Kazuhiko Kinosita

Direct Observation of the Rotation of ε Subunit in F1-ATPase

Rotation of the ε subunit in F1-ATPase from thermophilic Bacillusstrain PS3 (TF1) was observed under a fluorescence microscope by the method used for observation of the γ subunit rotation (Noji, H., Yasuda, R., Yoshida, M., and Kinosita, K., Jr. (1997) Nature 386, 299–302). The α3β3γε complex of TF1 was fixed to a solid surface, and fluorescently labeled actin filament was attached to the ε subunit through biotin-streptavidin. In the presence of ATP, the filament attached to ε subunit rotated in a unidirection. The direction of the rotation was the same as that observed for the γ subunit. The rotational velocity was slightly slower than the filament attached to the γ subunit, probably due to the experimental setup used. Thus, as suggested from biochemical studies (Aggeler, R., Ogilvie, I., and Capaldi, R. A. (1997)J. Biol. Chem. 272, 19621–19624), the ε subunit rotates with the γ subunit in F1-ATPase during catalysis.

Purine but Not Pyrimidine Nucleotides Support Rotation of F1-ATPase

The binding change model for the F1-ATPase predicts that its rotation is intimately correlated with the changes in the affinities of the three catalytic sites for nucleotides. If so, subtle differences in the nucleotide structure may have pronounced effects on rotation. Here we show by single-molecule imaging that purine nucleotides ATP, GTP, and ITP support rotation but pyrimidine nucleotides UTP and CTP do not, suggesting that the extra ring in purine is indispensable for proper operation of this molecular motor. Although the three purine nucleotides were bound to the enzyme at different rates, all showed similar rotational characteristics: counterclockwise rotation, 120° steps each driven by hydrolysis of one nucleotide molecule, occasional back steps, rotary torque of ∼40 piconewtons (pN)·nm, and mechanical work done in a step of ∼80 pN·nm. These latter characteristics are likely to be determined by the rotational mechanism built in the protein structure, which purine nucleotides can energize. With ATP and GTP, rotation was observed even when the free energy of hydrolysis was −80 pN·nm/molecule, indicating ∼100% efficiency. Reconstituted FoF1-ATPase actively translocated protons by hydrolyzing ATP, GTP, and ITP, but CTP and UTP were not even hydrolyzed. Isolated F1 very slowly hydrolyzed UTP (but not CTP), suggesting possible uncoupling from rotation.

8.3 Rotational Catalysis by F 1 -ATPase

F 1 -ATPase, a water-soluble portion of ATP synthase, is a fully reversible rotary molecular machine in which a central γ subunit rotates inside a cylinder made of three α and three β subunits alternately arranged. This motor rotates counterclockwise by hydrolyzing ATP in three catalytic sites but synthesizes ATP when forced to rotate clockwise by an external force. Single-molecule studies have revealed how the chemical reactions that occur in the three catalytic sites are coupled to mechanical rotation.