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SummaryThe radical AdoMet superfamily comprises a diverse set of >2800 enzymes that utilize iron-sulfur clusters and S-adenosylmethionine (SAM or AdoMet) to initiate a diverse set of radical-mediated reactions. The intricate control these enzymes exercise over the radical transformations they catalyze is an amazing feat of elegance and sophistication in biochemistry. This review focuses on the accumulating evidence for how these enzymes control this remarkable chemistry, including controlling the reactivity between the iron-sulfur cluster and AdoMet, and controlling the subsequent radical transformations. Radical AdoMet enzymes: Diverse functions with a common mechanistic threadThe radical SAM superfamily comprises a diverse set of >2800 enzymes that utilize iron-sulfur clusters and S-adenosylmethionine (SAM or AdoMet) to initiate radical transformations [1,2]. The enzymes are involved in such central pathways as the metabolism of glucose and the reduction of ribonucleotides, as well as in pathways for the biosynthesis of complex cofactors and metal clusters, the modification of tRNA, and the synthesis of antibiotics (Figure 1). Central to each of these diverse reactions are believed to be a common series of mechanistic steps that culminate in the abstraction of a hydrogen atom from substrate ( Figure 2). First, AdoMet binds to the unique iron site of the site-differentiated [4Fe-4S] 2+ radical AdoMet cluster via the α-carboxyl and α-amino groups of the methionine. This novel interaction between AdoMet and the [4Fe-4S] cluster was first revealed by detailed electron-nuclear double resonance (ENDOR) spectroscopic studies on one member of the radical AdoMet superfamily, the pyruvate formatelyase activating enzyme [3][4][5], and was subsequently corroborated for other members of the superfamily by ENDOR [6] and by X-ray crystallography [7][8][9][10][11][12][13] of radical AdoMet enzymes complexed to AdoMet. Second, an electron is transferred from an electron donor (in many cases, reduced flavodoxin plays this role in vivo) to the radical AdoMet cluster to reduce it to the [4Fe-4S] 1+ state, which has been shown to be the catalytically relevant state for these enzymes [14]. Third, inner-sphere electron transfer from the [4Fe-4S] 1+ cluster to AdoMet promotes the homolytic cleavage of the S-C5' bond of AdoMet to yield methionine bound to the unique iron site, and a 5'-deoxyadenosyl radical intermediate (dAdo•). The dAdo• abstracts a hydrogen atom (H•) from substrate to generate a substrate radical and 5'-deoxyadenosine (5'-dAdo). For one class of radical AdoMet enzymes, the glycyl radical enzyme activating enzymes (GRE-AEs), this is the final step in catalysis, with the glycyl radical and 5'-dAdo being products of the reaction. For most radical AdoMet enzymes, however, abstraction of H• from substrate