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

Abstract

Ribonucleotide reductases (RNRs) are the sole de novo source of deoxyribonucleotides for DNA synthesis and repair across all organisms and carry out their reaction via a radical mechanism. RNR from Chlamydia trachomatis 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 ²H-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.