Electron paramagnetic resonance investigations of a kinetically competent intermediate formed in ribonucleotide reduction: Evidence for a thiyl radical-cob(II)alamin interaction

Gary J. Gerfen, S. Licht, J. P. Willems, B. M. Hoffman, J. Stubbe

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85 Citations (Scopus)

Abstract

The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii requires adenosylcobalamin (AdoCbl) as a cofactor to catalyze the conversion of nucleotides to deoxynucleotides. RTPR has previously been shown to catalyze the homolytic cleavage of the carbon-cobalt bond of AdoCbl, and the resulting paramagnetic species has been characterized by rapid freeze-quench EPR spectroscopy (Orme-Johnson, W.H.; Beinert, H.; Blakley, R.L., J. Biol. Chem. 1974, 249, 2338-2343. Licht, S.; Gerfen, O.J.; Stubbe, J. Science 1996, 271, 477-481). This study presents simulations of X- and Q-band EPR spectra of this intermediate. Modeling this species as a thiyl radical coupled to cob(II)alamin by electron-electron exchange and dipolar interactions yields reasonable fits to spectra obtained at both microwave frequencies, whereas simulations that employ a single-spin model do not. This modeling provides support for the intermediacy of a thiyl radical in this system. The techniques employed here may prove generally useful in simulation of similar spectra observed in other B12-dependent enzyme systems.

Original languageEnglish (US)
Pages (from-to)8192-8197
Number of pages6
JournalJournal of the American Chemical Society
Volume118
Issue number35
DOIs
StatePublished - 1996
Externally publishedYes

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ribonucleoside-triphosphate reductase
Ribonucleotides
Electron Spin Resonance Spectroscopy
Paramagnetic resonance
Lactobacillus leichmannii
Electrons
Microwave frequencies
Nucleotides
Microwaves
Cobalt
Spectrum Analysis
Carbon
Enzymes
Spectroscopy
cob(II)alamin
cobamamide
Oxidoreductases

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

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title = "Electron paramagnetic resonance investigations of a kinetically competent intermediate formed in ribonucleotide reduction: Evidence for a thiyl radical-cob(II)alamin interaction",
abstract = "The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii requires adenosylcobalamin (AdoCbl) as a cofactor to catalyze the conversion of nucleotides to deoxynucleotides. RTPR has previously been shown to catalyze the homolytic cleavage of the carbon-cobalt bond of AdoCbl, and the resulting paramagnetic species has been characterized by rapid freeze-quench EPR spectroscopy (Orme-Johnson, W.H.; Beinert, H.; Blakley, R.L., J. Biol. Chem. 1974, 249, 2338-2343. Licht, S.; Gerfen, O.J.; Stubbe, J. Science 1996, 271, 477-481). This study presents simulations of X- and Q-band EPR spectra of this intermediate. Modeling this species as a thiyl radical coupled to cob(II)alamin by electron-electron exchange and dipolar interactions yields reasonable fits to spectra obtained at both microwave frequencies, whereas simulations that employ a single-spin model do not. This modeling provides support for the intermediacy of a thiyl radical in this system. The techniques employed here may prove generally useful in simulation of similar spectra observed in other B12-dependent enzyme systems.",
author = "Gerfen, {Gary J.} and S. Licht and Willems, {J. P.} and Hoffman, {B. M.} and J. Stubbe",
year = "1996",
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journal = "Journal of the American Chemical Society",
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TY - JOUR

T1 - Electron paramagnetic resonance investigations of a kinetically competent intermediate formed in ribonucleotide reduction

T2 - Evidence for a thiyl radical-cob(II)alamin interaction

AU - Gerfen, Gary J.

AU - Licht, S.

AU - Willems, J. P.

AU - Hoffman, B. M.

AU - Stubbe, J.

PY - 1996

Y1 - 1996

N2 - The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii requires adenosylcobalamin (AdoCbl) as a cofactor to catalyze the conversion of nucleotides to deoxynucleotides. RTPR has previously been shown to catalyze the homolytic cleavage of the carbon-cobalt bond of AdoCbl, and the resulting paramagnetic species has been characterized by rapid freeze-quench EPR spectroscopy (Orme-Johnson, W.H.; Beinert, H.; Blakley, R.L., J. Biol. Chem. 1974, 249, 2338-2343. Licht, S.; Gerfen, O.J.; Stubbe, J. Science 1996, 271, 477-481). This study presents simulations of X- and Q-band EPR spectra of this intermediate. Modeling this species as a thiyl radical coupled to cob(II)alamin by electron-electron exchange and dipolar interactions yields reasonable fits to spectra obtained at both microwave frequencies, whereas simulations that employ a single-spin model do not. This modeling provides support for the intermediacy of a thiyl radical in this system. The techniques employed here may prove generally useful in simulation of similar spectra observed in other B12-dependent enzyme systems.

AB - The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii requires adenosylcobalamin (AdoCbl) as a cofactor to catalyze the conversion of nucleotides to deoxynucleotides. RTPR has previously been shown to catalyze the homolytic cleavage of the carbon-cobalt bond of AdoCbl, and the resulting paramagnetic species has been characterized by rapid freeze-quench EPR spectroscopy (Orme-Johnson, W.H.; Beinert, H.; Blakley, R.L., J. Biol. Chem. 1974, 249, 2338-2343. Licht, S.; Gerfen, O.J.; Stubbe, J. Science 1996, 271, 477-481). This study presents simulations of X- and Q-band EPR spectra of this intermediate. Modeling this species as a thiyl radical coupled to cob(II)alamin by electron-electron exchange and dipolar interactions yields reasonable fits to spectra obtained at both microwave frequencies, whereas simulations that employ a single-spin model do not. This modeling provides support for the intermediacy of a thiyl radical in this system. The techniques employed here may prove generally useful in simulation of similar spectra observed in other B12-dependent enzyme systems.

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