ENDOR/HYSCORE studies of the common intermediate trapped during nitrogenase reduction of N ₂H ₂, CH ₃N ₂H, and N ₂H ₄ support an alternating reaction pathway for N ₂ reduction

Enzymatic N ₂ reduction proceeds along a reaction pathway composed of a sequence of intermediate states generated as a dinitrogen bound to the active-site iron-molybdenum cofactor (FeMo-co) of the nitrogenase MoFe protein undergoes six steps of hydrogenation (e ^-/H ⁺ delivery). There are two competing proposals for the reaction pathway, and they invoke different intermediates. In the 'Distal' (D) pathway, a single N of N ₂ is hydrogenated in three steps until the first NH ₃ is liberated, and then the remaining nitrido-N is hydrogenated three more times to yield the second NH ₃. In the 'Alternating' (A) pathway, the two N's instead are hydrogenated alternately, with a hydrazine-bound intermediate formed after four steps of hydrogenation and the first NH ₃ liberated only during the fifth step. A recent combination of X/Q-band EPR and ¹⁵N, ^1,2H ENDOR measurements suggested that states trapped during turnover of the α-70 ^Ala/α-195 ^Gln MoFe protein with diazene or hydrazine as substrate correspond to a common intermediate (here denoted I) in which FeMo-co binds a substrate-derived [N _xH _y] moiety, and measurements reported here show that turnover with methyldiazene generates the same intermediate. In the present report we describe X/Q-band EPR and ^14/15N, ^1,2H ENDOR/HYSCORE/ESEEM measurements that characterize the N-atom(s) and proton(s) associated with this moiety. The experiments establish that turnover with N ₂H ₂, CH ₃N ₂H, and N ₂H ₄ in fact generates a common intermediate, I, and show that the N-N bond of substrate has been cleaved in I. Analysis of this finding leads us to conclude that nitrogenase reduces N ₂H ₂, CH ₃N ₂H, and N ₂H ₄ via a common A reaction pathway, and that the same is true for N ₂ itself, with Fe ion(s) providing the site of reaction.