Tracing the incorporation of the "ninth sulfur" into the nitrogenase cofactor precursor with selenite and tellurite.

Abstract

The Mo-nitrogenase catalyzes the reduction of N2 to NH3 at its cofactor, an [(R-homocitrate)MoFe7S9C] cluster synthesized via the formation of a [Fe8S9C] L-cluster prior to the insertion of Mo and homocitrate. Previously, we have identified a [Fe8S8C] L*-cluster, which is homologous to the core structure of the L-cluster but lacks the ‘9th sulfur’ in the belt region. However, direct evidence and mechanistic details of the L*- to L-cluster conversion upon ‘9th sulfur’ insertion remain elusive. Here, we trace the ‘9th sulfur’ insertion using SeO32− and TeO32− as ‘labeled’ SO32−. Biochemical, EPR and XAS/EXAFS studies suggest a role of the ‘9th sulfur’ in cluster transfer during cofactor biosynthesis while revealing the incorporation of Se2−- and Te2−-like species into the L-cluster. DFT calculations further point to a plausible mechanism involving in-situ reduction of SO32− to S2−, thereby suggesting the utility of this reaction to label the catalytically-important belt region for mechanistic investigations of nitrogenase.