Trapping an Intermediate of Dinitrogen (N2) Reduction on Nitrogenase

Nitrogenase reduces dinitrogen (N-2) by six electrons and six protons at an active-site metal-locluster called FeMo cofactor, to yield two ammonia molecules, Insights into the mechanism of substrate reduction by nitrogenase have come from recent successes in trapping and characterizing intermediates generated during the reduction of protons as well as nitrogenous and alkyne substrates by MoFe proteins with amino acid substitutions. Here, we describe an intermediate generated at a high concentration during reduction of the natural nitrogenase substrate, N-2, by wild-type MoFe protein, providing evidence that it contains N-2 bound to the active-site FeMo cofactor. When MoFe protein was frozen at 77 K during steady-state turnover with N-2 the S = 3/2 EPR signal (g [4.3, 3.64, 2.00]) arising from the resting state of FeMo cofactor was observed to convert to a rhombic, S = 1/2, signal (g = [2.08, 1.99, 1.97]). The intensity of the N-2-dependent EPR signal increased with increasing N-2 partial pressure, reaching a maximum intensity of approximately 20% of that of the original FeMo cofactor signal at >= 0.2 atm N-2. An almost complete loss of resting FeMo cofactor signal in this sample implies that the remainder of the enzyme has been reduced to an EPR-silent intermediate state. The N-2-dependent EPR signal intensity also varied with the ratio of Fe protein to MoFe protein (electron flux through nitrogenase), with the maximum signal intensity observed with a ratio of 2:1 (1:1 Fe protein: FeMo cofactor) or higher. The pH optimum for the signal was 7.1. The N-2-dependent EPR signal intensity exhibited a linear dependence on the square root of the EPR microwave power in contrast to the nonlinear response of signal intensity observed for hydrazine-, diazene-, and methyldiazene-trapped states. N-15 ENDOR spectroscopic analysis of MoFe protein captured during turnover with N-15(2) revealed a N-15 nuclear spin coupled to the FeMo cofactor with a hyperfine tensor A = [0.9, 1.4, 0.45] MHz establishing that an N-2-derived species was trapped on the FeMo cofactor. The observation of a single type of N-15-coupled nucleus from the field dependence, along with the absence of ail associated exchangeable H-1 ENDOR signal, is consistent with an N-2 molecule bound end-on to the FeMo cofactor.