The kinetics of methyl viologen oxidation and reduction by the hydrogenase from Clostridium pasteurianium

Erbes, David L.; Burris, R. H.

doi:10.1016/0005-2744(78)90198-5

Cited by 66 publications

(30 citation statements)

References 19 publications

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“…To determine the apparent Km for H,, a phosphate buffer solution was saturated with H, gas at 20 "C and atmospheric pressure and then portions of this solution were added to the assay mixture in anaerobic cuvettes. The saturated H, solution was calculated to contain 0.8 mM-H, (Handbook of Chemistry and Physics; CRC Press) which was very close to values determined by Erbes & Burris (1978). Table 2 shows that values obtained for the apparent Km for H, did not vary significantly between whole bacteria and broken cell suspensions of the bacteria tested.…”

Section: Results a N D Discussionsupporting

confidence: 65%

The Influence of Extracellular Hydrogen on the Metabolism of Bacteroides ruminicola, Anaerovibrio lipolytica and Selenomonas ruminantium

Henderson¹

1980

Microbiology

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I N T R O D U C T I O NWhen inhibitors of rumen methanogenic bacteria are fed to ruminants the resulting depression of methane production is accompanied by an accumulation of H, and an increase in the proportion of propionic acid relative to acetic acid in the rumen (Demeyer & Van Nevel, 1975). It may be that the accumulating H, so decreases growth of those rumen bacteria which form acetate as their main product (Hungate, 1966) as to allow the propionate-producing bacteria to compete more successfully for fermentable substrate. Alternatively, the H, produced by the acetate-producing bacteria might be used directly by the propionate producers to increase the proportion of pyruvate which is reduced to propionate and thus increase the ratio of propionate to acetate produced.In the present study several important propionate-and succinate-producing rumen bacteria were tested for their ability to utilize extracellular H, as a source of reducing power. Such activity has been shown to be the main source of energy for the nonfermentative rumen bacterium Vibrio succinogenes which requires an exogenous supply of H, (or formate) and fumarate (Wolin et al., 1961). The reduction of fumarate to succinate by V. succinogenes is accompanied by electron transport-linked phosphorylation (Reddy & Peck, 1978). M E T H O D SAnaerovibrio lipolytica 5s was the strain of Hobson (1965); it was grown on the medium described by Henderson (1971) with fructose as energy source. Selenomonas ruminantiurn strains 17 and WPLl 51 j l were isolated at the Rowett Research Institute as described by Hobson & Mann (1961); they were grown on medium H5 which was based on medium 1 of Kurihara et al. (1968) with rumen fluid replaced by distilled t

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Section: Results a N D Discussionsupporting

confidence: 65%

The Influence of Extracellular Hydrogen on the Metabolism of Bacteroides ruminicola, Anaerovibrio lipolytica and Selenomonas ruminantium

Henderson¹

1980

Microbiology

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“…Anaerobic titrations with sodium dithionite were performed in the double septum seal apparatus previously described (19). Dithionite solutions were standardized by anaerobic titration with excess methyl viologen, monitored spectrophotometrically (20); samples were transferred anaerobically to EPR tubes and frozen within 2 min.…”

Section: Methodsmentioning

confidence: 99%

Evidence for a spin-coupled binuclear iron unit at the active site of the purple acid phosphatase from beef spleen.

Davis¹,

Averill²

1982

Proc. Natl. Acad. Sci. U.S.A.

120

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The purple acid phosphatase from beef spleen, which contains two iron atoms per molecule, is EPR silent in its native (oxidized) purple form. Treatment with mild reducing agents results in conversion to a pink, enzymatically active form, which exhibits an unusual EPR signal centered at g "'1.77; double integration ofthe EPR spectrum gives one spin per two iron atoms. A similar EPR spectrum is observed for enzyme reduced anaerobically by one electron, using sodium dithionite. Variable-temperature magnetic susceptibility measurements show that the oxidized and reduced proteins are both antiferromagnetically coupled systems, with S = 0 and /2 ground states, respectively. Replacement of one of the iron atoms by zinc produces an FeZn enzyme with full catalytic activity. The FeZn enzyme exhibits a highly temperature dependent g = 4.3 EPR signal, and magnetic susceptibility data are consistent with an S = 5/2 paramagnet. Treatment of the FeZn enzyme with phosphate, a competitive inhibitor, results in sharpening of the EPR spectrum; double integration at 77 K gives one spin per iron. These results strongly suggest the presence of a spin-coupled bimetallic unit at the active site of the enzyme. 505 nm) forms, the pink form is produced by treatment of the native purple form with mild reducing agents (1,14). Resonance Raman spectra of the purple form of the porcine enzyme (15) show evidence for coordination of tyrosine phenolate groups [in agreement with similar studies on the sweet potato enzyme (8 16)]. Low-temperature EPR spectra of the porcine enzyme e~hibita novel g' = 1.74 signal, which together with magnetic susceptibility results was interpreted in terms of mononuclear ferric iron in an unusual S = 1/2 spin state in both the purple and pink forms (17).In an attempt to resolve the questions of the nature of the chromophore and its role in catalysis, we have examined the purple acid phosphatase from beef spleen in some detail. We describe herein EPR spectra and magnetic susceptibility data on the native (purple) form, the reduced (pink) form, and a form in which halfthe iron has been replaced by zinc, with complete retention of enzymatic activity. Together with our earlier studies, the present results strongly suggest that the enzyme contains a binuclear spin-coupled iron unit at the active site.Purple metalloproteins with phosphatase activity have now been isolated from mammalian, plant, and microbial sources (1). All but that from Micrococcus sodonensis (2) are similar in being basic glycoproteins with optimal activity at low pH, and all exhibit an intense absorption band at 510-550 nm (E 4,000 M1 cm-') with a shoulder at 310-320 nm. Despite these obvious similarities, there exists substantial disagreement with regard to the metal content. Thus, the protein from beef spleen (3, 4) is reported to contain two iron atoms (5) and that from porcine uterine fluids either one (6) or two (5) iron atoms. The protein from kidney beans apparently contains iron as well (7), although the stoichiometry has not been dete...

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“…1). Kleiner and Burris (20) and Erbes and Burris (15) reported that the substitution of 2H2 for 'H2 in a reaction containing the reversible hydrogenase from C. pasteurianum had no significant effect on the maximum velocity at pH 7.0. The Km for 'H2, however, was only 63% of that for 'H2.…”

mentioning

confidence: 99%

Investigation of the H₂ Oxidation System in Rhizobium japonicum 122 DES Nodule Bacteroids

Emerich¹,

Ruiz-Argüeso²,

Russell³

et al. 1980

Plant Physiol.

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total electron flow to the nitrogenase reaction (16). The discovery (11-13, 25) of a H2-oxidizing system in legume nodules and other N2-fixing organisms (7, 8,19) has created considerable interest in the H2-recycling process. Recent evidence supports two of the benefits that Dixon (13) postulated might be derived from possession of the H2-oxidizing system. Walker and Yates (35) and preparation of bacteroids have been described elsewhere (28). Bacteroid suspensions were prepared daily from freshly harvested nodules. H2 and O2 were determined amperometrically as previously described (14). The specific activity of hydrogenase in the fresh bacteroid preparations ranged between 1.5 and 2.0 ,imol/h.

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The kinetics of methyl viologen oxidation and reduction by the hydrogenase from Clostridium pasteurianium

Cited by 66 publications

References 19 publications

The Influence of Extracellular Hydrogen on the Metabolism of Bacteroides ruminicola, Anaerovibrio lipolytica and Selenomonas ruminantium

The Influence of Extracellular Hydrogen on the Metabolism of Bacteroides ruminicola, Anaerovibrio lipolytica and Selenomonas ruminantium

Evidence for a spin-coupled binuclear iron unit at the active site of the purple acid phosphatase from beef spleen.

Investigation of the H₂ Oxidation System in Rhizobium japonicum 122 DES Nodule Bacteroids

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The kinetics of methyl viologen oxidation and reduction by the hydrogenase from Clostridium pasteurianium

Cited by 66 publications

References 19 publications

The Influence of Extracellular Hydrogen on the Metabolism of Bacteroides ruminicola, Anaerovibrio lipolytica and Selenomonas ruminantium

The Influence of Extracellular Hydrogen on the Metabolism of Bacteroides ruminicola, Anaerovibrio lipolytica and Selenomonas ruminantium

Evidence for a spin-coupled binuclear iron unit at the active site of the purple acid phosphatase from beef spleen.

Investigation of the H2 Oxidation System in Rhizobium japonicum 122 DES Nodule Bacteroids

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Investigation of the H₂ Oxidation System in Rhizobium japonicum 122 DES Nodule Bacteroids