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

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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.

Original languageEnglish
Article number3914
JournalInternational Journal of Molecular Sciences
Volume19
Issue number12
DOIs
Publication statusPublished - 1 Dec 2018

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NADPH-Ferrihemoprotein Reductase
hinges
cytochromes
Hinges
Cytochrome P-450 Enzyme System
Cytochromes
Oxidoreductases
Proteins
interactions
Cytochromes b5
Carcinogens
Electrons
Oxidation-Reduction
Protein Isoforms
carcinogens
Enzyme kinetics
Mutation
electron transfer
Ionic strength
Static Electricity

Keywords

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

Cite this

@article{59c5b53ffaf144618dcb9db5f0a4c59a,
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 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 = "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",
year = "2018",
month = "12",
day = "1",
doi = "10.3390/ijms19123914",
language = "English",
volume = "19",
journal = "International Journal of Molecular Sciences",
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number = "12",

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T1 - Probing the role of the hinge segment of cytochrome P450 oxidoreductase in the interaction with cytochrome P450

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 - 2018/12/1

Y1 - 2018/12/1

N2 - 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.

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 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.

KW - Cytochrome b5 (CYB5)

KW - Electron-transfer (ET)

KW - Microsomal cytochrome p450 (CYP)

KW - NADPH-cytochrome P450 reductase (CPR)

KW - Protein dynamics

KW - Protein-protein interaction

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U2 - 10.3390/ijms19123914

DO - 10.3390/ijms19123914

M3 - Article

VL - 19

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1422-0067

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