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

Danny Yun, Lana Saleh, Ricardo Garcia-Serres, Brandon M. Chicalese, Young H. An, Hanh Huynh Boi, Joseph M. Bollinger, Jr.

Research output: Contribution to journalArticle

4 Citations (Scopus)

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°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 ∼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.

Original languageEnglish (US)
Pages (from-to)13067-13073
Number of pages7
JournalBiochemistry
Volume46
Issue number45
DOIs
StatePublished - Nov 13 2007

Fingerprint

Ribonucleotide Reductases
Electron injection
Substitution reactions
Electrons
Oxygen
Escherichia coli
Proteins
Injections
Tryptophan
Paramagnetic resonance
Tyrosine
Cations
Chemical activation
Spectroscopy
Electron Spin Resonance Spectroscopy
Oxidation
Enzymes
Spectrum Analysis

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Yun, Danny ; Saleh, Lana ; Garcia-Serres, Ricardo ; Chicalese, Brandon M. ; An, Young H. ; Boi, Hanh Huynh ; Bollinger, Jr., Joseph M. / 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. In: Biochemistry. 2007 ; Vol. 46, No. 45. pp. 13067-13073.
@article{d05c6ec65f254296a2114d8377ba4ffc,
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°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 Gr{\"a}slund, A. (1998) J. Biol. Chem. 273, 21463-21472]. The crucial evidence was the observation that Y177• production is slowed by ∼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 M{\"o}ssbauer 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.",
author = "Danny Yun and Lana Saleh and Ricardo Garcia-Serres and Chicalese, {Brandon M.} and An, {Young H.} and Boi, {Hanh Huynh} and {Bollinger, Jr.}, {Joseph M.}",
year = "2007",
month = "11",
day = "13",
doi = "10.1021/bi7003747",
language = "English (US)",
volume = "46",
pages = "13067--13073",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "45",

}

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. / Yun, Danny; Saleh, Lana; Garcia-Serres, Ricardo; Chicalese, Brandon M.; An, Young H.; Boi, Hanh Huynh; Bollinger, Jr., Joseph M.

In: Biochemistry, Vol. 46, No. 45, 13.11.2007, p. 13067-13073.

Research output: Contribution to journalArticle

TY - JOUR

T1 - 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

AU - Yun, Danny

AU - Saleh, Lana

AU - Garcia-Serres, Ricardo

AU - Chicalese, Brandon M.

AU - An, Young H.

AU - Boi, Hanh Huynh

AU - Bollinger, Jr., Joseph M.

PY - 2007/11/13

Y1 - 2007/11/13

N2 - 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°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 ∼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.

AB - 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°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 ∼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.

UR - http://www.scopus.com/inward/record.url?scp=36049009113&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=36049009113&partnerID=8YFLogxK

U2 - 10.1021/bi7003747

DO - 10.1021/bi7003747

M3 - Article

C2 - 17941645

AN - SCOPUS:36049009113

VL - 46

SP - 13067

EP - 13073

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 45

ER -