KINETIC-PARAMETERS FOR HYDROGEN EVOLUTION BY THE NAD LINKED HYDROGENASE OF ALCALIGENES-EUTROPHUS

The hydrogen-evolving reaction of the purified soluble NAD-linked hydr ogenase of Alcaligenes eutrophus was used to determine kinetic paramet ers of the enzyme. The H-2-evolving activity with methyl viologen as e lectron mediator was 20-fold as compared to that with NADH. In the ass ay with dithionite-reduced methyl viologen (K-m 0.7 mM) the hydrogenas e 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 Hz evolu tion, was 6.2 nM. With electrochemically reduced methyl viologen the p H optimum was shifted to pH 6.0. Double-reciprocal plots of reaction r ates versus proton concentrations intercepted at the ordinate for diff erent methyl viologen concentrations. At different pH values such an i ntercept was also observed with the dye as the varied substrate. The k inetic data are diagnostic for an ordered bisubstrate mechanism where both substrates are bound before the product H-2 is released. Hydrogen ase coupled to thylakoid membranes resulted in a constant H-2 evolutio n rate over 6 h. The system appeared to be limited by the capacity of the thylakoid membranes.