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 language | English (US) |
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Pages (from-to) | 213-219 |
Number of pages | 7 |
Journal | Journal of Magnetic Resonance |
Volume | 203 |
Issue number | 2 |
DOIs | |
State | Published - Apr 2010 |
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