ATP-Independent Turnover of Dinitrogen Intermediates Captured on the Nitrogenase Cofactor.

Nitrogenase reduces N 2 to NH 3 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 /SO 3 2- -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 C 2 H 2 - and H + -reduction, and decreased formation of NH 3 by Av1* concomitant with the release of N 2 under H 2 ; moreover, they reveal a strict dependence of these activities on SO 3 2- . 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.