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Jean Legall - One of the best experts on this subject based on the ideXlab platform.

  • OXIDATIVE PHOSPHORYLATION IN THE OBLIGATE ANAEROBE, Desulfovibrio Gigas
    Horizons of Bioenergetics, 2014
    Co-Authors: Larry L. Barton, Jean Legall, Harry D. Peck
    Abstract:

    Publisher Summary This chapter discusses oxidative phosphorylation in the obligate anaerobe, Desulfovibrio Gigas (D. Gigas). The sulfate-reducing bacteria are the first non-photosynthetic obligate anaerobes in which phosphorylation coupled to electron transfer is clearly demonstrated. The nature of the oxidative phosphorylation system in D. Gigas can have some general implications for the possible occurrence of oxidative phosphorylation in anaerobic and facultatively anaerobic bacteria. As the ability to utilize sulfate as a terminal electron acceptor is the fundamental taxonomic criterion for the sulfate reducing bacteria, fumarate reductase is more intimately associated with the respiratory chain than the enzymes responsible for sulfate respiration. This suggests that the ability to reduce fumarate to succinate with molecular hydrogen may be characteristic of a phosphorylating electron transfer complex in anaerobic and facultatively anaerobic bacteria.

  • preparation and x ray crystallographic analysis of rubredoxin crystals from Desulfovibrio Gigas to beyond ultra high 0 68 a resolution
    Biochemical and Biophysical Research Communications, 2003
    Co-Authors: Chunjung Chen, Mingyih Liu, Yiting Chen, Jean Legall
    Abstract:

    Rubredoxin (D.g. Rd), a small non-heme iron-sulfur protein shown to function as a redox coupling protein from the sulfate reducing bacteria Desulfovibrio Gigas, has been crystallized using the hanging-drop vapor diffusion method and macroseeding method. Rubredoxin crystals diffract to an ultra-high resolution 0.68 A using synchrotron radiation X-ray, and belong to the space group P2(1) with unit-cell parameters a=19.44 A, b=41.24 A, c=24.10 A, and beta=108.46 degrees. The data set of single-wavelength anomalous dispersion signal of iron in the native crystal was also collected for ab initio structure re-determination. Preliminary analysis indicates that there is one monomer with a [Fe-4S] cluster in each asymmetric unit. The crystal structure at this ultra-high resolution will reveal the details of its biological function. The crystal character and data collection strategy for ultra-high resolution will also be discussed.

  • response of a strict anaerobe to oxygen survival strategies in Desulfovibrio Gigas
    Microbiology, 2003
    Co-Authors: Paula Fareleira, Antonio V Xavier, Jean Legall, Bruno Santos, Celia Antonio, Pedro Moradasferreira, Helena Santos
    Abstract:

    The biochemical response to oxygen of the strictly anaerobic sulfate-reducing bacterium Desulfovibrio Gigas was studied with the goal of elucidating survival strategies in oxic environments. Cultures of D. Gigas on medium containing lactate and sulfate were exposed to oxygen (concentration 5–120 mM). Growth was fully inhibited by oxygen, but the cultures resumed growth as soon as they were shifted back to anoxic conditions. Following 24 h exposure to oxygen the growth rate was as high as 70 % of the growth rates observed before oxygenation. Catalase levels and activity were enhanced by exposure to oxygen whereas superoxide-scavenging and glutathione reductase activities were not affected. The general pattern of cellular proteins as analysed by two-dimensional electrophoresis was altered in the presence of oxygen, the levels of approximately 12 % of the detected proteins being markedly increased. Among the induced proteins, a homologue of a 60 kDa eukaryotic heat-shock protein (Hsp60) was identified by immunoassay analysis. In the absence of external substrates, the steady-state levels of nucleoside triphosphates detected by in vivo 31 P-NMR under saturating concentrations of oxygen were 20 % higher than under anoxic conditions. The higher energy levels developed under oxygen correlated with a lower rate of substrate (glycogen) mobilization, but no experimental evidence for a contribution from oxidative phosphorylation was found. The hypothesis that oxygen interferes with ATP dissipation processes is discussed.

  • structure of dimeric cytochrome c3 from Desulfovibrio Gigas at 1 2 a resolution
    Acta Crystallographica Section D-biological Crystallography, 2003
    Co-Authors: David Aragao, Carlos Frazao, George M. Sheldrick, Jean Legall, L C Sieker, Maria Armenia Carrondo
    Abstract:

    The structure of dimeric cytochrome c(3) from the sulfate-reducing bacterium Desulfovibrio Gigas, diDg, obtained by ab initio methods was further refined to 1.2 A resolution, giving final reliability factors of R(free) = 14.8% and R = 12.4%. This cytochrome is a dimer of tetraheme cytochrome c(3) molecules covalently linked by two solvent-accessible disulfide bridges, a characteristic unique to members of the cytochrome c(3) superfamily. Anisotropic analysis using the semi-rigid TLS method shows different behaviour for analogous loops in each monomer arising from their different packing environments. A detailed sequence and structural comparison with all other known cytochrome c(3) domains in single- and multi-domain cytochromes c(3) shows the presence of structurally conserved regions in this family, despite the high variability of the amino-acid sequence. An internal water molecule is conserved in a common structural arrangement in all c(3) tetraheme domains, indicating a probable electron-transfer pathway between hemes I and II. Unique features of diDg are an internal methionine residue close to heme I and to one of the axial ligands of heme III, where all other structures of the cytochrome c(3) superfamily have a phenylalanine, and a rather unusual CXXXCH heme-binding motif only found so far in this cytochrome.

  • the quinol fumarate oxidoreductase from the sulphate reducing bacterium Desulfovibrio Gigas spectroscopic and redox studies
    Journal of Bioenergetics and Biomembranes, 2002
    Co-Authors: Rita S Lemos, Antonio V Xavier, Jean Legall, Claudio M Gomes, Miguel Teixeira
    Abstract:

    The membrane bound fumarate reductase (FRD) from the sulphate-reducer Desulfovibrio Gigas was purified from cells grown on a fumarate/sulphate medium and extensively characterized. The FRD is isolated with three subunits of apparent molecular masses of 71, 31, and 22 kDa. The enzyme is capable of both fumarate reduction and succinate oxidation, exhibiting a higher specificity toward fumarate (K m for fumarate is 0.02 and for succinate 2 mM) and a reduction rate 30 times faster than that for oxidation. Studies by Visible and EPR spectroscopies allowed the identification of two B-type haems and the three iron–sulphur clusters usually found in FRDs and succinate dehydrogenases: [2Fe-2S]2+/1+ (S1), [4Fe-4S]2+/1+ (S2), and [3Fe-4S]1+/0 (S3). The apparent macroscopic reduction potentials for the metal centers, at pH 7.6, were determined by redox titrations: −45 and −175 mV for the two haems, and +20 and −140 mV for the S3 and S1 clusters, respectively. The reduction potentials of the haem groups are pH dependent, supporting the proposal that fumarate reduction is associated with formation of the membrane proton gradient. Furthermore, co-reconstitution in liposomes of D. Gigas FRD, duroquinone, and D. Gigas cytochrome bd shows that this system is capable of coupling succinate oxidation with oxygen reduction to water.

José J. G. Moura - One of the best experts on this subject based on the ideXlab platform.

  • mo cu metal cluster formation and binding in an orange protein isolated from Desulfovibrio Gigas
    Journal of Biological Inorganic Chemistry, 2014
    Co-Authors: Marta S P Carepo, Sofia R Pauleta, José J. G. Moura, Anthony G Wedd, Isabel Moura
    Abstract:

    The orange protein (ORP) isolated from the sulfate-reducing bacterium Desulfovibrio Gigas (11.8 kDa) contains a mixed-metal sulfide cluster of the type [S2MoS2CuS2MoS2]3- noncovalently bound to the polypeptide chain. The D. Gigas ORP was heterologously produced in Escherichia coli in the apo form. Different strategies were used to reconstitute the metal cluster into apo-ORP and obtain insights into the metal cluster synthesis: (1) incorporation of a synthesized inorganic analogue of the native metal cluster and (2) the in situ synthesis of the metal cluster on the addition to apo-ORP of copper chloride and tetrathiomolybdate or tetrathiotungstate. This latter procedure was successful, and the visible spectrum of the Mo–Cu reconstituted ORP is identical to the one reported for the native protein with absorption maxima at 340 and 480 nm. The 1H–15N heteronuclear single quantum coherence spectra of the reconstituted ORP obtained by strategy 2, in contrast to strategy 1, exhibited large changes, which required sequential assignment in order to identify, by chemical shift differences, the residues affected by the incorporation of the cluster, which is stabilized inside the protein by both electrostatic and hydrophobic interactions.

  • zinc substituted Desulfovibrio Gigas desulforedoxins resolving subunit degeneracy with nonsymmetric pseudocontact shifts
    Protein Science, 2009
    Co-Authors: Brian J Goodfellow, Frank Rusnak, Isabel Moura, José J. G. Moura, Sofia Nunes, Carla Ascenso, Brian F Volkman, John L Markley
    Abstract:

    Desulfovibrio Gigas desulforedoxin (Dx) consists of two identical peptides, each containing one [Fe-4S] center per monomer. Variants with different iron and zinc metal compositions arise when desulforedoxin is produced recombinantly from Escherichia coli. The three forms of the protein, the two homodimers [Fe(III)/Fe(III)]Dx and [Zn(II)/Zn(II)]Dx, and the heterodimer [Fe(III)/Zn(II)]Dx, can be separated by ion exchange chromatography on the basis of their charge differences. Once separated, the desulforedoxins containing iron can be reduced with added dithionite. For NMR studies, different protein samples were prepared labeled with (15)N or (15)N + (13)C. Spectral assignments were determined for [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx from 3D (15)N TOCSY-HSQC and NOESY-HSQC data, and compared with those reported previously for [Zn(II)/Zn(II)]Dx. Assignments for the (13)C(alpha) shifts were obtained from an HNCA experiment. Comparison of (1)H-(15)N HSQC spectra of [Zn(II)/Zn(II)]Dx, [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx revealed that the pseudocontact shifts in [Fe(II)/Zn(II)]Dx can be decomposed into inter- and intramonomer components, which, when summed, accurately predict the observed pseudocontact shifts observed for [Fe(II)/Fe(II)]Dx. The degree of linearity observed in the pseudocontact shifts for residues >/=8.5 A from the metal center indicates that the replacement of Fe(II) by Zn(II) produces little or no change in the structure of Dx. The results suggest a general strategy for the analysis of NMR spectra of homo-oligomeric proteins in which a paramagnetic center introduced into a single subunit is used to break the magnetic symmetry and make it possible to obtain distance constraints (both pseudocontact and NOE) between subunits.

  • direct electrochemical study of the multiple redox centers of hydrogenase from Desulfovibrio Gigas
    Bioelectrochemistry, 2008
    Co-Authors: Cristina M Cordas, Isabel Moura, José J. G. Moura
    Abstract:

    Direct electrochemical response was first time observed for the redox centers of Desulfovibrio Gigas [NiFe]-Hase, in non-turnover conditions, by cyclic voltammetry, in solution at glassy carbon electrode. The activation of the enzyme was achieved by reduction with H(2) and by electrochemical control and electrocatalytic activity was observed. The inactivation of the [NiFe]-Hase was also attained through potential control. All electrochemical data was obtained in the absence of enzyme inhibitors. The results are discussed in the context of the proposed mechanism currently accepted for activation/inactivation of [NiFe]-Hases.

  • nmr assignment of the apo form of a Desulfovibrio Gigas protein containing a novel mo cu cluster
    Biomolecular Nmr Assignments, 2007
    Co-Authors: Sofia R Pauleta, Isabel Moura, Americo G Duarte, Marta S P Carepo, Alice S Pereira, Pedro Tavares, José J. G. Moura
    Abstract:

    We report the 98% assignment of the apo-form of an orange protein, containing a novel Mo-Cu cluster isolated from Desulfovibrio Gigas. This protein presents a region where backbone amide protons exchange fast with bulk solvent becoming undetectable. These residues were assigned using 13 C-detection experiments.

  • study of the spin spin interactions between the metal centers of Desulfovibrio Gigas aldehyde oxidoreductase identification of the reducible sites of the 2fe 2s 1 2 clusters
    Biochemistry, 2005
    Co-Authors: Claude More, José J. G. Moura, Bruno Guigliarelli, Marcel Asso, Jorge Caldeira, Guy Roger, Patrick Bertrand
    Abstract:

    The aldehyde oxidoreductase from Desulfovibrio Gigas belongs to the family of molybdenum hydroxylases. Besides a molybdenum cofactor which constitutes their active site, these enzymes contain two [2Fe-2S]2+,1+ clusters which are believed to transfer the electrons provided by the substrate to an acceptor which is either a FAD group or an electron-transferring protein. When the three metal centers of D. Gigas AOR are simultaneously paramagnetic, splittings due to intercenter spin−spin interactions are visible when the EPR spectra are recorded at low temperatures. By studying quantitatively these interactions with a model based on the X-ray crystal structure, which takes into consideration the interactions between the magnetic moments carried by all the metal sites of the system, it is possible to determine the location of the reducible sites of the [2Fe-2S] clusters. When combined with the electron-transfer pathways proposed on the basis of the X-ray crystal structure, the results provide a detailed descrip...

Antonio V Xavier - One of the best experts on this subject based on the ideXlab platform.

  • response of a strict anaerobe to oxygen survival strategies in Desulfovibrio Gigas
    Microbiology, 2003
    Co-Authors: Paula Fareleira, Antonio V Xavier, Jean Legall, Bruno Santos, Celia Antonio, Pedro Moradasferreira, Helena Santos
    Abstract:

    The biochemical response to oxygen of the strictly anaerobic sulfate-reducing bacterium Desulfovibrio Gigas was studied with the goal of elucidating survival strategies in oxic environments. Cultures of D. Gigas on medium containing lactate and sulfate were exposed to oxygen (concentration 5–120 mM). Growth was fully inhibited by oxygen, but the cultures resumed growth as soon as they were shifted back to anoxic conditions. Following 24 h exposure to oxygen the growth rate was as high as 70 % of the growth rates observed before oxygenation. Catalase levels and activity were enhanced by exposure to oxygen whereas superoxide-scavenging and glutathione reductase activities were not affected. The general pattern of cellular proteins as analysed by two-dimensional electrophoresis was altered in the presence of oxygen, the levels of approximately 12 % of the detected proteins being markedly increased. Among the induced proteins, a homologue of a 60 kDa eukaryotic heat-shock protein (Hsp60) was identified by immunoassay analysis. In the absence of external substrates, the steady-state levels of nucleoside triphosphates detected by in vivo 31 P-NMR under saturating concentrations of oxygen were 20 % higher than under anoxic conditions. The higher energy levels developed under oxygen correlated with a lower rate of substrate (glycogen) mobilization, but no experimental evidence for a contribution from oxidative phosphorylation was found. The hypothesis that oxygen interferes with ATP dissipation processes is discussed.

  • the quinol fumarate oxidoreductase from the sulphate reducing bacterium Desulfovibrio Gigas spectroscopic and redox studies
    Journal of Bioenergetics and Biomembranes, 2002
    Co-Authors: Rita S Lemos, Antonio V Xavier, Jean Legall, Claudio M Gomes, Miguel Teixeira
    Abstract:

    The membrane bound fumarate reductase (FRD) from the sulphate-reducer Desulfovibrio Gigas was purified from cells grown on a fumarate/sulphate medium and extensively characterized. The FRD is isolated with three subunits of apparent molecular masses of 71, 31, and 22 kDa. The enzyme is capable of both fumarate reduction and succinate oxidation, exhibiting a higher specificity toward fumarate (K m for fumarate is 0.02 and for succinate 2 mM) and a reduction rate 30 times faster than that for oxidation. Studies by Visible and EPR spectroscopies allowed the identification of two B-type haems and the three iron–sulphur clusters usually found in FRDs and succinate dehydrogenases: [2Fe-2S]2+/1+ (S1), [4Fe-4S]2+/1+ (S2), and [3Fe-4S]1+/0 (S3). The apparent macroscopic reduction potentials for the metal centers, at pH 7.6, were determined by redox titrations: −45 and −175 mV for the two haems, and +20 and −140 mV for the S3 and S1 clusters, respectively. The reduction potentials of the haem groups are pH dependent, supporting the proposal that fumarate reduction is associated with formation of the membrane proton gradient. Furthermore, co-reconstitution in liposomes of D. Gigas FRD, duroquinone, and D. Gigas cytochrome bd shows that this system is capable of coupling succinate oxidation with oxygen reduction to water.

  • molecular characterization of Desulfovibrio Gigas neelaredoxin a protein involved in oxygen detoxification in anaerobes
    Journal of Bacteriology, 2001
    Co-Authors: Gabriela Silva, Jean Legall, Antonio V Xavier, Miguel Teixeira, Claudina Rodriguespousada
    Abstract:

    Desulfovibrio Gigas neelaredoxin is an iron-containing protein of 15 kDa, having a single iron site with a His4Cys 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 ς70 recognition sequences were identified. Neelaredoxin is abundantly and constitutively expressed, and its expression is not further induced during treatment with O2 or H2O2. 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.

  • the strict anaerobe Desulfovibrio Gigas contains a membrane bound oxygen reducing respiratory chain
    FEBS Letters, 2001
    Co-Authors: Rita S Lemos, Antonio V Xavier, Jean Legall, Claudio M Gomes, Margarida Santana, Miguel Teixeira
    Abstract:

    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 O2 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.

  • purification and characterization of an iron superoxide dismutase and a catalase from the sulfate reducing bacterium Desulfovibrio Gigas
    Journal of Bacteriology, 2000
    Co-Authors: Wagner Dos G Santos, Antonio V Xavier, Jean Legall, Isabel Pacheco, Miguel Teixeira, Mingyih Liu
    Abstract:

    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 H2O2 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 H2O2 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 (gy,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.

Isabel Moura - One of the best experts on this subject based on the ideXlab platform.

  • mo cu metal cluster formation and binding in an orange protein isolated from Desulfovibrio Gigas
    Journal of Biological Inorganic Chemistry, 2014
    Co-Authors: Marta S P Carepo, Sofia R Pauleta, José J. G. Moura, Anthony G Wedd, Isabel Moura
    Abstract:

    The orange protein (ORP) isolated from the sulfate-reducing bacterium Desulfovibrio Gigas (11.8 kDa) contains a mixed-metal sulfide cluster of the type [S2MoS2CuS2MoS2]3- noncovalently bound to the polypeptide chain. The D. Gigas ORP was heterologously produced in Escherichia coli in the apo form. Different strategies were used to reconstitute the metal cluster into apo-ORP and obtain insights into the metal cluster synthesis: (1) incorporation of a synthesized inorganic analogue of the native metal cluster and (2) the in situ synthesis of the metal cluster on the addition to apo-ORP of copper chloride and tetrathiomolybdate or tetrathiotungstate. This latter procedure was successful, and the visible spectrum of the Mo–Cu reconstituted ORP is identical to the one reported for the native protein with absorption maxima at 340 and 480 nm. The 1H–15N heteronuclear single quantum coherence spectra of the reconstituted ORP obtained by strategy 2, in contrast to strategy 1, exhibited large changes, which required sequential assignment in order to identify, by chemical shift differences, the residues affected by the incorporation of the cluster, which is stabilized inside the protein by both electrostatic and hydrophobic interactions.

  • zinc substituted Desulfovibrio Gigas desulforedoxins resolving subunit degeneracy with nonsymmetric pseudocontact shifts
    Protein Science, 2009
    Co-Authors: Brian J Goodfellow, Frank Rusnak, Isabel Moura, José J. G. Moura, Sofia Nunes, Carla Ascenso, Brian F Volkman, John L Markley
    Abstract:

    Desulfovibrio Gigas desulforedoxin (Dx) consists of two identical peptides, each containing one [Fe-4S] center per monomer. Variants with different iron and zinc metal compositions arise when desulforedoxin is produced recombinantly from Escherichia coli. The three forms of the protein, the two homodimers [Fe(III)/Fe(III)]Dx and [Zn(II)/Zn(II)]Dx, and the heterodimer [Fe(III)/Zn(II)]Dx, can be separated by ion exchange chromatography on the basis of their charge differences. Once separated, the desulforedoxins containing iron can be reduced with added dithionite. For NMR studies, different protein samples were prepared labeled with (15)N or (15)N + (13)C. Spectral assignments were determined for [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx from 3D (15)N TOCSY-HSQC and NOESY-HSQC data, and compared with those reported previously for [Zn(II)/Zn(II)]Dx. Assignments for the (13)C(alpha) shifts were obtained from an HNCA experiment. Comparison of (1)H-(15)N HSQC spectra of [Zn(II)/Zn(II)]Dx, [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx revealed that the pseudocontact shifts in [Fe(II)/Zn(II)]Dx can be decomposed into inter- and intramonomer components, which, when summed, accurately predict the observed pseudocontact shifts observed for [Fe(II)/Fe(II)]Dx. The degree of linearity observed in the pseudocontact shifts for residues >/=8.5 A from the metal center indicates that the replacement of Fe(II) by Zn(II) produces little or no change in the structure of Dx. The results suggest a general strategy for the analysis of NMR spectra of homo-oligomeric proteins in which a paramagnetic center introduced into a single subunit is used to break the magnetic symmetry and make it possible to obtain distance constraints (both pseudocontact and NOE) between subunits.

  • direct electrochemical study of the multiple redox centers of hydrogenase from Desulfovibrio Gigas
    Bioelectrochemistry, 2008
    Co-Authors: Cristina M Cordas, Isabel Moura, José J. G. Moura
    Abstract:

    Direct electrochemical response was first time observed for the redox centers of Desulfovibrio Gigas [NiFe]-Hase, in non-turnover conditions, by cyclic voltammetry, in solution at glassy carbon electrode. The activation of the enzyme was achieved by reduction with H(2) and by electrochemical control and electrocatalytic activity was observed. The inactivation of the [NiFe]-Hase was also attained through potential control. All electrochemical data was obtained in the absence of enzyme inhibitors. The results are discussed in the context of the proposed mechanism currently accepted for activation/inactivation of [NiFe]-Hases.

  • crystal structure of the 16 heme cytochrome from Desulfovibrio Gigas a glycosylated protein in a sulphate reducing bacterium
    Journal of Molecular Biology, 2007
    Co-Authors: Teresa Santossilva, Joao M Dias, Luísa L. Gonçalves, Marieclaire Durand, Jorge Lampreia, Isabel Moura, Alain Dolla, Maria João Romão
    Abstract:

    Abstract Sulphate-reducing bacteria have a wide variety of periplasmic cytochromes involved in electron transfer from the periplasm to the cytoplasm. HmcA is a high molecular mass cytochrome of 550 amino acid residues that harbours 16 c-type heme groups. We report the crystal structure of HmcA isolated from the periplasm of Desulfovibrio Gigas. Crystals were grown using polyethylene glycol 8K and zinc acetate, and diffracted beyond 2.1 A resolution. A multiple-wavelength anomalous dispersion experiment at the iron absorption edge enabled us to obtain good-quality phases for structure solution and model building. DgHmcA has a V-shape architecture, already observed in HmcA isolated from Desulfovibrio vulgaris Hildenborough. The presence of an oligosaccharide molecule covalently bound to an Asn residue was observed in the electron density maps of DgHmcA and confirmed by mass spectrometry. Three modified monosaccharides appear at the highly hydrophobic vertex, possibly acting as an anchor of the protein to the cytoplasmic membrane.

  • nmr assignment of the apo form of a Desulfovibrio Gigas protein containing a novel mo cu cluster
    Biomolecular Nmr Assignments, 2007
    Co-Authors: Sofia R Pauleta, Isabel Moura, Americo G Duarte, Marta S P Carepo, Alice S Pereira, Pedro Tavares, José J. G. Moura
    Abstract:

    We report the 98% assignment of the apo-form of an orange protein, containing a novel Mo-Cu cluster isolated from Desulfovibrio Gigas. This protein presents a region where backbone amide protons exchange fast with bulk solvent becoming undetectable. These residues were assigned using 13 C-detection experiments.

Claudina Rodriguespousada - One of the best experts on this subject based on the ideXlab platform.

  • an hcpr paralog of Desulfovibrio Gigas provides protection against nitrosative stress
    FEBS Open Bio, 2015
    Co-Authors: Sofia M. Silva, Catarina Pimentel, Catarina Amaral, Susana S Neves, Catia Santos, Claudina Rodriguespousada
    Abstract:

    Desulfovibrio Gigas belongs to the group of sulfate reducing bacteria (SRB). These ubiquitous and metabolically versatile microorganisms are often exposed to reactive nitrogen species (RNS). Nonetheless, the mechanisms and regulatory elements involved in nitrosative stress protection are still poorly understood. The transcription factor HcpR has emerged as a putative regulator of nitrosative stress response among anaerobic bacteria. HcpR is known to orchestrate the expression of the hybrid cluster protein gene, hcp, proposed to be involved in cellular defense against RNS. According to phylogenetic analyses, the occurrence of hcpR paralog genes is a common feature among several Desulfovibrio species. Within the D. Gigas genome we have identified two HcpR-related sequences. One of these sequences, hcpR1, was found in the close vicinity of the hcp gene and this finding prompted us to proceed with its functional characterization. We observed that the growth of a D. Gigas strain lacking hcpR1 is severely impaired under nitrosative stress. An in silico search revealed several putative targets of HcpR1 that were experimentally validated. The fact that HcpR1 regulates several genes encoding proteins involved in nitrite and nitrate metabolism, together with the sensitive growth phenotype to NO displayed by an hcpR1 mutant strain, strongly supports a relevant role of this factor under nitrosative stress. Moreover, the finding that several Desulfovibrio species possess HcpR paralogs, which have been transmitted vertically in the evolution and diversification of the genus, suggests that these sequences may confer adaptive or survival advantage to these organisms, possibly by increasing their tolerance to nitrosative stress.

  • roles of hynab and ech the only two hydrogenases found in the model sulfate reducer Desulfovibrio Gigas
    Journal of Bacteriology, 2013
    Co-Authors: Fabio O Moraissilva, Catia I Santos, R. Rodrigues, Ines A C Pereira, Claudina Rodriguespousada
    Abstract:

    Sulfate-reducing bacteria are characterized by a high number of hydrogenases, which have been proposed to contribute to the overall energy metabolism of the cell, but exactly in what role is not clear. Desulfovibrio spp. can produce or consume H2 when growing on organic or inorganic substrates in the presence or absence of sulfate. Because of the presence of only two hydrogenases encoded in its genome, the periplasmic HynAB and cytoplasmic Ech hydrogenases, Desulfovibrio Gigas is an excellent model organism for investigation of the specific function of each of these enzymes during growth. In this study, we analyzed the physiological response to the deletion of the genes that encode the two hydrogenases in D. Gigas, through the generation of ΔechBC and ΔhynAB single mutant strains. These strains were analyzed for the ability to grow on different substrates, such as lactate, pyruvate, and hydrogen, under respiratory and fermentative conditions. Furthermore, the expression of both hydrogenase genes in the three strains studied was assessed through quantitative reverse transcription-PCR. The results demonstrate that neither hydrogenase is essential for growth on lactate-sulfate, indicating that hydrogen cycling is not indispensable. In addition, the periplasmic HynAB enzyme has a bifunctional activity and is required for growth on H2 or by fermentation of pyruvate. Therefore, this enzyme seems to play a dominant role in D. Gigas hydrogen metabolism.

  • roles of hynab and ech the only two hydrogenases found in the model sulfate reducer Desulfovibrio Gigas
    Journal of Bacteriology, 2013
    Co-Authors: Fabio O Moraissilva, Catia I Santos, R. Rodrigues, Ines A C Pereira, Claudina Rodriguespousada
    Abstract:

    Sulfate-reducing bacteria are characterized by a high number of hydrogenases, which have been proposed to contribute to the overall energy metabolism of the cell, but exactly in what role is not clear. Desulfovibrio spp. can produce or consume H2 when growing on organic or inorganic substrates in the presence or absence of sulfate. Because of the presence of only two hydrogenases encoded in its genome, the periplasmic HynAB and cytoplasmic Ech hydrogenases, Desulfovibrio Gigas is an excellent model organism for investigation of the specific function of each of these enzymes during growth. In this study, we analyzed the physiological response to the deletion of the genes that encode the two hydrogenases in D. Gigas, through the generation of ΔechBC and ΔhynAB single mutant strains. These strains were analyzed for the ability to grow on different substrates, such as lactate, pyruvate, and hydrogen, under respiratory and fermentative conditions. Furthermore, the expression of both hydrogenase genes in the three strains studied was assessed through quantitative reverse transcription-PCR. The results demonstrate that neither hydrogenase is essential for growth on lactate-sulfate, indicating that hydrogen cycling is not indispensable. In addition, the periplasmic HynAB enzyme has a bifunctional activity and is required for growth on H2 or by fermentation of pyruvate. Therefore, this enzyme seems to play a dominant role in D. Gigas hydrogen metabolism.

  • role of norr like transcriptional regulators under nitrosative stress of the δ proteobacterium Desulfovibrio Gigas
    Biochemical and Biophysical Research Communications, 2013
    Co-Authors: Ana Varelaraposo, Fabio O Moraissilva, Antonio Mauro Rezende, Catarina Pimentel, Jeronimo C Ruiz, Claudina Rodriguespousada
    Abstract:

    NorR protein was shown to be responsible for the transcriptional regulation of flavorubredoxin and its associated oxidoreductase in Escherichia coli. Since Desulfovibrio Gigas has a rubredoxin:oxygen oxidoreductase (ROO) that is involved in both oxidative and nitrosative stress response, a NorR-like protein was searched in D. Gigas genome. We have found two putative norR coding units in its genome. To study the role of the protein designated as NorR1-like (NorR1L) in the presence of nitrosative stress, a norR1L null mutant of D. Gigas was created and a phenotypic analysis was performed under the nitrosating agent GSNO. We show that under these conditions, the growth of both D. Gigas mutants Δroo and ΔnorR1-like is impaired. In order to confirm that D. Gigas NorR1-like may play identical function as the NorR of E. coli, we have complemented the E. coli ΔnorR mutant strain with the norR1-like gene and have evaluated growth when nitrosative stress was imposed. The growth phenotype of E. coli ΔnorR mutant strain was recovered under these conditions. We also found that induction of roo gene expression is completely abolished in the norR1L mutant strain of D. Gigas subjected to nitrosative stress. It is identified in δ-proteobacteria, for the first time a transcription factor that is involved in nitrosative stress response and regulates the rd-roo gene expression.

  • molecular determinants for fmn binding in Desulfovibrio Gigas flavoredoxin
    FEBS Letters, 2007
    Co-Authors: Manuela Broco, Solange Oliveira, Claudio M Soares, Stephen G Mayhew, Claudina Rodriguespousada
    Abstract:

    This work is dedicated to the memory of Professor Antonio Xavier deceased on May 7th 2006. Abstract Flavoredoxin participates in Desulfovibrio Gigas thio- sulfate reduction pathway. Its 3-dimensional model was gener- ated allowing the oxidized riboflavin-5 0 -phosphate (FMN) site to be predicted. Residues likely to be involved in FMN-binding were identified (N29, W35, T56, K92, H131 and F164) and mutated to alanine. Fluorescence titration with apoprotein showed that FMN is strongly bound in the wild-type protein. Comparison of Kd values for mutants suggests that interactions with the phosphate group of FMN, contribute more to binding than the interactions with the isoalloxazine ring. The redox potential of bound FMN determined for wild-type and mutants revealed shifts to less negative values. These findings were corre- lated with the protein structure in order to contribute to a better understanding of the structure-function relationships in flavore-