Escherichia coli ribonucleotide reductase is an alpha 2 beta 2 complex that catalyzes the conversion of nucleotides to deoxynucleotides and requires a diferric-tyrosyl radical (Y-center dot) cofactor to initiate catalysis. The initiation process requires long-range proton-coupled electron transfer (PCET) over 35 angstrom between the two subunits by a specific pathway (Y-122(center dot)-> W-48 -> Y-356 within beta to Y-731 -> Y-730 -> C-439 within alpha). 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 Y-122 in beta 2. The protein as isolated contained a diferric cluster but no nitrotyrosyl radical (NO2Y center dot) and was inactive. In the present paper we show that incubation of apo-Y122NO2Y-beta 2 with Fe2+ and O-2 generates a diferric-NO2Y center dot that has a half-life of 40 s at 25 degrees C. Sequential mixing experiments, in which the cofactor is assembled to 1.2 NO2Y center dot/beta 2 and then mixed with alpha 2, 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 center dot 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-center dot is produced at 97 s(-1). Studies with pathway mutants suggest that the new Y-center dot is predominantly located at 356 in beta 2. In consideration of these data and the crystal structure of Y122NO2Y-beta 2, a mechanism for PCET uncoupling in NO2Y center dot-RNR is proposed.