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

Abstract

Escherichia coli class Ia ribonucleotide reductase (RNR) converts ribonucleotides to deoxynucleotides. A diferric-tyrosyl radical (Y122•) in one subunit (β2) generates a transient thiyl radical in another subunit (α2) via long-range radical transport (RT) through aromatic amino acid residues (Y122 ⇆ [W48] ⇆ Y356 in β2 to Y731 ⇆ Y730 ⇆ C439 in α2). Equilibration of Y356•, Y731•, and Y730• was recently observed using site specifically incorporated unnatural tyrosine analogs; however, equilibration between Y122• and Y356• has not been detected. Our recent report of Y356• formation in a kinetically and chemically competent fashion in the reaction of β2 containing 2,3,5-trifluorotyrosine at Y122 (F3Y122•-β2) with α2, CDP (substrate), and ATP (effector) has now afforded the opportunity to investigate equilibration of F3Y122• and Y356•. Incubation of F3Y122•-β2, Y731F-α2 (or Y730F-α2), CDP, and ATP at different temperatures (2–37 °C) provides ΔE°′(F3Y122•–Y356•) of 20 ± 10 mV at 25 °C. The pH dependence of the F3Y122• ⇆ Y356• interconversion (pH 6.8–8.0) reveals that the proton from Y356 is in rapid exchange with solvent, in contrast to the proton from Y122. Insertion of 3,5-difluorotyrosine (F2Y) at Y356 and rapid freeze-quench EPR analysis of its reaction with Y731F-α2, CDP, and ATP at pH 8.2 and 25 °C shows F2Y356• generation by the native Y122•. FnY-RNRs (n = 2 and 3) together provide a model for the thermodynamic landscape of the RT pathway in which the reaction between Y122 and C439 is ∼200 meV uphill.

Formal Reduction Potentials of Difluorotyrosine and Trifluorotyrosine Protein Residues: Defining the Thermodynamics of Multistep Radical Transfer

“…62,63 Taken together these Δ E °’s sketch out the thermodynamic landscape associated with the intersubunit RT pathway in E. coli RNR (Figure S8). 63 …”

“…The modeled potentials are pH dependent since the proton released upon Y 356 oxidation is in rapid exchange with the bulk solvent. 63 The p K a values are based on the 0.4 increase in the p K a of NO 2 Y 356 . 65 The modeling of site 122 was more involved and is described in a stepwise manner below and in Figure 8B.…”

Properties of Site-Specifically Incorporated 3-Aminotyrosine in Proteins To Study Redox-Active Tyrosines: Escherichia coli Ribonucleotide Reductase as a Paradigm

“…The dCDP formation would occur 10 2 to 10 4 -fold faster than wt-RNR turnover (~5 s −1 ) which is conformationally gated. 35 …”

“…16, 35, 36 Based on measurements on aqueous amino acids, 4, 17, 37 incorporation of NH 2 Y at pathway Y sites was proposed to introduce a thermodynamic sink of −190 to −290 mV. 12, 13 …”

Photochemical Rescue of a Conformationally Inactivated Ribonucleotide Reductase

“…These results suggest that the primary role of E 350 is not as a base during either Y 356 [β] or Y 731 [α] oxidation, a conclusion corroborated by other studies. 20,34,35,36 Notwithstanding, the role of E 350 is obscured by our inability to induce RT such that pathway residue oxidation “downstream” from Y 122 (Y 356 [β], Y 731 [α], Y 730 [α], or C 439 [α]) may be examined. Thus, to address the functional implications of E 350 on the RT pathway, novel tools for generating radicals downstream from Y 122 are required.…”