Diazene (HNNH) Is a Substrate for Nitrogenase: Insights into the Pathway of N2 Reduction

Abstract

Nitrogenase catalyzes the sequential addition of six electrons and six protons to a N₂ 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 (HNNH, 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 N₂ reduction, is inhibited by H₂. This contrasts with the absence of H₂ inhibition when nitrogenase reduces hydrazine. These results support the existence of an intermediate early in the N₂ reduction pathway at the level of reduction of diazene. Freeze-quenching a MoFe protein variant with α-195^His substituted by Gln and α-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 g₁ = 2.09, g₂ = 2.01, and g₃ ∼ 1.98. ¹⁵N- and ¹H-ENDOR establish that this state consists of a diazene-derived [−NH_x] moiety bound to FeMo-cofactor. This moiety is indistinguishable from the hydrazine-derived [−NH_x] moiety bound to FeMo-cofactor when the same MoFe protein is trapped during turnover with hydrazine. These observations suggest that diazene joins the normal N₂-reduction pathway, and that the diazene- and hydrazine-trapped turnover states represent the same intermediate in the normal reduction of N₂ by nitrogenase. Implications of these findings for the mechanism of N₂ reduction by nitrogenase are discussed.