F1, a water-soluble portion of FoF1-ATP synthase, is an ATP hydrolysis-driven rotary motor. The central ␥-subunit rotates in the ␣3␤3 cylinder by repeating the following four stages of rotation: ATPbinding dwell, rapid 80°substep rotation, interim dwell, and rapid 40°substep rotation. At least two 1-ms catalytic events occur in the interim dwell, but it is still unclear which steps in the ATPase cycle, except for ATP binding, correspond to these events. To discover which steps, we analyzed rotations of F 1 subcomplex (␣3␤3␥) from thermophilic Bacillus PS3 under conditions where cleavage of ATP at the catalytic site is decelerated: hydrolysis of ATP by the catalytic-site mutant F 1 and hydrolysis of a slowly hydrolyzable substrate ATP␥S (adenosine 5-[␥-thio]triphosphate) by wild-type F 1. In both cases, interim dwells were extended as expected from bulk phase kinetics, confirming that cleavage of ATP takes place during the interim dwell. Furthermore, the results of ATP␥S hydrolysis by the mutant F 1 ensure that cleavage of ATP most likely corresponds to one of the two 1-ms events and not some other faster undetected event. Thus, cleavage of ATP on F 1 occurs in 1 ms during the interim dwell, and we call this interim dwell catalytic dwell. Fo F 1 -ATP synthase is an enzyme ubiquitous from bacteria to animals and plants. It synthesizes ATP from ADP and inorganic phosphate by using ⌬ H ϩ -driven proton flow through a membrane (1, 2). F 0 F 1 -ATP synthase can easily be separated into two major portions: water-soluble F 1 and membraneembedded F o . The isolated F 1 (␣ 3 ␤ 3 ␥␦) has an ATP hydrolysis activity and is often called F 1 -ATPase (3, 4). The crystal structure of F 1 shows that the rod-shaped ␥-subunit is surrounded by a cylinder made of three ␣-and three ␤-subunits arranged alternatively (5). The catalytic sites are located in ␤-subunits but residues from adjacent ␣-subunits also contribute. It has been thought that F o F 1 -ATP synthase is a complex of F o motor and F 1 motor that share a common rotor: a downhill proton flow through F o drives rotation of the rotor, causing conformational changes in F 1 that result in ATP synthesis. Conversely, ATP hydrolysis in F 1 causes a reverse rotation of the rotor that enforces F o to pump protons in the reverse direction (6). The rotor is made of a c-subunit ring of F o (7-11) and ␥ subunits of F 1 (12-15).We have visualized and analyzed the ATP-driven rotation of the ␥-subunit in the minimum assembly of F 1 motor, ␣ 3 ␤ 3 ␥ subcomplex (hereafter in this article, this subcomplex is called F 1 ) (13,16). To date, the following features have been established. The ␥-subunit makes a 120°step per one ATP consumption (17), which is further divided into 90°and 30°substeps (18). The dwelling time before the 90°substep rotation depends on ATP concentration and disappears beyond the limit of time resolution of the observation methods as ATP concentration ([ATP]) increases. Therefore, the dwell before the 90°substep rotation is a dwell for ATP binding, and the 90°substep rota...

The conformational fluctuation of enzymes has a crucial role in reaction acceleration. However, the contribution to catalysis enhancement of individual substates with conformations far from the average conformation remains unclear. We studied the catalytic power of the rotary molecular motor F(1)-ATPase from thermophilic Bacillus PS3 as it was stalled in transient conformations far from a stable pausing angle. The rate constants of ATP binding and hydrolysis were determined as functions of the rotary angle. Both rates exponentially increase with rotation, revealing the molecular basis of positive cooperativity among three catalytic sites: elementary reaction steps are accelerated via the mechanical rotation driven by other reactions on neighboring catalytic sites. The rate enhancement induced by ATP binding upon rotation was greater than that brought about by hydrolysis, suggesting that the ATP binding step contributes more to torque generation than does the hydrolysis step. Additionally, 9% of the ATP-driven rotary step was supported by thermal diffusion, suggesting that acceleration of the ATP docking process occurs via thermally agitated conformational fluctuations.

Motility often plays a decisive role in the survival of species. Five systems of motility have been studied in depth: those propelled by bacterial flagella, eukaryotic actin polymerization and the eukaryotic motor proteins myosin, kinesin and dynein. However, many organisms exhibit surprisingly diverse motilities, and advances in genomics, molecular biology and imaging have showed that those motilities have inherently independent mechanisms. This makes defining the breadth of motility nontrivial, because novel motilities may be driven by unknown mechanisms. Here, we classify the known motilities based on the unique classes of movement-producing protein architectures.Based on this criterion, the current total of independent motility systems stands at 18 types. In this perspective, we discuss these modes of motility relative to the latest phylogenetic Tree of Life and propose a history of motility. During the ~4 billion years since the emergence of life, motility arose in Bacteria with flagella and pili, and in Archaea with archaella. Newer modes of motility became possible in Eukarya with changes to the cell envelope. Presence or absence of a peptidoglycan layer, the acquisition of robust membrane dynamics, the enlargement of cells and environmental opportunities likely provided the context for the (co)evolution of novel types of motility. K E Y W O R D S appendage, cytoskeleton, flagella, membrane remodeling, Mollicutes, motor protein, peptidoglycan, three domains | 9Genes to Cells MIYATA eT Al.

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