Diazene (HN=NH) is a substrate for nitrogenase:: Insights into the pathway of N2 reduction

Nitrogenase catalyzes the sequential addition of six electrons and six protons to a N-2 that is bound to the active site metal cluster FeMo-cofactor, yielding two ammonia molecules. The nature of the intermediates bound to FeMo-cofactor along this reduction pathway remains unknown, although it has been suggested that there are intermediates at the level of reduction of diazene (HN=NH, also called diimide) and hydrazine (H2N-NH2). Through in situ generation of diazene during nitrogenase turnover, we show that diazene is a substrate for the wild-type nitrogenase and is reduced to NH3. Diazene reduction, like N-2 reduction, is inhibited by H-2. This contrasts with the absence of H-2 inhibition when nitrogenase reduces hydrazine. These results support the existence of an intermediate early in the N-2 reduction pathway at the level of reduction of diazene. Freeze-quenching a MoFe protein variant with (alpha-195(His) substituted by Gln and alpha-70(Val) substituted by Ala during steady-state turnover with diazene resulted in conversion of the S = 3 /2 resting state FeMo-cofactor to a novel S = 1/2 state with g(1) = 2.09, g(2) = 2.01, and g(3) similar to 1-98 N-15- and H-1-ENDOR establish that this state consists of a diazene-derived [-NHx] moiety bound to FeMo-cofactor. This moiety is indistinguishable from the hydrazine-derived [-NHx] moiety bound to FeMo-cofactor when the same MoFe protein is trapped during turnover with hydrazine. These observations suggest that diazene joins the normal N-2-reduction pathway, and that the diazene- and hydrazine-trapped turnover states represent the same intermediate in the normal reduction of N-2 by nitrogenase. Implications of these findings for the mechanism of N-2 reduction by nitrogenase are discussed.