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

Abstract

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 (C[subscript 439]) in α2 by a stable diferric tyrosyl radical (Y[subscript 122]•) 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[subscript 122]•→ W[subscript 48]→ Y[subscript 356] in β2 to Y[subscript 731]→ Y[subscript 730]→ C[subscript 439] in α2. To study the details of this process, 3-aminotyrosine (NH[subscript 2]Y) has been site-specifically incorporated in place of Y[subscript 356] of β. The resulting protein, Y[subscript 356]NH[subscript 2]Y-β2, and the previously generated proteins Y[subscript 731]NH[subscript 2]Y-α2 and Y[subscript 730]NH[subscript 2]Y-α2 (NH[subscript 2]Y-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[superscript –1]. Evidence acquired by three different methods indicates that the catalytic activity is inherent to NH[subscript 2]Y-RNRs and not the result of copurifying wt enzyme. The kinetics of formation of 3-aminotyrosyl radical (NH[subscript 2]Y•) at position 356, 731, and 730 have been measured with all S/E pairs. In all cases, NH[subscript 2]Y• formation is biphasic (k[subscript fast] of 9–46 s[superscript –1] and k[subscript slow] of 1.5–5.0 s[subscript –1]) and kinetically competent to be an intermediate in nucleotide reduction. The slow phase is proposed to report on the conformational gating of NH[subscript 2]Y• formation, while the k[subscript cat] of 0.5 s[superscript –1] is proposed to be associated with rate-limiting oxidation by NH[subscript 2]Y• of the subsequent amino acid on the pathway during forward PCET. The X-ray crystal structures of Y[subscript 730]NH[subscript 2]Y-α2 and Y[subscript 731]NH[subscript 2]Y-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.