ENDOR/HYSCORE Studies of the Common Intermediate Trapped During Nitrogenase Reduction of N2H2 CH3N2H and N2H4 Support an Alternating Reaction Pathway for N2 Reduction

摘要

Enzymatic N2 reduction proceeds along a reaction pathway comprised 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 N2 is hydrogenated in three steps until the first NH3 is liberated, then the remaining nitrido-N is hydrogenated three more times to yield the second NH3. 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 NH3 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 [NxHy] 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 N2H2, CH3N2H, and N2H4 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 N2H2, CH3N₂H, and N₂H₄ via a common >A reaction pathway, and that the same is true for N₂ itself, with Fe ion(s) providing the site of reaction.