ABSTRACTFoF1-ATP synthases are the ubiquitous membrane enzymes which catalyze ATP synthesis or ATP hydrolysis in reverse, respectively. Enzyme kinetics are controlled by internal subunit rotation, by substrate and product concentrations, by mechanical inhibitory mechanisms, but also by the electrochemical potential of protons across the membrane. By utilizing an Anti- Brownian electrokinetic trap (ABEL trap), single-molecule Förster resonance energy transfer (smFRET)-based subunit rotation monitoring was prolonged from milliseconds to seconds. The extended observation times for single proteoliposomes in solution allowed to observe fluctuating rotation rates of individual enzymes and to map the broad distributions of ATP-dependent catalytic rates in FoF1-ATP synthase. The buildup of an electrochemical potential of protons was confirmed to limit the maximum rate of ATP hydrolysis. In the presence of ionophores and uncouplers the fastest subunit rotation speeds measured in single reconstituted FoF1-ATP synthases were 180 full rounds per second, i.e. much faster than measured by biochemical ensemble averaging, but not as fast as the maximum rotational speed reported previously for isolated single F1 fragments without coupling to the membrane-embedded Fo domain of the enzyme.
Fast ATP-dependent subunit rotation in reconstituted FoF1-ATP synthase trapped in solution