ATP‐Independent Turnover of Dinitrogen Intermediates Captured on the Nitrogenase Cofactor

Nitrogenase reduces N-2 to NH3 at its active-site cofactor. Previous studies of an N-2-bound Mo-nitrogenase from Azotobacter vinelandii suggest binding of three N-2 species via asymmetric belt-sulfur displacements in the two cofactors of its catalytic component (designated Av1*), leading to the proposal of stepwise N-2 reduction involving all cofactor belt-sulfur sites; yet, the evidence for the existence of multiple N-2 species on Av1* remains elusive. Here we report a study of ATP-independent, Eu-II/SO32--driven turnover of Av1* using GC-MS and frequency-selective pulse NMR techniques. Our data demonstrate incorporation of D-2-derived D by Av1* into the products of C2H2- and H+-reduction, and decreased formation of NH3 by Av1* concomitant with the release of N-2 under H-2; moreover, they reveal a strict dependence of these activities on SO32-. These observations point to the presence of distinct N-2 species on Av1*, thereby providing strong support for our proposed mechanism of stepwise reduction of N-2 via belt-sulfur mobilization.