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 (Y₁₂₂˙) in subunit β2 to a cysteine (C₄₃₉) 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 (Y₁₂₂ ↔ [W₄₈?] ↔ Y₃₅₆ in β2 to Y₇₃₁ ↔ Y₇₃₀ ↔ C₄₃₉ 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 (NH₂Y₇₃₁˙)-α2 trapped by incubation of NH₂Y₇₃₁-α2/β2/CDP(substrate)/ATP(allosteric effector) suggested that R₄₁₁-α2, a residue close to the α2β2 interface, interacts with NH₂Y₇₃₁˙ and accounts in part for its perturbed EPR parameters. To examine its role, we further modified NH₂Y₇₃₁-α2 with a R₄₁₁A substitution. NH₂Y₇₃₁˙/R₄₁₁A generated upon incubation of NH₂Y₇₃₁/R₄₁₁A-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 NH₂Y₇₃₁˙/R₄₁₁A relative to the NH₂Y₇₃₁˙ single mutant. Particularly, the inter-spin distance from NH₂Y₇₃₁˙/R₄₁₁A in one αβ pair to Y₁₂₂˙ in a second αβ 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-α2 in an α2β2 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.