Coupling of Rotation and Catalysis in F1-ATPase Revealed by Single-Molecule Imaging and Manipulation

Abstract

F(1)-ATPase is a rotary molecular motor that proceeds in 120 degrees steps, each driven by ATP hydrolysis. How the chemical reactions that occur in three catalytic sites are coupled to mechanical rotation is the central question. Here, we show by high-speed imaging of rotation in single molecules of F(1) that phosphate release drives the last 40 degrees of the 120 degrees step, and that the 40 degrees rotation accompanies reduction of the affinity for phosphate. We also show, by single-molecule imaging of a fluorescent ATP analog Cy3-ATP while F(1) is forced to rotate slowly, that release of Cy3-ADP occurs at approximately 240 degrees after it is bound as Cy3-ATP at 0 degrees . This and other results suggest that the affinity for ADP also decreases with rotation, and thus ADP release contributes part of energy for rotation. Together with previous results, the coupling scheme is now basically complete.

Estimating the rotation rate in the vacuolar proton-ATPase in native yeast vacuolar membranes

“…Such modifications may add or remove barriers to the rotation not present in the native system. Consequently, the reported rotation rates vary greatly in such studies, depending on the gene-engineered construct and the details of the artificial environment of the enzyme (Panke and Rumberg 1997;Masaike et al 2000;Itoh et al 2004;Imamura et al 2005;Adachi et al 2007;Takeda et al 2009). Such studies are not possible on a native enzyme in a native membrane, but since the ATP/rotation stoichiometry is known, one could assay the inorganic phosphate liberated from ATP hydrolysis by V-ATPase in unit time and relate it to the concentration of V-ATPase.…”

What can be learned about the enzyme ATPase from single-molecule studies of its subunit F1?

“…Using this approach in the case of mammalian (human and bovine) F 1 -ATPase Walker and coworkers (Bason et al 2014) found three such states about 65°, 25°and 30°apart. A somewhat similar 80°, 10°and 30°division of the 120°for the dwells of the thermophilic Bacillus ATPase studied in current single-molecule experiments has been suggested (Adachi et al 2007) based on a mutant that retards the rate and permits an improved time-resolution. Kinosita and coworkers (Adachi et al 2007) suggested that the combined 10°and 30°substeps give rise to the 40°substep resolved in previous single-molecule experiments (Nishizaka et al 2004) and associated with hydrolysis and Pi release in a different subunit (Watanabe & Noji, 2014;Watanabe et al 2010).…”

“…A somewhat similar 80°, 10°and 30°division of the 120°for the dwells of the thermophilic Bacillus ATPase studied in current single-molecule experiments has been suggested (Adachi et al 2007) based on a mutant that retards the rate and permits an improved time-resolution. Kinosita and coworkers (Adachi et al 2007) suggested that the combined 10°and 30°substeps give rise to the 40°substep resolved in previous single-molecule experiments (Nishizaka et al 2004) and associated with hydrolysis and Pi release in a different subunit (Watanabe & Noji, 2014;Watanabe et al 2010). The 10°is presumably the hydrolysis step, a similar small 15°hydrolysis step is supported by a very approximate independent analysis (Volkán- Kacsó & Marcus, 2017b), but the latter can be improved when more accurate kinetic data in the stalling experiments become available.…”

“…3. ADP release occurs during an 80°step, namely from 240°t o 320°: Using a mixture of ATP and Cy3-ATP, fluorescent Cy3-ATP was seen to undergo hydrolysis (the Cy3-ATP in solution, like ATP, induced the spontaneous rotation in F 1 -ATPase) and the resulting fluorescent Cy3-ADP was released during 240°to 320°step (Adachi et al 2007). 4.…”

“…With the assumption of the reversible nature of the individual steps in the overall process one then learns details of the mechanism of the ATP synthesis in the system. There are nevertheless some single molecule studies described in Section 2 of ATP synthesis directly using controlled rotation (Adachi et al 2007(Adachi et al , 2012. In the absence of F O driven rotation, the F 1 shaft spontaneously rotates in the hydrolysis direction, but in these controlled rotation experiments the rotation and hence the function can be artificially reversed using magnetic tweezers by forcing the γ shaft to rotate in the opposite direction, thus effectively emulating the function of the F O .…”