Isolation of Two Hydrogenase Activities in Chromatium

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Abstract
Kinetic, chromatographic and electrophoretic studies of Chromatium hydrogenase show the existence in vitro of 2 different activities (I and II). The 2 hydrogenases exhibit different kinetic parameters and properties. Using reduced methyl viologen, Km and [S]0.5 values of .apprx. 20 and 360 .mu.M were calculated for hydrogenases I and II, respectively. Hill plots revealed that hydrogenase I followed classical hyperbolic (Michaelis-Menten) kinetics. A Hill coefficient (h = 0.68) indicating nonhyperbolic kinetics was shown for hydrogenase II. After several purification steps, hydrogenase II still showed kinetics typical of the action of either 2 enzymes, each of which shows Michaelis-Menten kinetics, but with different substrate affinities, or only 1 enzyme which shows apparent negative cooperative regulation. The MW of the hydrogenases were .apprx. 37,000 (I) and 55,000 (II) when determined by gel filtration. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed that both enzymes give a coincidental single protein band with the same relative mobility indicating a MW of 31,000. Both hydrogenases were able to catalyze the reversible activation of H2 in the presence of artificial electron carriers, but with different rates.sbd.hydrogenase II being much more active in the H2-uptake mode. The kinetic properties and MW of hydrogenase II are partially modified by high ionic strength resulting in an increased substrate affinity and Hill coefficient, and thus, resembling hydrogenase I. Monomeric and dimeric forms of Chromatium hydrogenase may exist in vitro.