Kinetics of radical intermediate formation and deoxynucleotide production in 3-aminotyrosine-substituted Escherichia coli ribonucleotide reductases

Escherichia coli ribonucleotide reductase is an α2β2 complex and catalyzes the conversion of nucleoside 5′-diphosphates (NDPs) to 2′-deoxynucleotides (dNDPs). The reaction is initiated by the transient oxidation of an active-site cysteine (C₄₃₉) in α2 by a stable diferric tyrosyl radical (Y₁₂₂•) cofactor in β2. This oxidation occurs by a mechanism of long-range proton-coupled electron transfer (PCET) over 35 Å through a specific pathway of residues: Y ₁₂₂•→ W₄₈→ Y₃₅₆ in β2 to Y₇₃₁→ Y₇₃₀→ C₄₃₉ in α2. To study the details of this process, 3-aminotyrosine (NH₂Y) has been site-specifically incorporated in place of Y₃₅₆ of β. The resulting protein, Y₃₅₆NH₂Y-β2, and the previously generated proteins Y₇₃₁NH₂Y-α2 and Y ₇₃₀NH₂Y-α2 (NH₂Y-RNRs) are shown to catalyze dNDP production in the presence of the second subunit, substrate (S), and allosteric effector (E) with turnover numbers of 0.2-0.7 s^-1. Evidence acquired by three different methods indicates that the catalytic activity is inherent to NH₂Y-RNRs and not the result of copurifying wt enzyme. The kinetics of formation of 3-aminotyrosyl radical (NH ₂Y•) at position 356, 731, and 730 have been measured with all S/E pairs. In all cases, NH₂Y• formation is biphasic (k _fast of 9-46 s^-1 and k_slow of 1.5-5.0 s ^-1) and kinetically competent to be an intermediate in nucleotide reduction. The slow phase is proposed to report on the conformational gating of NH₂Y• formation, while the k_cat of ∼0.5 s ^-1 is proposed to be associated with rate-limiting oxidation by NH₂Y• of the subsequent amino acid on the pathway during forward PCET. The X-ray crystal structures of Y₇₃₀NH₂Y-α2 and Y₇₃₁NH₂Y-α2 have been solved and indicate minimal structural changes relative to wt-α2. From the data, a kinetic model for PCET along the radical propagation pathway is proposed.