Methyl-coenzyme M reductase (MCR) is the key enzyme in the biological formation and anaerobic oxidation of methane (AOM). Methane is the major component of natural gas. Given the abundance of natural gas reserves in remote areas, there is great current interest in a scalable bio-based process for the conversion of methane to liquid fuel and other high-value chemicals. MCR holds much promise for use in such a methane bioconversion strategy. However, MCR cannot currently be produced in an active form in an industrially viable strain due to the lack of genetic and biochemical information about the formation of its unique nickel-containing coenzyme, F430. The coenzyme F430 biosynthesis (Cfb) pathway was recently elucidated, and the key step was found to involve an unprecedented reductive cyclization reaction that converts the intermediate Ni-sirohydrochlorin a,c-diamide to the immediate precursor of F430, 15,17³-seco-F430-17³-acid. This remarkable transformation, which involves a six-electron ring reduction, cyclization of an acetamide side chain to form a g-lactam ring, and the formation of seven stereocenters, is catalyzed by a primitive homolog of nitrogenase (CfbCD). Nitrogenase is a two-component metalloenzyme that catalyzes the adenosine triphosphate (ATP)-dependent reduction of nitrogen gas to ammonia and hydrogen gas (biological nitrogen fixation), a reaction of great industrial importance. Homologs of nitrogenase are also involved in the biosynthesis of the photosynthetic pigments chlorophyll and bacteriochlorophyll. Phylogenetic analysis of the CfbCD complex suggests that it is representative of a more ancient lineage of the nitrogenase superfamily, and a thorough investigation of its structure and function is likely to shed light on the mechanisms and evolution of these important metalloenzymes. Moreover, a detailed understanding of the mechanism of the CfbCD complex may be exploited for the production of MCR for use in methane bioconversion. Towards these goals, the objectives of this research are focused on 1) the identification of physiological electron donors and in vivo coenzyme F430 synthesis, 2) the analysis of the iron-sulfur centers, structure, and oligomerization state changes, and 3) the characterization of transient intermediates and the intercomponent electron transfer of the CfbCD complex.