Novel Reversible Inactivation of Cytochrome P450 2E1 T303A by tert-Butyl Acetylene: The Role of Threonine 303 in Proton Delivery to the Active Site of Cytochrome P450 2E1

Abstract

This report investigates and characterizes the mechanism for the novel reversible inactivation of a T303A mutant of rabbit cytochrome P450 (P450) 2E1 by tert-butyl acetylene (tBA). P450 2E1 T303A was inactivated in a time-, concentration-, and NADPH-dependent manner through the formation of two tBA adducts to the P450 heme. Interestingly, losses in enzymatic activity and in the reduced CO spectrum of the tBA-inactivated T303A mutant could be restored to the samples after an overnight incubation at 4°C. Removal of free tBA and NADPH from the tBA-inactivated T303A samples by spin column gel filtration demonstrated that the observed reversibility was time-dependent and was not significantly affected by the presence or absence of NADPH or tBA. Furthermore, the recovery of native heme was dependent on the native P450 enzyme structure. Electrospray ionization liquid chromatography-tandem mass spectrometry analysis under nondenaturing conditions of a preacidified tBA-inactivated T303A sample yielded two tBA adducts (m/z of 661 Da) with ion fragmentation patterns characteristic of a tBA adduct to the P450 heme. These adducts were absent in nonacidified samples subjected to the same conditions. In contrast, tandem mass spectrometry analysis of both non- and preacidified tBA-inactivated wild-type 2E1 samples yielded two tBA adducts (m/z of 661 Da) with ion fragmentation patterns similar to the preacidified T303A mutant adducts. These results lend insight into the reversible inactivation mechanism of the tBA-inactivated T303A mutant and suggest a role for the highly conserved threonine 303 residue in proton donation to the P450 2E1 active site and the stabilization of a reactive intermediate during substrate metabolism by P450.