Radical transfer in E. coli ribonucleotide reductase: a NH2Y731/R411A-α mutant unmasks a new conformation of the pathway residue 731

Abstract

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 (Y122c) in subunit b2 to a cysteine (C439) in the active site of subunit a2, which subsequently initiates nucleotide reduction. This oxidation occurs over 35 °A and involves a specific pathway of redox active amino acids (Y1224[W48?]4Y356 in b2 to Y7314Y7304C439 in a2). The mechanisms of the PCET steps at the interface of the a2b2 complex remain puzzling due to a lack of structural information for this region. Recently, DFT calculations on the 3-aminotyrosyl radical (NH2Y731c)-a2 trapped by incubation of NH2Y731-a2/b2/CDP(substrate)/ATP(allosteric effector) suggested that R411-a2, a residue close to the a2b2 interface, interacts with NH2Y731c and accounts in part for its perturbed EPR parameters. To examine its role, we further modified NH2Y731-a2 with a R411A substitution. NH2Y731c/ R411A generated upon incubation of NH2Y731/R411A-a2/b2/CDP/ATP was investigated using multifrequency (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 NH2Y731c/R411A relative to the NH2Y731c single mutant. Particularly, the inter-spin distance from NH2Y731c/R411A in one ab pair to Y122c in a second ab pair decreases by 3 °A in the presence of the R411A mutation. This is the first experimental evidence for the flexibility of pathway residue Y731-a2 in an a2b2 complex and suggests a role for R411 in the stacked Y731/Y730 conformation involved in collinear PCET. Furthermore, NH2Y731c/R411A serves as a probe of the PCET process across the subunit interface.