Mechanistic and spectroscopic investigations of radical generation and epimerization pathways in radical SAM enzymes

Abstract

Metalloproteins are ubiquitous biocatalysts that mediate diverse chemical transformations by exploiting the redox flexibility of metal cofactors. Among these, iron-sulfur (FeS) clusters are exceptionally versatile, enabling electron transfer, radical generation, and complex bond-forming reactions. The radical S-adenosyl-L-methionine (rSAM) superfamily represents one of the largest and most functionally diverse groups of enzymes that rely on [4Fe-4S] clusters to catalyze chemically challenging reactions via radical intermediates. In these enzymes, SAM coordinates to the unique iron of the [4Fe-4S] cluster, undergoing reductive cleavage to generate the potent 5'-deoxyadenosyl radical (5'-dAdo*). The work described in this chapter first examines the electronic basis of SAM activation, highlighting the substantial thermodynamic barrier between SAM reduction and cluster redox potentials, and presents evidence implicating the Jahn-Teller effect in determining the regioselectivity of S-C bond cleavage. The latter section focuses on the biochemical and spectroscopic characterization of OpgD, a newly identified radical SAM epimerase belonging to the origamin family. EPR analysis of OpgD reveals an auxiliary [4Fe-4S] cluster (AuxI) that may participate in electron transfer or radical quenching during catalysis. Together, these studies advance our understanding of sulfonium activation and the functional diversity of auxiliary clusters in radical SAM enzymes.