On chemomechanical coupling of the F1-ATPase molecular motor

F₁-ATPase catalyzes ATP hydrolysis to drive the central γ-shaft rotating inside a hexameric cylinder composed of alternating α and β subunits. Experiments showed that the rotation of γ-shaft proceeds in steps of 120° and each 120°-rotation is composed of an 80° substep and a 40° substep. Here, based on the previously proposed models, an improved physical model for chemomechanical coupling of F₁-ATPase is presented, with which the two-substep rotation is well explained. One substep is driven by the power stroke upon ATP binding, while the other one resulted from the passage of γ-shaft from previous to next adjacent β subunits via free diffusion. Using the model, the dynamics and kinetics of F₁-ATPase, such as the rotating time of each substep, the dwell time at each pause and the rotation rate, are analytically studied. The theoretical results obtained with only three adjustable parameters reproduce the available experimental data well.     

Single-molecule Study on the Temperature-sensitive Reaction of F1-ATPase with a Hybrid F1 Carrying a Single ��(E190D)

F₁-ATPase is a rotary molecular motor in which the γ-subunit rotates against the α₃β₃ cylinder. The unitary γ-rotation is a 120° step comprising 80 and 40° substeps, each of these initiated by ATP binding and ADP release and by ATP hydrolysis and inorganic phosphate release, respectively. In our previous study on γ-rotation at low temperatures, a highly temperature-sensitive (TS) reaction step of F₁-ATPase from thermophilic Bacillus PS3 was found below 9 °C as an intervening pause before the 80° substep at the same angle for ATP binding and ADP release. However, it remains unclear as to which reaction step the TS reaction corresponds. In this study, we found that the mutant F₁(βE190D) from thermophilic Bacillus PS3 showed a clear pause of the TS reaction below 18 °C. In an attempt to identify the catalytic state of the TS reaction, the rotation of the hybrid F₁, carrying a single copy of βE190D, was observed at 18 °C. The hybrid F₁ showed a pause of the TS reaction at the same angle as for the ATP binding of the incorporated βE190D, although kinetic analysis revealed that the TS reaction is not the ATP binding step. These findings suggest that the TS reaction is a structural rearrangement of β before or after ATP binding.F₁-ATPase (F₁)² is an ATP-driven rotary motor protein. The subunit composition of the bacterial F₁-ATPase is α₃β₃γδϵ, and the minimum complex of F₁-ATPase as a rotary motor is α₃β₃γ subcomplex. This motor protein forms the F_oF₁-ATP synthase complex by binding to another rotary motor, namely, F_o, which is driven by the proton flux resulting from the proton motive force across the membranes (1–4). Under physiological conditions, where the proton motive force is sufficiently large, F_o forcibly rotates F₁-ATPase in the reverse direction of F₁-ATPase, leading the reverse reaction of ATP hydrolysis, i.e. ATP synthesis from ADP and inorganic phosphate (P_i). When the proton motive force diminishes or F₁ is isolated from F_o, F₁-ATPase hydrolyzes ATP to rotate the γ-subunit against the α₃β₃ stator ring in the counterclockwise direction as viewed from the F_o side (5). The catalytic sites are located at the interface of the α- and β-subunits, predominantly on the β-subunit (6). Each β-subunit carries out a single turnover of ATP hydrolysis during the γ-rotation of 360° following the common catalytic reaction pathway, whereas they are 120° different in the catalytic phase. In this manner, the three β-subunits undergo different reaction steps of ATP hydrolysis upon each rotational step. The rotary motion of the γ-subunit has been demonstrated by biochemical (7) and spectroscopic methods (8) and directly proved in single-molecule observation studies (5).Since the establishment of the single-molecule rotation assay, the chemomechanical coupling scheme of F₁ has been studied extensively by resolving the rotation into discrete steps. The stepping rotation was first observed under an ATP-limiting condition where F₁ makes discrete 120° steps upon ATP binding (9). Then, high speed imaging of the rotation with a small probe of low friction was performed, which revealed that the 120° step comprises 80 and 40° substeps, each initiated by ATP binding, and two unknown consecutive reactions, respectively (10). This finding necessitated the identification of the two reactions that trigger the 40° substep. Hence, the rotation assay was performed using a mutant, namely F₁(βE190D), and a slowly hydrolyzed ATP analog, namely ATPγS (11). Glutamate 190 of the β-subunit of F₁, derived from thermophilic Bacillus PS3 and the corresponding glutamates from other F₁-ATPases (Glu-181 of F₁ from Escherichia coli and Glu-188 of F₁ from bovine mitochondria), has been identified as one of the most critical catalytic residues for ATP hydrolysis (6, 12–15). When this glutamate was substituted with aspartic acid, which has a shorter side chain than that of glutamate, the ATP cleavage step of F₁ was drastically slowed. In the rotation assay, this mutant showed a distinct long pause before the 40° substep. ATPγS also caused a long pause before the 40° substep. These observations established that the 40° substep is initiated by hydrolysis. Accordingly, the pause angles before the 80 and 40° substeps are referred to as to the binding angle and the catalytic angle, respectively. Then, the rotation assay was performed in the presence of a high amount of P_i in the solution. It was shown that P_i rebinding caused the long pause at the catalytic angle, suggesting that P_i is released before the 40° substep (16).However, the reaction scheme of F₁ cannot be established by simply assigning each reaction step to either the binding angle or the catalytic angle, because each reaction step must be assigned to one of the three binding or catalytic angles when considering the 360° cyclic reaction scheme of each β-subunit. Direct information about the timing of ADP release was obtained by simultaneous imaging of fluorescently labeled nucleotides and γ rotation, which showed that each β retains ADP until the γ rotates 240° after binding of the nucleotide as ATP and releases ADP between 240 and 320° (16, 17). Another powerful approach is the use of a hybrid F₁ carrying a mutant β that causes a characteristic pause during the rotation. In a previous study, the hybrid F₁ carrying a single copy of β(E190D), α₃β₂β(E190D)γ, showed a distinct pause caused by the slow hydrolysis of β(E190D) at +200° from the ATP binding angle of the mutant β (18). From this observation, it was confirmed that each β executes the chemical cleavage of the bound ATP at +200° from the angle where the ATP binds to β. The asymmetric feature of the pause of the hybrid F₁ was also utilized in other experiments as a marker in the rotational trajectory to correlate the rotational angle and the conformational state of β (19) or to determine the state of F₁ in the crystal structures as the pausing state at catalytic angle (20).Recently, we have found a new reaction intermediate of F₁ rotation as a clear intervening pause before the 80° substep in the rotation assay below 9 °C (21). Furuike et al. (22) also observed the TS reaction in a high speed imaging experiment. The rate constant of this reaction was remarkably sensitive to temperature, giving a Q₁₀ factor around 19. When ADP was added to solution, the pause before the 80° substep was prolonged, whereas the solution P_i caused a longer pause before the 40° substep (21). Although this result can be explained by assuming that the temperature-sensitive (TS) reaction is ADP release, it was not decisive for the identification of the TS reaction.In this study, we found that the mutant F₁(βE190D) also exhibits the distinct pause of the TS reaction but at a higher temperature than for the wild-type F₁, i.e. at 18 °C. This feature was advantageous in identifying the angle position of the TS reaction in the catalytic cycle for each β-subunit coupled with the 360° rotation. Taking advantage of the feature of the hybrid F₁, we analyzed the rotational behavior of the hybrid F₁ at 18 °C in order to assign the angle position of the TS reaction in the catalytic cycle of the 360° rotation, and we have shown that the TS reaction is not directly involved in the ADP release but in some conformational rearrangement before or after ATP binding step.     

An alternative reaction pathway of F1-ATPase suggested by rotation without 80 degrees/40 degrees substeps of a sluggish mutant at low ATP

F(1)-ATPase, a water-soluble portion of F(o)F(1)-ATP synthase, is a rotary motor driven by ATP hydrolysis. The central gamma-subunit rotates in the alpha(3)beta(3) cylinder by repeating four stages of rotation: ATP-binding dwell, rapid 80 degrees substep rotation, catalytic dwell, and rapid 40 degrees substep rotation. In the catalytic dwell, at least two catalytic reactions occur-cleavage of the enzyme-bound ATP and presumably release of the hydrolyzed product(s) from the enzyme-but we found that a slow ATP cleavage mutant of F(1)-ATPase from thermophilic Bacillus PS3 rotates at low ATP concentration without substeps and the catalytic dwell. Analysis indicates that in this alternative reaction pathway the two catalytic reactions occur during the preceding long ATP-binding dwell. Thus, F(1)-ATPase can operate through (at least) two competing reaction pathways, not necessarily through a simple consecutive reaction.     

Acceleration of the ATP-binding rate of F1-ATPase by forcible forward rotation

F₁-ATPase (F₁) is a reversible ATP-driven rotary motor protein. When its rotary shaft is reversely rotated, F₁ produces ATP against the chemical potential of ATP hydrolysis, suggesting that F₁ modulates the rate constants and equilibriums of catalytic reaction steps depending on the rotary angle of the shaft. Although the chemomechanical coupling scheme of F₁ has been determined, it is unclear how individual catalytic reaction steps depend on its rotary angle. Here, we report direct evidence that the ATP-binding rate of F₁ increases upon the forward rotation of the rotor, and its binding affinity to ATP is enhanced by rotation.     

Normal‐mode‐based modeling of allosteric couplings that underlie cyclic conformational transition in F1 ATPase

F₁ ATPase, a rotary motor comprised of a central stalk ( γ subunit) enclosed by three α and β subunits alternately arranged in a hexamer, features highly cooperative binding and hydrolysis of ATP. Despite steady progress in biophysical, biochemical, and computational studies of this fascinating motor, the structural basis for cooperative ATPase involving its three catalytic sites remains not fully understood. To illuminate this key mechanistic puzzle, we have employed a coarse‐grained elastic network model to probe the allosteric couplings underlying the cyclic conformational transition in F₁ ATPase at a residue level of detail. We will elucidate how ATP binding and product (ADP and phosphate) release at two catalytic sites are coupled with the rotation of γ subunit via various domain motions in α ₃ β ₃ hexamer (including intrasubunit hinge‐bending motions in β subunits and intersubunit rigid‐body rotations between adjacent α and β subunits). To this end, we have used a normal‐mode‐based correlation analysis to quantify the allosteric couplings of these domain motions to local motions at catalytic sites and the rotation of γ subunit. We have then identified key amino acid residues involved in the above couplings, some of which have been validated against past studies of mutated and γ ‐truncated F₁ ATPase. Our finding strongly supports a binding change mechanism where ATP binding to the empty catalytic site triggers a series of intra‐ and intersubunit domain motions leading to ATP hydrolysis and product release at the other two closed catalytic sites. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Thermodynamic Analyses of Nucleotide Binding to an Isolated Monomeric β Subunit and the α3β3γ Subcomplex of F1-ATPase

Rotation of the γ subunit of the F₁-ATPase plays an essential role in energy transduction by F₁-ATPase. Hydrolysis of an ATP molecule induces a 120° step rotation that consists of an 80° substep and 40° substep. ATP binding together with ADP release causes the first 80° step rotation. Thus, nucleotide binding is very important for rotation and energy transduction by F₁-ATPase. In this study, we introduced a βY341W mutation as an optical probe for nucleotide binding to catalytic sites, and a βE190Q mutation that suppresses the hydrolysis of nucleoside triphosphate (NTP). Using a mutant monomeric βY341W subunit and a mutant α₃β₃γ subcomplex containing the βY341W mutation with or without an additional βE190Q mutation, we examined the binding of various NTPs (i.e., ATP, GTP, and ITP) and nucleoside diphosphates (NDPs, i.e., ADP, GDP, and IDP). The affinity (1/K_d) of the nucleotides for the isolated β subunit and third catalytic site in the subcomplex was in the order ATP/ADP > GTP/GDP > ITP/IDP. We performed van’t Hoff analyses to obtain the thermodynamic parameters of nucleotide binding. For the isolated β subunit, NDPs and NTPs with the same base moiety exhibited similar ΔH⁰ and ΔG⁰ values at 25°C. The binding of nucleotides with different bases to the isolated β subunit resulted in different entropy changes. Interestingly, NDP binding to the α₃β(Y341W)₃γ subcomplex had similar K_d and ΔG⁰ values as binding to the isolated β(Y341W) subunit, but the contributions of the enthalpy term and the entropy term were very different. We discuss these results in terms of the change in the tightness of the subunit packing, which reduces the excluded volume between subunits and increases water entropy.     

One rotary mechanism for F1-ATPase over ATP concentrations from millimolar down to nanomolar

F₁-ATPase is a rotary molecular motor in which the central γ-subunit rotates inside a cylinder made of α₃β₃-subunits. The rotation is driven by ATP hydrolysis in three catalytic sites on the β-subunits. How many of the three catalytic sites are filled with a nucleotide during the course of rotation is an important yet unsettled question. Here we inquire whether F₁ rotates at extremely low ATP concentrations where the site occupancy is expected to be low. We observed under an optical microscope rotation of individual F₁ molecules that carried a bead duplex on the γ-subunit. Time-averaged rotation rate was proportional to the ATP concentration down to 200 pM, giving an apparent rate constant for ATP binding of 2 × 10⁷ M⁻¹s⁻¹. A similar rate constant characterized bulk ATP hydrolysis in solution, which obeyed a simple Michaelis-Menten scheme between 6 mM and 60 nM ATP. F₁ produced the same torque of ~40 pN·nm at 2 mM, 60 nM, and 2 nM ATP. These results point to one rotary mechanism governing the entire range of nanomolar to millimolar ATP, although a switchover between two mechanisms cannot be dismissed. Below 1 nM ATP, we observed less regular rotations, indicative of the appearance of another reaction scheme.     

Rotary catalysis of the stator ring of F1-ATPase

F₁-ATPase is a rotary motor protein in which 3 catalytic β-subunits in a stator α₃β₃ ring undergo unidirectional and cooperative conformational changes to rotate the rotor γ-subunit upon adenosine triphosphate hydrolysis. The prevailing view of the mechanism behind this rotary catalysis elevated the γ-subunit as a “dictator” completely controlling the chemical and conformational states of the 3 catalytic β-subunits. However, our recent observations using high-speed atomic force microscopy clearly revealed that the 3 β-subunits undergo cyclic conformational changes even in the absence of the rotor γ-subunit, thus dethroning it from its dictatorial position. Here, we introduce our results in detail and discuss the possible operating principle behind the F₁-ATPase, along with structurally related hexameric ATPases, also mentioning the possibility of generating hybrid nanomotors. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Single-Molecule Analysis of the Rotation of F1-ATPase under High Hydrostatic Pressure

F₁-ATPase is the water-soluble part of ATP synthase and is an ATP-driven rotary molecular motor that rotates the rotary shaft against the surrounding stator ring, hydrolyzing ATP. Although the mechanochemical coupling mechanism of F₁-ATPase has been well studied, the molecular details of individual reaction steps remain unclear. In this study, we conducted a single-molecule rotation assay of F₁ from thermophilic bacteria under various pressures from 0.1 to 140 MPa. Even at 140 MPa, F₁ actively rotated with regular 120° steps in a counterclockwise direction, showing high conformational stability and retention of native properties. Rotational torque was also not affected. However, high hydrostatic pressure induced a distinct intervening pause at the ATP-binding angles during continuous rotation. The pause was observed under both ATP-limiting and ATP-saturating conditions, suggesting that F₁ has two pressure-sensitive reactions, one of which is evidently ATP binding. The rotation assay using a mutant F₁(βE190D) suggested that the other pressure-sensitive reaction occurs at the same angle at which ATP binding occurs. The activation volumes were determined from the pressure dependence of the rate constants to be +100 Å³ and +88 Å³ for ATP binding and the other pressure-sensitive reaction, respectively. These results are discussed in relation to recent single-molecule studies of F₁ and pressure-induced protein unfolding.     

Binding of the inhibitor protein IF(1) to bovine F(1)-ATPase

In the structure of bovine F₁-ATPase inhibited with residues 1-60 of the bovine inhibitor protein IF₁, the α-helical inhibitor interacts with five of the nine subunits of F₁-ATPase. In order to understand the contributions of individual amino acid residues to this complex binding mode, N-terminal deletions and point mutations have been introduced, and the binding properties of each mutant inhibitor protein have been examined. The N-terminal region of IF₁ destabilizes the interaction of the inhibitor with F₁-ATPase and may assist in removing the inhibitor from its binding site when F₁F_o-ATPase is making ATP. Binding energy is provided by hydrophobic interactions between residues in the long α-helix of IF₁ and the C-terminal domains of the β_DP-subunit and β_TP-subunit and a salt bridge between residue E30 in the inhibitor and residue R408 in the C-terminal domain of the β_DP-subunit. Several conserved charged amino acids in the long α-helix of IF₁ are also required for establishing inhibitory activity, but in the final inhibited state, they are not in contact with F₁-ATPase and occupy aqueous cavities in F₁-ATPase. They probably participate in the pathway from the initial interaction of the inhibitor and the enzyme to the final inhibited complex observed in the structure, in which two molecules of ATP are hydrolysed and the rotor of the enzyme turns through two 120° steps. These findings contribute to the fundamental understanding of how the inhibitor functions and to the design of new inhibitors for the systematic analysis of the catalytic cycle of the enzyme.     

Thermodynamic analysis of F1‐ATPase rotary catalysis using high‐speed imaging

F₁-ATPase (F₁) is a rotary motor protein fueled by ATP hydrolysis. Although the mechanism for coupling rotation and catalysis has been well studied, the molecular details of individual reaction steps remain elusive. In this study, we performed high-speed imaging of F₁ rotation at various temperatures using the total internal reflection dark-field (TIRDF) illumination system, which allows resolution of the F₁ catalytic reaction into elementary reaction steps with a high temporal resolution of 72 µs. At a high concentration of ATP, F₁ rotation comprised distinct 80° and 40° substeps. The 80° substep, which exhibited significant temperature dependence, is triggered by the temperature-sensitive reaction, whereas the 40° substep is triggered by ATP hydrolysis and the release of inorganic phosphate (P_i). Then, we conducted Arrhenius analysis of the reaction rates to obtain the thermodynamic parameters for individual reaction steps, that is, ATP binding, ATP hydrolysis, P_i release, and TS reaction. Although all reaction steps exhibited similar activation free energy values, ΔG^‡ = 53–56 kJ mol⁻¹, the contributions of the enthalpy (ΔH^‡), and entropy (ΔS^‡) terms were significantly different; the reaction steps that induce tight subunit packing, for example, ATP binding and TS reaction, showed high positive values of both ΔH^‡ and ΔS^‡. The results may reflect modulation of the excluded volume as a function of subunit packing tightness at individual reaction steps, leading to a gain or loss in water entropy.     

F1-ATPase of Escherichia coli: THE ?-INHIBITED STATE FORMS AFTER ATP HYDROLYSIS,IS DISTINCT FROM THE ADP-INHIBITED STATE,AND RESPONDS DYNAMICALLY TO CATALYTIC SITE LIGANDS*

F₁-ATPase is the catalytic complex of rotary nanomotor ATP synthases. Bacterial ATP synthases can be autoinhibited by the C-terminal domain of subunit ϵ, which partially inserts into the enzyme''s central rotor cavity to block functional subunit rotation. Using a kinetic, optical assay of F₁·ϵ binding and dissociation, we show that formation of the extended, inhibitory conformation of ϵ (ϵ_X) initiates after ATP hydrolysis at the catalytic dwell step. Prehydrolysis conditions prevent formation of the ϵ_X state, and post-hydrolysis conditions stabilize it. We also show that ϵ inhibition and ADP inhibition are distinct, competing processes that can follow the catalytic dwell. We show that the N-terminal domain of ϵ is responsible for initial binding to F₁ and provides most of the binding energy. Without the C-terminal domain, partial inhibition by the ϵ N-terminal domain is due to enhanced ADP inhibition. The rapid effects of catalytic site ligands on conformational changes of F₁-bound ϵ suggest dynamic conformational and rotational mobility in F₁ that is paused near the catalytic dwell position.     

Chemo-Mechanical Coupling in F1-ATPase Revealed by Catalytic Site Occupancy during Catalysis

F₁-ATPase is a rotary molecular motor in which the central γ subunit rotates inside a cylinder made of α₃β₃ subunits. To clarify how ATP hydrolysis in three catalytic sites cooperate to drive rotation, we measured the site occupancy, the number of catalytic sites occupied by a nucleotide, while assessing the hydrolysis activity under identical conditions. The results show hitherto unsettled timings of ADP and phosphate releases: starting with ATP binding to a catalytic site at an ATP-waiting γ angle defined as 0°, phosphate is released at ∼200°, and ADP is released during quick rotation between 240° and 320° that is initiated by binding of a third ATP. The site occupancy remains two except for a brief moment after the ATP binding, but the third vacant site can bind a medium nucleotide weakly.     

Single-Molecule Analysis of the Rotation of F1-ATPase under High Hydrostatic Pressure

F₁-ATPase is the water-soluble part of ATP synthase and is an ATP-driven rotary molecular motor that rotates the rotary shaft against the surrounding stator ring, hydrolyzing ATP. Although the mechanochemical coupling mechanism of F₁-ATPase has been well studied, the molecular details of individual reaction steps remain unclear. In this study, we conducted a single-molecule rotation assay of F₁ from thermophilic bacteria under various pressures from 0.1 to 140 MPa. Even at 140 MPa, F₁ actively rotated with regular 120° steps in a counterclockwise direction, showing high conformational stability and retention of native properties. Rotational torque was also not affected. However, high hydrostatic pressure induced a distinct intervening pause at the ATP-binding angles during continuous rotation. The pause was observed under both ATP-limiting and ATP-saturating conditions, suggesting that F₁ has two pressure-sensitive reactions, one of which is evidently ATP binding. The rotation assay using a mutant F₁(βE190D) suggested that the other pressure-sensitive reaction occurs at the same angle at which ATP binding occurs. The activation volumes were determined from the pressure dependence of the rate constants to be +100 Å³ and +88 Å³ for ATP binding and the other pressure-sensitive reaction, respectively. These results are discussed in relation to recent single-molecule studies of F₁ and pressure-induced protein unfolding.     

Mechanical Modulation of ATP-binding Affinity of V1-ATPase

V₁-ATPase is a rotary motor protein that rotates the central shaft in a counterclockwise direction hydrolyzing ATP. Although the ATP-binding process is suggested to be the most critical reaction step for torque generation in F₁-ATPase (the closest relative of V₁-ATPase evolutionarily), the role of ATP binding for V₁-ATPase in torque generation has remained unclear. In the present study, we performed single-molecule manipulation experiments on V₁-ATPase from Thermus thermophilus to investigate how the ATP-binding process is modulated upon rotation of the rotary shaft. When V₁-ATPase showed an ATP-waiting pause, it was stalled at a target angle and then released. Based on the response of the V₁-ATPase released, the ATP-binding probability was determined at individual stall angles. It was observed that the rate constant of ATP binding (k_on) was exponentially accelerated with forward rotation, whereas the rate constant of ATP release (k_off) was exponentially reduced. The angle dependence of the k_off of V₁-ATPase was significantly smaller than that of F₁-ATPase, suggesting that the ATP-binding process is not the major torque-generating step in V₁-ATPase. When V₁-ATPase was stalled at the mean binding angle to restrict rotary Brownian motion, k_on was evidently slower than that determined from free rotation, showing the reaction rate enhancement by conformational fluctuation. It was also suggested that shaft of V₁-ATPase should be rotated at least 277° in a clockwise direction for efficient release of ATP under ATP-synthesis conditions.     

Thermodynamic Analyses of Nucleotide Binding to an Isolated Monomeric β Subunit and the α3β3γ Subcomplex of F1-ATPase

Rotation of the γ subunit of the F₁-ATPase plays an essential role in energy transduction by F₁-ATPase. Hydrolysis of an ATP molecule induces a 120° step rotation that consists of an 80° substep and 40° substep. ATP binding together with ADP release causes the first 80° step rotation. Thus, nucleotide binding is very important for rotation and energy transduction by F₁-ATPase. In this study, we introduced a βY341W mutation as an optical probe for nucleotide binding to catalytic sites, and a βE190Q mutation that suppresses the hydrolysis of nucleoside triphosphate (NTP). Using a mutant monomeric βY341W subunit and a mutant α₃β₃γ subcomplex containing the βY341W mutation with or without an additional βE190Q mutation, we examined the binding of various NTPs (i.e., ATP, GTP, and ITP) and nucleoside diphosphates (NDPs, i.e., ADP, GDP, and IDP). The affinity (1/K_d) of the nucleotides for the isolated β subunit and third catalytic site in the subcomplex was in the order ATP/ADP > GTP/GDP > ITP/IDP. We performed van’t Hoff analyses to obtain the thermodynamic parameters of nucleotide binding. For the isolated β subunit, NDPs and NTPs with the same base moiety exhibited similar ΔH⁰ and ΔG⁰ values at 25°C. The binding of nucleotides with different bases to the isolated β subunit resulted in different entropy changes. Interestingly, NDP binding to the α₃β(Y341W)₃γ subcomplex had similar K_d and ΔG⁰ values as binding to the isolated β(Y341W) subunit, but the contributions of the enthalpy term and the entropy term were very different. We discuss these results in terms of the change in the tightness of the subunit packing, which reduces the excluded volume between subunits and increases water entropy.     

A change in the radius of rotation of F1-ATPase indicates a tilting motion of the central shaft

F₁-ATPase is a water-soluble portion of F_oF₁-ATP synthase and rotary molecular motor that exhibits reversibility in chemical reactions. The rotational motion of the shaft subunit γ has been carefully scrutinized in previous studies, but a tilting motion of the shaft has never been explicitly postulated. Here we found a change in the radius of rotation of the probe attached to the shaft subunit γ between two different intermediate states in ATP hydrolysis: one waiting for ATP binding, and the other waiting for ATP hydrolysis and/or subsequent product release. Analysis of this radial difference indicates a ∼4° outward tilting of the γ-subunit induced by ATP binding. The tilt angle is a new parameter, to our knowledge, representing the motion of the γ-subunit and provides a new constraint condition of the ATP-waiting conformation of F₁-ATPase, which has not been determined as an atomic structure from x-ray crystallography.     

Torque Transmission Mechanism via DELSEED Loop of F1-ATPase

F₁-ATPase (F₁) is an ATP-driven rotary motor in which the three catalytic β subunits in the stator ring sequentially induce the unidirectional rotation of the rotary γ subunit. Many lines of evidence have revealed open-to-closed conformational transitions in the β subunit that swing the C-terminal domain inward. This conformational transition causes a C-terminal protruding loop with conserved sequence DELSEED to push the γ subunit. Previous work, where all residues of DELSEED were substituted with glycine to disrupt the specific interaction with γ and introduce conformational flexibility, showed that F₁ still rotated, but that the torque was halved, indicating a remarkable impact on torque transmission. In this study, we conducted a stall-and-release experiment on F₁ with a glycine-substituted DELSEED loop to investigate the impact of the glycine substitution on torque transmission upon ATP binding and ATP hydrolysis. The mutant F₁ showed a significantly reduced angle-dependent change in ATP affinity, whereas there was no change in the equilibrium for ATP hydrolysis. These findings indicate that the DELSEED loop is predominantly responsible for torque transmission upon ATP binding but not for that upon ATP hydrolysis.     

Catalysis-Enhancement via Rotary Fluctuation of F1-ATPase

Protein conformational fluctuations modulate the catalytic powers of enzymes. The frequency of conformational fluctuations may modulate the catalytic rate at individual reaction steps. In this study, we modulated the rotary fluctuation frequency of F₁-ATPase (F₁) by attaching probes with different viscous drag coefficients at the rotary shaft of F₁. Individual rotation pauses of F₁ between rotary steps correspond to the waiting state of a certain elementary reaction step of ATP hydrolysis. This allows us to investigate the impact of the frequency modulation of the rotary fluctuation on the rate of the individual reaction steps by measuring the duration of rotation pauses. Although phosphate release was significantly decelerated, the ATP-binding and hydrolysis steps were less sensitive or insensitive to the viscous drag coefficient of the probe. Brownian dynamics simulation based on a model similar to the Sumi-Marcus theory reproduced the experimental results, providing a theoretical framework for the role of rotational fluctuation in F₁ rate enhancement.     

Catalysis-Enhancement via Rotary Fluctuation of F1-ATPase

Protein conformational fluctuations modulate the catalytic powers of enzymes. The frequency of conformational fluctuations may modulate the catalytic rate at individual reaction steps. In this study, we modulated the rotary fluctuation frequency of F₁-ATPase (F₁) by attaching probes with different viscous drag coefficients at the rotary shaft of F₁. Individual rotation pauses of F₁ between rotary steps correspond to the waiting state of a certain elementary reaction step of ATP hydrolysis. This allows us to investigate the impact of the frequency modulation of the rotary fluctuation on the rate of the individual reaction steps by measuring the duration of rotation pauses. Although phosphate release was significantly decelerated, the ATP-binding and hydrolysis steps were less sensitive or insensitive to the viscous drag coefficient of the probe. Brownian dynamics simulation based on a model similar to the Sumi-Marcus theory reproduced the experimental results, providing a theoretical framework for the role of rotational fluctuation in F₁ rate enhancement.