EPR characterization of ascorbyl and sulfur dioxide anion radicals trapped during the reaction of bovine Cytochrome c Oxidase with molecular oxygen.
The reaction intermediates of reduced bovine Cytochrome c Oxidase (CcO) were trapped following its reaction with oxygen at 50 micros-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(-.)), respectively. The X-band spectra show a linewidth of 12 G for the ascorbyl radical and 11 G for the SO2(-.) 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 g(x)=2.0068, g(y)=2.0066, and g(z)=2.0023. The SO2(-.) radical has rhombic symmetry with g-values of g(x)=2.0089, g(y)=2.0052, and g(z)=2.0017. When the contributions from the ascorbyl and SO2(-.) radicals were removed, no protein-based radical on CcO could be identified in the EPR spectra.
Yu
Michelle A
MA
Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA.
Egawa
Tsuyoshi
T
Yeh
Syun-Ru
SR
Rousseau
Denis L
DL
Gerfen
Gary J
GJ
D000818:Animals; D000838:Anions; D001665:Binding Sites; D003683:Dehydroascorbic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D003576:Electron Transport Complex IV / Q000737:chemistry*; D004789:Enzyme Activation; D005609:Free Radicals / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry*; D011485:Protein Binding; D013458:Sulfur Dioxide / Q000737:chemistry*; D013552:Swine
D000838:Anions; D005609:Free Radicals; D013458:Sulfur Dioxide; C000820:semidehydroascorbic acid; D003576:Electron Transport Complex IV; D010100:Oxygen; D003683:Dehydroascorbic Acid