A TP synthase of mitochondria, chloroplasts, and bacteria catalyzes ATP synthesis coupled with a transmembrane proton flow (1-4). The enzyme consists of a membraneembedded, proton-conducting portion (F 0 ) and a protruding portion (F 1 ) in which catalytic sites for ATP synthesis͞hydrolysis exist. The isolated F 1 portion has ATPase activity; hence, it is often called F 1 -ATPase. It is composed of five different subunits with a stoichiometry of ␣ 3 ␤ 3 ␥␦. The ␣ 3 ␤ 3 ␥ subcomplex is the minimum ATPase-active complex, which has catalytic features similar to F 1 -ATPase. In the crystal structure (5), the central ␥ subunit is surrounded by an ␣ 3 ␤ 3 cylinder where three ␣ and three ␤ subunits are arranged alternately, and the six nucleotide binding sites are located at the ␣͞␤ subunit interfaces. Three of the binding sites are catalytic, and the ␤ subunits provide most of the catalytic residues. The other three are noncatalytic, and the ␣ subunits provide most residues contributing nucleotide binding.It has been postulated that the energy of the proton flow liberated at F 0 is transformed into the energy of ATP synthesis at F 1 through rotation of the central ␥ subunit and vice versathe energy of ATP hydrolysis can be converted into the energy of proton pumping through reverse rotation of the ␥ subunit (6). By using an ␣ 3 ␤ 3 ␥ subcomplex of thermophilic F 1 -ATPase (F 1 -ATPase) immobilized on a glass surface, we have observed ATP hydrolysis-driven rotation of the fluorescent actin filament attached to the ␥ subunit (7).At nanomolar ATP concentration, F 1 -ATPase binds and hydrolyzes a single ATP molecule, makes a 120°rotation, and waits for the next ATP molecule. As the ATP concentration increases, the ATP-waiting period becomes shorter until it is finally undetectable, and rotation of the actin filament becomes apparently continuous over hundreds of revolutions (8). However, when the rotation was observed for long periods, occasional pauses of rotation were recognized, even at high ATP concentrations (7, 9). Here, we show that these pauses occur at an intermediate step of rotation and mostly correspond to the ADP-Mg inhibition, which has been observed in bulk-phase kinetics as a general feature of the F 1 -ATPases (and ATP synthases). Slow interconversion between rotating and pausing states thus contributes to the attenuation of ATPase during steady-state catalysis. Materials and MethodsProtein Preparation. Escherichia coli strains used were JM109 (10) for preparation of plasmids, CJ236 (11) for generating uracilcontaining single-stranded plasmids for site-directed mutagenesis, and JM103⌬ (uncB-uncD) for expression of the mutant complexes of F 1 from the thermophilic Bacillus PS3. Plasmids M13mp18 and pKAGB1 (12), which carried genes for the ␣, ␤, and ␥ subunits of F 1 from the thermophilic Bacillus PS3, were used for mutagenesis and for gene expression, respectively. Site-directed mutagenesis was accomplished as described by Kunkel et al. (11). The plasmid pKAGB1͞␣C193S͞␥S107C͞ ␤His10tag has been described ...

F1-ATPase is a rotary molecular motor driven by ATP hydrolysis that rotates the ␥-subunit against the ␣3␤3 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 ␤E391 and ␥R84 of F1 from thermophilic Bacillus PS3. In the crystal structures of the mitochondrial F1, the corresponding residues in the ADPbound ␤ (␤DP) and ␥ were in direct contact. The ␤E190D mutation was additionally introduced into the ␤ to slow ATP hydrolysis. By incorporating a single copy of the mutant ␤-subunit, the chimera F1, ␣3␤2␤(E190D/E391C)␥(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 ␤(E190D/E391C). When the mutant ␤ and ␥ were cross-linked through a disulfide bond between ␤E391C and ␥R84C, F1 paused the rotation at the catalytic dwell angle of ␤(E190D/E391C), indicating that the crystal structure represents the catalytic dwell state and that ␤DP is the catalytically active form. The former point was again confirmed in experiments where F1 rotation was inhibited by adenosine-5-(␤,␥-imino)-triphosphate and/or azide, the most commonly used inhibitors for the crystallization of F1. ATP synthase ͉ cross-link

A rotary motor F1, a catalytic part of ATP synthase, makes a 120°s tep rotation driven by hydrolysis of one ATP, which consists of 80°a nd 40°substeps initiated by ATP binding and probably by ADP and͞or Pi dissociation, respectively. During active rotations, F1 spontaneously fails in ADP release and pauses after a 80°substep, which is called the ADP-inhibited form. In the present work, we found that, when pushed >؉40°with magnetic tweezers, the pausing F1 resumes its active rotation after releasing inhibitory ADP. The rate constant of the mechanical activation exponentially increased with the pushed angle, implying that F1 weakens the affinity of its catalytic site for ADP as the angle goes forward. This finding explains not only its unidirectional nature of rotation, but also its physiological function in ATP synthesis; it would readily bind ADP from solution when rotated backward by an Fo motor in the ATP synthase. Furthermore, the mechanical work for the forced rotation was efficiently converted into work for expelling ADP from the catalytic site, supporting the tight coupling between the rotation and catalytic event.ADP inhibition ͉ ATP synthase ͉ F1-ATPase ͉ magnetic tweezers ͉ single-molecule observation