KINETICS OF CO BINDING TO CYTOCHROMES P450 IN THE ENDOPLASMIC-RETICULUM

The kinetics of CO binding to cytochromes P450 in rat liver microsomes were examined using the flash photolysis technique. Modulation of the kinetics by P450 form-specific effecters such as anti-P450 monoclonal antibodies and substrates was used to elucidate the kinetic behavior of individual P450s within the endoplasmic reticulum. The problem of a ttributing a kinetic parameter to a single P450 in the presence of mul tiple microsomal P450s was overcome with a difference method that empl oys the difference of the kinetic profiles obtained in the presence an d absence of a P450 effector. Applying this approach to study the conf ormation/dynamics of P450 2B1 in microsomes from phenobarbital-treated rats revealed that the substrate benzphetamine enhances while testost erone inhibits the rate of CO binding to this P450. Similar experiment s with P450 1A1 in microsomes from 3-methylcholanthrene-treated rats s howed that the substrate benzo[alpha]pyrene accelerates CO binding. Th ese results show that the access channel between solvent and heme in t he P450 interior can be altered in a substrate- and P450-dependent man ner to either hinder or facilitate CO diffusion to the heme iron. Thes e results also demonstrate that analytical difference methods may be e mployed to characterize the conformation of individual P450s in their native membrane environment in the endoplasmic reticulum.