Mechanism of rapid electron transfer during oxygen activation in the R2 subunit of Escherichia coli ribonucleotide reductase. 1. Evidence for a transient tryptophan radical

J. Baldwin, Carsten Krebs, B. A. Ley, D. E. Edmondson, B. H. Huynh, Joseph M. Bollinger, Jr.

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Abstract

Activation of dioxygen at the carboxylate-bridged diiron(II) cluster in the R2 subunit of Escherichia coli class I ribonucleotide reductase produces the enzyme's catalytically essential stable tyrosyl radical by one-electron oxidation of tyrosine 122. An intermediate in the reaction, the formally Fe(IV)Fe(III) cluster X, can oxidize Y122 in the final and rate-limiting step. During formation of X, an "extra" electron must be transferred to an as-yet-uncharacterized adduct between O2 and the diiron(II) cluster. It was previously shown that a transient, broad absorption band centered near 560 nm develops when the reaction is carried out without an obvious exogenous source of the extra electron, and this band was ascribed to a tryptophan cation radical (W) resulting from temporary donation of the electron by the near-surface tryptophan residue 48 during formation of X [Bollinger, J. M., Jr.; Tong, W. H.; Ravi, N.; Huynh, B. H.; Edmondson, D. E.; Stubbe, J. J. Am. Chem. Soc. 1994, 116, 8024-8032]. In this work, we provide more definitive evidence for the W assignment by showing that (1) the absorbing species reacts rapidly with reductants, (2) the species is associated with a g = 2.0 EPR signal and perturbs the EPR and Mössbauer spectra of X, and (3) most definitively, the absorption spectrum of the species from 310 to 650 nm closely matches the very distinctive spectrum of the tryptophan cation radical previously determined in pulse radiolysis studies [Solar, S.; Getoff, N.; Surdhar, P. S.; Armstrong, D. A.; Sing, A. J. Phys. Chem. 1991, 95, 3639-3643]. Quantitation of species at short reaction times by optical, EPR, and Mössbauer spectroscopies is consistent with the rapid formation of an intermediate containing both X and the W (an X-Wdiradical species). Formation of the W (and presumably of X) is kinetically first order in both O2 and Fe(II)-R2 complex, even at the highest reactant concentrations examined, which give a formation rate constant approaching 200 s-1. This observation implies that precursors to the diradical species must not accumulate to greater than ∼10% of the initial Fe(II)-R2 reactant concentration and that the immediate precursor must generate the highly oxidizing W with a rate constant of at least 400 s-1 at 5 °C.

Original languageEnglish (US)
Pages (from-to)12195-12206
Number of pages12
JournalJournal of the American Chemical Society
Volume122
Issue number49
DOIs
StatePublished - Dec 13 2000

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Ribonucleotide Reductases
Tryptophan
Escherichia coli
Chemical activation
Electrons
Oxygen
Paramagnetic resonance
Cations
Absorption spectra
Rate constants
Positive ions
Pulse Radiolysis
Radiolysis
Reducing Agents
Tyrosine
Spectrum Analysis
Enzymes
Spectroscopy
Oxidation
Oxidoreductases

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

@article{30af4582e601450084e7c550f8b4bc65,
title = "Mechanism of rapid electron transfer during oxygen activation in the R2 subunit of Escherichia coli ribonucleotide reductase. 1. Evidence for a transient tryptophan radical",
abstract = "Activation of dioxygen at the carboxylate-bridged diiron(II) cluster in the R2 subunit of Escherichia coli class I ribonucleotide reductase produces the enzyme's catalytically essential stable tyrosyl radical by one-electron oxidation of tyrosine 122. An intermediate in the reaction, the formally Fe(IV)Fe(III) cluster X, can oxidize Y122 in the final and rate-limiting step. During formation of X, an {"}extra{"} electron must be transferred to an as-yet-uncharacterized adduct between O2 and the diiron(II) cluster. It was previously shown that a transient, broad absorption band centered near 560 nm develops when the reaction is carried out without an obvious exogenous source of the extra electron, and this band was ascribed to a tryptophan cation radical (W+·) resulting from temporary donation of the electron by the near-surface tryptophan residue 48 during formation of X [Bollinger, J. M., Jr.; Tong, W. H.; Ravi, N.; Huynh, B. H.; Edmondson, D. E.; Stubbe, J. J. Am. Chem. Soc. 1994, 116, 8024-8032]. In this work, we provide more definitive evidence for the W+· assignment by showing that (1) the absorbing species reacts rapidly with reductants, (2) the species is associated with a g = 2.0 EPR signal and perturbs the EPR and M{\"o}ssbauer spectra of X, and (3) most definitively, the absorption spectrum of the species from 310 to 650 nm closely matches the very distinctive spectrum of the tryptophan cation radical previously determined in pulse radiolysis studies [Solar, S.; Getoff, N.; Surdhar, P. S.; Armstrong, D. A.; Sing, A. J. Phys. Chem. 1991, 95, 3639-3643]. Quantitation of species at short reaction times by optical, EPR, and M{\"o}ssbauer spectroscopies is consistent with the rapid formation of an intermediate containing both X and the W+· (an X-W+·diradical species). Formation of the W+· (and presumably of X) is kinetically first order in both O2 and Fe(II)-R2 complex, even at the highest reactant concentrations examined, which give a formation rate constant approaching 200 s-1. This observation implies that precursors to the diradical species must not accumulate to greater than ∼10{\%} of the initial Fe(II)-R2 reactant concentration and that the immediate precursor must generate the highly oxidizing W+· with a rate constant of at least 400 s-1 at 5 °C.",
author = "J. Baldwin and Carsten Krebs and Ley, {B. A.} and Edmondson, {D. E.} and Huynh, {B. H.} and {Bollinger, Jr.}, {Joseph M.}",
year = "2000",
month = "12",
day = "13",
doi = "10.1021/ja001278u",
language = "English (US)",
volume = "122",
pages = "12195--12206",
journal = "Journal of the American Chemical Society",
issn = "0002-7863",
publisher = "American Chemical Society",
number = "49",

}

TY - JOUR

T1 - Mechanism of rapid electron transfer during oxygen activation in the R2 subunit of Escherichia coli ribonucleotide reductase. 1. Evidence for a transient tryptophan radical

AU - Baldwin, J.

AU - Krebs, Carsten

AU - Ley, B. A.

AU - Edmondson, D. E.

AU - Huynh, B. H.

AU - Bollinger, Jr., Joseph M.

PY - 2000/12/13

Y1 - 2000/12/13

N2 - Activation of dioxygen at the carboxylate-bridged diiron(II) cluster in the R2 subunit of Escherichia coli class I ribonucleotide reductase produces the enzyme's catalytically essential stable tyrosyl radical by one-electron oxidation of tyrosine 122. An intermediate in the reaction, the formally Fe(IV)Fe(III) cluster X, can oxidize Y122 in the final and rate-limiting step. During formation of X, an "extra" electron must be transferred to an as-yet-uncharacterized adduct between O2 and the diiron(II) cluster. It was previously shown that a transient, broad absorption band centered near 560 nm develops when the reaction is carried out without an obvious exogenous source of the extra electron, and this band was ascribed to a tryptophan cation radical (W+·) resulting from temporary donation of the electron by the near-surface tryptophan residue 48 during formation of X [Bollinger, J. M., Jr.; Tong, W. H.; Ravi, N.; Huynh, B. H.; Edmondson, D. E.; Stubbe, J. J. Am. Chem. Soc. 1994, 116, 8024-8032]. In this work, we provide more definitive evidence for the W+· assignment by showing that (1) the absorbing species reacts rapidly with reductants, (2) the species is associated with a g = 2.0 EPR signal and perturbs the EPR and Mössbauer spectra of X, and (3) most definitively, the absorption spectrum of the species from 310 to 650 nm closely matches the very distinctive spectrum of the tryptophan cation radical previously determined in pulse radiolysis studies [Solar, S.; Getoff, N.; Surdhar, P. S.; Armstrong, D. A.; Sing, A. J. Phys. Chem. 1991, 95, 3639-3643]. Quantitation of species at short reaction times by optical, EPR, and Mössbauer spectroscopies is consistent with the rapid formation of an intermediate containing both X and the W+· (an X-W+·diradical species). Formation of the W+· (and presumably of X) is kinetically first order in both O2 and Fe(II)-R2 complex, even at the highest reactant concentrations examined, which give a formation rate constant approaching 200 s-1. This observation implies that precursors to the diradical species must not accumulate to greater than ∼10% of the initial Fe(II)-R2 reactant concentration and that the immediate precursor must generate the highly oxidizing W+· with a rate constant of at least 400 s-1 at 5 °C.

AB - Activation of dioxygen at the carboxylate-bridged diiron(II) cluster in the R2 subunit of Escherichia coli class I ribonucleotide reductase produces the enzyme's catalytically essential stable tyrosyl radical by one-electron oxidation of tyrosine 122. An intermediate in the reaction, the formally Fe(IV)Fe(III) cluster X, can oxidize Y122 in the final and rate-limiting step. During formation of X, an "extra" electron must be transferred to an as-yet-uncharacterized adduct between O2 and the diiron(II) cluster. It was previously shown that a transient, broad absorption band centered near 560 nm develops when the reaction is carried out without an obvious exogenous source of the extra electron, and this band was ascribed to a tryptophan cation radical (W+·) resulting from temporary donation of the electron by the near-surface tryptophan residue 48 during formation of X [Bollinger, J. M., Jr.; Tong, W. H.; Ravi, N.; Huynh, B. H.; Edmondson, D. E.; Stubbe, J. J. Am. Chem. Soc. 1994, 116, 8024-8032]. In this work, we provide more definitive evidence for the W+· assignment by showing that (1) the absorbing species reacts rapidly with reductants, (2) the species is associated with a g = 2.0 EPR signal and perturbs the EPR and Mössbauer spectra of X, and (3) most definitively, the absorption spectrum of the species from 310 to 650 nm closely matches the very distinctive spectrum of the tryptophan cation radical previously determined in pulse radiolysis studies [Solar, S.; Getoff, N.; Surdhar, P. S.; Armstrong, D. A.; Sing, A. J. Phys. Chem. 1991, 95, 3639-3643]. Quantitation of species at short reaction times by optical, EPR, and Mössbauer spectroscopies is consistent with the rapid formation of an intermediate containing both X and the W+· (an X-W+·diradical species). Formation of the W+· (and presumably of X) is kinetically first order in both O2 and Fe(II)-R2 complex, even at the highest reactant concentrations examined, which give a formation rate constant approaching 200 s-1. This observation implies that precursors to the diradical species must not accumulate to greater than ∼10% of the initial Fe(II)-R2 reactant concentration and that the immediate precursor must generate the highly oxidizing W+· with a rate constant of at least 400 s-1 at 5 °C.

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U2 - 10.1021/ja001278u

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JO - Journal of the American Chemical Society

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