Study of human Cytochrome P450 reductase by Electron Paramagnetic Resonance

Human cytochrome P450 reductase is a membrane protein (73 kDa) located on the cytoplasmic side of the endoplasmic reticulum. It contains two binding domains for FAD and FMN. These two domains are connected by a flexible region. CPR transfers electrons from NADPH (hydride transfer) to cytochromes P450 (CYPs) through flavin sites. Two crystallographic structures were obtained for Rattus norvegicus showing different conformations for the FMN binding domain. Indeed, it is proposed that for inter-flavin Electronic Transfer ET, the two FAD and FMN domains must be nearby (a « closed » conformation), but inter-protein ET (FMN to CYPs) requires a more « open » conformation allowing a rapprochement of these two partners. The « closed » conformation was isolated on the WT protein and the « open » conformation was obtained after a deletion of 4 residues on the flexible region (ΔTGEE). These two structures show a different distance between the flavin cofactors. The understanding and characterization of domain movement associated with ET is essential. First results on its study by measuring distances between two paramagnetic centers will be discussed. Two approaches were followed : 1) by using endogenous probes by isolating the radical (semi-quinone) forms of the FMN and 2) by EPR-coupled Site-Directed Spin Labeling, which involves the insertion of paramagnetic nitroxide probes. Classically, these paramagnetic probes (nitroxides) are specific to cysteines. However, the cysteines of the CPR are involved in the enzymatic activity and cannot be modified. The incorporation of non-natural amino acids (nnaa) and their labelling with specific nitroxides has been shown to be a powerful alternative. First experiments on human soluble CPR (truncated) concerning the labelling of non-natural amino acids, as well as the trapping of flavin centers have been performed and will be presented. Those results will be fundamental to be able to study full length human (membrane) CPR.