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

[Image: see text] 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 (Y(122)(•)) that generates a thiyl radical (C(439)(•)) in α2 where dNDPs are made. This oxidation occurs over 35 Å through a pathway of amino acid radical intermediates (Y(122) → [W(48)] → Y(356) in β2 to Y(731) → Y(730) → C(439) 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 (F(3)Y(•)-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 F(3)Y(•)-β2, α2, 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 F(3)Y 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 [Y(356)(•)] does not change, consistent with rate-limiting F(3)Y 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 α2β2 complex to complete the catalytic cycle.