Study of human Cytochrome P450 reductase by Electron Paramagnetic Resonance - Archive ouverte HAL
Article Dans Une Revue Biochimica biophysica acta (BBA) - Bioenergetics Année : 2022

Study of human Cytochrome P450 reductase by Electron Paramagnetic Resonance

Résumé

Human cytochrome P450 reductase is a membrane protein located on the cytoplasmic side of the endoplasmic reticulum. It contains two binding domains for FAD and FMN, which are connected by a flexible region [1]. CPR transfers electrons from NADPH to cytochromes P450 through flavins. Two crystallographic structures were obtained for Rattus norvegicus showing "closed" and "open" conformations [2] These two structures show different distances between the flavin cofactors. Indeed, for inter-flavin Electronic Transfer (ET), the two FAD and FMN domains must be nearby (a "closed" conformation), but inter-protein ET (FMN to CYPs) requires a more "open" conformation. The understanding and characterization of domain movement associated with ET is essential and will be studied by measuring distances between two paramagnetic centers via two approaches: 1) using endogenous probes by isolating the flavin radical, and 2) by Site-Directed Spin Labeling and EPR, based on the insertion of paramagnetic nitroxide probes. Classically, these are specific to cysteines [3]. However, C566 is involved in the enzymatic activity and cannot be modified. The incorporation of non-natural amino acids and their labelling with specific nitroxides has been shown to be a powerful alternative [4] First experiments on human soluble CPR concerning the labelling of non-natural amino acids, as well as the trapping of flavin centers will be presented.
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Dates et versions

hal-04050498 , version 1 (29-03-2023)

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Maxime Bizet, Deborah Byrne, Guillaume Gerbaud, Frédéric Biaso, Pierre Dorlet, et al.. Study of human Cytochrome P450 reductase by Electron Paramagnetic Resonance. Biochimica biophysica acta (BBA) - Bioenergetics, 2022, 1863, pp.148752. ⟨10.1016/j.bbabio.2022.148752⟩. ⟨hal-04050498⟩
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