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

Research output: Contribution to journalArticle

14 Scopus citations

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
Publication statusPublished - Apr 1 2010

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Keywords

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

ASJC Scopus subject areas

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

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