title: 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] ⇆ Y356in β2 to Y731⇆ Y730⇆ C439in 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 Y356is in rapid exchange with solvent, in contrast to the proton from Y122. Insertion of 3,5-difluorotyrosine (F2Y) at Y356and 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 Y122and C439is ∼200 meV uphill.
authors.forename: Kanchana R
authors.initials: KR
authors.lastname: Ravichandran
authors.forename: Alexander T
authors.initials: AT
authors.lastname: Taguchi
authors.forename: Yifeng
authors.initials: Y
authors.lastname: Wei
authors.affiliation: Department of Biochemistry and Biophysics, University of Pennsylvania , Philadelphia, Pennsylvania 19104, United States.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Tommos
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University , 12 Oxford Street, Cambridge, Massachusetts 02138, United States.
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Nocera
authors.forename: JoAnne
authors.initials: J
authors.lastname: Stubbe
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D003565:Cytidine Diphosphate / Q000378:metabolism; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology*; D005609:Free Radicals / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011522:Protons; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000378:metabolism*; D012997:Solvents / Q000737:chemistry; D013696:Temperature; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry
chemical_list: D005609:Free Radicals; D011522:Protons; D012997:Solvents; C025200:3,5-difluorotyrosine; D014443:Tyrosine; D003565:Cytidine Diphosphate; D000255:Adenosine Triphosphate; D012264:Ribonucleotide Reductases