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A V Xavier

Publications and source records attributed to A V Xavier.

At least 19 recordsLinked to original sources
PubMed2001

A membrane-bound cytochrome c3: a type II cytochrome c3 from Desulfovibrio vulgaris Hildenborough.

A new tetraheme cytochrome c3 was isolated from the membranes of Desulfovibrio vulgaris Hildenborough (DvH). This cytochrome has a molecular mass of 13.4 kDa and a pI of 5.5 and contains four heme c groups with apparent reduction potentials of -170 mV, -235 mV, -260 mV and -325 mV at pH 7.6. The complete sequence of the new cytochrome, retrieved from the preliminary data of the DvH genome, shows that this cytochrome is homologous to the "acidic" cytochrome c3 from Desulfovibrio africanus (Da). A model for the structure of the DvH cytochrome was built based on the structure of the Da cytochrome. Both cytochromes share structural features that distinguish them from other cytochrome c3 proteins, such as a solvent-exposed heme 1 surrounded by an acidic surface area, and a heme 4 which lacks most of the surface lysine patch proposed to be the site of hydrogenase interaction in other cytochrome c3 proteins. Furthermore, in contrast to previously discovered cytochrome c3 proteins, the genes coding for these two cytochromes are adjacent to genes coding for two membrane-associated FeS proteins, which indicates that they may be part of membrane-bound oxidoreductase complexes. Altogether these observations suggest that the DvH and Da cytochromes are a new type of cytochrome c3 proteins (Type II: TpII-c3) with different redox partners and physiological function than the other cytochrome c3 proteins (Type I: TpI-c3). The DvH TpII-c3 is reduced at considerable rates by the two membrane-bound [NiFe] and [NiFeSe] hydrogenases, but catalytic amounts of TpI-c3 increase these rates two- and fourfold, respectively. With the periplasmic [Fe] hydrogenase TpII-c3 is reduced much slower than TpI-c3, and no catalytic effect of TpI-c3 is observed.

Amino Acid Sequence↗
PubMed2001

Cooperativity between electrons and protons in a monomeric cytochrome c(3): the importance of mechano-chemical coupling for energy transduction.

To fully understand the structural bases for the mechanisms of biological energy transduction, it is essential to determine the microscopic thermodynamic parameters which describe the properties of each centre involved in the reactions, as well as its interactions with the others. These interactions between centres can then be interpreted in the light of structural features of the proteins. Redox titrations of cytochrome c(3) from Desulfovibrio desulfuricans ATCC 27774 followed by NMR and visible spectroscopy were analysed by using an equilibrium thermodynamic model. The network of homotropic and heterotropic cooperativities results in the coupled transfer of electrons and protons under physiological conditions. The microscopic characterisation allows the identification of several pairs of centres for which there are clear conformational (non-Coulombic) contributions to their coupling energies, thus establishing the existence of localised redox- and acid-base-linked structural modifications in the protein (mechano-chemical coupling). The modulation of interactions between centres observed for this cytochrome may be an important general phenomenon and is discussed in the framework of its physiological function and of the current focus of energy transduction research.

Acid-Base Equilibrium↗
PubMed2001

Conformational component in the coupled transfer of multiple electrons and protons in a monomeric tetraheme cytochrome.

Cell metabolism relies on energy transduction usually performed by complex membrane-spanning proteins that couple different chemical processes, e.g. electron and proton transfer in proton-pumps. There is great interest in determining at the molecular level the structural details that control these energy transduction events, particularly those involving multiple electrons and protons, because tight control is required to avoid the production of dangerous reactive intermediates. Tetraheme cytochrome c(3) is a small soluble and monomeric protein that performs a central step in the bioenergetic metabolism of sulfate reducing bacteria, termed "proton-thrusting," linking the oxidation of molecular hydrogen with the reduction of sulfate. The mechano-chemical coupling involved in the transfer of multiple electrons and protons in cytochrome c(3) from Desulfovibrio desulfuricans ATCC 27774 is described using results derived from the microscopic thermodynamic characterization of the redox and acid-base centers involved, crystallographic studies in the oxidized and reduced states of the cytochrome, and theoretical studies of the redox and acid-base transitions. This proton-assisted two-electron step involves very small, localized structural changes that are sufficient to generate the complex network of functional cooperativities leading to energy transduction, while using molecular mechanisms distinct from those established for other Desulfovibrio sp. cytochromes from the same structural family.

Cytochrome c Group↗
PubMed2001

Effect of hydrogen-bond networks in controlling reduction potentials in Desulfovibrio vulgaris (Hildenborough) cytochrome C3 probed by site-specific mutagenesis.

Cytochromes C3 isolated from Desulfovibrio spp. are periplasmic proteins that play a central role in energy transduction by coupling the transfer of electrons and protons from hydrogenase. Comparison between the oxidized and reduced structures of cytochrome C3 isolated from Desulfovibrio vulgaris (Hildenborough) show that the residue threonine 24, located in the vicinity of heme III, reorients between these two states [Messias, A. C., Kastrau, D. H. W., Costa, H. S., LeGall, J., Turner, D. L., Santos, H., and Xavier, A. V. (1998) J. Mol. Biol. 281, 719-739]. Threonine 24 was replaced with valine by site-directed mutagenesis to elucidate its effect on the redox properties of the protein. The NMR spectra of the mutated protein are very similar to those of the wild type, showing that the general folding and heme core architecture are not affected by the mutation. However, thermodynamic analysis of the mutated cytochrome reveals a large alteration in the microscopic reduction potential of heme III (75 and 106 mV for the protonated forms of the fully reduced and oxidized states, respectively). The redox interactions involving this heme are also modified, while the remaining heme-heme interactions and the redox-Bohr interactions are less strongly affected. Hence, the order of oxidation of the hemes in the mutated cytochrome is different from that in the wild type, and it has a higher overall affinity for electrons. This is consistent with the replacement of threonine 24 by valine preventing the formation of a network of hydrogen bonds, which stabilizes the oxidized state. The mutated protein is unable to perform a concerted two-electron step between the intermediate oxidation stages, 1 and 3, which can occur in the wild-type protein. Thus, replacing a single residue unbalances the global network of cooperativities tuned to control thermodynamically the directionality of the stepwise electron transfer and may affect the functionality of the protein.

Cytochrome c Group↗
PubMed2001

In the facultative sulphate/nitrate reducer Desulfovibrio desulfuricans ATCC 27774, the nine-haem cytochrome c is part of a membrane-bound redox complex mainly expressed in sulphate-grown cells.

The bacterium Desulfovibrio desulfuricans ATCC 27774 belongs to the group of sulphate reducers also capable of utilising nitrate as its terminal electron acceptor for anaerobic growth. One of the complex multihaem proteins found in nitrate- or sulphate-grown cells of Desulfovibrio desulfuricans ATCC 27774 is the nine-haem cytochrome c. The present work shows that the gene encoding for Desulfovibrio desulfuricans ATCC 27774 nine-haem cytochrome c is part of an operon formed by the gene cluster 9hcA-D. Besides 9hcA, the gene encoding for the nine-haem cytochrome c, genes 9hcB to D encode for a protein containing four [4Fe-4S](2+/1+) centres, for a dihaem transmembrane cytochrome b and for an unknown hydrophobic protein, respectively. The four proteins have a predicted topology that is in accordance with the formation of a membrane-bound redox complex. Furthermore, the transcriptional studies show that not only the expression of the 9HcA-D complex is dependent on the growth phase, but also is markedly increased in sulphate-grown cells.

Amino Acid Sequence↗
PubMed2001

The 'strict' anaerobe Desulfovibrio gigas contains a membrane-bound oxygen-reducing respiratory chain.

Sulfate-reducing bacteria are considered as strict anaerobic microorganisms, in spite of the fact that some strains have been shown to tolerate the transient presence of dioxygen. This report shows that membranes from Desulfovibrio gigas grown in fumarate/sulfate contain a respiratory chain fully competent to reduce dioxygen to water. In particular, a membrane-bound terminal oxygen reductase, of the cytochrome bd family, was isolated, characterized, and shown to completely reduce oxygen to water. This oxidase has two subunits with apparent molecular masses of 40 and 29 kDa. Using NADH or succinate as electron donors, the oxygen respiratory rates of D. gigas membranes are comparable to those of aerobic organisms (3.2 and 29 nmol O(2) min(-1) mg protein(-1), respectively). This 'strict anaerobic' bacterium contains all the necessary enzymatic complexes to live aerobically, showing that the relationships between oxygen and anaerobes are much more complex than originally thought.

Anaerobiosis↗
PubMed2001

NMR structure of the haem core of a novel tetrahaem cytochrome isolated from Shewanella frigidimarina: identification of the haem-specific axial ligands and order of oxidation.

The tetrahaem cytochrome isolated during anaerobic growth of Shewanella frigidimarina NCIMB400 is a small protein (86 residues) involved in electron transfer to Fe(III), which can be used as a terminal respiratory oxidant by this bacterium. A 3D solution structure model of the reduced form of the cytochrome has been determined using NMR data in order to determine the relative orientation of the haems. The haem core architecture of S. frigidimarina tetrahaem cytochrome differs from that found in all small tetrahaem cytochromes c(3) so far isolated from strict anaerobes, but has some similarity to the N-terminal cytochrome domain of flavocytochrome c(3) isolated from the same bacterium. NMR signals obtained for the four haems of S. frigidimarina tetrahaem cytochrome at all stages of oxidation were cross-assigned to the solution structure using the complete network of chemical exchange connectivities. Thus, the order in which each haem in the structure becomes oxidised was determined.

Amino Acid Sequence↗
PubMed2001

Analysis of the Desulfovibrio gigas transcriptional unit containing rubredoxin (rd) and rubredoxin-oxygen oxidoreductase (roo) genes and upstream ORFs.

Rubredoxin-oxygen oxidoreductase, an 86-kDa homodimeric flavoprotein, is the final component of a soluble electron transfer chain that couples NADH oxidation with oxygen reduction to water from the sulfate-reducing bacterium Desulfovibrio gigas. A 4.2-kb fragment of D. gigas chromosomal DNA containing the roo gene and the rubredoxin gene was sequenced. Additional open reading frames designated as ORF-1, ORF-2, and ORF-3 were also identified in this DNA fragment. ORF-1 encodes a protein exhibiting homology to several proteins of the short-chain dehydrogenase/reductase family of enzymes. The N-terminal coenzyme-binding pattern and the active-site pattern characteristic of short chain dehydrogenase/reductase proteins are conserved in ORF-1 product. ORF-2 does not show any significant homology with any known protein, whereas ORF-3 encodes a protein having significant homologies with the branched-chain amino acid transporter AzlC protein family. Northern blot hybridization analysis with rd and roo-specific probes identified a common 1.5-kb transcript, indicating that these two genes are cotranscribed. The transcription start site was identified by primer extension analysis to be a guanidine 87 bp upstream the ATG start codon of rubredoxin. The transcript size indicates that the rd-roo mRNA terminates downstream the roo-coding unit. Putative -10 and -35 regulator regions of a sigma(70)-type promoter, having similarity with E. coli sigma(70) promoter elements, are found upstream the transcription start site. Rubredoxin-oxygen oxidoreductase and rubredoxin genes are shown to be constitutively and abundantly expressed. Using the data available from different prokaryotic genomes, the rubredoxin genomic organization and the first tentative to understand the phylogenetic relationships among the flavoprotein family are reported in this study.

Amino Acid Sequence↗
PubMed2001

The genetic organization of Desulfovibrio desulphuricans ATCC 27774 bacterioferritin and rubredoxin-2 genes: involvement of rubredoxin in iron metabolism.

The anaerobic bacterium Desulfovibrio desulphuricans ATCC 27774 contains a unique bacterioferritin, isolated with a stable di-iron centre and having iron-coproporphyrin III as its haem cofactor, as well as a type 2 rubredoxin with an unusual spacing of four amino acid residues between the first two binding cysteines. The genes encoding for these two proteins were cloned and sequenced. The deduced amino acid sequence of the bacterioferritin shows that it is among the most divergent members of this protein family. Most interestingly, the bacterioferritin and rubredoxin-2 genes form a dicistronic operon, which reflects the direct interaction between the two proteins. Indeed, bacterioferritin and rubredoxin-2 form a complex in vitro, as shown by the significant increase in the anisotropy and decay times of the fluorescence of rubredoxin-2 tryptophan(s) when mixed with bacterioferritin. In addition, rubredoxin-2 donates electrons to bacterioferritin. This is the first identification of an electron donor to a bacterioferritin and shows the involvement of rubredoxin-2 in iron metabolism. Furthermore, analysis of the genomic data for anaerobes suggests that rubredoxins play a general role in iron metabolism and oxygen detoxification in these prokaryotes.

Amino Acid Sequence↗
PubMed2001

Molecular characterization of Desulfovibrio gigas neelaredoxin, a protein involved in oxygen detoxification in anaerobes.

Desulfovibrio gigas neelaredoxin is an iron-containing protein of 15 kDa, having a single iron site with a His(4)Cys coordination. Neelaredoxins and homologous proteins are widespread in anaerobic prokaryotes and have superoxide-scavenging activity. To further understand its role in anaerobes, its genomic organization and expression in D. gigas were studied and its ability to complement Escherichia coli superoxide dismutase deletion mutant was assessed. In D. gigas, neelaredoxin is transcribed as a monocistronic mRNA of 500 bases as revealed by Northern analysis. Putative promoter elements resembling sigma(70) recognition sequences were identified. Neelaredoxin is abundantly and constitutively expressed, and its expression is not further induced during treatment with O(2) or H(2)O(2). The neelaredoxin gene was cloned by PCR and expressed in E. coli, and the protein was purified to homogeneity. The recombinant neelaredoxin has spectroscopic properties identical to those observed for the native one. Mutations of Cys-115, one of the iron ligands, show that this ligand is essential for the activity of neelaredoxin. In an attempt to elucidate the function of neelaredoxin within the cell, it was expressed in an E. coli mutant deficient in cytoplasmic superoxide dismutases (sodA sodB). Neelaredoxin suppresses the deleterious effects produced by superoxide, indicating that it is involved in oxygen detoxification in the anaerobe D. gigas.

Anaerobiosis↗
PubMed2000

Iron-coproporphyrin III is a natural cofactor in bacterioferritin from the anaerobic bacterium Desulfovibrio desulfuricans.

A bacterioferritin was recently isolated from the anaerobic sulphate-reducing bacterium Desulfivibrio desulfuricans ATCC 27774 [Romão et al. (2000) Biochemistry 39, 6841-6849]. Although its properties are in general similar to those of the other bacterioferritins, it contains a haem quite distinct from the haem B, found in bacterioferritins from aerobic organisms. Using visible and NMR spectroscopies, as well as mass spectrometry analysis, the haem is now unambiguously identified as iron-coproporphyrin III, the first example of such a prosthetic group in a biological system. This unexpected finding is discussed in the framework of haem biosynthetic pathways in anaerobes and particularly in sulphate-reducing bacteria.

Anaerobiosis↗
PubMed2000

Characterization of a heme c nitrite reductase from a non-ammonifying microorganism, Desulfovibrio vulgaris Hildenborough.

A cytochrome c nitrite reductase (NiR) was purified for the first time from a microorganism not capable of growing on nitrate, the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough. It was isolated from the membranes as a large heterooligomeric complex of 760 kDa, containing two cytochrome c subunits of 56 and 18 kDa. This complex has nitrite and sulfite reductase activities of 685 micromol NH(4)(+)/min/mg and 1.0 micromol H(2)/min/mg. The enzyme was studied by UV-visible and electron paramagnetic resonance (EPR) spectroscopies. The overall redox behavior was determined through a visible redox titration. The data were analyzed with a set of four redox transitions, with an E(0)' of +160 mV (12% of total absorption), -5 mV (38% of total absorption), -110 mV (38% of total absorption) and -210 mV (12% of total absorption) at pH 7.6. The EPR spectra of oxidized and partially reduced NiR show a complex pattern, indicative of multiple heme-heme magnetic interactions. It was found that D. vulgaris Hildenborough is not capable of using nitrite as a terminal electron acceptor. These results indicate that in this organism the NiR is not involved in the dissimilative reduction of nitrite, as is the case with the other similar enzymes isolated so far. The possible role of this enzyme in the detoxification of nitrite and/or in the reduction of sulfite is discussed.

Amino Acid Sequence↗
PubMed2000

A bacterioferritin from the strict anaerobe Desulfovibrio desulfuricans ATCC 27774.

A bacterioferritin was isolated from the anaerobic bacterium Desulfovibrio desulfuricans ATCC 27774, grown with nitrate as the terminal electron acceptor, which is the first example of a bacterioferritin from a strict anaerobic organism. This new bacterioferritin was isolated mainly as a 24-mer of 20 kDa identical subunits, containing 0.5 noncovalently bound heme and 2 iron atoms per monomer. Although its N-terminal sequence is significantly homologous with ferritins from other microorganisms and the ligands to the di-iron ferroxidase center are conserved, it is one of the most divergent bacterioferritins so far characterized. Also, in contrast to all other known bacterioferritins, its heme is not of the B type; its chromatographic behavior is identical to that of iron uroporphyrin. Thus, D. desulfuricans bacterioferritin appears to be the second example of a protein unexpectedly containing this heme cofactor, or a closely related porphyrin, after its finding in Desulfovibrio gigas rubredoxin:oxygen oxidoreductase ¿Timkovich, R., Burkhalter, R. S., Xavier, A. V., Chen, L., and Le Gall, J. (1994) Bioorg. Chem. 22, 284-293. The oxidized form of the protein has a visible spectrum characteristic of low-spin ferric hemes, exhibiting a weak absorption band at 715 nm, indicative of bis-methionine heme axial coordination; upon reduction, the alpha-band appears at 550 nm and a splitting of the Soret band occurs, with two maxima at 410 and 425 nm. The heme center has a reduction potential of 140 +/- 10 mV (pH 7.6), a value unusually high compared to that of other bacterioferritins (ca. -200 mV).

Amino Acid Sequence↗
PubMed2000

Structural basis for the network of functional cooperativities in cytochrome c(3) from Desulfovibrio gigas: solution structures of the oxidised and reduced states.

Cytochrome c(3) is a 14 kDa tetrahaem protein that plays a central role in the bioenergetic metabolism of Desulfovibrio spp. This involves an energy transduction mechanism made possible by a complex network of functional cooperativities between redox and redox/protolytic centres (the redox-Bohr effect), which enables cytochrome c(3) to work as a proton activator. The three-dimensional structures of the oxidised and reduced Desulfovibrio gigas cytochrome c(3) in solution were solved using 2D (1)H-NMR data. The reduced protein structures were calculated using INDYANA, an extended version of DYANA that allows automatic calibration of NOE data. The oxidised protein structure, which includes four paramagnetic centres, was solved using the program PARADYANA, which also includes the structural paramagnetic parameters. In this case, initial structures were used to correct the upper and lower volume restraints for paramagnetic leakage, and angle restraints derived from (13)C Fermi contact shifts of haem moiety substituents were used for the axial histidine ligands. Despite the reduction of the NOE intensities by paramagnetic relaxation, the final family of structures is of similar precision and accuracy to that obtained for the reduced form. Comparison of the two structures shows that, although the global folds of the two families of structures are similar, significant localised differences occur upon change of redox state, some of which could not be detected by comparison with the X-ray structure of the oxidised state: (1) there is a redox-linked concerted rearrangement of Lys80 and Lys90 that results in the stabilisation of haem moieties II and III when both molecules are oxidised or both are reduced, in agreement with the previously measured positive redox cooperativity between these two haem moieties. This cooperativity regulates electron transfer, enabling a two-electron step adapted to the function of cytochromes c(3) as the coupling partner of hydrogenase; and (2) the movement of haem I propionate 13 towards the interior of the protein upon reduction explains the positive redox-Bohr effect, establishing the structural basis for the redox-linked proton activation mechanism necessary for energy conservation, driving ATP synthesis.

Allosteric Regulation↗
PubMed2000

Correlation of empirical magnetic susceptibility tensors and structure in low-spin haem proteins.

Experimental magnetic susceptibility tensors are reported for eight haems c with bis-His coordination. These data, obtained by fitting the dipolar shifts of backbone protons in the tetrahaem cytochromes c(3) from Desulfovibrio vulgaris and D. gigas, are analysed together with published values for other haem proteins. The x and y axes are found to rotate in the opposite sense to the axial ligands and are also counter-rotated with respect to the frontier molecular orbitals of the haem. The magnetic z-axis is close to the normal to the haem plane in each case. The magnitudes of the magnetic anisotropies are used to derive crystal field parameters and the rhombic splitting, V, is correlated with the dihedral angle between the axial ligands. Hence, it is apparent that the axial ligands are the dominant factor in determining the variation in magnetic properties between haems, and it is confirmed that "high g(max)" EPR signals are a reliable indicator of near-perpendicular ligands. These results are in full agreement with the analysis of non-Curie effects and electronic structure in the His-Met coordinated cytochromes c and C(551). Collectively, they show that the orientations of axial ligands to the haem may be estimated from single-crystal EPR data, from (13)C NMR shifts of the haem substituents, or from NMR dipolar shifts of the polypeptide.

Cytochrome c Group↗
PubMed2000

Structure of a dioxygen reduction enzyme from Desulfovibrio gigas.

Desulfovibrio gigas is a strict anaerobe that contains a well-characterized metabolic pathway that enables it to survive transient contacts with oxygen. The terminal enzyme in this pathway, rubredoxin:oxygen oxidoreductase (ROO) reduces oxygen to water in a direct and safe way. The 2.5 A resolution crystal structure of ROO shows that each monomer of this homodimeric enzyme consists of a novel combination of two domains, a flavodoxin-like domain and a Zn-beta-lactamase-like domain that contains a di-iron center for dioxygen reduction. This is the first structure of a member of a superfamily of enzymes widespread in strict and facultative anaerobes, indicating its broad physiological significance.

Amino Acid Sequence↗
PubMed2000

Purification and characterization of an iron superoxide dismutase and a catalase from the sulfate-reducing bacterium Desulfovibrio gigas.

The iron-containing superoxide dismutase (FeSOD; EC 1.15.1.1) and catalase (EC 1.11.1.6) enzymes constitutively expressed by the strictly anaerobic bacterium Desulfovibrio gigas were purified and characterized. The FeSOD, isolated as a homodimer of 22-kDa subunits, has a specific activity of 1,900 U/mg and exhibits an electron paramagnetic resonance (EPR) spectrum characteristic of high-spin ferric iron in a rhombically distorted ligand field. Like other FeSODs from different organisms, D. gigas FeSOD is sensitive to H(2)O(2) and azide but not to cyanide. The N-terminal amino acid sequence shows a high degree of homology with other SODs from different sources. On the other hand, D. gigas catalase has an estimated molecular mass of 186 +/- 8 kDa, consisting of three subunits of 61 kDa, and shows no peroxidase activity. This enzyme is very sensitive to H(2)O(2) and cyanide and only slightly sensitive to sulfide. The native enzyme contains one heme per molecule and exhibits a characteristic high-spin ferric-heme EPR spectrum (g(y,x) = 6.4, 5.4); it has a specific activity of 4,200 U/mg, which is unusually low for this class of enzyme. The importance of these two enzymes in the context of oxygen utilization by this anaerobic organism is discussed.

Amino Acid Sequence↗