Use of 2,3,5-F₃Y-α2 and 3-NH₂Y-α2 to study proton-coupled electron transfer in escherichia coli ribonucleotide reductase

Escherichia coli ribonucleotide reductase is an β2β2 complex that catalyzes the conversion of nucleoside 5′-diphosphates (NDPs) to deoxynucleotides (dNDPs). The active site for NDP reduction resides in β2, and the essential diferric-tyrosyl radical (Y₁₂₂^•) cofactor that initiates transfer of the radical to the active site cysteine in β2 (C₄₃₉), 35 Å removed, is in β2. The oxidation is proposed to involve a hopping mechanism through aromatic amino acids (Y ₁₂₂ → W₄₈ → Y₃₅₆ in β2 to Y ₇₃₁ → Y₇₃₀ → C₄₃₉ in β2) and reversible proton-coupled electron transfer (PCET). Recently, 2,3,5-F ₃Y (F₃Y) was site-specifically incorporated in place of Y₃₅₆ in β2 and 3-NH₂Y (NH₂Y) in place of Y₇₃₁ and Y₇₃₀ in β2. A pH-rate profile with F ₃Y₃₅₆-β2 suggested that as the pH is elevated, the rate-determining step of RNR can be altered from a conformational change to PCET and that the altered driving force for F₃Y oxidation, by residues adjacent to it in the pathway, is responsible for this change. Studies with NH₂Y_731(730)-β2, β2, CDP, and ATP resulted in detection of NH₂Y radical (NH₂Y^•) intermediates capable of dNDP formation. In this study, the reaction of F ₃Y₃₅₆-β2, β2, CDP, and ATP has been examined by stopped-flow (SF) absorption and rapid freeze quench electron paramagnetic resonance spectroscopy and has failed to reveal any radical intermediates. The reaction of F₃Y₃₅₆-β2, CDP, and ATP has also been examined with NH₂Y₇₃₁-β2 (or NH₂Y ₇₃₀-β2) by SF kinetics from pH 6.5 to 9.2 and exhibited rate constants for NH₂Y^• formation that support a change in the rate-limiting step at elevated pH. The results together with kinetic simulations provide a guide for future studies to detect radical intermediates in the pathway.