Interaction of Polycyclic Aromatic Hydrocarbons and Flavones with Cytochromes P450 in the Endoplasmic Reticulum: Effect on CO Binding Kinetics

Abstract

The flash photolysis technique was used to examine the kinetics of CO binding to cytochromes P450 in rat liver microsomes. The effect of polycyclic aromatic hydrocarbons (PAHs) and flavones was used to distinguish the kinetic behavior of the PAH-metabolizing P450 1A1 from that of the remaining multiple microsomal P450s. Applying this approach to microsomes from 3-methylcholanthrene-treated rats showed that although all tested PAHs accelerated CO binding to P450 1A1, the extent varied markedly for different PAHs. The tricyclic PAHs phenanthrene and anthracene enhanced CO binding by 37- and 49-fold, respectively, while several tetracyclic and pentacyclic PAHs increased the rate by 3-16-fold. The results indicate that PAHs exert a dual effect on the rate of CO binding to P450 1A1: a general enhancement via widening of the CO access channel and a reduction that is dependent on PAH size. Although 5,6-benzoflavone increased the rate of CO binding to P450 1A1 by 3.5-fold, it additionally decelerated binding to a constitutive P450 by 15-fold. This flavone thus exerts markedly different effects on two P450s within the same microsomal sample. In contrast, the sole effect of 7,8-benzoflavone was acceleration of CO binding to P450 1A1 by 18-fold. The divergent effects of these isomeric flavones, which only differ in positioning of an aromatic ring, illustrate the sensitivity of CO binding to substrate structure. The varying effects of these PAHs and flavones on CO binding kinetics show that they differentially modulate P450 conformation and access of ligands to the P450 heme and demonstrate that binding of carcinogens to a specific target P450 can be evaluated in its native microsomal milieu.