EPR characterization of ascorbyl and sulfur dioxide anion radicals trapped during the reaction of bovine Cytochrome c Oxidase with molecular oxygen

Michelle A. Yu, Tsuyoshi Egawa, Syun-Ru Yeh, Denis L. Rousseau, Gary J. Gerfen

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14 Citations (Scopus)

Abstract

The reaction intermediates of reduced bovine Cytochrome c Oxidase (CcO) were trapped following its reaction with oxygen at 50 μs-6 ms by innovative freeze-quenching methods and studied by EPR. When the enzyme was reduced with either ascorbate or dithionite, distinct radicals were generated; X-band (9 GHz) and D-band (130 GHz) CW-EPR measurements support the assignments of these radicals to ascorbyl and sulfur dioxide anion radical (SO2 - {radical dot}), respectively. The X-band spectra show a linewidth of 12 G for the ascorbyl radical and 11 G for the SO2 - {radical dot} radical and an isotropic g-value of 2.005 for both species. The D-band spectra reveal clear distinctions in the g-tensors and powder patterns of the two species. The ascorbyl radical spectrum displays approximate axial symmetry with g-values of gx = 2.0068, gy = 2.0066, and gz = 2.0023. The SO2 - {radical dot} radical has rhombic symmetry with g-values of gx = 2.0089, gy = 2.0052, and gz = 2.0017. When the contributions from the ascorbyl and SO2 - {radical dot} radicals were removed, no protein-based radical on CcO could be identified in the EPR spectra.

Original languageEnglish (US)
Pages (from-to)213-219
Number of pages7
JournalJournal of Magnetic Resonance
Volume203
Issue number2
DOIs
StatePublished - Apr 2010

Fingerprint

Sulfur Dioxide
Molecular oxygen
sulfur dioxides
oxidase
cytochromes
Electron Transport Complex IV
Anions
Paramagnetic resonance
Oxygen
anions
oxygen
Dithionite
Reaction intermediates
Linewidth
Powders
Tensors
Quenching
Enzymes
superhigh frequencies
semidehydroascorbic acid

Keywords

  • Ascorbate
  • Cytochrome c Oxidase
  • Dithionite
  • High-field EPR
  • Radicals
  • Time-resolved EPR

ASJC Scopus subject areas

  • Nuclear and High Energy Physics
  • Biochemistry
  • Biophysics
  • Condensed Matter Physics

Cite this

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title = "EPR characterization of ascorbyl and sulfur dioxide anion radicals trapped during the reaction of bovine Cytochrome c Oxidase with molecular oxygen",
abstract = "The reaction intermediates of reduced bovine Cytochrome c Oxidase (CcO) were trapped following its reaction with oxygen at 50 μs-6 ms by innovative freeze-quenching methods and studied by EPR. When the enzyme was reduced with either ascorbate or dithionite, distinct radicals were generated; X-band (9 GHz) and D-band (130 GHz) CW-EPR measurements support the assignments of these radicals to ascorbyl and sulfur dioxide anion radical (SO2 - {radical dot}), respectively. The X-band spectra show a linewidth of 12 G for the ascorbyl radical and 11 G for the SO2 - {radical dot} radical and an isotropic g-value of 2.005 for both species. The D-band spectra reveal clear distinctions in the g-tensors and powder patterns of the two species. The ascorbyl radical spectrum displays approximate axial symmetry with g-values of gx = 2.0068, gy = 2.0066, and gz = 2.0023. The SO2 - {radical dot} radical has rhombic symmetry with g-values of gx = 2.0089, gy = 2.0052, and gz = 2.0017. When the contributions from the ascorbyl and SO2 - {radical dot} radicals were removed, no protein-based radical on CcO could be identified in the EPR spectra.",
keywords = "Ascorbate, Cytochrome c Oxidase, Dithionite, High-field EPR, Radicals, Time-resolved EPR",
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T1 - EPR characterization of ascorbyl and sulfur dioxide anion radicals trapped during the reaction of bovine Cytochrome c Oxidase with molecular oxygen

AU - Yu, Michelle A.

AU - Egawa, Tsuyoshi

AU - Yeh, Syun-Ru

AU - Rousseau, Denis L.

AU - Gerfen, Gary J.

PY - 2010/4

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N2 - The reaction intermediates of reduced bovine Cytochrome c Oxidase (CcO) were trapped following its reaction with oxygen at 50 μs-6 ms by innovative freeze-quenching methods and studied by EPR. When the enzyme was reduced with either ascorbate or dithionite, distinct radicals were generated; X-band (9 GHz) and D-band (130 GHz) CW-EPR measurements support the assignments of these radicals to ascorbyl and sulfur dioxide anion radical (SO2 - {radical dot}), respectively. The X-band spectra show a linewidth of 12 G for the ascorbyl radical and 11 G for the SO2 - {radical dot} radical and an isotropic g-value of 2.005 for both species. The D-band spectra reveal clear distinctions in the g-tensors and powder patterns of the two species. The ascorbyl radical spectrum displays approximate axial symmetry with g-values of gx = 2.0068, gy = 2.0066, and gz = 2.0023. The SO2 - {radical dot} radical has rhombic symmetry with g-values of gx = 2.0089, gy = 2.0052, and gz = 2.0017. When the contributions from the ascorbyl and SO2 - {radical dot} radicals were removed, no protein-based radical on CcO could be identified in the EPR spectra.

AB - The reaction intermediates of reduced bovine Cytochrome c Oxidase (CcO) were trapped following its reaction with oxygen at 50 μs-6 ms by innovative freeze-quenching methods and studied by EPR. When the enzyme was reduced with either ascorbate or dithionite, distinct radicals were generated; X-band (9 GHz) and D-band (130 GHz) CW-EPR measurements support the assignments of these radicals to ascorbyl and sulfur dioxide anion radical (SO2 - {radical dot}), respectively. The X-band spectra show a linewidth of 12 G for the ascorbyl radical and 11 G for the SO2 - {radical dot} radical and an isotropic g-value of 2.005 for both species. The D-band spectra reveal clear distinctions in the g-tensors and powder patterns of the two species. The ascorbyl radical spectrum displays approximate axial symmetry with g-values of gx = 2.0068, gy = 2.0066, and gz = 2.0023. The SO2 - {radical dot} radical has rhombic symmetry with g-values of gx = 2.0089, gy = 2.0052, and gz = 2.0017. When the contributions from the ascorbyl and SO2 - {radical dot} radicals were removed, no protein-based radical on CcO could be identified in the EPR spectra.

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