Radical transfer in E. coli ribonucleotide reductase: a NH2Y731/R411A-? 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 (Y122?) in subunit ?2 to a cysteine (C439) in the active site of subunit ?2, which subsequently initiates nucleotide reduction. This oxidation occurs over 35 Å and involves a specific pathway of redox active amino acids (Y122 ? [W48?] ? Y356 in ?2 to Y731 ? Y730 ? C439 in ?2). The mechanisms of the PCET steps at the interface of the ?2?2 complex remain puzzling due to a lack of structural information for this region. Recently, DFT calculations on the 3-aminotyrosyl radical (NH2Y731?)-?2 trapped by incubation of NH2Y731-?2/?2/CDP(substrate)/ATP(allosteric effector) suggested that R411-?2, a residue close to the ?2?2 interface, interacts with NH2Y731? and accounts in part for its perturbed EPR parameters. To examine its role, we further modified NH2Y731-?2 with a R411A substitution. NH2Y731?/R411A generated upon incubation of NH2Y731/R411A-?2/?2/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 NH2Y731?/R411A relative to the NH2Y731? single mutant. Particularly, the inter-spin distance from NH2Y731?/R411A in one ?? pair to Y122? in a second ?? pair decreases by 3 Å in the presence of the R411A mutation. This is the first experimental evidence for the flexibility of pathway residue Y731-?2 in an ?2?2 complex and suggests a role for R411 in the stacked Y731/Y730 conformation involved in collinear PCET. Furthermore, NH2Y731?/R411A serves as a probe of the PCET process across the subunit interface.

SUBMITTER: Kasanmascheff M 

PROVIDER: S-EPMC5968753 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature