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 (C c O) 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 ( SO 2 - ), respectively. The X-band spectra show a linewidth of 12 G for the ascorbyl radical and 11 G for the SO 2 - 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 SO 2 - 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 SO 2 - radicals were removed, no protein-based radical on C c O could be identified in the EPR spectra.