A Hot Oxidant, 3-NO2Y122 Radical, Unmasks Conformational Gating in Ribonucleotide Reductase

摘要

Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides and requires a diferric-tyrosyl radical (Y•) cofactor to initiate catalysis. The initiation process requires long-range proton-coupled electron transfer (PCET) over 35 Å between the two subunits by a specific pathway (Y₁₂₂•→W₄₈→Y₃₅₆ within β to Y₇₃₁→Y₇₃₀→C₄₃₉ within α). The rate-limiting step in nucleotide reduction is the conformational gating of the PCET process, which masks the chemistry of radical propagation. 3-Nitrotyrosine (NO₂Y) has recently been incorporated site-specifically in place of Y₁₂₂ in β2. The protein as isolated contained a diferric cluster but no nitrotyrosyl radical (NO₂Y•) and was inactive. In the present paper we show that incubation of apo-Y₁₂₂NO₂Y-β2 with Fe2+ and O₂ generates a diferric-NO₂Y• that has a half-life of 40 s at 25 °C. Sequential mixing experiments, in which the cofactor is assembled to 1.2 NO₂Y•/β2 and then mixed with α2, CDP, and ATP, have been analyzed by stopped-flow absorption spectroscopy, rapid freeze quench EPR spectroscopy, and rapid chemical quench methods. These studies have, for the first time, unmasked the conformational gating. They reveal that the NO₂Y• is reduced to the nitrotyrosinate with biphasic kinetics (283 and 67 s−1), that dCDP is produced at 107 s−1, and that a new Y• is produced at 97 s−1. Studies with pathway mutants suggest that the new Y• is predominantly located at 356 in β2. In consideration of these data and the crystal structure of Y₁₂₂NO₂Y-β2, a mechanism for PCET uncoupling in NO₂Y•-RNR is proposed.