Reverse Electron Transfer Completes the Catalytic Cycle in a 2,3,5-Trifluorotyrosine-Substituted Ribonucleotide Reductase

<i>Escherichia coli</i> 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