Residue 249 in subunit beta regulates ADP inhibition and its phosphate modulation in Escherichia coli ATP synthase

Fast ATP-dependent Subunit Rotation in Reconstituted FoF1-ATP Synthase Trapped in Solution

“…the mean turnover was twofold faster than in the presence of 100 µM ATP solely. A twofold increase of average ATP hydrolysis rates in biochemical assays in the presence of valinomycin and nigericin was observed previously using E. coli F o F 1 -ATP synthases reconstituted in liposomes 13 .…”

“…The average ATP hydrolysis rate from all enzymes was 295 ATP·s -1 , i.e., the mean turnover was twofold faster than in the presence of 100 µM ATP only. A twofold increase of average ATP hydrolysis rates in biochemical assays in the presence of valinomycin and nigericin had been reported recently using EF o F 1 reconstituted in liposomes 12 . However, a concentration of 300 µM K + in the ABEL trap buffer here might be insufficient to dissipate a pmf quickly by the combination of K + carrier valinomycin and H + /K + antiporter nigericin.…”

“…the buildup of a counteracting pmf . Accordingly, addition of protonophores or uncouplers to dissipate a ΔpH or Δψ increases the maximum ATP hydrolysis rates of reconstituted F o F 1 -ATP synthases in liposomes by factors of two 13 to five 11 , in good agreement to the rates of solubilized enzymes in detergent micelles.…”

“…These V (max) values of reconstituted F o F 1 -ATP synthases were smaller than V (max) =651 ATP·s -1 or V (max) =516 ATP·s -1 reported for single enzymes solubilized in detergent, attached to a glass surface, and measured at room temperature using dark-field microscopy 51 . The smFRET- based V (max) and K m were in agreement with biochemical assays of E. coli F o F 1 -ATP synthase using native membrane vesicles (V (max) ∼200 ATP·s -1 7 , V (max) =336 ATP·s -1 at 30°C 52 ), purified enzymes in detergent (V (max) =285 ATP·s -1 and K m =78 µM at 24°C 5 , V (max) =217 ATP·s -1 and K m =140 µM at 30°C 53 , V (max) =700 ATP·s -1 with 10 mM ATP and 5 mM Mg 2+ at 37°C 54 ), single enzymes reconstituted into lipid nanodiscs (V (max) =140 ATP·s -1 at 25°C 12 ) or purified enzymes reconstituted into liposomes (v=65 ATP·s -1 at 100 µM ATP, increasing to v=210 ATP·s -1 in the presence of uncouplers valinomycin and nigericin 11, 13 ). V (max) obtained from Figure 5 F was about twice as fast as the mean turnover of ∼70 ATP·s -1 at 1 mM ATP reported previously in single-molecule FRET experiments with reconstituted F o F 1 -ATP synthase in solution 35b, 39, 55 .…”

“…The slower rate for EF o F 1 in sealed liposomes is largely due to inhibition by the pmf generated by ATP hydrolysis-driven proton pumping across the lipid bilayer. Accordingly, addition of protonophores or uncouplers to dissipate the counteracting pmf increased the maximum ATP hydrolysis rates of reconstituted F o F 1 -ATP synthases in liposomes by factors of two 12 to five 9 .…”