title: Addition of oxygen to the diiron(II/II) cluster is the slowest step in formation of the tyrosyl radical in the W103Y variant of ribonucleotide reductase protein R2 from mouse.
abstract: Activation of O2 by the diiron(II/II) cluster in protein R2 of class I ribonucleotide reductase generates the enzyme's essential tyrosyl radical. A crucial step in this reaction is the transfer of an electron from solution to a diiron(II/II)-O2 adduct during formation of the radical-generating, diiron(III/IV) intermediate X. In the reaction of R2 from Escherichia coli, this electron injection is initiated by the rapid (>400 s-1 at 5 degrees C), transient oxidation of the near-surface residue, tryptophan 48, to a cation radical and is blocked by substitution of W48 with F, A, G, Y, L, or Q. By contrast, a study of the cognate reaction in protein R2 from mouse suggested that electron injection might be the slowest step in generation of its tyrosyl radical, Y177* [Schmidt, P. P., Rova, U., Katterle, B., Thelander, L., and Gräslund, A. (1998) J. Biol. Chem. 273, 21463-21472]. The crucial evidence was the observation that Y177* production is slowed by approximately 30-fold upon substitution of W103, the cognate of the electron-shuttling W48 in E. coli R2, with tyrosine. In this work, we have applied stopped-flow absorption and freeze-quench electron paramagnetic resonance and Mössbauer spectroscopies to the mouse R2 reaction to evaluate the possibility that an already sluggish electron-transfer step is slowed by 30-fold by substitution of this key residue. The drastically reduced accumulation of cluster X, failure of precursors to the intermediate to accumulate, and, most importantly, first-order dependence of the rate of Y177* formation on the concentration of O2 prove that addition of O2 to the diiron(II/II) cluster, rather than electron injection, is the slowest step in the R2-W103Y reaction. This finding indicates that the basis for the slowing of Y177* formation by the W103Y substitution is an unexpected secondary effect on the structure or dynamics of the protein, its diiron(II/II) cluster, or both rather than the expected chemical effect on the electron injection step.
authors.affiliation: Department of Biochemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
authors.forename: Danny
authors.initials: D
authors.lastname: Yun
authors.forename: Lana
authors.initials: L
authors.lastname: Saleh
authors.forename: Ricardo
authors.initials: R
authors.lastname: García-Serres
authors.forename: Brandon M
authors.initials: BM
authors.lastname: Chicalese
authors.forename: Young H
authors.initials: YH
authors.lastname: An
authors.forename: Boi Hanh
authors.initials: BH
authors.lastname: Huynh
authors.forename: J Martin
authors.initials: JM
authors.lastname: Bollinger
mesh_terms: D000818:Animals; D004578:Electron Spin Resonance Spectroscopy; D005296:Ferrous Compounds / Q000737:chemistry*; D005609:Free Radicals / Q000138:chemical synthesis*; D007700:Kinetics; D051379:Mice; D010100:Oxygen / Q000737:chemistry*; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000235:genetics; D015204:Spectroscopy, Mossbauer; D014443:Tyrosine / Q000031:analogs & derivatives
chemical_list: D005296:Ferrous Compounds; D005609:Free Radicals; D014443:Tyrosine; D012264:Ribonucleotide Reductases; C108427:ribonucleotide reductase R2 subunit; D010100:Oxygen