Escherichia coli ribonucleotide reductase is an α2α2 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. [ABSTRACT FROM AUTHOR]