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Article

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

by
Diana Campelo
1,
Francisco Esteves
1,
Bernardo Brito Palma
1,
Bruno Costa Gomes
1,
José Rueff
1,
Thomas Lautier
2,
Philippe Urban
2,
Gilles Truan
2 and
Michel Kranendonk
1,*
1
Center for Toxicogenomics and Human Health, Genetics, Oncology and Human Toxicology, NOVA Medical School, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1150-082 Lisbon, Portugal
2
LISBP, Université de Toulouse, CNRS, INRA, INSA, 31077 Toulouse CEDEX 04, France
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2018, 19(12), 3914; https://doi.org/10.3390/ijms19123914
Submission received: 15 November 2018 / Revised: 4 December 2018 / Accepted: 5 December 2018 / Published: 6 December 2018
(This article belongs to the Special Issue Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity 2.0)
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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.
Keywords: NADPH-cytochrome P450 reductase (CPR); microsomal cytochrome P450 (CYP); Cytochrome b5 (CYB5); protein dynamics; electron-transfer (ET); protein–protein interaction NADPH-cytochrome P450 reductase (CPR); microsomal cytochrome P450 (CYP); Cytochrome b5 (CYB5); protein dynamics; electron-transfer (ET); protein–protein interaction

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MDPI and ACS Style

Campelo, D.; Esteves, F.; Brito Palma, B.; Costa Gomes, B.; 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. https://doi.org/10.3390/ijms19123914

AMA Style

Campelo D, Esteves F, Brito Palma B, Costa Gomes B, 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. https://doi.org/10.3390/ijms19123914

Chicago/Turabian Style

Campelo, Diana, Francisco Esteves, Bernardo Brito Palma, Bruno Costa Gomes, José Rueff, Thomas Lautier, Philippe Urban, Gilles Truan, and Michel Kranendonk. 2018. "Probing the Role of the Hinge Segment of Cytochrome P450 Oxidoreductase in the Interaction with Cytochrome P450" International Journal of Molecular Sciences 19, no. 12: 3914. https://doi.org/10.3390/ijms19123914

APA Style

Campelo, D., Esteves, F., Brito Palma, B., Costa Gomes, B., Rueff, J., Lautier, T., Urban, P., Truan, G., & Kranendonk, M. (2018). Probing the Role of the Hinge Segment of Cytochrome P450 Oxidoreductase in the Interaction with Cytochrome P450. International Journal of Molecular Sciences, 19(12), 3914. https://doi.org/10.3390/ijms19123914

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