Probing the role of the hinge segment of cytochrome P450 oxidoreductase in the interaction with cytochrome P450

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

doi:10.3390/ijms19123914

Probing the role of the hinge segment of cytochrome P450 oxidoreductase in the interaction with cytochrome P450

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

Research output: Contribution to journal › Article › peer-review

18 Citations (Scopus)

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 b ₅ (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.

Original language	English
Article number	3914
Journal	International Journal of Molecular Sciences
Volume	19
Issue number	12
DOIs	https://doi.org/10.3390/ijms19123914
Publication status	Published - Dec 2018
Externally published	Yes

Bibliographical note

Publisher Copyright:
© 2018 by the authors. Licensee MDPI, Basel, Switzerland.

Funding

Funding: This work was in part financed by a joint project, funded by the Portuguese Funda\u00E7\u00E3o para a Ci\u00EAncia e a Tecnologia [Grant FCT-ANR/ BEXBCM/0002/2013, as well as Grant UID/BIM/0009/2016 of the Research Center for Toxicogenomics and Human Health (ToxOmics)] and the L\u2019Agence Nationale de la Recherche [Grant ANR-13-ISV5-0001]. F.E. was supported with a post-doctoral fellowship grant of the Portuguese Funda\u00E7\u00E3o para a Ci\u00EAncia e a Tecnologia [Grant SFRH/BPD/110633/2015]. This work was in part financed by a joint project, funded by the Portuguese Funda\u00E7\u00E3o para a Ci\u00EAncia e a Tecnologia [Grant FCT-ANR/ BEXBCM/0002/2013, as well as Grant UID/BIM/0009/2016 of the Research Center for Toxicogenomics and Human Health (ToxOmics)] and the L\u2019Agence Nationale de la Recherche [Grant ANR-13-ISV5-0001]. F.E. was supported with a post-doctoral fellowship grant of the Portuguese Funda\u00E7\u00E3o para a Ci\u00EAncia e a Tecnologia [Grant SFRH/BPD/110633/2015].

Funders	Funder number
Portuguese Fundação para a Ciência e a Tecnologia
Fundação para a Ciência e a Tecnologia	SFRH/BPD/110633/2015, FCT-ANR/ BEXBCM/0002/2013, UID/BIM/0009/2016
Agence Nationale de la Recherche	ANR-13-ISV5-0001

Keywords

Cytochrome b5 (CYB5)
Electron-transfer (ET)
Microsomal cytochrome p450 (CYP)
NADPH-cytochrome P450 reductase (CPR)
Protein dynamics
Protein-protein interaction

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10.3390/ijms19123914

Cite this

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title = "Probing the role of the hinge segment of cytochrome P450 oxidoreductase in the interaction with cytochrome P450",

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 b 5 (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 = "Cytochrome b5 (CYB5), Electron-transfer (ET), Microsomal cytochrome p450 (CYP), NADPH-cytochrome P450 reductase (CPR), Protein dynamics, Protein-protein interaction",

author = "Diana Campelo and Francisco Esteves and Palma, {Bernardo Brito} and Gomes, {Bruno Costa} and Jos{\'e} Rueff and Thomas Lautier and Philippe Urban and Gilles Truan and Michel Kranendonk",

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AU - Campelo, Diana

AU - Esteves, Francisco

AU - Palma, Bernardo Brito

AU - Gomes, Bruno Costa

AU - Rueff, José

AU - Lautier, Thomas

AU - Urban, Philippe

AU - Truan, Gilles

AU - Kranendonk, Michel

PY - 2018/12

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AB - 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 b 5 (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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KW - Protein dynamics

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ER -

Probing the role of the hinge segment of cytochrome P450 oxidoreductase in the interaction with cytochrome P450

Abstract

Bibliographical note

Funding

Keywords

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