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 50μs–6ms 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 (9GHz) and D-band (130GHz) 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 12G for the ascorbyl radical and 11G 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 gx=2.0068, gy=2.0066, and gz=2.0023. The SO2- 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- radicals were removed, no protein-based radical on CcO could be identified in the EPR spectra.