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A V Xavier

Publications and source records attributed to A V Xavier.

At least 73 records · Page 4Linked to original sources
PubMed1992

Revision of the haem-core architecture in the tetraheam cytochrome c3 from Desulfovibrio baculatus by two-dimensional 1H NMR.

The haem-core architecture in cytochrome c3 isolated from Desulfovibrio baculatus (Norway 4) was probed using two-dimensional 1H NMR. Interhaem connectivities detected in NOE spectroscopy experiments performed at short mixing times are incompatible with the structure of the protein determined by X-ray crystallography, but agree instead with the haem arrangement found in cytochrome c3 from Desulfovibrio vulgaris (Miyazaki). These experiments show unequivocally that the relative orientation of the four haems in the two proteins is the same and does not involve the 180 degrees rotation of haems I and IV indicated in the X-ray structure determined for the cytochrome c3 from D. baculatus (Norway 4).

Cytochrome c Group↗
PubMed1992

Involvement of a labile axial histidine in coupling electron and proton transfer in Methylophilus methylotrophus cytochrome c''.

Methylophilus methylotrophus cytochrome c'' is an unusual monohaem protein (15 kDa) undergoing a redox-linked spin-state transition [Santos, H. & Turner, D. L. (1988) Biochim. Biophys. Acta 954, 277-286]. The midpoint redox potential of cytochrome c" was measured over the pH range 4-10. The pH dependence of the midpoint redox potential was interpreted in terms of a model that considers the redox-state dependence of the ionization of two distinct and non-interacting protonated groups in the protein. This analysis led to the following pKa values within the pH range studied: pKa10 = 6.4, pKa1r = 5.4 and pKa2r = 8.1. Proton-NMR spectroscopy was used to assist the characterization of the two ionizing groups responsible for the observed redox-Bohr effect: the group ionizing with a lower pKar was assigned to a haem propionic acid substituent and the other to the axial histidine ligand which becomes detached upon reduction, which has a pKa0 too low to be measured. It is shown that M. methylotrophus cytochrome c" is able to couple electron and proton transfer in the physiological pH range through a mechanism involving reversible change in the haem-iron coordination. Possible implications for the physiological role of the protein are discussed.

Amino Acids↗
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↗
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

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↗
PubMed1989

Electron transport in sulfate-reducing bacteria. Molecular modeling and NMR studies of the rubredoxin--tetraheme-cytochrome-c3 complex.

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.

Binding Sites↗
PubMed1989

Redox intermediates of Desulfovibrio gigas [NiFe] hydrogenase generated under hydrogen. Mössbauer and EPR characterization of the metal centers.

The hydrogenase (EC 1.2.2.1) of Desulfovibrio gigas is a complex enzyme containing one nickel center, one [3Fe-4S] and two [4Fe-4S] clusters. Redox intermediates of this enzyme were generated under hydrogen (the natural substrate) using a redox-titration technique and were studied by EPR and Mössbauer spectroscopy. In the oxidized states, the two [4Fe-4S]2+ clusters exhibit a broad quadrupole doublet with parameters (apparent delta EQ = 1.10 mm/s and delta = 0.35 mm/s) typical for this type of cluster. Upon reduction, the two [4Fe-4S]1+ clusters are spectroscopically distinguishable, allowing the determination of their midpoint redox potentials. The cluster with higher midpoint potential (-290 +/- 20 mV) was labeled Fe-S center I and the other with lower potential (-340 +/- 20 mV), Fe-S center II. Both reduced clusters show atypical magnetic hyperfine coupling constants, suggesting structural differences from the clusters of bacterial ferredoxins. Also, an unusually broad EPR signal, labeled Fe-S signal B', extending from approximately 150 to approximately 450 mT was observed concomitantly with the reduction of the [4Fe-4S] clusters. The following two EPR signals observed at the weak-field region were tentatively attributed to the reduced [3Fe-4S] cluster: (i) a signal with crossover point at g approximately 12, labeled the g = 12 signal, and (ii) a broad signal at the very weak-field region (approximately 3 mT), labeled the Fe-S signal B. The midpoint redox potential associated with the appearance of the g = 12 signal was determined to be -70 +/- 10 mV. At potentials below -250 mV, the g = 12 signal began to decrease in intensity, and simultaneously, the Fe-S signal B appeared. The transformation of the g = 12 signal into the Fe-S signal B was found to parallel the reduction of the two [4Fe-4S] clusters indicating that the [3Fe-4S]o cluster is sensitive to the redox state of the [4Fe-4S] clusters. Detailed redox profiles for the previously reported Ni-signal C and the g = 2.21 signal were obtained in this study, and evidence was found to indicate that these two signals represent two different oxidation states of the enzyme. Finally, the mechanistic implications of our results are discussed.

Desulfovibrio↗
PubMed1989

In vivo 31P- and 13C-NMR studies of ATP synthesis and methane formation by Methanosarcina barkeri.

Carbon and phosphorus metabolism of cell suspensions of Methanosarcina barkeri strain MS (DSM 800), grown on methanol, were probed in vivo by NMR. The experimental conditions, which involved thick cell suspensions, did not significantly affect the efficiency of the rate of methanol uptake by cells. Following exposure to methanol an acidification of both the intracellular and the extracellular spaces was observed and a gradient of 0.5 pH units across the cytoplasmic membrane was determined from the 31P-NMR data. High levels of intracellular ATP up to 4 mM were detected. The ADP concentration determined in a suspension of starved cells was only 2 mM, suggesting that a significant amount of ADP may be immobilized and is thus not detectable by NMR. In the presence of the protonophore, 3,3',4',5-tetrachlorosalicylanilide, the proton gradient was dissipated and the synthesis of ATP stopped. The inhibitor of the ATP synthase, N,N'-dicyclohexylcarbodiimide, was rather inefficient in inhibiting ATP synthesis. High concentrations of N,N'-dicyclohexylcarbodiimide (corresponding to 300 nmol/mg protein-1) were required to decrease the ATP content by approximately 60%, and, under these conditions, formation of acetyl phosphate was detected. However, the methanol consumption rate was not affected.

Adenosine Diphosphate↗
PubMed1988

Spectroscopic characterization of a high-potential monohaem cytochrome from Wolinella succinogenes, a nitrate-respiring organism. Redox and spin equilibria studies.

When purified, a high-potential c-type monohaem cytochrome from the nitrate-respiring organism, Wollinella succinogenes (VPI 10659), displayed a minimum molecular mass of 8.2 kDa and 0.9 mol iron and 0.95 mol haem groups/mol protein. Visible light spectroscopy suggested the presence of an equilibrium between two ligand arrangements around the haem, i.e. an absorption band at 695 nm characteristic of haem-methionine coordination (low-spin form) coexisting with a high-spin form revealed by a band at 619 nm and a shoulder at 498 nm. The mid-point redox potential measured by visible redox titration of the low-spin form was approximately +100 mV. Binding cyanide (Ka = 5 x 10(5) M-1) resulted in the displacement of the methionyl axial residue, and full conversion to a low-spin, cyanide-bound form. Structural features were studied by 300-MHz 1H-NMR spectroscopy. In the oxidized state, the pH dependence of the haem methyl resonances (pH range 5-10) and the magnetic susceptibility measurements (using an NMR method) were consistent with the visible light spectroscopic data for the presence of a high-spin/low-spin equilibrium with a transition pKa of 7.3. The spin equilibrium was fast on the NMR time scale. The haem methyl resonances presented large downfield chemical shifts. An unusually broad methyl resonance at around 35 ppm (pH = 7.5, 25 degrees C) was extremely temperature-dependent [delta(323 K) - delta(273 K) = 7.2 ppm] and was assigned to the S-CH3 group of the axial methionine. In the ferrous state only a low-spin form is present. The haem meso protons, the methyl group and the methylene protons from the axial methionine were identified in the reduced form. The resonances from the aromatic residues (three tyrosines and one phenylalanine) were also assigned. Detailed monitoring of the NMR-redox pattern of the monohaem cytochrome from the fully reduced up to the fully oxidized state revealed that the rate of the intermolecular electronic exchange process was approximately 6 x 10(6) M-1 s-1 at 303 K and pH = 6.31. A dihaem cytochrome also present in the crude cell extract and purified to a homogeneous state, exhibited a molecular mass of 11 kDa and contained 2.43 mol iron and 1.89 mol haem c moieties/mol cytochrome. The absorption spectrum in the visible region exhibited no band at 695 nm, suggesting that methione is not a ligand for either of the two haems. Recovery of only small amounts of this protein prevented more detailed structural analyzes.

Bacteroidaceae↗
PubMed1988

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.

Cytochrome c Group↗
PubMed1988

Isolation and characterization of rubrerythrin, a non-heme iron protein from Desulfovibrio vulgaris that contains rubredoxin centers and a hemerythrin-like binuclear iron cluster.

A new non-heme iron protein from the periplasmic fraction of Desulfovibrio vulgaris (Hildenbourough NCIB 8303) has been purified to homogeneity, and its amino acid composition, molecular weight, redox potential, iron content, and optical, EPR, and Mössbauer spectroscopic properties have been determined. This new protein is composed of two identical subunits with subunit molecular weight of 21,900 and contains four iron atoms per molecule. The as-purified oxidized protein exhibits an optical spectrum with absorption maxima at 492, 365, and 280 nm, and its EPR spectrum shows resonances at g = 4.3 and 9.4, characteristic of oxidized rubredoxin. The Mössbauer data indicate the presence of approximately equal amounts of two types of iron; we named them the Rd-like and the Hr-like iron due to their similarity to the iron centers of rubredoxins (Rds) and hemerythrins (Hrs), respectively. For the Rd-like iron, the measured fine and hyperfine parameters (D = 1.5 cm-1, E/D = 0.26, delta EQ = -0.55 mm/s, delta = 0.27 mm/s, Axx/gn beta n = -16.5 T, Ayy/gn beta n = -15.6 T, and Azz/gn beta n = -17.0 T) are almost identical with those obtained for the rubredoxin from Clostridium pasteurianum. Redox-titration studies monitored by EPR, however, showed that these Rd-like centers have a midpoint redox potential of +230 +/- 10 mV, approximately 250 mV more positive than those reported for rubredoxins. Another unusual feature of this protein is the presence of the Hr-like iron atoms.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1988

Cytochrome components of nitrate- and sulfate-respiring Desulfovibrio desulfuricans ATCC 27774.

Three multiheme c-type cytochromes--the tetraheme cytochrome c3 (molecular weight [MW] 13,500), a dodecaheme cytochrome c (MW 40,800), and a "split-Soret" cytochrome c (MW 51,540), which is a dimer with 2 hemes per subunit (MW 26,300)--were isolated from the soluble fraction of Desulfovibrio desulfuricans (ATCC 27774) grown under nitrate- or sulfate-respiring conditions. Two of them, the dodecaheme and the split-Soret cytochromes, showed no similarities to any of the c-type cytochromes isolated from other sulfate-reducing bacteria, while the tetraheme cytochrome c3 appeared to be analogous to the cytochrome c3 found in other sulfate-reducing bacteria. For all three multiheme c-type cytochromes isolated, the homologous proteins from nitrate- and sulfate-grown cells were indistinguishable in amino acid composition, physical properties, and spectroscopic characteristics. It therefore appears that the same c-type cytochrome components are present when D. desulfuricans ATCC 27774 cells are grown under either condition. This is in contrast to the considerable difference found in Pseudomonas perfectomarina (Liu et al., J. Bacteriol. 154:278-286, 1983), a marine denitrifier, when the cells are grown on nitrate or oxygen as the terminal electron acceptor. In addition, two spectroscopy methods capable of revealing minute structural variations in proteins provided identical information about the tetraheme cytochrome c3 from nitrate-grown and sulfate-grown cells.

Amino Acids↗