Escherichia coli class Ia ribonucleotide reductase is composed of two subunits (α and β), which form an α2β2 complex that catalyzes the conversion of nucleoside 5'-diphosphates to deoxynucleotides (dNDPs). β2 contains the essential tyrosyl radical (Y122(•)) that generates a thiyl radical (C439(•)) in α2 where dNDPs are made. This oxidation occurs over 35 Å through a pathway of amino acid radical intermediates (Y122 → [W48] → Y356 in β2 to Y731 → Y730 → C439 in α2). 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 β2 (F3Y(•)-β2) 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(•)-β2, α2, CDP, and ATP (effector) reaction show generation of 0.5 equiv of Y356(•) and 0.5 equiv of dCDP, both at 30 s(-1). In the absence of an external reducing system, Y356(•) reduction occurs concomitant with F3Y reoxidation (0.4 s(-1)) and subsequent to oxidation of all α2s. 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 [Y356(•)] does not change, consistent with rate-limiting F3Y reoxidation. The data support a mechanism where Y122(•) is reduced and reoxidized on each turnover and demonstrate for the first time the ability of a pathway radical in an active α2β2 complex to complete the catalytic cycle.