Intermediates trapped during nitrogenase reduction of N≡N, CH ₃-N=NH, and H₂N-NH₂

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-N=NH by the α-195^Gln variant and during reduction of H₂N-NH₂ by the α-70^Ala/α-195^Gln 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.