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

Complex I is a crucial respiratory enzyme that conserves the energy from NADH oxidation by ubiquinone‐10 (Q 10 ) in proton transport across a membrane. Studies of its energy transduction mechanism are hindered by the extreme hydrophobicity of Q 10 , and they have so far relied on native membranes with many components or on hydrophilic Q 10 analogues that partition into membranes and undergo side reactions. Herein, we present a self‐assembled system without these limitations: proteoliposomes containing mammalian complex I, Q 10 , and a quinol oxidase (the alternative oxidase, AOX) to recycle Q 10 H 2 to Q 10 . AOX is present in excess, so complex I is completely rate determining and the Q 10 pool is kept oxidized under steady‐state catalysis. The system was used to measure a fully‐defined K M value for Q 10 . 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.