F₁-ATPase Rotation Mechanism
F₁ can be purified from Fₒ and the membrane, and studied as a soluble ATPase. Each catalytic site steps through successive steps of ATP binding, ATP hydrolysis, and product release. The steps of the 3 catalytic sites are staggered and correspond to their position relative to the rotor. Each catalytic cycle involves a catalytic dwell when hydrolysis occurs, followed by a 120° power stroke.
Two of the catalytic sites do the heavy lifting during a given catalytic cycle when they are in the βE and βD conformations, while the third βT site is in a conformation with tightly bound ATP (not shown). A cross-section of F₁ of the rotor surrounded by the two catalytic sites that change conformation during one catalytic cycle show that the lever domains are open and closed in the empty and product release conformations, respectively.
We used the AuNR rotation assay to measure the 120° power strokes. The angular velocity averaged from thousands of power strokes shows that the velocity changes with rotational position.
We observed similar angular velocity profiles in:
F₁-ATPase from: E. coli, thermophilic G. stearothermophilus bacteria, and Mycobacterium smegmatis (not shown)
A₁-ATPase from: M. mazei Gö1
V₁-ATPase from: S. cerevisiae (not shown).
While these profiles look similar, these motors differ in the rotary positions where substrate binding and product release occur. To date, the reason for these differences is unknown.
When we carried out the first single-molecule Årrhenius analysis on the E. coli F1-ATPase power stroke, we got the unexpected result that the activation energy (EA) was negative during the first 60° indicating that this rotation was powered by elastic energy. An elastic spring constant of 50 kBT rad-1 was calculated from the parabolic dependence of EA vs rotational position (red).
This parabolic dependence also correlated to the probability of ATP binding to the empty catalytic site (optimal at ~34°), and a second parabolic dependence of EA on rotational position (green) correlated to the dissociation of ADP (optimal at ~95°).
The Elastic Coupling Mechanism of F₁-ATPase Driven Rotation
This mechamism is based on these and many other results.
A. Catalytic dwell 0° after hydrolysis with tightly wound rotor coiled-coil tethered to βE catalytic site (*). Power stroke starts by βE-Pi release to allow coiled-coil unwinding.
B. Phase-1 elastic powered rotation to equilibrium position of 34° where ATP binds. Binding energy of ATP closes βE-lever to rotate further.
C. Tether of rotor coiled-coil switches to βD catalytic site for Phase-2 rotation to dissociate ADP at ~95°.
D. Catalytic dwell begins when conformations change (βE to βT and βD to βE), and coiled-coil is rewound.
Publications
Martin, J., Hornung, T., Ishmukhametov, R., Spetzler, D., and Frasch, W. D. (2018) “Elastic Coupling Power Stroke Mechanism of the F1-ATPase”, Proc. Natl. Acad. Sci. USA, 115, 5750-5755.
Ragunathan, R., Sielaff, J., Sundararaman, L., Biukovic, G., Sony, M., Manimekalai, S., Singh, D., Kundu, S., Wohland, T., Frasch, W.D., Dick, T., and Grüber, G. (2017) “The Uniqueness of Subunit a of Mycobacterial F-ATP synthases: An evolutionary Variant for Niche Adaptation”, J. Biol. Chem. 292:11262-11279
Sielaff, H., Martin, J., Grüber, G., and Frasch, W. D. (2016) “Power Stroke Angular Velocity Profiles of Archaeal A-ATP Synthase Versus Thermophilic and Mesophilic F-ATP Synthase Molecular Motors” J. Biol. Chem. 291: 25351-25363.
Martin, J., Ishmukhametov, R., Hornung, T., Ahmad, Z., and Frasch, W. D. (2014) “Anatomy of F1-ATPase Powered Rotation” Proc. Natl. Acad. Sci. USA 111, 3715-3720.
Hornung, T., Martin, J., Ishmukhametov, R., Spetzler, D., and Frasch, W. D. (2011) “Microsecond Resolution of Single Molecule Rotation Catalyzed by Molecular Motors”, Methods in Molecular Biology 778, 273-289
Spetzler, D., Ishmukhametov, R., Day, L. J., Hornung, T., Martin, J., and Frasch, W. D. (2009) “Single Molecule Measurements of F1-ATPase Reveal an Interdependence between the Power Stroke and the Dwell Duration”, Biochemistry 49, 7979-7985.
Hornung, T., Ishmukhametov, R., Spetzler, D., Martin, J., and Frasch, W. D. (2008) “Determination of Torque Generation from the Power Stroke of Escherichia coli F1-ATPase.” Biochim. Biophys. Acta- Bioenergetics 1777, 579-582.
York, J., Spetzler, D., Hornung, T., Ishmukhametov, R., Martin, J., and Frasch, W.D. (2007) “Abundance of Escherichia coli F1-ATPase Molecules Observed to Rotate via Single-Molecule Microscopy with Gold Nanorod Probes”, J. Bioenergetics and Biomembranes, 39, 435-439.
Spetzler, D., York, J., Lowry, D., Daniel, D., Fromme, R. and Frasch, W. D. (2006) “Microsecond Time Resolution of Single Molecule F1-ATPase Rotation”, Biochemistry 45, 3117-3124. DOI: 10.1021/bi052363n. Accelerated publication designated a Hot Article by the ACS based on top-10 down loads from the journal.