In vitro reconstructed minimal respiratory chains are powerful tools to investigate molecular interactions between the different enzyme components and how they are influenced by their environment. One such system is the coreconstitution of the terminal cytochrome bo 3 oxidase and the ATP synthase from Escherichia coli into liposomes, where the ATP synthase activity is driven through a proton motive force (pmf) created by the bo 3 oxidase. The proton pumping activity of the bo 3 oxidase is initiated using the artificial electron mediator short-chain ubiquinone and electron source DTT. Here, we extend this system and use either complex II or NDH-2 and succinate or NADH, respectively, as electron entry points employing the natural long-chain ubiquinone Q 8 or Q 10 . By testing different lipid compositions, we identify that negatively charged lipids are a prerequisite to allow effective NDH-2 activity. Simultaneously, negatively charged lipids decrease the overall pmf formation and ATP synthesis rates. We find that orientation of the bo 3 oxidase in liposomal membranes is governed by electrostatic interactions between enzyme and membrane surface, where positively charged lipids yield the desired bo 3 oxidase orientation but hinder reduction of the quinone pool by NDH-2. To overcome this conundrum, we exploit ionizable lipids, which are either neutral or positively charged depending on the pH value. We first coreconstituted bo 3 oxidase and ATP synthase into temporarily positively charged liposomes, followed by fusion with negatively charged empty liposomes at low pH. An increase of the pH to physiological values renders these proteoliposomes overall negatively charged, making them compatible with quinone reduction via NDH-2. Using this strategy, we not only succeeded in orienting the bo 3 oxidase essentially unidirectionally into liposomes but also found up to 3-fold increased ATP synthesis rates through the usage of natural, long-chain quinones in combination with the substrate NADH compared to the synthetic electron donor/mediator pair.