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.