The Elusive 5′-Deoxyadenosyl Radical: Captured and Characterized by EPR and ENDOR Spectroscopies

The 5'-deoxyadenosyl radical (5'-dAdo•) abstracts a substrate H-atom as the first step in radical-based transformations catalyzed by adenosylcobalamin-dependent and radical S-adenosyl-l-methionine (RS) enzymes. Notwithstanding its central biological role, 5'-dAdo• has eluded characterization despite efforts spanning more than a half-century. Here we report generation of 5'-dAdo• in a RS enzyme active site at 12 K, using a novel approach involving cryogenic photoinduced electron transfer from the [4Fe-4S]⁺cluster to the coordinated S-adenosylmethionine (SAM) to induce homolytic S-C' bond cleavage.