Escherichia coli class Ia ribonucleotide reductase (RNR) converts ribonucleotides to deoxynucleotides. A diferric-tyrosyl radical (Y-122 center dot) in one subunit (beta 2) generates a transient thiyl radical in another subunit (alpha 2) via long-range radical transport (RT) through aromatic amino acid residues (Y-122 reversible arrow [W-48] reversible arrow Y-356 in beta 2 to Y-731 reversible arrow Y-730 reversible arrow C-439 in alpha 2). Equilibration of Y-356 center dot, Y-731 center dot, and Y-730 center dot was recently observed using site specifically incorporated unnatural tyrosine analogs; however, equilibration between Y-122 center dot and Y-356 center dot has not been detected. Our recent report of Y-356 center dot formation in a kinetically and chemically competent fashion in the reaction of beta 2 containing 2,3,5-trifluorotyrosine at Y-122 (F3Y122 center dot-beta 2) with alpha 2, CDP (substrate), and ATP (effector) has now afforded the opportunity to investigate equilibration of F3Y122 center dot and Y-356 center dot. Incubation of F3Y122 center dot-beta 2, Y731F-alpha 2 (or Y730F-alpha 2), CDP, and ATP at different temperatures (2-37 degrees C) provides Delta E degrees'(F3Y122 center dot-Y-356 center dot) of 20 +/- 10 mV at 25 degrees C. The pH dependence of the F3Y122 center dot reversible arrow Y356 center dot interconversion (pH 6.88.0) reveals that the proton from Y-356 is in rapid exchange with solvent, in contrast to the proton from Y-122. Insertion of 3,5-difluorotyrosine (F2Y) at Y-356 and rapid freeze-quench EPR analysis of its reaction with Y731F-alpha 2, CDP, and ATP at pH 8.2 and 25 degrees C shows F2Y356 center dot generation by the native Y-122 center dot. FnY-RNRs (n = 2 and 3) together provide a model for the thermodynamic landscape of the RT pathway in which the reaction between Y-122 and C-439 is similar to 200 meV uphill.