The Elusive 5′-Deoxyadenosyl Radical: Captured and Characterized by Electron Paramagnetic Resonance and Electron Nuclear Double Resonance 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-C5′ bond cleavage. We unequivocally reveal the structure of this long-sought radical species through the use of electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies with isotopic labeling, complemented by density-functional computations: a planar C5′ (2pπ) radical (∼70% spin occupancy); the C5′(H)₂ plane is rotated by ∼37° (experiment)/39° (DFT) relative to the C5′-C4′-(C4′-H) plane, placing a C5′-H antiperiplanar to the ribose-ring oxygen, which helps stabilize the radical against elimination of the 4′-H. The agreement between from experiment and in vacuo DFT indicates that the conformation is intrinsic to 5-dAdo· itself, and not determined by its environment.