Escherichia coli class Ia ribonucleotide reductase is composed of two subunits (alpha and beta), which form an alpha 2 beta 2 complex that catalyzes the conversion of nucleoside 5'-diphosphates to deoxynucleotides (dNDPs). beta 2 contains the essential tyrosyl radical (Y-122) that generates a thiyl radical (C-439) in alpha 2 where dNDPs are made. This oxidation occurs over 35 angstrom through a pathway of amino acid radical intermediates (Y-122 -> [W-48] -> Y-356 in beta 2 to Y-731 -> Y-730 -> C-439 in alpha 2). However, chemistry is preceded by a slow protein conformational change(s) that prevents observation of these intermediates. 2,3,5-Trifluorotyrosine site-specifically inserted at position 122 of beta 2 (F3Y-beta 2) perturbs its conformation and the driving force for radical propagation, while maintaining catalytic activity (1.7 s(-1)). Rapid freeze-quench electron paramagnetic resonance spectroscopy and rapid chemical-quench analysis of the F3Y-beta 2, alpha 2, CDP, and ATP (effector) reaction show generation of 0.5 equiv of Y-356 and 0.5 equiv of dCDP, both at 30 s(-1). In the absence of an external reducing system, Y-356 reduction occurs concomitant with F3Y reoxidation (0.4 s(-1)) and subsequent to oxidation of all alpha 2s. In the presence of a reducing system, a burst of dCDP (0.4 equiv at 22 s(-1)) is observed prior to steady-state turnover (1.7 s(-1)). The [Y-356] does not change, consistent with rate-limiting F3Y reoxidation. The data support a mechanism where Y-122 is reduced and reoxidized on each turnover and demonstrate for the first time the ability of a pathway radical in an active alpha 2 beta 2 complex to complete the catalytic cycle.