Escherichia coil ribonucleotide reductase is an alpha 2 beta 2 complex that catalyzes the conversion of nucleotides to deoxynucleotides using a diferric tyrosyl radical (Y-122(center dot)) cofactor in beta 2 to initiate catalysis in alpha 2. Each turnover requires reversible long-range proton-coupled electron transfer (PCET) over 35 angstrom between the two subunits by a specific pathway (Y-122(center dot) reversible arrow [W-48?] reversible arrow Y-356 within beta to Y-731 reversible arrow Y-730 reversible arrow C-439 within alpha). Previously, we reported that beta 2 mutant with 3-nitrotyrosyl radical (NO2Y.; 1.2 radicals/beta 2) in place of Y-122. in the presence of alpha 2, CDP, and ATP catalyzes formation of 0.6 equiv of dCDP and accumulates 0.6 equiv of a new Y. proposed to be located on Y-356 in beta 2. We now report three independent methods that establish that Y-356 is the predominant location (85-90%) of the radical, with the remaining 10-15% delocalized onto Y-731 and Y-730 in alpha 2. Pulsed electron-electron double-resonance spectroscopy on samples prepared by rapid freeze quench (RFQ) methods identified three distances: 30 +/- 0.4 angstrom (88% +/- 3%) and 33 +/- 0.4 and 38 +/- 0.5 angstrom (12% +/- 3%) indicative of NO2Y122 center dot,-Y-356(center dot), NO2Y122 center dot, and NO2Y122 center dot-Y-731(730)(center dot), respectively. Radical distribution in alpha 2 was supported by RFQelectron paramagnetic resonance (EPR) studies usinig Y-731(3,5-F2Y) or Y-730(3,5- F2Y)-alpha 2, which revealed F2Y center dot, studies using globally incorporated [beta-H-2(2)]Y-alpha 2, and analysis using parameters obtained from 140 GHz EPR spectroscopy. The amount of Y. delocalized in alpha 2 from these two studies varied from 6% to 15%. The studies together give the first insight into the relative redox potentials of the three transient Y. radicals in the PCET pathway and their conformations.