Desulfovibrio gigas flavodiiron protein (FDP), rubredoxin:oxygen oxidoreductase (ROO), was proposed to be the terminal oxidase of a soluble electron transfer chain coupling NADH oxidation to oxygen reduction. However, several members from the FDP family, to which ROO belongs, revealed nitric oxide (NO) reductase activity. Therefore, the protection afforded by ROO against the cytotoxic effects of NO was here investigated. The NO and oxygen reductase activities of recombinant ROO in vitro were tested by amperometric methods, and the enzyme was shown to effectively reduce NO and O 2 . Functional complementation studies of an Escherichia coli mutant strain lacking the ROO homologue flavorubredoxin, an NO reductase, showed that ROO restores the anaerobic growth phenotype of cultures exposed to otherwise-toxic levels of exogenous NO. Additional studies in vivo using a D. gigas roo-deleted strain confirmed an increased sensitivity to NO of the mutant strain in comparison to the wild type. This effect is more pronounced when using the nitrosating agent S-nitrosoglutathione (GSNO), which effectively impairs the growth of the D. gigas ⌬roo strain. roo is constitutively expressed in D. gigas under all conditions tested. However, real-time reverse transcription-PCR analysis revealed a twofold induction of mRNA levels upon exposure to GSNO, suggesting regulation at the transcription level by NO. The newly proposed role of D. gigas ROO as an NO reductase combined with the O 2 reductase activity reveals a versatility which appears to afford protection to D. gigas at the onset of both oxidative and nitrosative stresses.Desulfovibrio gigas is a sulfate-reducing bacterium considered to be a strict anaerobe. However, there is growing evidence that this organism can survive and probably even profit energetically from transient exposure to oxygen (8,23). Like other isolates from the Desulfovibrio genus, D. gigas has metalbinding enzymes for the detoxification of molecular oxygen (rubredoxin:oxygen oxidoreductase [ROO] and cytochrome bd) (8, 23), its reactive species (catalase and superoxide dismutase) (9), and superoxide reductase (34). The first proposed D. gigas defense mechanism against O 2 involved a flavometalloprotein, ROO (8), which accomplishes full oxygen reduction to water by accepting electrons from reduced rubredoxin (Rd). Rd is in turn reduced by NADH:rubredoxin oxidoreductase (7,8).ROO belongs to the flavodiiron protein (FDP) family, whose members are widespread in strict and facultative anaerobic Archaea and Bacteria members, as well as in anaerobic protozoa (1, 29, 30). The resolution of its crystallographic structure (12) revealed that each monomer of this homodimeric protein consists of two structural modules, a C-terminal flavin mononucleotide-binding flavodoxin-like domain and an N-terminal metallo-␤-lactamase-like domain. More recently, the structure of the FDP from Moorella thermoacetica was also solved (33).Although a role in oxygen reduction was initially assigned to the FDP family, recent reports contributed...

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Abstract:In the present study, we report the identification of an operon with six coding regions for a multisubunit membrane-bound [NiFe] hydrogenase in the genome of Desulfovibrio gigas. Sequence analysis of the deduced polypeptides reveals a high similarity to subunits of proteins belonging to the family of Ech hydrogenases. The operon is organised similarly to the operon coding for the Ech hydrogenase from Methanosarcina barkeri, suggesting that both encode very similar hydrogenases. Expression of the operon was detected by Northern blot and RT-PCR analyses, and the presence of the encoded proteins was examined by Western blotting. The possible role of this hydrogenase is discussed, relating it with a potential function in the H-2 cycling as a mechanism for energy conservation in D. gigas.

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 H 2 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 ⌬echBC and ⌬hynAB 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 H 2 or by fermentation of pyruvate. Therefore, this enzyme seems to play a dominant role in D. gigas hydrogen metabolism. Hydrogenases are key enzymes in the hydrogen metabolism of Desulfovibrio spp. that catalyze the reversible oxidation of molecular hydrogen into protons and electrons (1). However, their role during sulfate respiration has not been clearly established. Odom and Peck proposed a hydrogen cycling model to explain energy conservation during growth on lactate and sulfate by Desulfovibrio spp., which belong to the deltaproteobacteria subgroup of the sulfate-reducing bacteria (SRB) (2). The model predicts that protons and electrons produced in the oxidation of lactate are used for the production of molecular hydrogen by a cytoplasmic hydrogenase. This hydrogen then diffuses across the membrane to the periplasm, where it is reoxidized by a periplasmic hydrogenase. Electrons are transferred back to the cytoplasm for sulfate reduction, thus creating a proton gradient across the membrane that leads to ATP formation. In this model, the presence of at least two hydrogenases on opposite sides of the membrane is a requirement for growth. In contrast, other studies suggested that the physiological role of these enzymes was to regulate the redox potential of the cell, controlling the flow of protons and electrons and generating a proton motive force (3). More recent models, proposed for Desulfovibrio vulgaris, suggested dual pathways for electron transfer from lactate to sulfate, one involving the cycling of H 2 and the other a route involving a membrane-associated electron transfer chain (4, 5). Several membrane complexes have been identified in SRB that could be involved in this proce...

Although classified as anaerobic, Desulfovibrio gigas contains a functional canonical membrane respiratory chain, including a cytochrome bd quinol oxidase as its terminal element. In the present study, we report the identification of the operon cydAB encoding the two subunits of cytochrome bd from this bacterium. Two hypothetical promoter regions and sequences resembling transcriptional regulators-binding sites have been identified. Amino acid sequence analysis revealed a high similarity to cytochrome bd from other organisms, presenting the conserved residues typical from these proteins. Reverse transcription polymerase chain reaction (RT-PCR) and Northern blot analysis confirmed the operon transcription. Gene expression was assessed by real-time RT-PCR in cells grown in different media and under exposure to oxygen and nitric oxide. mRNA levels were slightly enhanced in the presence of 150 mu M NO. However, in the presence of 10 mu M NO, a decrease was observed of the steady-state population of cydAB mRNA. No considerable effect was observed in the presence of fumarate/sulfate medium, 60 mu M O-2 or 10 mu M NO. Document Type: Article

The interaction and electron transfer (ET) between rubredoxin (Rd) and rubredoxin:oxygen oxidoreductase (ROO) from Desulfovibrio gigas is studied by molecular modelling techniques. Experimental kinetic assays using recombinant proteins show that the Rd reoxidation by ROO displays a bell-shaped dependence on ionic strength, suggesting a non-trivial electrostatic dependence of the interaction between these two proteins. Rigid docking studies reveal a prevalence for Rd to interact, in a very specific way, with the surface of the ROO dimer near its FMN cofactors. The optimization of the lowest energy complexes, using molecular dynamics simulation, shows a very tight interaction between the surface of the two proteins, with a high probability for Rd residues (but not the iron centre directly) to be in direct contact with the FMN cofactors of ROO. Both electrostatics and van der Waals interactions contribute to the final energy of the complex. In these complexes, the major contributions for complex formation are polar interactions between acidic residues of Rd and basic residues of ROO, plus substantial non-polar interactions between different groups. Important residues for this process are identified. ET estimates (using the Pathways model), in the optimized lowest energy complexes, suggest that these configurations are efficient for transferring electrons. The experimental bell-shaped dependence of kinetics on ionic strength is analysed in view of the molecular modelling results, and hypotheses for the molecular basis of this phenomenon are discussed.

O orçamento público em saúde era da ordem de 53 bilhões de Reais em 2003. Dentro desse orçamento, qual é a parcela referente ao tratamento de vítimas da violência? Devido às limitações dos dados disponíveis não é possível calcular diretamente essa parcela. Neste artigo propomos uma metodologia para estimar o custo da violência para o sistema público de saúde que utiliza informações do Sistema Único de Saúde, dos orçamentos estaduais e municipais e estimativas sobre a demanda por atendimento ambulatorial da Pesquisa Nacional por Amostra de Domicílios. Segundo nossos resultados, em 2004 o tratamento de vítimas de causas externas, de agressões e de acidentes de transporte teria custado ao setor público R$ 2,2 bilhões, R$ 119 milhões e R$ 453 milhões, respectivamente. Esses valores são cerca de quatro vezes maiores que aqueles verificados em trabalhos que computaram apenas os custos com internações.

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