Tracing the Incorporation of the “9(th) Sulfur” into the Nitrogenase Cofactor Precursor with Selenite and Tellurite

The Mo-nitrogenase catalyzes the reduction of N(2) to NH(3) at its cofactor, an [(R-homocitrate)MoFe(7)S(9)C] cluster synthesized via the formation of a [Fe(8)S(9)C] L-cluster prior to the insertion of Mo and homocitrate. Previously, we have identified a [Fe(8)S(8)C] L*-cluster, which is homologous to the core structure of the L-cluster but lacks the ‘9(th) sulfur’ in the belt region. However, direct evidence and mechanistic details of the L*- to L-cluster conversion upon ‘9(th) sulfur’ insertion remain elusive. Here, we trace the ‘9(th) sulfur’ insertion using SeO(3)(2−) and TeO(3)(2−) as ‘labeled’ SO(3)(2−). Biochemical, EPR and XAS/EXAFS studies suggest a role of the ‘9(th) sulfur’ in cluster transfer during cofactor biosynthesis while revealing the incorporation of Se(2−)- and Te(2−)-like species into the L-cluster. DFT calculations further point to a plausible mechanism involving in-situ reduction of SO(3)(2−) to S(2−), thereby suggesting the utility of this reaction to label the catalytically-important belt region for mechanistic investigations of nitrogenase.