Facile electron transfer during formation of cluster X and kinetic competence of X for tyrosyl radical production in protein R2 of ribonucleotide reductase from mouse

Danny Yun, Carsten Krebs, Govind P. Gupta, David F. Iwig, Boi Hanh Huynh, J. Martin Bollinger

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

The kinetics and mechanism of formation of the tyrosyl radical and μ-(oxo)diiron(III) cluster in the R2 subunit of ribonucleotide reductase from mouse have been examined by stopped-flow absorption and freeze-quench electron paramagnetic resonance and Mössbauer spectroscopies. The reaction comprises (1) acquisition of Fe(II) ions by the R2 apo protein, (2) activation of dioxygen at the resulting carboxylate-bridged diiron(II) cluster to form oxidized intermediate diiron species, and (3) univalent oxidation of Y177 by one of these intermediates to form the stable radical, with concomitant or subsequent formation of the adjacent μ-(oxo)diiron(III) cluster. The data establish that an oxidized diiron intermediate spectroscopically similar to the well-characterized, formally Fe(III)Fe(IV) cluster X from the reaction of the Escherichia coli R2 protein precedes the Y177 radical in the reaction sequence and is probably the Y177 oxidant. As formation of the X intermediate (1) requires transfer of an "extra" reducing equivalent to the buried diiron cluster following the addition of dioxygen and (2) is observed to be rapid relative to other steps in the reaction, the present data indicate that the transfer of this reducing equivalent is not rate-limiting for Y177 radical formation, in contrast to what was previously proposed (Schmidt, P. P., Rova, U., Katterle, B., Thelander, L., and Gräslund, A. (1998) J. Biol. Chem. 273, 21463-21472). Indeed, the formation of X (kobs = 13 ± 3 s-1 at 5 °C and 0.95 mM O2) and the decay of the intermediate to give the Y177 radical (kobs = 5 ± 2 s-1) are both considerably faster than the formation of the reactive Fe(II)-R2 complex from the apo protein and Fe(II)aq (kobs = 0.29 ± 0.03 s-1), which is the slowest step overall. The conclusions that cluster X is an intermediate in Y177 radical formation and that transfer of the reducing equivalent is relatively facile imply that the mouse R2 and E. coli R2 reactions are mechanistically similar.

Original languageEnglish (US)
Pages (from-to)981-990
Number of pages10
JournalBiochemistry
Volume41
Issue number3
DOIs
StatePublished - Jan 22 2002

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Ribonucleotide Reductases
Mental Competency
Electrons
Oxygen
Escherichia coli
Kinetics
Escherichia coli Proteins
Electron Spin Resonance Spectroscopy
Oxidants
Spectrum Analysis
Proteins
Ions
Paramagnetic resonance
Chemical activation
Spectroscopy
Oxidation
ribonucleotide reductase R2 subunit

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

@article{99e8bd8bc2974859bfe505195d850f22,
title = "Facile electron transfer during formation of cluster X and kinetic competence of X for tyrosyl radical production in protein R2 of ribonucleotide reductase from mouse",
abstract = "The kinetics and mechanism of formation of the tyrosyl radical and μ-(oxo)diiron(III) cluster in the R2 subunit of ribonucleotide reductase from mouse have been examined by stopped-flow absorption and freeze-quench electron paramagnetic resonance and M{\"o}ssbauer spectroscopies. The reaction comprises (1) acquisition of Fe(II) ions by the R2 apo protein, (2) activation of dioxygen at the resulting carboxylate-bridged diiron(II) cluster to form oxidized intermediate diiron species, and (3) univalent oxidation of Y177 by one of these intermediates to form the stable radical, with concomitant or subsequent formation of the adjacent μ-(oxo)diiron(III) cluster. The data establish that an oxidized diiron intermediate spectroscopically similar to the well-characterized, formally Fe(III)Fe(IV) cluster X from the reaction of the Escherichia coli R2 protein precedes the Y177 radical in the reaction sequence and is probably the Y177 oxidant. As formation of the X intermediate (1) requires transfer of an {"}extra{"} reducing equivalent to the buried diiron cluster following the addition of dioxygen and (2) is observed to be rapid relative to other steps in the reaction, the present data indicate that the transfer of this reducing equivalent is not rate-limiting for Y177 radical formation, in contrast to what was previously proposed (Schmidt, P. P., Rova, U., Katterle, B., Thelander, L., and Gr{\"a}slund, A. (1998) J. Biol. Chem. 273, 21463-21472). Indeed, the formation of X (kobs = 13 ± 3 s-1 at 5 °C and 0.95 mM O2) and the decay of the intermediate to give the Y177 radical (kobs = 5 ± 2 s-1) are both considerably faster than the formation of the reactive Fe(II)-R2 complex from the apo protein and Fe(II)aq (kobs = 0.29 ± 0.03 s-1), which is the slowest step overall. The conclusions that cluster X is an intermediate in Y177 radical formation and that transfer of the reducing equivalent is relatively facile imply that the mouse R2 and E. coli R2 reactions are mechanistically similar.",
author = "Danny Yun and Carsten Krebs and Gupta, {Govind P.} and Iwig, {David F.} and Huynh, {Boi Hanh} and Bollinger, {J. Martin}",
year = "2002",
month = "1",
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doi = "10.1021/bi011797p",
language = "English (US)",
volume = "41",
pages = "981--990",
journal = "Biochemistry",
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Facile electron transfer during formation of cluster X and kinetic competence of X for tyrosyl radical production in protein R2 of ribonucleotide reductase from mouse. / Yun, Danny; Krebs, Carsten; Gupta, Govind P.; Iwig, David F.; Huynh, Boi Hanh; Bollinger, J. Martin.

In: Biochemistry, Vol. 41, No. 3, 22.01.2002, p. 981-990.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Facile electron transfer during formation of cluster X and kinetic competence of X for tyrosyl radical production in protein R2 of ribonucleotide reductase from mouse

AU - Yun, Danny

AU - Krebs, Carsten

AU - Gupta, Govind P.

AU - Iwig, David F.

AU - Huynh, Boi Hanh

AU - Bollinger, J. Martin

PY - 2002/1/22

Y1 - 2002/1/22

N2 - The kinetics and mechanism of formation of the tyrosyl radical and μ-(oxo)diiron(III) cluster in the R2 subunit of ribonucleotide reductase from mouse have been examined by stopped-flow absorption and freeze-quench electron paramagnetic resonance and Mössbauer spectroscopies. The reaction comprises (1) acquisition of Fe(II) ions by the R2 apo protein, (2) activation of dioxygen at the resulting carboxylate-bridged diiron(II) cluster to form oxidized intermediate diiron species, and (3) univalent oxidation of Y177 by one of these intermediates to form the stable radical, with concomitant or subsequent formation of the adjacent μ-(oxo)diiron(III) cluster. The data establish that an oxidized diiron intermediate spectroscopically similar to the well-characterized, formally Fe(III)Fe(IV) cluster X from the reaction of the Escherichia coli R2 protein precedes the Y177 radical in the reaction sequence and is probably the Y177 oxidant. As formation of the X intermediate (1) requires transfer of an "extra" reducing equivalent to the buried diiron cluster following the addition of dioxygen and (2) is observed to be rapid relative to other steps in the reaction, the present data indicate that the transfer of this reducing equivalent is not rate-limiting for Y177 radical formation, in contrast to what was previously proposed (Schmidt, P. P., Rova, U., Katterle, B., Thelander, L., and Gräslund, A. (1998) J. Biol. Chem. 273, 21463-21472). Indeed, the formation of X (kobs = 13 ± 3 s-1 at 5 °C and 0.95 mM O2) and the decay of the intermediate to give the Y177 radical (kobs = 5 ± 2 s-1) are both considerably faster than the formation of the reactive Fe(II)-R2 complex from the apo protein and Fe(II)aq (kobs = 0.29 ± 0.03 s-1), which is the slowest step overall. The conclusions that cluster X is an intermediate in Y177 radical formation and that transfer of the reducing equivalent is relatively facile imply that the mouse R2 and E. coli R2 reactions are mechanistically similar.

AB - The kinetics and mechanism of formation of the tyrosyl radical and μ-(oxo)diiron(III) cluster in the R2 subunit of ribonucleotide reductase from mouse have been examined by stopped-flow absorption and freeze-quench electron paramagnetic resonance and Mössbauer spectroscopies. The reaction comprises (1) acquisition of Fe(II) ions by the R2 apo protein, (2) activation of dioxygen at the resulting carboxylate-bridged diiron(II) cluster to form oxidized intermediate diiron species, and (3) univalent oxidation of Y177 by one of these intermediates to form the stable radical, with concomitant or subsequent formation of the adjacent μ-(oxo)diiron(III) cluster. The data establish that an oxidized diiron intermediate spectroscopically similar to the well-characterized, formally Fe(III)Fe(IV) cluster X from the reaction of the Escherichia coli R2 protein precedes the Y177 radical in the reaction sequence and is probably the Y177 oxidant. As formation of the X intermediate (1) requires transfer of an "extra" reducing equivalent to the buried diiron cluster following the addition of dioxygen and (2) is observed to be rapid relative to other steps in the reaction, the present data indicate that the transfer of this reducing equivalent is not rate-limiting for Y177 radical formation, in contrast to what was previously proposed (Schmidt, P. P., Rova, U., Katterle, B., Thelander, L., and Gräslund, A. (1998) J. Biol. Chem. 273, 21463-21472). Indeed, the formation of X (kobs = 13 ± 3 s-1 at 5 °C and 0.95 mM O2) and the decay of the intermediate to give the Y177 radical (kobs = 5 ± 2 s-1) are both considerably faster than the formation of the reactive Fe(II)-R2 complex from the apo protein and Fe(II)aq (kobs = 0.29 ± 0.03 s-1), which is the slowest step overall. The conclusions that cluster X is an intermediate in Y177 radical formation and that transfer of the reducing equivalent is relatively facile imply that the mouse R2 and E. coli R2 reactions are mechanistically similar.

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