FoF1-ATP synthases are the ubiquitous membrane enzymes which catalyze ATP synthesis or ATP hydrolysisin 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 F\"orster 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 calatytic 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 previoulsy for isolated single F1 fragments without coupling to the membrane-embedded Fo domain of the enzyme .
FoF1-ATP synthases are ubiquitous membrane-bound, rotary motor enzymes that can catalyze ATP synthesis and hydrolysis. Their 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. Single-molecule F\"orster resonance energy transfer (smFRET) has been used to detect subunit rotation within FoF1-ATP synthases embedded in freely diffusing liposomes. We now report that kinetic monitoring of functional rotation can be prolonged from milliseconds to seconds by utilizing an Anti-Brownian electrokinetic trap (ABEL trap). These extended observation times allowed us to observe fluctuating rates of functional rotation for individual FoF1-liposomes in solution. Broad distributions of ATP-dependent catalytic rates were revealed . The buildup of an electrochemical potential of protons was confirmed to limit the maximum rate of ATP hydrolysis. In the presence of ionophores or uncouplers, the fastest subunit rotation speeds measured in single reconstituted FoF1-ATP synthases were 180 full rounds per second . This was much faster than measured by biochemical ensemble averaging, but not as fast as the maximum rotational speed reported previously for isolated single F1 complexes uncoupled from the membrane-embedded Fo complex. Further application of ABEL trap measurements should help resolve the mechanistic causes of such fluctuating rates of subunit rotation .
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the ubiquitous membrane enzymes which
ubiquitous membrane-bound, rotary motor enzymes that can
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By utilizing an Anti-Brownian electrokinetic trap (ABEL trap), single-molecule
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-based subunit rotation monitoring was
has been used to detect subunit rotation within FoF1-ATP synthases embedded in freely diffusing liposomes. We now report that kinetic monitoring of functional rotation can be
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. The
by utilizing an Anti-Brownian electrokinetic trap (ABEL trap). These
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for single proteoliposomes in solution allowed
allowed us
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rotation rates of individual enzymes and to map the broad
rates of functional rotation for individual FoF1-liposomes in solution. Broad
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calatytic rates in FoF1-ATP synthase
catalytic rates were revealed
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and uncouplers
or uncouplers,
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, i. e.
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previoulsy
previously
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fragments without coupling to
complexes uncoupled from
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domain of the enzyme
complex. Further application of ABEL trap measurements should help resolve the mechanistic causes of such fluctuating rates of subunit rotation