Comparison of isotope exchange, H 2 evolution, and H 2 oxidation activities of Azotobacter vinelandii hydrogenase

Abstract

Azotobacter vinelandii hydrogenase was purified aerobically with a 35% yield. The purified enzyme catalyzed H 2 oxidation at much greater velocity than H 2 evolution. There was a large difference in activation energy for the two reactions. E A was 10 kcal/mol for H 2 oxidation and 22 kcal/mol for evolution. This difference in activation energies between the two reactions means that the ratio of oxidation velocity to evolution velocity drops from 70 at 33°C to 8 at 488°C. With D 2 and H 2O as substrates, both membranes and purified enzyme produced only H 2 and no HD in the isotope exchange reaction. The velocity of isotope exchange was equal to the velocity of H 2 evolution from reduced methyl viologen, indicating that the two reactions share the same rate-limiting step. D 2 and H 2 inhibited H 2 evolution, but D 2 did not inhibit isotope exchange. We conclude that H 2 and D 2 do not inhibit H 2 evolution by competing with H + for the active site of the reduced enzyme. The K m for D 2 in isotope exchange is 40-times greater than its K m in D 2 oxidation. The difference in K m cannot be accounted for by differences in k cat. We propose that redox environment regulates hydrogenase's affinity for D 2 (and likely H 2 as well).