THEORETICAL INVESTIGATION OF THE PROTON ASSISTED PATHWAY TO FORMATIONOF CYTOCHROME-P450 COMPOUND-I

Functional and dysfunctional enzymatic pathways of cytochrome P450s af ter formation of the reduced ferrous dioxygen species have been invest igated using nonlocal density functional quantum chemical methods, emp loying a methyl mercapto iron porphine model of the cytochrome P450 he me complex. The goal of this study was to assess the validity of propo sed pathways to both compound I and peroxide involving protonation of the distal and proximal oxygen atoms of the reduced ferrous dioxygen s pecies. Optimized geometries, energies, and electrostatic and electron ic properties of each putative heme intermediate in these pathways wer e calculated and these properties examined for consistency with the pr oposed role of the intermediate in compound I or peroxide formation. S ingle protonation of the distal oxygen resulted in significant weakeni ng of the O-O bond. Addition of a second proton to the distal oxygen a nd energy optimization led directly to compound I and water products, without any apparent activation barrier or formation of a diprotonated intermediate. These results provide direct robust support for the pro ton-assisted mechanism of dioxygen bond cleavage to form compound I. T he dysfunctional pathway to the formation of peroxide was explored by examining the properties of the distal and proximal singly protonated species. The proximal tautomer is thermodynamically less favorable tha n the distal species by 18.4 kcal/mol. Electrostatic features of both singly protonated species suggest preferred proton delivery to the rem aining unprotonated oxygen in each case, favoring peroxide formation. Moreover, addition of a second proton to either of these singly proton ated species results in formation of a stable hydrogen peroxide heme c omplex. These results, taken together, suggest that the simultaneous a vailability of two protons on the distal oxygen is a requirement for P 450 enzymatic efficacy, while asynchronous delivery of protons to the dioxygen site favors decoupling.