Probing the Role of the Hinge Segment of Cytochrome P450 Oxidoreductase in the Interaction with Cytochrome P450.

Diana Campelo,Michel Kranendonk,Thomas Lautier,Bruno Costa Gomes,Philippe Urban,Francisco Esteves,Gilles Truan,Bernardo Brito Palma,José Rueff

doi:10.3390/ijms19123914

Abstract

NADPH-cytochrome P450 reductase (CPR) is the unique redox partner of microsomal cytochrome P450s (CYPs). CPR exists in a conformational equilibrium between open and closed conformations throughout its electron transfer (ET) function. Previously, we have shown that electrostatic and flexibility properties of the hinge segment of CPR are critical for ET. Three mutants of human CPR were studied (S243P, I245P and R246A) and combined with representative human drug-metabolizing CYPs (isoforms 1A2, 2A6 and 3A4). To probe the effect of these hinge mutations different experimental approaches were employed: CYP bioactivation capacity of pre-carcinogens, enzyme kinetic analysis, and effect of the ionic strength and cytochrome b5 (CYB5) on CYP activity. The hinge mutations influenced the bioactivation of pre-carcinogens, which seemed CYP isoform and substrate dependent. The deviations of Michaelis-Menten kinetic parameters uncovered tend to confirm this discrepancy, which was confirmed by CYP and hinge mutant specific salt/activity profiles. CPR/CYB5 competition experiments indicated a less important role of affinity in CPR/CYP interaction. Overall, our data suggest that the highly flexible hinge of CPR is responsible for the existence of a conformational aggregate of different open CPR conformers enabling ET-interaction with structural varied redox partners.

Highlights

Microsomal cytochrome P450 (CYP) metabolism requires a coupled supply of electrons, which are donated by the auxiliary protein NADPH cytochrome P450 oxidoreductase (CPR)
Wild-type cytochrome P450 reductase (CPR) and CPR hinge mutants S243P, I245P and R246A were separately introduced in the E. coli BTC strain and co-expressed with CYP1A2, 2A6 or 3A4, using methods described
Microsomal CYP-mediated metabolism is dependent on electron transfer (ET) through protein:protein interaction with its primary redox partner CPR

Summary

Introduction

Microsomal cytochrome P450 (CYP) metabolism requires a coupled supply of electrons, which are donated by the auxiliary protein NADPH cytochrome P450 oxidoreductase (CPR). CPR mediates a two-electron transfer (ET) per reaction cycle, originated from NADPH enabling CYP-mediated metabolism of many compounds. These include endobiotics, e.g., steroids, bile acids, vitamins and arachidonic acid metabolites, as well as many xenobiotics, including therapeutic drugs and environmental toxins [2,3]. CPR is the unique electron supplier of heme oxygenase, squalene monooxygenase and fatty acid elongase [4], sustaining exclusively the activity of these enzymes. Cytochrome b5 (CYB5) can donate the second electron to CYP, competing with CPR for the binding site on the proximal side of CYP [5]. CYB5’s interaction may have either a stimulating, inhibiting or having no effect over CYP catalytic activity, which seems to be CYP isoform and even substrate dependent [6]

Methods

Results

Conclusion