Isolation of two hydrogenase activities in Chromatium.
Kinetic, chromatographic and electrophoretic studies of Chromatium hydrogenase show the existence in vitro of two different activities (I and II). The two hydrogenases exhibit different kinetic parameters and properties. Using reduced methyl viologen, Km and [S]0.5 values of about 20 microM and 360 microM were calculated for the hydrogenases I and II, respectively. Hill plots revealed that hydrogenase I followed classical hyperbolic (Michaelis-Menten) kinetics. However, a Hill coefficient (h = 0.68) indicating non-hyperbolic kinetics could be shown for hydrogenase II. After several purification steps hydrogenase II still showed kinetics typical of the action of either (a) two enzymes each of which shows Michaelis-Menten kinetics but with different substrate affinities or (b) only one enzyme which shows apparent negative cooperative regulation. The molecular weights of the hydrogenases were about 37,000 (I) and 55,000 (II) when determined by gel filtration. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis revealed that both enzymes give a coincidental single protein band with the same relative mobility indicating a molecular weight of 31,000. Both hydrogenases were able to catalyse the reversible activation of H2 in the presence of artificial electron carriers but with different rates, hydrogenase II being much more active in the H2-uptake mode. The kinetic properties and molecular weight of hydrogenase II are partially modified by high ionic strength resulting in an increased substrate affinity and Hill coefficient and thus resembling hydrogenase I. These results are interpreted as due to the existence in vitro of monomeric and dimeric forms of Chromatium hydrogenase.
D002844:Chromatium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006859:Hydrogen / Q000378:metabolism; D006864:Hydrogenase; D007700:Kinetics; D008970:Molecular Weight; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism
D006859:Hydrogen; D010088:Oxidoreductases; D006864:Hydrogenase