Protein/Protein Interactions in the Human Cytochrome P450 System

Abstract

Cytochrome P450 enzymes are the major superfamily of enzymes involved in xenobiotic metabolism and steroid biosynthesis. In vivo, human P450 enzymes are embedded in the lipid membrane and require a membrane-bound redox partner protein for catalysis. This study focuses on P450 enzyme interactions with their redox partner, cytochrome P450 reductase. This protein is the required redox partner for 50 of the 57 human P450 enzymes. It accepts electrons from NADPH via its FAD domain, transfers them to its FMN domain, and then supplies those electrons to the P450 heme to support P450 catalysis. The FMN domain binds directly to the proximal face of P450 enzymes, but this interaction is transient and little is known about the details of the interactions of the FMN domain and different human P450 enzymes. Thus, this study sought to better define the structural interactions and the functional implications of the reductase FMN domain binding to multiple human P450 enzymes, including P450 enzymes responsible for both steroidogenic and xenobiotic metabolism. To facilitate interactions of the reductase FMN domain and the different P450 enzymes, artificial fusion proteins were generated. The fusion proteins were successfully expressed in E. coli and purified. Binding studies and substrate metabolism assays with typical ligands were used to compare the functions of these fusion proteins with the respective isolated P450 enzyme. These fusions are also being used to generate X-ray structures to reveal the specific interactions between the reductase FMN domain and each different P450 enzyme. While the P450 proximal surface is thought to be the general binding surface for the reductase FMN domain, we expect to observe variations in the specific FMN domain/P450 interactions that may correlate with functional differences between the enzymes.