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 proton-driven membrane enzymes that supply the cells with ATP made from ADP and phosphate. Different control mechanisms exist to monitor and prevent the enzymes’ reverse chemical reaction of fast wasteful ATP hydrolysis, including mechanical or redox-based blockade of catalysis and ADP inhibition. In general, product inhibition is expected to slow down the mean catalytic turnover. Biochemical assays are ensemble measurements and cannot discriminate between a mechanism affecting all enzymes equally or individually. For example, all enzymes could work more slowly at a decreasing substrate/product ratio, or an increasing number of individual enzymes could be completely blocked. Here, we examined the effect of increasing amounts of ADP on ATP hydrolysis of single Escherichia coli FoF1-ATP synthases in liposomes. We observed the individual catalytic turnover of the enzymes one after another by monitoring the internal subunit rotation using single-molecule Förster resonance energy transfer (smFRET). Observation times of single FRET-labeled FoF1-ATP synthases in solution were extended up to several 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 remaining active enzymes that operated at similar catalytic rates for varying substrate/product ratios.

Single NV in nanodiamond for quantum sensing of protein dynamics in an ABEL trap

“…Our FPGA-based confocal ABEL trap setup was described earlier [17,18,31,[53][54][55] and was modified here. Briefly, a fiber-coupled linearly polarized pulsed laser diode emitting at 531 nm with a beam waist of 700 m (LDH-D-FA-530DL, Picoquant, Berlin, Germany) was used for excitation.…”

“…We have taken an alternative approach to monitor subunit rotation of a single FoF1-ATP synthase in real time during both ATP-driven hydrolysis as well as proton-driven ATP synthesis [4,[13][14][15][16][17][18]. Attaching one fluorophore on a rotating subunit and a second different fluorophore on a non-rotating static subunit allowed us to measure the distance between these two markers by Förster resonance energy transfer within the enzyme (smFRET).…”

“…A proton motive force could be generated by a difference in proton concentrations inside and outside of the liposome plus a directional electric membrane potential comprising a [K + ] diffusion potential [19]. Thus, we analyzed rotary step sizes, direction of rotation during ATP synthesis, internal twisting of subunits [20], ATP concentration dependence [17], and the mechanism of ADP inhibition [18].…”

“…The latest implementation of this confocal ABEL trap with smFRET yields very narrow FRET efficiency distributions, laser power-dependent single-molecule time traces of tens of seconds, and provides diffusion coefficients and electric charge information as well [30]. We could obtain time traces of the catalytic turnover from FRET-labeled single FoF1-ATP synthase reconstituted in a liposome for several seconds using ABEL-FRET [17,18,31].…”

Transcriptional and Metabolic Response of a Strain of Escherichia coli PTS− to a Perturbation of the Energetic Level by Modification of [ATP]/[ADP] Ratio

“…Central and peripheric stalks link F 0 and F 1 subunits together. The central stalk and a ring of the subunit c of the F 0 complex form a rotating structure whose rotation is driven by extracellular proton translocation across the F 0 complex, resulting in structural changes in the F 1 complex, leading in the synthesis of ATP from ADP and phosphate [ 1 , 2 , 3 , 4 , 5 ]. ATP plays a fundamental role in the cell’s physiology by providing energy for essential processes such as active transport, central carbon metabolism, biosynthetic pathways, DNA, RNA, protein synthesis, signal transduction, motility, cellular division, and stress responses [ 6 ].…”