Use of 2,3,5-F[subscript 3]Y-beta 2 and 3-NH[subscript 2]Y-alpha 2 To Study Proton-Coupled Electron Transfer in Escherichia coli Ribonucleotide Reductase

Abstract

Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleoside 5′-diphosphates (NDPs) to deoxynucleotides (dNDPs). The active site for NDP reduction resides in α2, and the essential diferric-tyrosyl radical (Y[subscript 122][superscript •]) cofactor that initiates transfer of the radical to the active site cysteine in α2 (C[subscript 439]), 35 Å removed, is in β2. The oxidation is proposed to involve a hopping mechanism through aromatic amino acids (Y[subscript 122] → W[subscript 48] → Y[subscript 356] in β2 to Y[subscript 731] → Y[subscript 730] → C[subscript 439] in α2) and reversible proton-coupled electron transfer (PCET). Recently, 2,3,5-F[subscript 3]Y (F[subscript 3]Y) was site-specifically incorporated in place of Y[subscript 356] in β2 and 3-NH[subscript 2]Y (NH[subscript 2]Y) in place of Y[subscript 731] and Y[subscript 730] in α2. A pH−rate profile with F[subscript 3]Y[subscript 356-]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 F[subscript 3]Y oxidation, by residues adjacent to it in the pathway, is responsible for this change. Studies with NH[subscript 2]Y[subscript 731(730)-]α2, β2, CDP, and ATP resulted in detection of NH[subscript 2]Y radical (NH[subscript 2]Y•) intermediates capable of dNDP formation. In this study, the reaction of F[subscript 3]Y[subscript 356-]β2, α2, 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 F[subscript 3]Y[subscript 356-]β2, CDP, and ATP has also been examined with NH[subscript 2]Y[subscript 731-]α2 (or NH[subscript 2]Y[subscript 730-]α2) by SF kinetics from pH 6.5 to 9.2 and exhibited rate constants for NH[subscript 2]Y• 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.