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Abstract

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides in all organisms. In all Class Ia RNRs, initiation of nucleotide diphosphate (NDP) reduction requires a reversible oxidation over 35 Å by a tyrosyl radical (Y₁₂₂•, Escherichia coli) in subunit β of a cysteine (C₄₃₉) in the active site of subunit α. This radical transfer (RT) occurs by a specific pathway involving redox active tyrosines (Y₁₂₂ ⇆ Y₃₅₆ in β to Y₇₃₁ ⇆ Y₇₃₀ ⇆ C₄₃₉ in α); each oxidation necessitates loss of a proton coupled to loss of an electron (PCET). To study these steps, 3-aminotyrosine was site-specifically incorporated in place of Y₃₅₆-β, Y₇₃₁- and Y₇₃₀-α, and each protein was incubated with the appropriate second subunit β(α), CDP and effector ATP to trap an amino tyrosyl radical (NH₂Y•) in the active α2β2 complex. High-frequency (263 GHz) pulse electron paramagnetic resonance (EPR) of the NH₂Y•s reported the gx values with unprecedented resolution and revealed strong electrostatic effects caused by the protein environment. ²H electron–nuclear double resonance (ENDOR) spectroscopy accompanied by quantum chemical calculations provided spectroscopic evidence for hydrogen bond interactions at the radical sites, i.e., two exchangeable H bonds to NH₂Y₇₃₀•, one to NH₂Y₇₃₁• and none to NH₂Y₃₅₆•. Similar experiments with double mutants α-NH₂Y₇₃₀/C₄₃₉A and α-NH₂Y₇₃₁/Y₇₃₀F allowed assignment of the H bonding partner(s) to a pathway residue(s) providing direct evidence for colinear PCET within α. The implications of these observations for the PCET process within α and at the interface are discussed.