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.
Sekiya
Mizuki
M
Department of Biochemistry, Faculty of Pharmaceutical Sciences, and Futai Special Laboratory, Iwate Medical University, Yahaba, Iwate 028-3694, Japan.
Hosokawa
Hiroyuki
H
Nakanishi-Matsui
Mayumi
M
Al-Shawi
Marwan K
MK
Nakamoto
Robert K
RK
Futai
Masamitsu
M
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