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Reactions of substrate-free ferric cytochrome P450cam with peracids to generate Fe=O intermediates have previously been investigated with contradictory results. Using stopped-flow spectrophotometry, the reaction with m-chloroperoxybenzoic acid demonstrated an Fe(IV)=O + porphyrin pi-cation radical (Cpd I) (Egawa, T., Shimada, H., and Ishimura, Y. (1994) Biochem. Biophys. Res. Commun. 201, 1464-1469). By contrast, with peracetic acid, Fe(IV)=O plus a tyrosyl radical were observed by freeze-quench Mossbauer and EPR spectroscopy (Schunemann, V., Jung, C., Trautwein, A. X., Mandon, D., and Weiss, R. (2000) FEBS Lett. 479, 149-154). Our detailed kinetic studies have resolved these contradictory results. At pH >7, a significant fraction of Cpd I is formed transiently, whereas at low pH only a species with a Soret band at 406 nm, presumably Fe(IV)=O + tyrosyl radical, is observed. Evidence for formation of an acylperoxo complex en route to Cpd I was obtained. Because of rapid heme destruction, steps subsequent to formation of the highly oxidized forms could not be fully characterized. Heme destruction was avoided by including peroxidase substrates (e.g. guaiacol), which were oxidized to characteristic peroxidase products as the Fe(III)-P450 was regenerated. Addition of ascorbate to either of the high valent species also reforms the Fe(III) state with only a small loss of heme absorbance. These results indicate that typical peroxidase chemistry occurs with P450cam and offer an explanation for the contrasting results reported earlier. The delineation of improved conditions (pH, temperature, choice of peracid) for generating highly oxidized species with P450cam should be valuable for their further characterization.
Spolitak et al. (Tue,) studied this question.