Abstract
The hydrogen-evolving reaction of the purified soluble NAD-linked hydrogenase of Alcaligenes eutrophus was used to determine kinetic parameters of the enzyme. The H2-evolving activity with methyl viologen as electron mediator was 20-fold as compared to that with NADH. In the assay with dithionite-reduced methyl viologen (K m 0.7 mM) the hydrogenase was most active at a redox potential of −560 mV and exhibited a pH optimum of 7.0. The K m for protons, the second substrate for H2 evolution, was 6.2 nM. With electrochemically reduced methyl viologen the pH optimum was shifted to pH 6.0. Double-reciprocal plots of reaction rates versus proton concentrations intercepted at the ordinate for different methyl viologen concentrations. At different pH values such an intercept was also observed with the dye as the varied substrate. The kinetic data are diagnostic for an ordered bisubstrate mechanism where both substrates are bound before the product H2 is released. Hydrogenase coupled to thylakoid membranes resulted in a constant H2 evolution rate over 6 h. The system appeared to be limited by the capacity of the thylakoid membranes.
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References
Adams MWW, Mortenson LE, Chen J-S (1981) Hydrogenase. Biochim Biophys Acta 594:105–176
Aristarkhov AI, Nikandrov, VV, Krasnovskii AA (1986) Conditions for the photoproduction of hydrogen by subchloroplast fragments containing photosystem I. Mol Biol 20:1344–1355
Bisswanger H (1994) Enzymkinetik: Theorie und Methoden, 2nd edn. VCH, Weinheim, New York
Cammack R, Fernández VM, Hatchikian EC (1994) Nickel-iron hydrogenase. Methods Enzymol 243:43–68
Cuendet P, Rao KK, Grätzel M, Hall DO (1986) Light induces H2 evolution in a hydrogenase-TiO2 particle system by direct electron transfer or via rhodium complexes. Biochimie 68:217–221
Eng LH, Lewin MB-M, Neujahr HY (1993) Light-driven H2 production with proflavin and hydrogenase: comparison of cytochrome c 3 and methyl viologen as e− mediators. Photochem Photobiol 58:594–599
Erbes DL, Burris RH (1978) The kinetics of methyl viologen oxidation and reduction by the hydrogenase from Clostridium pasteurianum. Biochim Biophys Acta 525:45–54
Fernández VM (1983) An electrochemical cell for reduction of biochemicals: its application to the study of the effect of pH and redox potential on the activity of hydrogenases. Biochim Biophys Acta:487–499
Fernández VM, Munilla R, Ballesteros A (1981) Influence of the redox potential on the activity of Clostridium pasteurinium and Chromatium hydrogenases. Arch Biochem Biophys 215:129–135
Friedrich CG, Friedrich B, Bowien B (1981) Formation of enzymes of autotrophic metabolism during heterotrophic growth of Alcaligenes eutrophus. J Gen Microbiol 122:69–78
Fry I, Papageorgiou G, Tel-Or E, Packer L (1977) Reconstitution of a system for H2 evolution with chloroplast, ferredoxin, and hydrogenase. Z Naturforsch 32:110–117
Hoffmann D, Thauer R, Trebst A (1977) Photosynthetic hydrogen evolution by spinach chloroplasts coupled to a Clostridium hydrogenase. Z Naturforsch 32:257–262
Keefe RG, Axley MJ, Harabin AL (1995) Kinetic mechanism studies of the soluble hydrogenase of Alcaligenes eutrophus H16, Arch Biochem Biophys 317:449–456
Lissolo R, Cocquempot M-F, Thomas D, Le Gall J, Schneider K, Schlegel HG (1983) Hydrogen production using chloroplast membranes without oxygen scavengers: an assay with hydrogenases from aerobic hydrogen-oxidizing bacteria and flavodoxins from Desulfovibrio sp. Eur J Appl Microbiol Biotechnol 17:158–162
Mayhew SG (1978) The redox potential of dithionite and SO −2 from equilibrium reactions with flavodoxins, methyl viologens and hydrogen plus hydrogenase. Eur J Biochem 85:535–547
Payen B, Segui M, Monsan P, Schneider K, Friedrich CG, Schlegel HG (1983) Use of cytoplasmic hydrogenase from Alcaligenes eutrophus for NADH regeneration. Biotechnol Lett 5:463–468
Schneider K, Schlegel HG (1976) Purification and properties of soluble hydrogenase from Alcaligenes eutrophus H16. Biochim Biophys Acta 452:66–80
Schneider K, Schlegel HG (1981) Production of superoxide radicals by soluble hydrogenase from Alcaligenes eutrophus H16. Biochem J 193:99–107
Schneider K, Pinkwart M, Jochim K (1983) Purification of hydrogenases by affinity chromatography on Procion red-agarose. Biochem J 213:391–398
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Haverkamp, G.K., Ranke, H. & Friedrich, C.G. Kinetic parameters for hydrogen evolution by the NAD-linked hydrogenase of Alcaligenes eutrophus . Appl Microbiol Biotechnol 44, 514–518 (1995). https://doi.org/10.1007/BF00169953
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DOI: https://doi.org/10.1007/BF00169953