Intermediates Trapped during Nitrogenase Reduction of N⋮N, CH3−NNH, and H2N−NH2

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Abstract
A high population intermediate has been trapped on the nitrogenase active site FeMo cofactor during reduction of N2. In addition, intermediates have been trapped during reduction of CH3−NNH by the α-195Gln variant and during reduction of H2N−NH2 by the α-70Ala/α-195Gln variant. Each of these trapped states shows an EPR signal arising from an S = 1/2 state of the FeMo cofactor. 15N ENDOR shows that each intermediate has a nitrogenous species bound to the FeMo cofactor, with a single type of N seen for each bound intermediate. The g tensors are unique to each intermediate, g(e) = [2.084, 1.993, 1.969], g(m) = [2.083, 2.021, 1.993], g(l) = [2.082, 2.015, 1.987], as are the 15N hyperfine couplings at g1, which suggests that three distinct stages of N⋮N reduction may have been trapped. The 1H ENDOR spectra show that the N2 intermediate is at a distinct and earlier stage of reduction from the other two, so at least two stages of N⋮N reduction have been trapped. Some possible structures of the hydrazine intermediate are presented.