A >200 meV Uphill Thermodynamic Landscape for Radical Transport in Escherichia coli Ribonucleotide Reductase Determined Using Fluorotyrosine-Substituted Enzymes.
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abstract
Escherichia coli class Ia ribonucleotide reductase (RNR) converts ribonucleotides to deoxynucleotides. A diferric-tyrosyl radical (Y122) in one subunit (2) generates a transient thiyl radical in another subunit (2) via long-range radical transport (RT) through aromatic amino acid residues (Y122 [W48] Y356 in 2 to Y731 Y730 C439 in 2). 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 2 containing 2,3,5-trifluorotyrosine at Y122 (F3Y122-2) with 2, CDP (substrate), and ATP (effector) has now afforded the opportunity to investigate equilibration of F3Y122 and Y356. Incubation of F3Y122-2, Y731F-2 (or Y730F-2), CDP, and ATP at different temperatures (2-37 C) provides E'(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-2, 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.
