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

Escherichia coli class Ia ribonucleotide reductase (RNR) converts ribonucleotides to deoxynucleotides. A diferric-tyrosyl radical (Y•) 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 ⇆ [W] ⇆ Y in β2 to Y ⇆ Y ⇆ C in α2). Equilibration of Y•, Y•, and Y• was recently observed using site specifically incorporated unnatural tyrosine analogs; however, equilibration between Y• and Y• has not been detected. Our recent report of Y• formation in a kinetically and chemically competent fashion in the reaction of β2 containing 2,3,5-trifluorotyrosine at Y (FY•-β2) with α2, CDP (substrate), and ATP (effector) has now afforded the opportunity to investigate equilibration of FY• and Y•. Incubation of FY•-β2, YF-α2 (or YF-α2), CDP, and ATP at different temperatures (2-37 °C) provides ΔE°'(FY•-Y•) of 20 ± 10 mV at 25 °C. The pH dependence of the FY• ⇆ Y• interconversion (pH 6.8-8.0) reveals that the proton from Y is in rapid exchange with solvent, in contrast to the proton from Y. Insertion of 3,5-difluorotyrosine (FY) at Y and rapid freeze-quench EPR analysis of its reaction with YF-α2, CDP, and ATP at pH 8.2 and 25 °C shows FY• generation by the native Y•. FY-RNRs (n = 2 and 3) together provide a model for the thermodynamic landscape of the RT pathway in which the reaction between Y and C is ∼200 meV uphill.