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F1-ATPase is a rotary molecular motor driven by ATP hydrolysis that rotates the γ-subunit against the α3β3 ring. The crystal structures of F1, which provide the structural basis for the catalysis mechanism, have shown essentially 1 stable conformational state. In contrast, single-molecule studies have revealed that F1 has 2 stable conformational states: ATP-binding dwell state and catalytic dwell state. Although structural and single-molecule studies are crucial for the understanding of the molecular mechanism of F1, it remains unclear as to which catalytic state the crystal structure represents. To address this issue, we introduced cysteine residues at βE391 and γR84 of F1 from thermophilic Bacillus PS3. In the crystal structures of the mitochondrial F1, the corresponding residues in the ADP-bound β (βDP) and γ were in direct contact. The βE190D mutation was additionally introduced into the β to slow ATP hydrolysis. By incorporating a single copy of the mutant β-subunit, the chimera F1, α3β2β(E190D/E391 C)β(R84C), was prepared. In single-molecule rotation assay, chimera F1 showed a catalytic dwell pause in every turn because of the slowed ATP hydrolysis of β(E190D/E391C). When the mutant β and γ were cross-linked through a disulfide bond between βE391C and γR84C, F1 paused the rotation at the catalytic dwell angle of β(E190D/E391C), indicating that the crystal structure represents the catalytic dwell state and that βDP is the catalytically active form. The former point was again confirmed in experiments where F1 rotation was inhibited by adenosine-5'- (β,γ-imino)-triphosphate and/or azide, the most commonly used inhibitors for the crystallization of F1. [ABSTRACT FROM AUTHOR]

V-ATPases are rotary motor proteins that convert the chemical energy of ATP into the electrochemical potential of ions across cell membranes. V-ATPases consist of two rotary motors, V o and V 1 , and Enterococcus hirae V-ATPase (EhV o V 1 ) actively transports Na + in V o (EhV o ) by using torque generated by ATP hydrolysis in V 1 (EhV 1 ). Here, we observed ATP-driven stepping rotation of detergent-solubilized EhV o V 1 wild-type, aE634A, and BR350K mutants under various Na + and ATP concentrations ([Na + ] and [ATP], respectively) by using a 40-nm gold nanoparticle as a low-load probe. When [Na + ] was low and [ATP] was high, under the condition that only Na + binding to EhV o is rate limiting, wild-type and aE634A exhibited 10 pausing positions reflecting 10-fold symmetry of the EhV o rotor and almost no backward steps. Duration time before the forward steps was inversely proportional to [Na + ], confirming that Na + binding triggers the steps. When both [ATP] and [Na + ] were low, under the condition that both Na + and ATP bindings are rate limiting, aE634A exhibited 13 pausing positions reflecting 10- and 3-fold symmetries of EhV o and EhV 1 , respectively. The distribution of duration time before the forward step was fitted well by the sum of two exponential decay functions with distinct time constants. Furthermore, occasional backward steps smaller than 36° were observed. Small backward steps were also observed during three long ATP cleavage pauses of BR350K. These results indicate that EhV o and EhV 1 do not share pausing positions, Na + and ATP bindings occur at different angles, and the coupling between EhV o and EhV 1 has a rigid component.