Positive cooperativity in catalysis of pyrene 1‐hydroxylation by cytochrome P450 1A2

Abstract

Cytochrome P450s (P450s) are a class of heme‐containing enzymes that oxidize endogenous and exogenous compounds such as steroids and drugs. P450s generally show Michaelis‐Menten kinetics, but several P450s display non‐hyperbolic behavior for some substrates. Prior work in this laboratory identified cooperativity in oxidation of 1‐alkoxy‐4‐nitrobenzenes by rabbit P450 1A2. We analyzed the kinetics associated with the oxidation of P450 1A2 substrates α‐naphthoflavone (αNF) and pyrene. While rates of formation of the 5,6‐epoxide from αNF generate hyperbolic plots, rates of pyrene 1‐hydroxylation yield sigmoidal behavior. Homology modeling of rabbit P450 1A2 using a human P450 1A2 crystal structure shows that only one αNF molecule fits but two pyrenes may be stacked in the P450 1A2 active site, possibly explaining the positive cooperativity. Binding rates measured using stopped‐flow kinetics show that interactions with the ligand and P450 1A2, as indicated by ligand fluorescence quenching, precede changes associated with the heme spectrum. Circular dichroism studies suggest rapid conformational changes upon ligand binding. Kinetic fitting of these data suggests a possible three‐step mechanism for pyrene oxidation in which pyrene monomer binds followed by dimer formation and oxidation. (Supported by USPHS R37 CA090426, T32 ES007028).