Probing the Role of the Hinge Segment of Cytochrome P450 Oxidoreductase in the Interaction with Cytochrome P450
AbstractNADPH-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.
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Campelo, D.; Esteves, F.; Palma, B.B.; Gomes, B.C.; Rueff, J.; Lautier, T.; Urban, P.; Truan, G.; Kranendonk, M. Probing the Role of the Hinge Segment of Cytochrome P450 Oxidoreductase in the Interaction with Cytochrome P450. Int. J. Mol. Sci. 2018, 19, 3914.
Campelo D, Esteves F, Palma BB, Gomes BC, Rueff J, Lautier T, Urban P, Truan G, Kranendonk M. Probing the Role of the Hinge Segment of Cytochrome P450 Oxidoreductase in the Interaction with Cytochrome P450. International Journal of Molecular Sciences. 2018; 19(12):3914.Chicago/Turabian Style
Campelo, Diana; Esteves, Francisco; Palma, Bernardo Brito; Gomes, Bruno Costa; Rueff, José; Lautier, Thomas; Urban, Philippe; Truan, Gilles; Kranendonk, Michel. 2018. "Probing the Role of the Hinge Segment of Cytochrome P450 Oxidoreductase in the Interaction with Cytochrome P450." Int. J. Mol. Sci. 19, no. 12: 3914.
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