Structural Elucidation of the Reduced Mn(III)/Fe(III) Intermediate of the Radical-Initiating Metallocofactor in Ribonucleotide Reductase.

Ribonucleotide reductases (RNRs) are the sole source of deoxyribonucleotides for DNA synthesis and repair across all organisms and carry out their reaction via a radical mechanism. RNR from generates its turnover-initiating cysteinyl radical by long-range reduction of a Mn(IV)/Fe(III) cofactor, producing a Mn(III)/Fe(III) intermediate. Herein, we characterize the protonation states of the inorganic ligands in this reduced state using advanced pulse electron paramagnetic resonance (EPR) spectroscopy and H-isotope labeling. A strongly coupled deuteron is observed by hyperfine sublevel correlation (HYSCORE) spectroscopy experiments and indicates the presence of a bridging hydroxo ligand. Isotope-dependent EPR line broadening analysis and the magnitude of the estimated Mn-Fe exchange coupling constant together suggest a μ-oxo/μ-hydroxo core. Two distinct signals detected in electron-nuclear double resonance (ENDOR) spectra are attributable to less strongly coupled hydrons of a terminal water ligand to Mn(III). Together, these experiments imply that the reduced cofactor has a mixed μ-oxo/μ-hydroxo core with a terminal water ligand on Mn(III). This structural assignment sheds light generally on the reactivity of Mn/Fe heterobimetallic sites and, more specifically, on the proton-coupling in the electron transfer that initiates ribonucleotide reduction in this subclass of RNRs.