Radical Transfer in Ribonucleotide Reductase: a NHY/RA-α Mutant Unmasks a New Conformation of the Pathway Residue 731

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides in all living organisms. The catalytic cycle of RNR involves a long-range proton-coupled electron transfer (PCET) from a tyrosyl radical (Y˙) in subunit β2 to a cysteine (C) in the active site of subunit α2, which subsequently initiates nucleotide reduction. This oxidation occurs over 35 Å and involves a specific pathway of redox active amino acids (Y ↔ [W?] ↔ Y in β2 to Y ↔ Y ↔ C in α2). The mechanisms of the PCET steps at the interface of the α2β2 complex remain puzzling due to a lack of structural information for this region. Recently, DFT calculations on the 3-aminotyrosyl radical (NHY˙)-α2 trapped by incubation of NHY-α2/β2/CDP(substrate)/ATP(allosteric effector) suggested that R-α2, a residue close to the α2β2 interface, interacts with NHY˙ and accounts in part for its perturbed EPR parameters. To examine its role, we further modified NHY-α2 with a RA substitution. NHY˙/RA generated upon incubation of NHY/RA-α2/β2/CDP/ATP was investigated using multi-frequency (34, 94 and 263 GHz) EPR, 34 GHz pulsed electron-electron double resonance (PELDOR) and electron-nuclear double resonance (ENDOR) spectroscopies. The data indicate a large conformational change in NHY˙/RA relative to the NHY˙ single mutant. Particularly, the inter-spin distance from NHY˙/RA in one αβ pair to Y˙ in a second αβ pair decreases by 3 Å in the presence of the RA mutation. This is the first experimental evidence for the flexibility of pathway residue Y-α2 in an α2β2 complex and suggests a role for R in the stacked Y/Y conformation involved in collinear PCET. Furthermore, NHY˙/RA serves as a probe of the PCET process across the subunit interface.