfate-reducing bacterium is presented.

Amino Acid Sequence↗
PubMed1997

Multiheme cytochromes from the sulfur-reducing bacterium Desulfuromonas acetoxidans.

Two new multiheme cytochromes were isolated from the anaerobic sulfur reducing bacterium Desulfuromonas acetoxidans. They have monomeric molecular masses of 50 and 65 kDa and contain six and eight hemes, respectively. Visible and EPR spectroscopies, in the as-isolated (oxidised) cytochromes, show the presence of only low-spin hemes in the 50-kDa cytochrome, and of high-spin and low-spin hemes in the 65-kDa cytochrome. The EPR spectra of the native 65-kDa cytochrome indicate multiple heme-heme interactions, including integer-spin systems as judged by parallel-mode EPR. The 50-kDa cytochrome has a complex redox pattern, as shown by EPR redox titrations, and contains one heme with unusual characteristics. Both cytochromes cover an extremely wide range of reduction potentials, which go from +100 mV to -375 mV for the 50-kDa cytochrome, and +185 mV to -235 mV for the 65-kDa cytochrome. The two cytochromes were tested for hydroxylamine oxidoreductase activity and polysulfide reductase activity, but neither displayed any activity. In contrast, it was found for the first time that the previously characterised cytochrome c551.5, from the same bacterium is very active in the reduction of polysulfide, which suggests that it acts as a terminal reductase in D. acetoxidans.

Amino Acid Sequence↗
PubMed1997

Use of paramagnetic NMR probes for structural analysis in cytochrome c3 from Desulfovibrio vulgaris.

The dipolar field generated by each of the four haems in the tetrahaem ferricytochrome c3 from Desulfovibrio vulgaris (Hildenborough) (c3DvH) is determined by means of a novel procedure. In this method the 13C chemical shifts of the nuclei directly bound to the haems are used to determine the in-plane orientations of the rhombic perturbation in each of the four haems with respect to a model of molecular orbitals of e(g) symmetry which are subject to a rhombic perturbation [Turner, D. L., Salgueiro, C. A., Schenkels, P., LeGall, J. & Xavier, A. V. (1995) Biochim. Biophys. Acta 1246, 24-281. These orientations, together with the components of the magnetic susceptibility tensors obtained from the EPR g values and the crystal structure of c3DvH, can be used to calculate the dipolar shifts induced by each haem throughout the protein. Thus the observed 13C paramagnetic shifts of the c3DvH haem substituents were fitted considering both the pseudocontact and contact shifts of each haem simultaneously. The dipolar shifts calculated by this method were tested against the observed dipolar shifts for some amino acid residues strategically placed in the protein and also for the haem propionate groups. The effect of considering the calculated dipolar extrinsic shifts on the behaviour of the chemical shifts of the haem methyl groups in the intermediate stages of oxidation at different pH values was also analysed. The several tests applied to the calculated dipolar shifts have shown that the method is extremely useful for predicting chemical shifts as an aid to complete proton assignment, and to add further constraints in the refinement of solution structures of paramagnetic proteins and hence to probe subtle structural rearrangements around the haem pocket.

Amino Acids↗
PubMed1997

Assignment of the ligand geometry and redox potentials of the trihaem ferricytochrome c3 from Desulfuromonas acetoxidans.

Cytochrome c551.5 is a trihaem cytochrome of the cytochrome c3 family isolated from Desulfuromonas acetoxidans. Although several X-ray structures are available for tetrahaem cytochromes of this family, there is no X-ray structure for trihaem cytochromes. Cytochrome C551.5 was studied in the oxidized form by means of two-dimensional NMR. The pattern of observed interhaem NOESY connectivities is in agreement with the haem core structure previously determined by NMR for the reduced protein [Coutinho, I. B., Turner, D. L., Liu, M. Y., LeGall, J. & Xavier, A. V. (1996) J. Biol. Inorg. Chem. 1, 305-311]. The similarities found between the haem core structure and the amino acid sequence of cytochrome c551.5 and those of tetrahaem cytochromes c3 allows each of the haems to be specifically assigned in the polypeptide sequence, and the attribution of the midpoint redox potentials to the individual haems. This also allows individual redox potentials to be assigned to each haem in the NMR spectrum. The paramagnetic shifts of the 13C resonances of the haem substituents were analyzed in terms of pi molecular orbitals with perturbed D4h symmetry. The parameters of this analysis have been shown to be controlled by the orientation of the axial ligands in several other bis-His-coordinated haems and hence the ligand geometry was deduced for cytochrome C551.5. The structural analogy between the relative haem plane orientations in cytochrome c551.5 and the tetrahaem cytochromes c3 is found to extend to the axial ligands with the largest differences being in the vicinity of the deleted fourth haem, using the numbering of cytochrome c3 haems.

Bacteria↗
PubMed1997

Pathways for utilization of carbon reserves in Desulfovibrio gigas under fermentative and respiratory conditions.

The sulfate-reducing bacterium Desulfovibrio gigas accumulates large amounts of polyglucose as an endogenous carbon and energy reserve. In the absence of exogenous substrates, the intracellular polysaccharide was utilized, and energy was conserved in the process (H. Santos, P. Fareleira, A. V. Xavier, L. Chen, M.-Y. Liu, and J. LeGall, Biochem. Biophys. Res. Commun. 195:551-557, 1993). When an external electron acceptor was not provided, degradation of polyglucose by cell suspensions of D. gigas yielded acetate, glycerol, hydrogen, and ethanol. A detailed investigation of the metabolic pathways involved in the formation of these end products was carried out, based on measurements of the activities of glycolytic enzymes in cell extracts, by either spectrophotometric or nuclear magnetic resonance (NMR) assays. All of the enzyme activities associated with the glycogen cleavage and the Embden-Meyerhof pathway were determined as well as those involved in the formation of glycerol from dihydroxyacetone phosphate (glycerol-3-phosphate dehydrogenase and glycerol phosphatase) and the enzymes that catalyze the reactions leading to the production of ethanol (pyruvate decarboxylase and ethanol dehydrogenase). The key enzymes of the Entner-Doudoroff pathway were not detected. The methylglyoxal bypass was identified as a second glycolytic branch operating simultaneously with the Embden-Meyerhof pathway. The relative contribution of these two pathways for polyglucose degradation was 2:3. 13C-labeling experiments with cell extracts using isotopically enriched glucose and 13C-NMR analysis supported the proposed pathways. The information on the metabolic pathways involved in polyglucose catabolism combined with analyses of the end products formed from polyglucose under fermentative conditions provided some insight into the role of NADH in D. gigas. In the presence of electron acceptors, NADH resulting from polyglucose degradation was utilized for the reduction of sulfate, thiosulfate, or nitrite, leading to the formation of acetate as the only carbon end product besides CO2. Evidence supporting the role of NADH as a source of reducing equivalents for the production of hydrogen is also presented.

Desulfovibrio↗
PubMed1996

NMR studies of cooperativity in the tetrahaem cytochrome c3 from Desulfovibrio vulgaris.

The thermodynamic properties of the Desulfovibrio vulgaris (Hildenborough) tetrahaem cytochrome c3 (Dvc3) are rationalised by a model which involves both homotropic (e-/e-) and heterotropic (e-/H+) cooperativity. The paramagnetic shifts of a methyl group from each haem of the Dvc3 have been determined in each stage of oxidation at several pH values by means of two-dimensional exchange NMR. The thermodynamic parameters are obtained by fitting the model to the NMR data and to redox titrations followed by visible spectroscopy. They show significant positive cooperativity between two of the haems whereas the remaining interactions appear to be largely electrostatic in origin. These parameters imply that the protein undergoes a proton-assisted two-electron transfer which can be used for energy transduction. Comparison with the crystal structure together with measurement of the kinetics of proton exchange suggest that the pH dependence is mediated by a charged residue(s) readily acessible to the solvent and close to haem I.

Allosteric Regulation↗
PubMed1996

Nitrite reductase from Desulfovibrio desulfuricans (ATCC 27774)--a heterooligomer heme protein with sulfite reductase activity.

The membrane bound cytochrome c nitrite reductase from the sulfate reducer Desulfovibrio desulfuricans (ATCC 27774) was found to have a high specific activity in the reduction of sulfite, producing stoichiometric amounts of sulfide. The K(m) for sulfite in the MV+.:sulfite oxidoreductase assay is 0.75 mM, and the specific activity 2.06 mumolH2/min/mg. Visible and EPR spectroscopies studies indicate that the enzyme high-spin heme reacts with sulfite in the oxidised state, and that sulfide partially reduces the enzyme. The redoxcycled enzyme, using H2/Hydrogenase/MV+. as a reductant, is identical to the resting enzyme. This is the first time that a c-type nitrite reductase has been shown to reduce sulfite. These findings, besides revealing a new function for the nitrite reductase, raise a major question regarding the sulfur metabolism in the sulfate reducing bacteria as well as the cellular localization of the enzymatic activities involved in the dissimilatory reduction of sulfate. The purified nitrite reductase is a heterooligomer, containing two types of subunits of 62 kDa (+/- 5 kDa) and 18.8 kDa (+/- 1 kDa), and forms a complex or aggregate with a molecular mass of approximately 750 kDa.

Chromatography, Gel↗
PubMed1996

Structural and functional characterization of cytochrome c3 from D. desulfuricans ATCC 27774 by 1H-NMR.

Cooperativity between redox and protonation centres is known to be crucial for the function of complex proteins, but it is often difficult to describe in terms of thermodynamic parameters. Cytochrome c3 is a good model for these studies since, while retaining the overall complexity of larger systems, it is suitable for detailed crystallographic and spectroscopic studies. Assignment of the haem substituent NMR resonances, together with NMR redox titrations of cytochrome c3 from D. desulfuricans ATCC 27774, was used to correlate relative redox potentials to specific haems in the structure: haem II approximately equal to haem I < haem IV < haem III. This order is different from that determined for the homologous proteins studied and in disagreement with that previously reported for this cytochrome (Morais, J., Palma, N., Frazäo, C., Caldeira, J., LeGall, J., Moura, I., Moura, J.J.G. and Carrondo, M.A. (1995) Biochemistry 34, 12830-12841).

Cytochrome c Group↗
PubMed1995

NMR studies and redox titration of the tetraheme cytochrome c3 from Desulfomicrobium baculatum. Identification of the low-potential heme.

The tetraheme cytochromes c3 isolated from two strains of Desulfomicrobium baculatum were studied by monitoring the spectral changes undergone during redox titrations followed by 1H NMR. The evolution of the three-protein intensity signals at low field allowed the partial identification of the heme methyl resonances in the spectrum of the fully oxidized state. The chemical shift variation shown by the protons of the aromatic sidechains as well as of the substituents of the higher-potential heme HIII [Coutinho, I. B., Turner, D. L., LeGall, J. & Xavier, A. V. (1993) Biochem. J. 294, 899-908] yielded the assignment of the lower midpoint redox potential to heme HII in the three-dimensional structure. This cross-assignment is achieved by comparing the chemical shifts of the resonances in the spectra obtained at intermediate oxidation levels with the pseudocontact shifts predicted to arise from the three lower-potential hemes. The cross-assignment for the cytochromes from these two strains is different from that of the cytochromes from Desulfovibrio vulgaris and Desulfovibrio gigas.

Cytochrome c Group↗