The purification and some equilibrium properties of the nitrite reductase of the bacterium Wolinella succinogenes.
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ABSTRACT:
SUBMITTER: Blackmore R
PROVIDER: S-EPMC1153060 | biostudies-other | 1986 Jan
REPOSITORIES: biostudies-other
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Project description:The six haem groups of the nitrite reductase enzyme isolated from Wolinella succinogenes are rapidly reduced by the addition of dithionite (S2O4(2-)). The reduction, however, is not homogeneous. Two of the haem groups, namely those that show spectral characteristics typical of five-co-ordinated haem groups, are reduced in a dithionite-concentration-dependent fashion with a rate limit of 1.5 S-1. The other four haem groups, which show spectral characteristics very similar to those of normal six-co-ordinate c-haem groups, reduce in a linear dithionite-concentration-dependent manner with a second-order rate constant of 150 M-1/2 X S-1. The ratio of the amplitudes of the two reduction phases observed in stopped-flow studies is found to be dependent on the concentration of dithionite used. A model is proposed to account for these observations, and computer simulations show that the model represents a good fit to the experimental data. The two haem groups with five-co-ordinate spectral characteristics bind CO. Flash photolysis of the CO complex exhibits one major recombination process with a linear dependence in rate on CO concentration with a second-order rate constant of 2 X 10(6) M-1 X S-1. By contrast, stopped-flow mixing of the reduced protein with CO shows a very complex pattern of combination, with most of the observed absorbance change associated with a concentration-independent step. These findings are rationalized in terms of structural changes in the protein consequent to ligand binding.
| S-EPMC1153061 | biostudies-other
Project description:It is shown that the oxidized form of the hexa-haem nitrite reductase of Wolinella succinogenes exists in two structurally and functionally distinct forms, termed 'resting' and 'redox-cycled'. The nitrite reductase as initially isolated, termed 'resting', has five low-spin ferrihaem groups and one high-spin ferrihaem group. The reduction of these haem groups by Na2S2O4 occurs in two kinetically and spectrally distinct phases. In the slower phase the haem groups are reduced by dithionite with a limiting rate of 4 s-1. If the enzyme is re-oxidized after reduction with dithionite or with methyl viologen, the resulting ferric form, termed 'redox-cycled', possesses only low-spin haem centres and a rate of reduction in the slower phase that is no longer limited. In the resting form of the enzyme the high-spin ferrihaem group is weakly exchange-coupled to a low-spin haem group. It is proposed that in the redox-cycled form the exchange coupling occurs between two low-spin ferric haem groups. This change in spin state allows a more rapid rate of electron transfer to the coupled pair.
| S-EPMC1149542 | biostudies-other
Project description:The reduction kinetics of both the resting and redox-cycled forms of the nitrite reductase from the anaerobic rumen bacterium Wolinella succinogenes were studied by stopped-flow reaction techniques. Single-turnover reduction of the enzyme by dithionite occurs in two kinetic phases for both forms of the enzyme. When the resting form of the enzyme is subjected to a single-turnover reduction by dithionite, the slower of the two kinetic phases exhibits a hyperbolic dependence of the rate constant on the square root of the reductant concentration, the limiting value of which (approximately 4 s-1) is assigned to a slow internal electron-transfer process. In contrast, when the redox-cycled form of the enzyme is reduced by dithionite in a single-turnover experiment, both kinetic phases exhibit linear dependences of the rate on the square root of dithionite concentration, with associated rate constants of 150 M-1/2.s-1 and 6 M-1/2.s-1. Computer simulations of both the reduction processes shows that no unique set of rate constants can account for the kinetics of both forms, although the kinetics of the redox-cycled species is consistent with a much enhanced rate of internal electron transfer. Under turnover conditions the time course for reduction of the enzyme, in the presence of millimolar levels of nitrite and 100 mM-dithionite, is extremely complex. A working model for the mechanism of the turnover activity of the enzyme is proposed which very closely describes the reaction kinetics over a wide range of substrate concentrations, as shown by computer simulation. The similarity in the action of the nitrite reductase enzyme and mammalian cytochrome c oxidase is commented upon.
| S-EPMC1149576 | biostudies-other
Project description:The nitrite reductase of Wolinella succinogenes containing six covalently bound haem groups has one haem group that will not reduce fully in the presence of excess Na2S2O4. The effect of the extrinsic ligands CO and cyanide on the redox state of this haem was studied by e.p.r. and magnetic c.d. spectroscopy. It was found that both ligands increased the extent of reduction of this haem group, and that in the case of CO binding the level of reduction was correlated with the extent of CO saturation of the enzyme. Stopped-flow studies of the effect of cyanide binding on the rate of dithionite reduction showed that the rate of reduction of the ligand-binding site was increased in the presence of cyanide. This suggests that reduction of the haem groups at the active site is thermodynamically unfavourable in the absence of an extrinsic ligand. The role of the 'non-reducing' haem group and the effect of ligands on this centre and on the rate of reduction are discussed in relation to the reduction of nitrite by this enzyme.
| S-EPMC1149541 | biostudies-other
Project description:C(4)-dicarboxylate transport is a prerequisite for anaerobic respiration with fumarate in Wolinella succinogenes, since the substrate site of fumarate reductase is oriented towards the cytoplasmic side of the membrane. W. succinogenes was found to transport C(4)-dicarboxylates (fumarate, succinate, malate, and aspartate) across the cytoplasmic membrane by antiport and uniport mechanisms. The electrogenic uniport resulted in dicarboxylate accumulation driven by anaerobic respiration. The molar ratio of internal to external dicarboxylate concentration was up to 10(3). The dicarboxylate antiport was either electrogenic or electroneutral. The electroneutral antiport required the presence of internal Na(+), whereas the electrogenic antiport also operated in the absence of Na(+). In the absence of Na(+), no electrochemical proton potential (delta p) was measured across the membrane of cells catalyzing fumarate respiration. This suggests that the proton potential generated by fumarate respiration is dissipated by the concomitant electrogenic dicarboxylate antiport. Three gene loci (dcuA, dcuB, and dctPQM) encoding putative C(4)-dicarboxylate transporters were identified on the genome of W. succinogenes. The predicted gene products of dcuA and dcuB are similar to the Dcu transporters that are involved in the fumarate respiration of Escherichia coli with external C(4)-dicarboxylates. The genes dctP, -Q, and -M probably encode a binding-protein-dependent secondary uptake transporter for dicarboxylates. A mutant (DcuA(-) DcuB(-)) of W. succinogenes lacking the intact dcuA and dcuB genes grew by nitrate respiration with succinate as the carbon source but did not grow by fumarate respiration with fumarate, malate, or aspartate as substrates. The DcuA(-), DcuB(-), and DctQM(-) mutants grew by fumarate respiration as well as by nitrate respiration with succinate as the carbon source. Cells of the DcuA(-) DcuB(-) mutant performed fumarate respiration without generating a proton potential even in the presence of Na(+). This explains why the DcuA(-) DcuB(-) mutant does not grow by fumarate respiration. Growth by fumarate respiration appears to depend on the function of the Na(+)-dependent, electroneutral dicarboxylate antiport which is catalyzed exclusively by the Dcu transporters. Dicarboxylate transport via the electrogenic uniport is probably catalyzed by the DctPQM transporter and by a fourth, unknown transporter that may also operate as an electrogenic antiporter.
| S-EPMC94697 | biostudies-literature
Project description:To understand the origin and emergence of pathogenic bacteria, knowledge of the genetic inventory from their nonpathogenic relatives is a prerequisite. Therefore, the 2.11-megabase genome sequence of Wolinella succinogenes, which is closely related to the pathogenic bacteria Helicobacter pylori and Campylobacter jejuni, was determined. Despite being considered nonpathogenic to its bovine host, W. succinogenes holds an extensive repertoire of genes homologous to known bacterial virulence factors. Many of these genes have been acquired by lateral gene transfer, because part of the virulence plasmid pVir and an N-linked glycosylation gene cluster were found to be syntenic between C. jejuni and genomic islands of W. succinogenes. In contrast to other host-adapted bacteria, W. succinogenes does harbor the highest density of bacterial sensor kinases found in any bacterial genome to date, together with an elaborate signaling circuitry of the GGDEF family of proteins. Because the analysis of the W. succinogenes genome also revealed genes related to soil- and plant-associated bacteria such as the nif genes, W. succinogenes may represent a member of the epsilon proteobacteria with a life cycle outside its host.
| S-EPMC208819 | biostudies-literature
Project description:Thiocarboxylated proteins are important intermediates in a variety of biochemical sulfide transfer reactions. Here we identify a protein thiocarboxylate-dependent methionine biosynthetic pathway in Wolinella succinogenes. In this pathway, the carboxy terminal alanine of a novel sulfur transfer protein, HcyS-Ala, is removed in a reaction catalyzed by a metalloprotease, HcyD. HcyF, an ATP-utilizing enzyme, catalyzes the adenylation of HcyS. HcyS acyl-adenylate then undergoes nucleophilic substitution by bisulfide produced by Sir to give the HcyS thiocarboxylate. This adds to O-acetylhomoserine to give HcyS-homocysteine in a PLP-dependent reaction catalyzed by MetY. HcyD-mediated hydrolysis liberates homocysteine. A final methylation completes the biosynthesis. The biosynthetic gene cluster also encodes the enzymes involved in the conversion of sulfate to sulfide suggesting that sulfate is the sulfur source for protein thiocarboxylate formation in this system.
| S-EPMC3089676 | biostudies-literature
Project description:Quinol:fumarate reductase (QFR) is a membrane protein complex that couples the reduction of fumarate to succinate to the oxidation of quinol to quinone, in a reaction opposite to that catalyzed by the related enzyme succinate:quinone reductase (succinate dehydrogenase). In the previously determined structure of QFR from Wolinella succinogenes, the site of fumarate reduction in the flavoprotein subunit A of the enzyme was identified, but the site of menaquinol oxidation was not. In the crystal structure, the acidic residue Glu-66 of the membrane spanning, diheme-containing subunit C lines a cavity that could be occupied by the substrate menaquinol. Here we describe that, after replacement of Glu-C66 with Gln by site-directed mutagenesis, the resulting mutant is unable to grow on fumarate and the purified enzyme lacks quinol oxidation activity. X-ray crystal structure analysis of the Glu-C66-->Gln variant enzyme at 3.1-A resolution rules out any major structural changes compared with the wild-type enzyme. The oxidation-reduction potentials of the heme groups are not significantly affected. We conclude that Glu-C66 is an essential constituent of the menaquinol oxidation site. Because Glu-C66 is oriented toward a cavity leading to the periplasm, the release of two protons on menaquinol oxidation is expected to occur to the periplasm, whereas the uptake of two protons on fumarate reduction occurs from the cytoplasm. Thus our results indicate that the reaction catalyzed by W. succinogenes QFR generates a transmembrane electrochemical potential.
| S-EPMC27176 | biostudies-literature
Project description:The epsilon-proteobacteria Helicobacter pylori and Campylobacter jejuni are both human pathogens. They colonize mucosal surfaces causing severe diseases. The membrane protein complex QFR (quinol:fumarate reductase) from H. pylori has previously been established as a potential drug target, and the same is likely for the QFR from C. jejuni. In the present paper, we describe the cloning of the QFR operons from the two pathogenic bacteria H. pylori and C. jejuni and their expression in Wolinella succinogenes, a non-pathogenic -proteobacterium. To our knowledge, this is the first documentation of heterologous membrane protein production in W. succinogenes. We demonstrate that the replacement of the homologous enzyme from W. succinogenes with the heterologous enzymes yields mutants where fumarate respiration is fully functional. We have isolated and characterized the heterologous QFR enzymes. The high quality of the enzyme preparation enabled us to determine unequivocally by analytical ultracentrifugation the homodimeric state of the three detergent-solubilized heterotrimeric QFR enzymes, to accurately determine the different oxidation-reduction ('redox') midpoint potentials of the six prosthetic groups, the Michaelis constants for the quinol substrate, maximal enzymatic activities and the characterization of three different anti-helminths previously suggested to be inhibitors of the QFR enzymes from H. pylori and C. jejuni. This characterization allows, for the first time, a detailed comparison of the QFR enzymes from C. jejuni and H. pylori with that of W. succinogenes.
| S-EPMC1409705 | biostudies-literature