Detection of an oxyferryl porphyrin .pi.-cation-radical intermediate in the reaction between hydrogen peroxide and a mutant yeast cytochrome c peroxidase. Evidence for tryptophan-191 involvement in the radical site of compound I

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Abstract
Peroxide oxidation of a mutant cytochrome c peroxidase, in which Trp-191 has been replaced by Phe through site-directed mutagenesis, produces an oxidized intermediate whose stable UV/visible absorption spectrum is very similar to that of compound I of the native yeast enzyme. This spectrum is characteristic of an oxyferryl, Fe(IV), heme. Stopped-flow studies reveal that the reaction between the mutant enzyme and hydrogen peroxide is biphasic with the transient formation of an intermediate whose absorption spectrum is quite distinct from that of either the native ferric enzyme of the final product. Rapid spectral scanning of the intermediate provides a spectrum characteristic of an oxyferryl prophyrin .pi.-cation-radical species. At pH 6, 100 mM ionic strength, and 25.degree.C, the rate of constant for formation of the oxyferryl .pi.-cation radical has a lower limit of 6 .times. 107 M-1 s-1 and the rate of conversion of the transient intermediate to the final oxidized product is 51 .+-. 4 s-1. Evidence is presented indicating that Trp-191 either is the site of the radical in CcP compound I or is intimately involved in formation of the radical.