A Self-Assembled Respiratory Chain that Catalyzes NADH Oxidation by Ubiquinone-10 Cycling between Complex I and the Alternative Oxidase

• Published in: Angewandte Chemie International Edition 2016-01, Vol.55 (2), p.728-731
• Main Authors: Jones, Andrew J. Y., Blaza, James N., Bridges, Hannah R., May, Benjamin, Moore, Anthony L., Hirst, Judy
• Format: Article
• Language: English
• Subjects: Catalysis; Communication; Communications; Electron Transport; electron transport chain; Electron Transport Complex I - chemistry; Mitochondrial Proteins - chemistry; NAD - chemistry; NADH; Oxidation-Reduction; oxidoreductases; Oxidoreductases - chemistry; Plant Proteins - chemistry; proteoliposomes; quinones; Ubiquinone - chemistry
• ISSN: 1433-7851; 1521-3773
• DOI: 10.1002/anie.201507332

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 KM 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. Chained together: Mitochondrial complex I is crucial for respiration, but its reactions with ubiquinone‐10 (Q10) are poorly understood because Q10 is extremely hydrophobic. An artificial electron transport chain comprising complex I, Q10, and a quinol oxidase was self‐assembled in synthetic vesicles and used to study Q10 reduction in a fully defined environment. This self‐assembled system is suitable for studying any enzyme that uses a quinone/quinol substrate.