INTERACTION OF POLYCYCLIC AROMATIC-HYDROCARBONS WITH HUMAN CYTOCHROME-P450 1A1 - A CO FLASH-PHOTOLYSIS STUDY

The kinetics of CO binding to human cytochrome P450 1A1 was used to pr obe the interaction of polycyclic aromatic hydrocarbons (PAHs) with th e membrane-bound P450 expressed in baculovirus-infected SF9 insect cel ls. Biexponential kinetics was observed, indicating that P450 1A1 is c omposed of at least two kinetically distinguishable species. To define the substrate specificity of the individual species, we evaluated the effect of a series of PAHs of varying sizes and shapes on the CO bind ing kinetics of P450 1A1. The overall rate of CO binding was increased in the presence of the tricyclic PAHs phenanthrene and anthracene and the tetracyclic PAHs pyrene and 1,2-benzanthracene, but was decreased by the pentacyclic PAHs benzo[a]pyrene and 1,2:3,4-dibenzanthracene. A kinetic difference method was applied to kinetically define the indi vidual P450 1A1 species. Two species differing in their PAH specificit ies were identified: a slowly reacting species sensitive to the smalle r PAHs, and a rapidly reacting species responsive to larger PAHs. Upon PAH binding, CO binding to these species was accelerated and decelera ted, respectively. The results furthermore suggest that the two specie s are interconvertable. In addition to PAHs, the interactions of P450 1A1 with 7-ethoxy- and 7-pentoxyresorufin were likewise examined for t heir effect on the CO binding kinetics. These compounds interacted wit h and decreased the rate of the rapidly and slowly reacting P450 1A1 s pecies, respectively. The markedly variable effects of these PAHs and resorufins on the CO binding kinetics indicate differential modes of i nteraction with the two target P450 1A1 species, resulting in differen tial modulation of their conformations. These results demonstrate that multiple P450 1A1 species with distinct conformations and substrate r ecognition profiles coexist in a biological membrane and are resolvabl e using a rapid kinetic technique. (C) 1996 Academic Press, Inc.