Project Summary/Abstract Iron-sulfur enzymes perform some of the most challenging transformations in biology. Of these, [Fe4S4] enzymes are the most ubiquitous and catalyze dozens of known transformations (and likely many more) that play direct and indirect roles in human health and disease. This proposal concerns the mechanisms of the > 100,000 members of the radical S-adenosyl-L-methionine (SAM) superfamily of enzymes. Understanding their reaction mechanisms on a molecular level is critical for identifying disease targets and designing mechanism- based inhibitors. An emerging theme in mechanistic studies of these enzymes is the intermediacy of species containing Fe–alkyl bonds whereby generation of the reactive 5’-deoxyadenosyl radical may proceed by homolytic Fe–C bond cleavage. However, the exact electronic and geometric structure of this intermediate, its function in catalysis, the strength of its Fe–C bond, and the mechanisms by which these steps might occur are not clear, and there is no precedent in synthetic Fe–S clusters for this structure type or reactivity. We therefore propose to address these questions using structurally and functionally faithful synthetic [Fe4S4]–alkyl complexes. We will prepare [Fe4S4]–alkyl complexes with rationally tunable properties and formulate and test hypotheses concerning the geometric and electronic structure requirements for achieving Fe–C bond homolysis. These requirements will be elucidated through systematic kinetic and spectroscopic studies. Overall, this work will reveal the role of organometallic chemistry in catalysis by radical SAM enzymes and yield insights into how Nature utilizes reactive Fe–C bonds in human health and disease.