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

• Published in: Angewandte Chemie 2016-01, Vol.128 (2), p.738-741
• Main Authors: Jones, Andrew J. Y., Blaza, James N., Bridges, Hannah R., May, Benjamin, Moore, Anthony L., Hirst, Judy
• Format: Article
• Language: English
• Subjects: Chinone; Elektronentransportkette; NADH; Oxidoreduktasen; Proteoliposome
• ISSN: 0044-8249; 1521-3757
• DOI: 10.1002/ange.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. Bildet eine Kette! Der mitochondriale Komplex I ist lebenswichtig für die Atmung, doch seine Reaktionen mit dem stark hydrophoben Ubichinon‐10 (Q10) sind nur schlecht verstanden. Eine künstliche Elektronentransportkette aus Komplex I, Q10 und einer Chinol‐Oxidase wurde in synthetischen Vesikeln aufgebaut, um die Reduktion von Q10 in einer definierten Umgebung zu untersuchen. Das selbstorganisierte System eignet sich für Studien an allen Enzymen, die Chinone/Chinole als Substrate nutzen.