Mechanism of ADP-inhibited ATP hydrolysis in single proton-pumping FoF1-ATP synthase trapped in solution

Abstract

FoF1-ATP synthases in mitochondria, in chloroplasts and in most bacteria are the proton-driven membrane enzymes supplying the cells with ATP made from ADP and phosphate. To monitor and prevent the reverse chemical reaction of fast wasteful ATP hydrolysis by the enzymes, different control mechanisms exist including mechanical or redox-based blockade of catalysis and ADP inhibition. In general product inhibition is expected to slow down the mean catalytic turnover. However, biochemical assays are ensemble measurements and cannot discriminate between a mechanism affecting all enzymes equally or individually. For example, all enzymes could work slower at a decreasing substrate/product ratio, or more and more individual enzymes are blocked completely. Here, we examined how increasing amounts of ADP affected ATP hydrolysis of single Escherichia coli FoF1-ATP synthases in liposomes. We observed individual catalytic turnover of the enzymes one after another by monitoring the internal subunit rotation using single-molecule Forster resonance energy transfer (smFRET). Observation times of single FRET-labeled FoF1-ATP synthase in solution were increased up to seconds using a confocal Anti-Brownian electrokinetic trap (ABEL trap). By counting active versus inhibited enzymes we revealed that ADP inhibition did not decrease the catalytic turnover of all FoF1-ATP synthases equally. Instead, increasing ADP in the ADP/ATP mixture reduced the number of the remaining active enzymes which were operating at similar catalytic rates for varying substrate/product ratios.