The Elusive 5′-Deoxyadenosyl Radical: Captured and Characterized by Electron Paramagnetic Resonance and Electron Nuclear Double Resonance Spectroscopies

The 5'-deoxyadenosyl radical (5'-dAdo center dot) 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 center dot has eluded characterization despite efforts spanning more than a half century. Here, we report generation of 5'-dAdo center dot 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' (2p pi) radical (similar to 70% spin occupancy); the C5'(H)(2) plane is rotated by similar to 37 degrees (experiment)/39 degrees (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 phi from experiment and in vacuo DFT indicates that the conformation is intrinsic to 5-dAdo center dot itself, and not determined by its environment.