PROJECT SUMMARY Nitrogenase reaction represents a major source of the usable form of nitrogen that supports the existence of human population. As such, understanding how small building blocks are assembled into a functional nitrogenase entity is of significant relevance to human health. Using combined genetic, biochemical, spectroscopic and structural approaches, we propose to investigate how M-cluster, the unique metallocofactor of molybdenum nitrogenase, is assembled into a functional unit. Specifically, we will investigate how an interstitial carbide is inserted via a radical SAM-dependent mechanism concomitant with fusion of two 4Fe modules into an 8Fe core of the M-cluster, what is the in vivo donor/carrier of the “9th sulfur” and how this sulfur is inserted into the catalytically important belt region of the 8Fe core, and how Mo is mobilized and inserted into the 8Fe core to form a mature M-cluster. Through our proposed studies, we expect to further refine the biosynthetic pathway of the unique metallocofactor of nitrogenase, which will provide crucial insights into the structural-functional relationship of this important enzyme and reveal some general principles of the assembly mechanisms of complex metalloclusters in biological systems.