Inhibition of nitrogenase-catalyzed NH3 formation by H2

We have investigated the inhibition by H2 (D2) of NH3 formation by nitrogenase from Klebsiella pneumoniae and have confirmed that the inhibition is competitive vs. N2. D2 inhibits NH3 formation by diverting nitrogenase from production of NH3 to production of HD (one electron per HD). By careful exclusion of N2 from the reaction mixture, we have been able to place an upper limit on N2-independent HD formation by nitrogenase, under 1 atm of D2, at 1% of the total electron flux. Formation of NH3 and formation of HD were inhibited identically by CO. We observed that as the ratio of dinitrogenase to dinitrogenase reductase is increased, the ratio of HD formed to NH3 formed rises, and D2 becomes a stronger inhibitor of N2 reduction. This may be caused in part by an accompanying increase that is observed in the Km of nitrogenase for N2. We propose a model for D2 inhibition of NH3 formation in which D2 and N2 complete for the same form of nitrogenase. According to our proposal, when N2 reacts with nitrogenase, either N2 reduction proceeds to completion if H2 (D2) is absent or, if D2 already is bound to nitrogenase, N2 reduction is aborted and two molecules of HD are produced at the net expense of one electron per HD. Key consequences of the model are that it predicts that H2 (D2) is a competitive inhibitor of NH3 formation and that the apparent Km (N2) for formation of HD and NH3 may differ.