Polarographic measurement of H2 in aqueous solutions

Sweet, William J.; Houchins, Jeffrey P.; Rosen, Paul R.; Arp, Daniel J.

doi:10.1016/0003-2697(80)90393-0

Cited by 41 publications

(17 citation statements)

References 5 publications

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“…Formic hydrogen lyase (EC 1.2.1.1) was assayed by polarographic measurement of H2 production (Sweet et al, 1980) following addition of 10 mM-sodium formate to reaction mixtures that contained (in 1.5 ml) 100 mM-MES, pH 5.7-6.0,2 pg extract protein, and various electron carriers, including NAD, methyl viologen, benzyl viologen, or horse heart cytochrome c.…”

Section: Methodsmentioning

confidence: 99%

Control of Product Formation During Glucose Fermentation by Bacillus macerans

Weimer¹

1984

Microbiology

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The anaerobic fermentation of glucose by Bacillus macerans ATCC 7068 was studied in batch culture with and without pH control. The fermentation was characterized by two distinct metabolic phases. In the primary growth phase, the concentrations of ethanol and acetic acid increased exponentially, and formate was detected as a minor product. The secondary phase was marked by a slowing and eventual cessation of growth, along with the disappearance of formate and acetate, and the appearance of H2, C02, and acetone. Exogenously-added substrates were converted with stoichiometries of 1 formate -+ 1 H2 + 1 C o t , and approximately 2 acetate + 1 acetone. Consumption of > 8 g glucose 1-required exogenous pH control, and acetone formation was strongly pH-dependent. Glucose was fermented by the Embden-Meyerhof pathway. Cell extracts contained pyruvate formate-lyase and formate dehydrogenase activities, but only low pyruvate dehydrogenase activity. A balanced fermentation pathway is presented which is consistent with reaction stoichiometries and [ 14C]glucose labelling data in whole cultures, and with enzyme activities in extracts. The pathway is compared with those of other facultative anaerobes and the acetone-producing Clostridium acetobutylicum.

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Section: Methodsmentioning

confidence: 99%

Control of Product Formation During Glucose Fermentation by Bacillus macerans

Weimer¹

1984

Microbiology

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“…Half-saturated KCI solution served as the electrolyte bathing the electrode, and the electrode was separated from the water-jacketed glass reaction chamber by a 0.5-mil Teflon membrane (22).…”

Section: Methodsmentioning

confidence: 99%

Hydrogen burst associated with nitrogenase-catalyzed reactions.

Liang

Burris

1988

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

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We have used a membrane-leak mass spectrometer to follow the time courses of H2 evolution and substrate reduction by nitrogenase [reduced ferredoxin:dinitrogen oxidoreductase (ATP-hydrolyzing), EC 1.18.6.1]. In the absence of added substrates, dinitrogenase passes all of its electrons to protons to form H2, but when a reducible substrate is added the electrons from dinitrogenase are shared between protons and the added substrate so that the steady-state rate of H2 production is decreased. If a reducible substrate is added before the nitrogenase reaction is initiated, a pre-steady-state burst of H2 is evident upon initiation of the reaction. This burst is associated with all the substrates of nitrogenase examinedi.e., N2, N20, C2H2, NaN3, and NaCN. The H2 burst is stoichiometric with dinitrogenase, but not with dinitrogenase reductase. In the H2 burst phase, 1 H2 is evolved per dinitrogenase molybdenum. Although a change in the ratio of nitrogenase components changed the initial rate of the H2 burst, the stoichiometry was not affected. Production of H2 by the burst in the presence of a high concentration of substrate is terminated after production of 1 H2 per dinitrogenase molybdenum, and a steady-state rate of H2 production is established. This response suggests that the H2 burst is not a catalytic event but a result of a once-only activation process.Nitrogenase [reduced ferredoxin:dinitrogen oxidoreductase (ATP-hydrolyzing), EC 1.18.6.1] is composed of two electron-transferring proteins, dinitrogenase (MoFe protein) and dinitrogenase reductase (Fe protein). Reduced dinitrogenase reductase transfers a single electron at a time to dinitrogenase with the concomitant hydrolysis of MgATP. Dinitrogenase, with its multiple iron-sulfur centers and its "FeMoco" (FeMo cofactor) prosthetic groups, serves as an electron sink capable of reducing all ofthe substrates ofnitrogenase. In addition to reducing its physiological substrate N2 to NH3, nitrogenase also is capable of reducing a variety of low molecular weight compounds such as N20, C2H2, N-, CN-, methyl isocyanide, cyclopropene, and analogs of some ofthese compounds (1,2). Dihydrogen is intimately involved in nitrogenase catalysis (3). In the absence of other reducible substrates, nitrogenase passes electrons to protons in the aqueous medium to produce molecular hydrogen in an ATPdependent H2 evolution reaction (4). This H2 evolution is inhibited by various nitrogenase substrates in ways that differ both qualitatively and quantitatively. Whereas high concentrations of the alternative substrates such as C2H2, CN-, and N20 (5-7) will completely suppress H2 evolution, the physiological substrate of nitrogenase, N2, cannot abolish H2 evolution even at 50 atmospheres (1 atm = 101.3 kPa) of N2 (8). Studies of H2 evolution under various partial pressures of N2 and N20 indicate that at a relatively high PN20, H2 evolution becomes N2-dependent (6). Therefore, H2 evolution is an inherent part of the N2 reduction reaction. Dihydrogen is not only a product of nitrogenase ...

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“…H 2 oxidation and evolution were measured amperometrically as described earlier (17). All assays were carried out at a temperature of 42ЊC and with 50 mM MES (morpholineethanesulfonic acid)-NaOH, pH 6.0, unless otherwise stated.…”

Section: Methodsmentioning

confidence: 99%

Substitution of Azotobacter vinelandii hydrogenase small-subunit cysteines by serines can create insensitivity to inhibition by O2 and preferentially damages H2 oxidation over H2 evolution

1995

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Mutants in which conserved cysteines 294, 297 or 64 and 65 of the Azotobacter vinelandii hydrogenase small subunit were replaced by serines were studied. Cysteines 294 and 297 are homologous to cysteines 246 and 249 of the Desulfovibrio gigas hydrogenase, and these cysteines are ligands to the [3Fe-4S] clusters (A. Volbeda, M.-H. Charon, C. Piras, E. C. Hatchikian, M. Frey, and J. C. Fontecilla-Camps, Nature (London) 373:580-587, 1995). Cysteine 65 is homologous to cysteine 20 of the D. gigas hydrogenase, and this cysteine is a ligand to the proximal [4Fe-4S] cluster. All three mutants retained some hydrogenase activity. All three mutants studied had H 2 oxidation-to-H 2 evolution activity ratios with whole cells of approximately 1.5, compared with 46 for the wild type. The changes preferentially deplete H 2 oxidation activity, while having less effect on evolution. The K64,65C3S hydrogenase was partially purified and had a specific activity for the evolution reaction that was 22% that of the wild type, while the oxidation-specific activity was 2% that of the wild type. Because cysteine 65 provides a ligand to the proximal [4Fe-4S] cluster, this cluster can be altered without entirely eliminating enzyme activity. Likewise, the detection of H 2 evolution and H 2 oxidation activities with whole cells and membranes of the K294C3S and K297C3S mutants indicates that the [3Fe-4S] cluster can also be altered or possibly eliminated without entirely eliminating enzyme activity. Membranes with K294C3S or K297C3S hydrogenase were uninhibited by O 2 in H 2 oxidation and uninhibited by H 2 in H 2 evolution. Wild-type membranes and membranes with K64,65C3S hydrogenase were both sensitive to these inhibitors. These data indicate that the [3Fe-4S] cluster controls the reversible inhibition of hydrogenase activity by O 2 or H 2 .Hydrogenase catalyzes the reversible activation of two electrons from electron carriers and two protons to form molecular hydrogen (1, 2, 7, 11). Azotobacter vinelandii is a free-living, nitrogen-fixing, aerobic, heterotrophic bacterium whose hydrogenase functions in energy conservation by oxidizing H 2 that is evolved by nitrogenase in the course of nitrogen fixation (6,14). A. vinelandii hydrogenase is a member of the [NiFe] hydrogenases and contains approximately one Ni and several Fe atoms per dimer of 67-and 31-kDa polypeptides (6). Ni is thought to be at the active site in [NiFe] hydrogenases (4, 11). The primary evidence for this is electron paramagnetic resonance evidence that hydrogen and CO, a competitive inhibitor against H 2 , both bind the Ni (20, 21). The cysteine ligands to Ni and to a second metal atom close to the Ni (probably Fe) are all on the large polypeptide subunit (5,8,22,23). At least one ligand to Ni, a selenocysteine in some hydrogenases and presumably the homologous cysteine in other hydrogenases, is contributed by the large polypeptide subunit (5,8,23). Other evidence that the active site is on the large subunit is the fact that radioactive acetylene, a competitive inhibitor ...

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Polarographic measurement of H2 in aqueous solutions

Cited by 41 publications

References 5 publications

Control of Product Formation During Glucose Fermentation by Bacillus macerans

Control of Product Formation During Glucose Fermentation by Bacillus macerans

Hydrogen burst associated with nitrogenase-catalyzed reactions.

Substitution of Azotobacter vinelandii hydrogenase small-subunit cysteines by serines can create insensitivity to inhibition by O2 and preferentially damages H2 oxidation over H2 evolution

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