Substrate modulates compound I formation in peroxide shunt pathway of Pseudomonas putida cytochrome P450_cam

Abstract

The active oxygenating intermediate, a ferryl-oxo-(II) porphyrin cation radical (compound I), in substrate-bound cytochrome P450_cam (P450_cam) has eluded detection and kinetic analysis for several decades. Upon rapid mixing of peroxides-H₂O₂ and m-CPBA with substrate-bound forms of P450_cam, we observed an intermediate with spectral features characteristic of compound I. Unlike in H₂O₂, kinetic investigation on the reaction of m-CPBA with various substrate (camphor, adamantone, and norcamphor)-bound P450_cam and its Y96A mutant shows a preferential binding of the aromatic end group of m-CPBA to the active-site of the enzyme and modulation of compound I formation by the local environment of heme active-site. The results presented in this paper describe the importance of heme environment in modulating formation of compound I, and form the first kinetic analysis of this intermediate in the peroxide shunt pathway of substrate-bound P450_cam.