A >200 meV Uphill Thermodynamic Landscape for Radical Transport in Escherichia coli Ribonucleotide Reductase Determined Using Fluorotyrosine-Substituted Enzymes

[Image: see text] Escherichia coli class Ia ribonucleotide reductase (RNR) converts ribonucleotides to deoxynucleotides. A diferric-tyrosyl radical (Y(122)•) in one subunit (β2) generates a transient thiyl radical in another subunit (α2) via long-range radical transport (RT) through aromatic amino acid residues (Y(122) ⇆ [W(48)] ⇆ Y(356) in β2 to Y(731) ⇆ Y(730) ⇆ C(439) in α2). Equilibration of Y(356)•, Y(731)•, and Y(730)• was recently observed using site specifically incorporated unnatural tyrosine analogs; however, equilibration between Y(122)• and Y(356)• has not been detected. Our recent report of Y(356)• formation in a kinetically and chemically competent fashion in the reaction of β2 containing 2,3,5-trifluorotyrosine at Y(122) (F(3)Y(122)•-β2) with α2, CDP (substrate), and ATP (effector) has now afforded the opportunity to investigate equilibration of F(3)Y(122)• and Y(356)•. Incubation of F(3)Y(122)•-β2, Y(731)F-α2 (or Y(730)F-α2), CDP, and ATP at different temperatures (2–37 °C) provides ΔE°′(F(3)Y(122)•–Y(356)•) of 20 ± 10 mV at 25 °C. The pH dependence of the F(3)Y(122)• ⇆ Y(356)• interconversion (pH 6.8–8.0) 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 (F(2)Y) at Y(356) and rapid freeze-quench EPR analysis of its reaction with Y(731)F-α2, CDP, and ATP at pH 8.2 and 25 °C shows F(2)Y(356)• generation by the native Y(122)•. F(n)Y-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 ∼200 meV uphill.