title: Comparison of isotope exchange, H2 evolution, and H2 oxidation activities of Azotobacter vinelandii hydrogenase.
abstract: Azotobacter vinelandii hydrogenase was purified aerobically with a 35% yield. The purified enzyme catalyzed H2 oxidation at much greater velocity than H2 evolution. There was a large difference in activation energy for the two reactions. EA was 10 kcal/mol for H2 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 D2 and H2O as substrates, both membranes and purified enzyme produced only H2 and no HD in the isotope exchange reaction. The velocity of isotope exchange was equal to the velocity of H2 evolution from reduced methyl viologen, indicating that the two reactions share the same rate-limiting step. D2 and H2 inhibited H2 evolution, but D2 did not inhibit isotope exchange. We conclude that H2 and D2 do not inhibit H2 evolution by competing with H+ for the active site of the reduced enzyme. The Km for D2 in isotope exchange is 40-times greater than its Km in D2 oxidation. The difference in Km cannot be accounted for by differences in kcat. We propose that redox environment regulates hydrogenase's affinity for D2 (and likely H2 as well).
authors.affiliation: Department of Botany and Plant Pathology, Oregon State University, Corvallis 97331-2902, USA.
authors.forename: H
authors.initials: H
authors.lastname: McTavish
authors.forename: L A
authors.initials: LA
authors.lastname: Sayavedra-Soto
authors.forename: D J
authors.initials: DJ
authors.lastname: Arp
mesh_terms: D000332:Aerobiosis; D016948:Azotobacter vinelandii / Q000201:enzymology*; D002151:Calorimetry; D002852:Chromatography, Ion Exchange; D003903:Deuterium; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006859:Hydrogen / Q000378:metabolism*; D006864:Hydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D011865:Radioisotope Dilution Technique; D013816:Thermodynamics
chemical_list: D006859:Hydrogen; D003903:Deuterium; D006864:Hydrogenase