The interplay of lipids and respiratory enzymes in synthetic ATP producing systems

Deutschmann, Sabina (2022). The interplay of lipids and respiratory enzymes in synthetic ATP producing systems (Unpublished). (Dissertation, Universität Bern, Philosophisch-naturwissenschaftliche Fakultät)

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All organisms require energy to maintain vital macroscopic and microscopic processes. Primary energy supplied in form of light or as nutrients has to be converted to the universal energy currency ATP before it can be used by the cells. The majority of ATP is produced at energy-converting membranes where a series of respiratory enzyme complexes couple electron transport reactions to the generation of an electrochemical proton gradient across the membrane. Similar to power generation at a hydroelectric power turbine through water pressure, the electrochemical proton gradient energizes the production of ATP by driving rotation of the molecular turbine of the ATP synthase. While the structures and mech-anisms of most respiratory enzymes and the ATP synthase are well understood, relatively little is known how these respiratory complexes interact with each other, including the influence of the environment on the complexes and their interactions. Here, we used a bottom-up approach, in which the individual enzymes are purified and reinserted into a lipid bilayer to investigate the functional interplay of these enzymes. In the simplest scenario, the terminal respiratory bo3 oxidase and the ATP synthase from E. coli are coreconstituted into liposomes to form a minimal respiratory chain system, where proton pumping by bo3 oxidase is initiated by adding its reduced substrate ubiquinol Q1, and ATP synthesis is monitored in real-time under steady-state conditions. Nilsson et al. recently found a strong influence of the lipid composition on the ATP synthesis rate in this system 1 where the stepwise insertion of neg-atively charged lipids into zwitterionic liposomes dramatically reduced ATP synthesis. The data indicate lipid-dependent changes in the lateral distance between the two enzymes. In this PhD thesis, we aimed to test this hypothesis by reversibly coupling bo3 oxidase and ATP synthase, reconstituting the complex subsequently into liposomes of varying lipid composition and comparing coupled ATP synthesis rates of the coupled complex with rates of freely floating enzymes. We describe three different approaches for coupling of these two large complexes and the different challenges. While perfect stoichiometric coupling of the two enzymes has not yet been achieved here, the results are promising and are the basis for current experiments.
Instead of using the artificial DTT/Q1 electron donor system, we have expanded the minimal respiratory chain by the monotopic complex I analogue NDH-2 to supply the system with reduced quinol Q8, thereby generating a more natural alternative. If this alternative system was subjected to a lipid screen-ing, we observed an inverted lipid dependency than with the Q1 system, with a strong requirement of negatively charged lipids for coupled ATP synthesis. We were able to pinpoint this effect to an increased NADH:ubiquinone oxidoreductase activity in presence of anionic liposomes in experiments with solu-bilized NDH-2. We identified negatively charged liposomes to be essential for proper NDH-2 activity, indicating a charge-mediated binding of NDH-2 to the membrane.
The inverted lipid dependency of the two systems created doubt that a lipid-dependent lateral differ-ence of the proteins is the only reason for the observed lipid effect. We thus wanted to analyze the effect of the lipid composition on the orientation of the bo3 oxidase. To this end, we established a novel method to determine the relative orientation of membrane proteins in liposomes that is independent of protein function. Instead, the membrane protein is site-specifically labeled with a fluorophore that is quenched stepwise after reconstitution into liposomes with membrane-impermeable quencher. A strongly lipid-dependent orientation of bo3 oxidase in liposomes with lower fraction of desired inside-out orientation in liposomes carrying a net negative charge compared to uncharged liposomes was in-deed observed, in good agreement with the reduced ATP synthesis activity in negatively charged lipo-somes described above. Furthermore, we show that the fraction of inside-out orientation could be in-creased when reconstitution was carried out in presence of salt, suggesting an electrostatic-mediated insertion of bo3 oxidase into liposomes.
To suppress this unwanted effect on orientation, we aimed to achieve unidirectional inside-out orien-tation of bo3 oxidase. Based on the observation that the large head group of ATP synthase is unable to cross the membrane during reconstitution, a large soluble protein (~100 kDa) was coupled to bo3 oxi-dase using the SpyTag/SpyCatcher methodology to guide its insertion in the favored inside-out orien-tation. We present the successful procedure to produce the desired product keeping its native func-tionality and preliminary experiments with ATP synthase look very promising and are the basis for on-going trials of unidirectional bo3 oxidase reconstitution.

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