Roles of the axial push effect in cytochrome P450cam studied with the site-directed mutagenesis at the heme proximal site

To examine the roles of the axial thiolate in cytochrome P450-catalyzed rea ctions, a mutant of cytochrome P450cam, L358P, was prepared to remove one o f the conserved amide protons that are proposed to neutralize the negative charge of the thiolate sulfur. The increased push effect of the thiolate in L358P was evidenced by the reduced reduction potential of the heme. The N- 15-NMR and resonance Raman spectra of the mutant in the ferric-CN- and in t he ferrous-CO forms, respectively, also supported the increased push effect . The maintenance of stereo- and regioselectivities for d-camphor hydroxyla tion by the mutant suggests the minimum structural change at the distal sit e. The heterolysis/homolysis ratios of cumene hydroperoxide were the same f or wild-type and L358P. However, we observed the enhanced monooxygenations of the unnatural substrates using dioxygen and electrons supplied from the reconstituted system, which indicate the significant role of the push effec t in dioxygen activation. We interpret that the enhanced push effect inhibi ts the protonation of the inner oxygen atom and/or promotes the protonation of the outer oxygen atom in the putative iron-hydroperoxo intermediate (Fe 3+-O-OH) of P450cam. This work is the first experimental indication of the significance of the axial cysteine for the P450 reactivity. (C) 2000 Elsevi er Science S.A. All rights reserved.