title: A Self-Assembled Respiratory Chain that Catalyzes NADH Oxidation by Ubiquinone-10 Cycling between Complex I and the Alternative Oxidase.
abstract: Complex I is a crucial respiratory enzyme that conserves the energy from NADH oxidation by ubiquinone-10 (Q10) in proton transport across a membrane. Studies of its energy transduction mechanism are hindered by the extreme hydrophobicity of Q10, and they have so far relied on native membranes with many components or on hydrophilic Q10 analogues that partition into membranes and undergo side reactions. Herein, we present a self-assembled system without these limitations: proteoliposomes containing mammalian complex I, Q10, and a quinol oxidase (the alternative oxidase, AOX) to recycle Q10H2 to Q10. AOX is present in excess, so complex I is completely rate determining and the Q10 pool is kept oxidized under steady-state catalysis. The system was used to measure a fully-defined K(M) value for Q10. The strategy is suitable for any enzyme with a hydrophobic quinone/quinol substrate, and could be used to characterize hydrophobic inhibitors with potential applications as pharmaceuticals, pesticides, or fungicides.
authors.affiliation: Medical Research Council Mitochondrial Biology Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 0XY (UK).
authors.forename: Andrew J Y
authors.initials: AJ
authors.lastname: Jones
authors.affiliation: Medical Research Council Mitochondrial Biology Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 0XY (UK).
authors.forename: James N
authors.initials: JN
authors.lastname: Blaza
authors.affiliation: Medical Research Council Mitochondrial Biology Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 0XY (UK).
authors.forename: Hannah R
authors.initials: HR
authors.lastname: Bridges
authors.affiliation: School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG (UK).
authors.forename: Benjamin
authors.initials: B
authors.lastname: May
authors.affiliation: School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG (UK).
authors.forename: Anthony L
authors.initials: AL
authors.lastname: Moore
authors.affiliation: Medical Research Council Mitochondrial Biology Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 0XY (UK). jh@mrc-mbu.cam.ac.uk.
authors.forename: Judy
authors.initials: J
authors.lastname: Hirst
mesh_terms: D002384:Catalysis; D004579:Electron Transport; D042967:Electron Transport Complex I / Q000737:chemistry*; D024101:Mitochondrial Proteins / Q000737:chemistry*; D009243:NAD / Q000737:chemistry*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry*; D014451:Ubiquinone / Q000737:chemistry*; D000099188:Alternative Oxidase
chemical_list: D042967:Electron Transport Complex I; D024101:Mitochondrial Proteins; D009243:NAD; D010088:Oxidoreductases; D010940:Plant Proteins; D014451:Ubiquinone; D000099188:Alternative Oxidase; C025204:Ubiquinone Q2
keywords: NADH; electron transport chain; oxidoreductases; proteoliposomes; quinones