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

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 degrees C to 8 at 48 degrees C. With D-2 and H2O 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).