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

Authors

Hao Yang, Elizabeth C McDaniel, Stella Impano, Amanda S. Byer, Richard J Jodts, Kenichi Yokoyama, William E Broderick, Joan B Broderick, Brian M Hoffman

Publication

Journal of the American Chemical Society

Abstract

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? (2p?) 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.

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