title: A well-balanced preexisting equilibrium governs electron flux efficiency of a multidomain diflavin reductase.
abstract: Diflavin reductases are bidomain electron transfer proteins in which structural reorientation is necessary to account for the various intramolecular and intermolecular electron transfer steps. Using small-angle x-ray scattering and nuclear magnetic resonance data, we describe the conformational free-energy landscape of the NADPH-cytochrome P450 reductase (CPR), a typical bidomain redox enzyme composed of two covalently-bound flavin domains, under various experimental conditions. The CPR enzyme exists in a salt- and pH-dependent rapid equilibrium between a previously described rigid, locked state and a newly characterized, highly flexible, unlocked state. We further establish that maximal electron flux through CPR is conditioned by adjustable stability of the locked-state domain interface under resting conditions. This is rationalized by a kinetic scheme coupling rapid conformational sampling and slow chemical reaction rates. Regulated domain interface stability associated with fast stochastic domain contacts during the catalytic cycle thus provides, to our knowledge, a new paradigm for improving our understanding of multidomain enzyme function.
authors: Institut de Chimie des Substances Naturelles, CNRS, UPR 2301, Centre de Recherche de Gif, Gif-sur-Yvette, France.
authors: Oriane
authors: O
authors: Frances
authors: Institut de Chimie des Substances Naturelles, CNRS, UPR 2301, Centre de Recherche de Gif, Gif-sur-Yvette, France.
authors: Fataneh
authors: F
authors: Fatemi
authors: Université de Toulouse, INSA, UPS, INP, LISBP, Toulouse, France; INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, Toulouse, France; CNRS, UMR5504, Toulouse, France.
authors: Denis
authors: D
authors: Pompon
authors: Institut de Chimie des Substances Naturelles, CNRS, UPR 2301, Centre de Recherche de Gif, Gif-sur-Yvette, France.
authors: Eric
authors: E
authors: Guittet
authors: Institut de Chimie des Substances Naturelles, CNRS, UPR 2301, Centre de Recherche de Gif, Gif-sur-Yvette, France.
authors: Christina
authors: C
authors: Sizun
authors: SWING beamline, Synchrotron SOLEIL, Gif-sur-Yvette, France.
authors: Javier
authors: J
authors: Pérez
authors: Institut de Chimie des Substances Naturelles, CNRS, UPR 2301, Centre de Recherche de Gif, Gif-sur-Yvette, France. Electronic address: ewen.lescop@cnrs.fr.
authors: Ewen
authors: E
authors: Lescop
authors: Université de Toulouse, INSA, UPS, INP, LISBP, Toulouse, France; INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, Toulouse, France; CNRS, UMR5504, Toulouse, France. Electronic address: gilles.truan@insa-toulouse.fr.
authors: Gilles
authors: G
authors: Truan
mesh_terms: D004548:Elasticity; D004583:Electrons; D005415:Flavins / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D053838:Scattering, Small Angle; D012996:Solutions; D014965:X-Rays
chemical_list: D005415:Flavins; D012996:Solutions; D009251:NADPH-Ferrihemoprotein Reductase