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- and P-clusters of molybdenum nitrogenase are assembled via unique biochemical reactions into functional units. Specifically, we will investigate how two 4Fe modules are rearranged and coupled into an 8Fe core of M-cluster via radical SAM-dependent carbide insertion concomitant with the incorporation of a “9th” sulfur, how two 4Fe modules are rearranged and coupled into an 8Fe P-cluster via unique redox reactions concomitant with the removal of an “8th” sulfur, and how various assembly proteins interact with one another to facilitate the maturation of M- and P-clusters. Through our proposed studies, we expect to further refine the biosynthetic pathways of the unique metalloclusters 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.