Reaction of bovine cytochrome c oxidase with hydrogen peroxide produces a tryptophan cation radical and a porphyrin cation radical

Oxidized bovine cytochrome c oxidase reacts with hydrogen peroxide to gener ate two electron paramagnetic resonance (EPR) free radical signals (Fabian, M., and Palmer, G. (1995) Biochemistry 34, 13802-13810). These radicals ar e associated with the binuclear center and give rise to two overlapped EPR signals, one signal being narrower in line width (Delta Hptp = 12 G) than t he other (Delta Hptp = 45 G). We have used electron nuclear double resonanc e (ENDOR) spectrometry to identify the two different chemical species givin g rise to these two EPR signals. Comparison of the ENDOR spectrum associate d with the narrow signal with that of compound I of horseradish peroxidase (formed by reaction of that enzyme with hydrogen peroxide) demonstrates tha t the two species are virtually identical. The chemical species giving rise to the narrow signal is therefore identified as an exchange-coupled porphy rin cation radical similar to that formed in horseradish peroxidase compoun d I. Comparison of the ENDOR spectrum of compound ES (formed by the reactio n of hydrogen peroxide with cytochrome c peroxidase) with that of the broad signal indicates that the chemical species giving rise to the broad EPR si gnal in cytochrome c oxidase is probably an exchange coupled tryptophan cat ion radical. This is substantiated using H2O/ D2O solvent exchange experime nts where the ENDOR difference spectrum of the broad EPR signal of cytochro me c oxidase shows a feature consistent with hyperfine coupling to the exch angeable N(1) proton of a tryptophan cation radical.