Hydrogen reactions of nitrogenase. Formation of the molecule HD by nitrogenase and by an inorganic model.

Abstract HD formation from either D2 and H2O or H2 and D2O is shown to be catalyzed by N2-fixing extracts of Azotobacter vinelandii. The activity parallels nitrogenase but not hydrogenase activity and is not catalyzed by the sites of nitrogenase responsible for either ATP-dependent H2 evolution or competitive inhibition by H2 of N2 fixation. Requirements in addition to nitrogenase are N2, ATP, and reductant. HD formation occurs only while N2 is being reduced and is inhibited by CO. Other reducible substrates of nitrogenase including N3-, N2O, C2H2, HCN, CH2CHCN, and CH3NC do not support HD formation. The ratio of HD to NH3 formed is directly related to pD2 (0.8 and 4.5 HD per NH3 formed at 0.1 and 0.6 atm of D2, respectively) but not pN2. An exchange reaction is also catalyzed by intermediates of an inorganic model of nitrogenase. An equilibrium mixture of D2, HD, and H2 is formed from D2 and hydrogen atoms of either the aryldiimide- or arylhydrazine-platinum complexes of this model. These biological and model results provide the first evidence for nitrogenase-complexed diimide, hydrazine, and NH3 as intermediates of N2 fixation. It is proposed that protons of intermediates of N2 fixation bound via transiion metal or metals of nitrogenase, e.g. N2H+·nitrogenase, N2H3+·nitrogenase, NH3+·nitrogenase, exchange with D2 bounded to the transition metal or metals. Both D2 and H2 are equally effective competitive inhibitors of N2 fixation with a Ki of 0.2 atm. Protons are the ultimate source of the H2 evolved by nitrogenase based on the ratios of H2:HD:D2 evolved from H2O-D2O mixtures, but appear to be non-rate-limiting since there is no isotope effect (D2O versus H2O) on rates of either reductant-dependent adenosine triphosphatase or N2-fixing activities of nitrogenase.