PROJECT SUMMARY Nitrogenase catalyzes the ambient conversion of N2 to NH3 at its M-cluster site. This reaction represents a major source of the usable form of nitrogen that supports the existence of human population. As such, understanding how this enzyme effects ambient conversion of N2 to NH3 is of significant relevance to human health. Our recent structural observation of asymmetric belt sulfur displacements with distinct dinitrogen species in the two M-clusters of Mo-nitrogenase invokes a novel mechanism of N2 reduction that necessitates dynamic structural rearrangements of the M-clusters during catalysis. Using combined genetic, biochemical, spectroscopic and structural approaches, we propose to investigate this novel mechanism of N2 reduction by demonstrating the stepwise mechanism of N2 reduction via cluster rotation, assigning individual catalytic events at the three belt-sulfur sites of the M-cluster, and illustrating the alternate binding of Fe protein to the two MoFe protein dimers that drives the asynchronous cluster rotation in these dimers. Through our proposed studies, we hope to illustrate the dynamic structural rearrangements of the active site of Mo-nitrogenase during N2 reduction and establish a framework for further mechanistic exploration of this intricate reaction. Investigations along this line could prove instrumental in (re)calibrating the mechanistic thinking of nitrogenase, as all mechanistic studies to date have assumed that N2 reduction occurs at a single site of a uniform M-cluster species that is structurally `static' during catalysis. These efforts, if successful, will not only lead to a molecular depiction of the intricate catalytic mechanism of nitrogenase, but also facilitate development of nitrogenase-based applications in the long run.