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

Nitrogenase reduces N₂ to NH₃ at its active-site cofactor. Previous studies of an N₂-bound Mo-nitrogenase from Azotobacter vinelandii suggest binding of three N₂ species via asymmetric belt-sulfur displacements in the two cofactors of its catalytic component (designated Av1*), leading to the proposal of stepwise N₂ reduction involving all cofactor belt-sulfur sites; yet, the evidence for the existence of multiple N₂ species on Av1* remains elusive. Here we report a study of ATP-independent, Eu^II/SO₃ ^2--driven turnover of Av1* using GC-MS and frequency-selective pulse NMR techniques. Our data demonstrate incorporation of D₂-derived D by Av1* into the products of C₂H₂- and H⁺-reduction, and decreased formation of NH₃ by Av1* concomitant with the release of N₂ under H₂; moreover, they reveal a strict dependence of these activities on SO₃ ^2-. These observations point to the presence of distinct N₂ species on Av1*, thereby providing strong support for our proposed mechanism of stepwise reduction of N₂ via belt-sulfur mobilization.