Protein/protein interactions in the human cytochrome P450 system

Abstract

Human cytochrome P450 enzymes are heme‐containing monooxygenases that can act on diverse substrates including drugs, steroids, fatty acids, and vitamins. Most of these are microsomal enzymes that require electrons from NADPH‐cytochrome P450 reductase for catalysis. Specifically, the reductase FMN transfers electrons to the P450 heme, but little is known about the details of this transient interaction. Herein the reductase FMN domain was fused to various human P450 enzymes to promote their interaction, and the effects were measured on P450 ligand binding and catalysis occurring in the P450 active site some 15‐20 Å away from the FMN domain/P450 interaction. These FMN domain‐P450 fusion proteins were generated for both steroidogenic (CYP17A1 and CYP21A2) and drug‐metabolizing (CYP3A4, CYP2A6, and CYP2D6) P450 enzymes and compared with the respective isolated P450 enzyme. Fusion of the FMN domain does not appear to change the mode in which ligands bind in the P450 active site. Compounds that bound in the P450 active site either by displacing the heme water (type I interactions) or by directly binding the heme iron (type II interactions) in the isolated P450 enzyme, also did so in the corresponding FMN domain‐P450 fusion enzyme. However, among drug‐metabolizing enzymes fusion of the FMN domain can 1) alter the percentage of the P450 population that binds a particular ligand and 2) increase or decrease the ligand binding affinity, in a P450 and ligand‐specific way. The FMN domain appeared to have significantly less effects on steroidogenic enzymes, regardless of the ligand. Similar catalytic studies are being performed to relate these observed ligand binding trends to substrate metabolism. These current results demonstrate that the effect of the FMN domain is both isoform and ligand dependent, revealing an intricate communication between the P450 surface where the FMN domain binds and the buried P450 active site where substrates and inhibitors bind. This variability suggests that different P450 enzymes interact with the same reductase FMN domain in distinct ways. Since electron delivery is required for catalysis, this knowledge could potentially be exploited to selectively modulate the function of individual P450 enzymes to treat disease.