title: Use of 2,3,5-F(3)Y-β2 and 3-NH(2)Y-α2 to study proton-coupled electron transfer in Escherichia coli ribonucleotide reductase.
abstract: Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleoside 5'-diphosphates (NDPs) to deoxynucleotides (dNDPs). The active site for NDP reduction resides in α2, and the essential diferric-tyrosyl radical (Y(122)(•)) cofactor that initiates transfer of the radical to the active site cysteine in α2 (C(439)), 35 Å removed, is in β2. The oxidation is proposed to involve a hopping mechanism through aromatic amino acids (Y(122) → W(48) → Y(356) in β2 to Y(731) → Y(730) → C(439) in α2) and reversible proton-coupled electron transfer (PCET). Recently, 2,3,5-F(3)Y (F(3)Y) was site-specifically incorporated in place of Y(356) in β2 and 3-NH(2)Y (NH(2)Y) in place of Y(731) and Y(730) in α2. A pH-rate profile with F(3)Y(356)-β2 suggested that as the pH is elevated, the rate-determining step of RNR can be altered from a conformational change to PCET and that the altered driving force for F(3)Y oxidation, by residues adjacent to it in the pathway, is responsible for this change. Studies with NH(2)Y(731(730))-α2, β2, CDP, and ATP resulted in detection of NH(2)Y radical (NH(2)Y(•)) intermediates capable of dNDP formation. In this study, the reaction of F(3)Y(356)-β2, α2, CDP, and ATP has been examined by stopped-flow (SF) absorption and rapid freeze quench electron paramagnetic resonance spectroscopy and has failed to reveal any radical intermediates. The reaction of F(3)Y(356)-β2, CDP, and ATP has also been examined with NH(2)Y(731)-α2 (or NH(2)Y(730)-α2) by SF kinetics from pH 6.5 to 9.2 and exhibited rate constants for NH(2)Y(•) formation that support a change in the rate-limiting step at elevated pH. The results together with kinetic simulations provide a guide for future studies to detect radical intermediates in the pathway.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139-4307, USA.
authors.forename: Mohammad R
authors.initials: MR
authors.lastname: Seyedsayamdost
authors.forename: Cyril S
authors.initials: CS
authors.lastname: Yee
authors.forename: JoAnne
authors.initials: J
authors.lastname: Stubbe
mesh_terms: D000042:Absorption; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D020134:Catalytic Domain; D002417:Cattle; D003565:Cytidine Diphosphate / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; 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*
chemical_list: D005609:Free Radicals; D011522:Protons; D003565:Cytidine Diphosphate; D000255:Adenosine Triphosphate; D012264:Ribonucleotide Reductases