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

HD formation from either Da and Hz0 or Hz and DzO is shown to be catalyzed by Nz-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 Hz evolution or competitive inhibition by Hz of Nz fixation. Requirements in addition to nitrogenase are NB, ATP, and reductant. HD formation occurs only while Nz is being reduced and is inhibited by CO. Other reducible substrates of nitrogenase including N3-, NzO, CzH2, HCN, CHCHCN, and CHsNC do not support HD formation. The ratio of HD to NH8 formed is directly related to pDz (0.8 and 4.5 HD per NH3 formed at 0.1 and 0.6 atm of DB, respectively) but not pN,. An exchange reaction is also catalyzed by intermediates of an inorganic model of nitrogenase. An equilibrium mixture of Dz, HD, and Hz is formed from Dz and hydrogen atoms of either the aryldiimideor 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 Nz fixation. It is proposed that protons of intermediates of NB fkation bound via transition metal or metals of nitrogenase, e.g. NzH+ .nitrogenase, NzH3+ .nitrogenase, NH3+ .nitrogenase, exchange with D2 bonded to the transition metal or metals. Both Dz and HZ are equally effective competitive inhibitors of Nz 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:Dz evolved from H20-D20 mixtures, but appear to be non-rate-limiting since there is no isotope effect (DzO versus HzO) on rates of either reductant-dependent adenosine triphosphatase or N&king activities of nitrogenase.