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

Molybdenum nitrogenase catalyses the reduction of N-2 to NH3 at its cofactor, an [(R-homocitrate)MoFe7S9C] cluster synthesized via the formation of a [Fe8S9C] L-cluster prior to the insertion of molybdenum and homocitrate. We have previously identified a [Fe8S8C] L*-cluster, which is homologous to the core structure of the L-cluster but lacks the 'ninth sulfur' in the belt region. However, direct evidence and mechanistic details of the L*- to L-cluster conversion upon 'ninth sulfur' insertion remain elusive. Here we trace the 'ninth sulfur' insertion using SeO32- and TeO32- as 'labelled' SO32-. Biochemical, electron paramagnetic resonance and X-ray absorption spectroscopy/extended X-ray absorption fine structure studies suggest a role of the 'ninth sulfur' in cluster transfer during cofactor biosynthesis while revealing the incorporation of Se2-- and Te2--like species into the L-cluster. Density functional theory 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.

Automated summary

The study investigates the insertion of the 'ninth sulfur' in the biosynthesis of cofactors, revealing its role in cluster transfer and the incorporation of Se2- and Te2- species into the cluster. The research also proposes a mechanism involving the reduction of SO32- to S2- for labeling the catalytically important belt region for nitrogenase mechanistic investigations.