Kinetics of CO Binding to Cytochromes P450 in the Endoplasmic Reticulum

Abstract

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 effectors 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 attributing a kinetic parameter to a single P450 in the presence of multiple microsomal P450s was overcome with a difference method that employs the difference of the kinetic profiles obtained in the presence and absence of a P450 effector. Applying this approach to study the conformation/dynamics of P450 2B1 in microsomes from phenobarbital-treated rats revealed that the substrate benzphetamine enhances while testosterone inhibits the rate of CO binding to this P450. Similar experiments with P450 1A1 in microsomes from 3-methylcholanthrene-treated rats showed that the substrate benzo[a]pyrene accelerates CO binding. These results show that the access channel between solvent and heme in the P450 interior can be altered in a substrate- and P450-dependent manner to either hinder or facilitate CO diffusion to the heme iron. These results also demonstrate that analytical difference methods may be employed to characterize the conformation of individual P450s in their native membrane environment in the endoplasmic reticulum.

Formation of P450·P450 complexes and their effect on P450 function

“…Two protein conformations were involved in the substrate-dependent spin equilibrium, whereas enzyme in the third position of the aggregate was not affected by substrate and may have corresponded to the population of CYP3A4 not reduced by the flavin domain of P450 BM3 (Davydov et al, 2010). This response is consistent with the findings from a flash photolysis study by Friedman’s group showing that CYP3A4 existed in at least three conformations that were affected by the presence of substrate (Koley, Robinson, Markowitz, & Friedman, 1994; Koley, Buters, Robinson, Markowitz, & Friedman, 1995; Koley, Robinson, & Friedman, 1996). …”

Differential Mechanisms of Cytochrome P450 Inhibition and Activation by α-Naphthoflavone

“…Where indicated, lower concentrations of ANF were used. The mixture was then incubated for 20 min prior to addition of CO. Photodissociation of the P450 heme Fe-CO complex and monitoring of CO binding kinetics at 450 nm were performed as described (32). When both ANF and BP were present, identical curves were obtained regardless of their order of addition.…”

“…Data Analysis-A kinetic difference method was applied to kinetically distinguish the individual P450 species from the total P450 species (32). This approach evaluates the difference between the kinetic profiles * The costs of publication of this article were defrayed in part by the payment of page charges.…”

“…We have previously shown that P450s 3A4 (34) and 1A1 (35) are each comprised of multiple, kinetically distinguishable species that are unresolvable by standard multiexponential analysis. The kinetics of the individual P450 species, however, can be resolved using a kinetic difference method (32,34,35), which entails analysis of a curve generated by subtracting the kinetic data in the absence of effector from that in the presence of effector. The resulting kinetic difference profile thus reflects the kinetic properties of the effector-specific P450 species.…”

“…Thus, a higher rate of CO binding indicates a wide ligand access channel and/or a flexible protein, whereas a lower rate suggests a restricted access channel and/or rigid protein conformation. This kinetic approach was previously used to define P450-substrate interactions using both liver microsomes (32,33) and individual P450s (34,35).…”