Single molecule behavior of inhibited and active states of Escherichia coli ATP synthase F1 rotation.
ATP hydrolysis-dependent rotation of the F(1) sector of the ATP synthase is a successive cycle of catalytic dwells (∼0.2 ms at 24 °C) and 120° rotation steps (∼0.6 ms) when observed under V(max) conditions using a low viscous drag 60-nm bead attached to the γ subunit (Sekiya, M., Nakamoto, R. K., Al-Shawi, M. K., Nakanishi-Matsui, M., and Futai, M. (2009) J. Biol. Chem. 284, 22401-22410). During the normal course of observation, the γ subunit pauses in a stochastic manner to a catalytically inhibited state that averages ∼1 s in duration. The rotation behavior with adenosine 5'-O-(3-thiotriphosphate) as the substrate or at a low ATP concentration (4 μM) indicates that the rotation is inhibited at the catalytic dwell when the bound ATP undergoes reversible hydrolysis/synthesis. The temperature dependence of rotation shows that F(1) requires ∼2-fold higher activation energy for the transition from the active to the inhibited state compared with that for normal steady-state rotation during the active state. Addition of superstoichiometric ε subunit, the inhibitor of F(1)-ATPase, decreases the rotation rate and at the same time increases the duration time of the inhibited state. Arrhenius analysis shows that the ε subunit has little effect on the transition between active and inhibited states. Rather, the ε subunit confers lower activation energy of steady-state rotation. These results suggest that the ε subunit plays a role in guiding the enzyme through the proper and efficient catalytic and transport rotational pathway but does not influence the transition to the inhibited state.
Department of Biochemistry, Faculty of Pharmaceutical Sciences, and Futai Special Laboratory, Iwate Medical University, Yahaba, Iwate 028-3694, Japan.
Mizuki
M
Sekiya
Hiroyuki
H
Hosokawa
Mayumi
M
Nakanishi-Matsui
Marwan K
MK
Al-Shawi
Robert K
RK
Nakamoto
Masamitsu
M
Futai
D000255:Adenosine Triphosphate / Q000737:chemistry; D001671:Biochemistry / Q000379:methods; D001703:Biophysics / Q000379:methods; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D025261:Mitochondrial Proton-Translocating ATPases / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D013696:Temperature; D014783:Viscosity
D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; C474451:F1F0-ATP synthase; C496259:F0F1-ATPase epsilon subunit, E coli; D025261:Mitochondrial Proton-Translocating ATPases; C444660:gamma subunit, F(1) ATPase; D006180:Proton-Translocating ATPases; D008274:Magnesium