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 Q10 H2 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.