Radical transfer in E. coli ribonucleotide reductase: a NH(2)Y(731)/R(411)A-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 E. coli RNR involves a long-range proton-coupled electron transfer (PCET) from a tyrosyl radical (Y(122)˙) in subunit β2 to a cysteine (C(439)) 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(122) ↔ [W(48)?] ↔ Y(356) in β2 to Y(731) ↔ Y(730) ↔ C(439) 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 (NH(2)Y(731)˙)-α2 trapped by incubation of NH(2)Y(731)-α2/β2/CDP(substrate)/ATP(allosteric effector) suggested that R(411)-α2, a residue close to the α2β2 interface, interacts with NH(2)Y(731)˙ and accounts in part for its perturbed EPR parameters. To examine its role, we further modified NH(2)Y(731)-α2 with a R(411)A substitution. NH(2)Y(731)˙/R(411)A generated upon incubation of NH(2)Y(731)/R(411)A-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 NH(2)Y(731)˙/R(411)A relative to the NH(2)Y(731)˙ single mutant. Particularly, the inter-spin distance from NH(2)Y(731)˙/R(411)A in one αβ pair to Y(122)˙ in a second αβ pair decreases by 3 Å in the presence of the R(411)A mutation. This is the first experimental evidence for the flexibility of pathway residue Y(731)-α2 in an α2β2 complex and suggests a role for R(411) in the stacked Y(731)/Y(730) conformation involved in collinear PCET. Furthermore, NH(2)Y(731)˙/R(411)A serves as a probe of the PCET process across the subunit interface.