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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]

V1, a water-soluble portion of vacuole-type ATPase (V-ATPase), is an ATP-driven rotary motor, similar to F1-ATPase. Hydrolysis of ATP is coupled to unidirectional rotation of the central rotor D and F subunits relative to the A3B3 cylinder. In this study, we analyzed the rotation kinetics of V1 in detail. At low ATP concentrations, the D subunit rotated stepwise, pausing every 120°. The dwell time between steps revealed that V1 consumes one ATP per 120° step. V1 generated torque of ≈35 pN nm, slightly lower than the ≈46 pN nm measured for F1. Noticeably, the angles for both ATP cleavage and binding were apparently the same in V1. in sharp contrast to F1, which cleaves ATP at 80° posterior to the binding of ATP. Thus, the mechanochemical cycle of V1 has marked differences to that of F1. [ABSTRACT FROM AUTHOR]