Inhibition of F1-ATPase Rotational Catalysis by the Carboxyl-terminal Domain of the ϵ Subunit

Escherichia coli ATP synthase (F0F1) couples catalysis and proton transport through subunit rotation. The ϵ subunit, an endogenous inhibitor, lowers F1-ATPase activity by decreasing the rotation speed and extending the duration of the inhibited state (Sekiya, M., Hosokawa, H., Nakanishi-Matsui, M., Al-Shawi, M. K., Nakamoto, R. K., and Futai, M. (2010) Single molecule behavior of inhibited and active states of Escherichia coli ATP synthase F1 rotation. J. Biol. Chem. 285, 42058–42067). In this study, we constructed a series of ϵ subunits truncated successively from the carboxyl-terminal domain (helix 1/loop 2/helix 2) and examined their effects on rotational catalysis (ATPase activity, average rotation rate, and duration of inhibited state). As expected, the ϵ subunit lacking helix 2 caused about ½-fold reduced inhibition, and that without loop 2/helix 2 or helix 1/loop 2/helix 2 showed a further reduced effect. Substitution of ϵSer108 in loop 2 and ϵTyr114 in helix 2, which possibly interact with the β and γ subunits, respectively, decreased the inhibitory effect. These results suggest that the carboxyl-terminal domain of the ϵ subunit plays a pivotal role in the inhibition of F1 rotation through interaction with other subunits.

Stochastic rotational catalysis of proton pumping F-ATPase

F-ATPases synthesize ATP from ADP and phosphate coupled with an electrochemical proton gradient in bacterial or mitochondrial membranes and can hydrolyse ATP to form the gradient. F-ATPases consist of a catalytic F 1 and proton channel F 0 formed from the α 3 β 3 γδϵ and ab 2 c 10 subunit complexes, respectively. The rotation of γϵ c 10 couples catalyses and proton transport. Consistent with the threefold symmetry of the α 3 β 3 catalytic hexamer, 120° stepped revolution has been observed, each step being divided into two substeps. The ATP-dependent revolution exhibited stochastic fluctuation and was driven by conformation transmission of the β subunit (phosphate-binding P-loop/α-helix B/loop/β-sheet4). Recent results regarding mechanically driven ATP synthesis finally proved the role of rotation in energy coupling.