Continuous monitoring, by mass spectrometry, of H2 production and recycling in Rhodopseudomonas capsulata

Jouanneau, Yves; Kelley, Bruce C.; Berlier, Y.; Lespinat, Paul A.; Vignais, Paulette M.

doi:10.1128/jb.143.2.628-636.1980

Cited by 72 publications

(25 citation statements)

References 25 publications

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“…The method used to determine the isotope-exchange reaction was described by Jouanneau et al (1980) and Vignais et al (1982), and has been used to study Desulfovibrio hydrogenases (Lespinat et al, 1986;Teixeira et al, 1987;Fauque et al, 1987). Based on the continuous recording of changes in concentration of gases D,, HD and H,, dissolved in the incubation medium, it is a specific and very sensitive technique for the measurement of isotope exchange within the first seconds after the mixing of the enzyme with D,-saturated medium (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The H-D exchange measured at 30°C was followed continuously in the aqueous phase by a mass-spectrometric method (Jouanneau et al, 1980). The reaction vessel, maintained under strict anaerobic conditions, was connected by a membrane inlet to the ion source of the mass spectrometer MM 8-80 (VG Instruments) equipped with a computer data-acquisition system.…”

Section: Assay Of Hydrogenase Activity Hydrogenase Activity Wasmentioning

confidence: 99%

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Inhibition by Iodoacetamide and Acetylene of the H‐D‐Exchange Reaction Catalyzed by Thiocapsa Roseopersicina Hydrogenase

Зорин

Dimon

Gagnon

et al. 1996

European Journal of Biochemistry

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The kinetics of H-D isotope exchange catalyzed by the thermostable hydrogenase from Thiocapsa roseopersicina have been studied by analysis of the exchange between D, and H,O. The pH dependence of the exchange reaction was examined between pH 2.5 and pH 11. Over the whole pH range, HD was produced at a higher initial velocity than H,, with a marked optimum at pH 5.5; a second peak in the pH profile was observed at around pH 8.5. The rapid formation of H, with respect to HD in the D,/H,O system is consistent with a heterolytic cleavage of D, into D' and an enzyme hydride that can both exchange with the solvent. The H-D-exchange activity was lower in the H,/D,O system than in the D,/ H,O system. The other reactions catalyzed by the hydrogenase, H, oxidation and H, evolution, are pH dependent; the optimal pH were 9.5 for H, uptake and 4.0 for H, production. Treatment of the active form of hydrogenase by iodoacetamide led to a slow and irreversible inhibition of the H-D exchange. When i~do[l-'~C]acetamide was incubated with hydrogenase, the radioactive labeling of the large subunit was higher for the enzyme activated under H, than for the inactive oxidized form. Cysteine residues were identified as the alkylated derivative by amino acid analysis. Acetylene, which inhibits H-D exchange and abolishes the Ni-C EPR signal, protected the enzyme from irreversible inhibition by iodoacetamide. These data indicate that iodoacetamide can reach the active site of the H,-activated hydrogenase from 7: roseopersicina. This was not found to be the case with the seleno hydrogenase from Desulfovibrio baculatus (now Desulfornicrobiurn baculatus). Cysteine modification by iodoacetamide upon activation of the enzyme concomitant with loss of H-D exchange indicates that reductive activation makes at least one Cys residue of the active site available for alkylation.

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Section: Discussionmentioning

confidence: 99%

Section: Assay Of Hydrogenase Activity Hydrogenase Activity Wasmentioning

confidence: 99%

Inhibition by Iodoacetamide and Acetylene of the H‐D‐Exchange Reaction Catalyzed by Thiocapsa Roseopersicina Hydrogenase

Зорин

Dimon

Gagnon

et al. 1996

European Journal of Biochemistry

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“…The bottom of the vessel consisted of a stainless steel grid covered with a 12.5-pm-thick teflon membrane. For details of the construction, the reader is referred to the description by Jouanneau et al (1980). The vessel was filled with 7 ml anaerobic 120 mM potassium phosphate pH (pD) 6.7 containing 120 nmol CH-H,MPT' and was equilibrated with H,, HD, or D,.…”

Section: Methodsmentioning

confidence: 99%

Hydrogen Isotope Effects in the Reactions Catalyzed by H₂‐forming N⁵,N¹⁰‐Methylenetetrahydromethanopterin Dehydrogenase from Methanogenic Archaea

Klein

Hartmann

Thauer

1995

European Journal of Biochemistry

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H,-forming N',N"'-methylenetetrahydromethanopterin dehydrogenase from methanogenic Archaea, which is a novel hydrogenase containing neither nickel nor iron-sulfur clusters, catalyzes the reversible reduction of N5,N"'-methenyltetrahydomethanopterin (CH=H,MPT.') with H, to N',N"'-methylenetetrahydromethanopterin (CH,=H,MPT) and a proton (AGO' = -5.5 kJ/mol). The enzyme also catalyzes a CH-H,MPT+-dependent H,/H' exchange. We report here on kinetic deuterium isotope effects in these reactions. When CH=H,MPT' reduction was performed with DZ instead of H2, V,,,., and the K,,, did not change. A primary isotope effect of 1 was found at all pH and temperatures tested and independent of whether H,O or DZO was the solvent. The findings indicate that a step other than the activation of H, was rate-determining in CH=H,MPT+ reduction with H,. This was substantiated by the observation that also the CH=H,MPT+-dependent H,/H+ exchange reaction did not exhibit an appreciable deuterium isotope effect. V,,,,, for CH,=H,MPT dehydrogenation to CH-H,MPT+ and H2 was only 2-3 times higher than for CD2=H,MPT dehydrogenation to CD-H,MPT' and HD. Such a small primary isotope effect indicates that the breakage of the C-H bond in the methylene group of CH2=H,MPT was only rate-limiting when hydrogen was substituted by a deuterium.

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“…Proton-deuterium exchange activity was measured in whole-cell suspensions from D. fructosovorans wild-type and mutant strains grown in 20 mM fructose/20 mM fumarate obtained as described previously [21] by a mass spectrometric method [23]. Fumarate, rather than sulfate, was used as an electron acceptor to prevent formation of sulfur precipitates.…”

Section: Enzyme Assaysmentioning

confidence: 99%

Construction and physiological studies of hydrogenase depleted mutants of Desulfovibrio fructosovorans

Casalot¹

2002

FEMS Microbiology Letters

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Desulfovibrio fructosovorans possesses two periplasmic hydrogenases (a nickel-iron and an iron hydrogenase) and a cytoplasmic NADPdependent hydrogenase. The hydAB genes encoding the periplasmic iron hydrogenase were replaced, in the wild-type strain as well as in single mutants depleted of one of the other two hydrogenases, by the acc1 gene encoding resistance to gentamycin. Molecular characterization and remaining activity measurements of the resulting single and double mutants were performed. All mutated strains exhibited similar growth when H 2 was the electron donor but they grew differently on fructose, lactate or pyruvate as electron donors. Our results indicate that the loss of one enzyme might be compensated by another even though hydrogenases have different localization in the cells. ß

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Continuous monitoring, by mass spectrometry, of H2 production and recycling in Rhodopseudomonas capsulata

Cited by 72 publications

References 25 publications

Inhibition by Iodoacetamide and Acetylene of the H‐D‐Exchange Reaction Catalyzed by Thiocapsa Roseopersicina Hydrogenase

Inhibition by Iodoacetamide and Acetylene of the H‐D‐Exchange Reaction Catalyzed by Thiocapsa Roseopersicina Hydrogenase

Hydrogen Isotope Effects in the Reactions Catalyzed by H₂‐forming N⁵,N¹⁰‐Methylenetetrahydromethanopterin Dehydrogenase from Methanogenic Archaea

Construction and physiological studies of hydrogenase depleted mutants of Desulfovibrio fructosovorans

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Continuous monitoring, by mass spectrometry, of H2 production and recycling in Rhodopseudomonas capsulata

Cited by 72 publications

References 25 publications

Inhibition by Iodoacetamide and Acetylene of the H‐D‐Exchange Reaction Catalyzed by Thiocapsa Roseopersicina Hydrogenase

Inhibition by Iodoacetamide and Acetylene of the H‐D‐Exchange Reaction Catalyzed by Thiocapsa Roseopersicina Hydrogenase

Hydrogen Isotope Effects in the Reactions Catalyzed by H2‐forming N5,N10‐Methylenetetrahydromethanopterin Dehydrogenase from Methanogenic Archaea

Construction and physiological studies of hydrogenase depleted mutants of Desulfovibrio fructosovorans

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Hydrogen Isotope Effects in the Reactions Catalyzed by H₂‐forming N⁵,N¹⁰‐Methylenetetrahydromethanopterin Dehydrogenase from Methanogenic Archaea