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.     

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.     

Chemomechanical coupling in F1-ATPase revealed by simultaneous observation of nucleotide kinetics and rotation

F(1)-ATPase is a rotary molecular motor in which unidirectional rotation of the central gamma subunit is powered by ATP hydrolysis in three catalytic sites arranged 120 degrees apart around gamma. To study how hydrolysis reactions produce mechanical rotation, we observed rotation under an optical microscope to see which of the three sites bound and released a fluorescent ATP analog. Assuming that the analog mimics authentic ATP, the following scheme emerges: (i) in the ATP-waiting state, one site, dictated by the orientation of gamma, is empty, whereas the other two bind a nucleotide; (ii) ATP binding to the empty site drives an approximately 80 degrees rotation of gamma; (iii) this triggers a reaction(s), hydrolysis and/or phosphate release, but not ADP release in the site that bound ATP one step earlier; (iv) completion of this reaction induces further approximately 40 degrees rotation.     

Temperature-sensitive reaction intermediate of F1-ATPase

F(1)-ATPase is a rotary molecular motor that makes 120 degrees stepping rotations, with each step being driven by a single-ATP hydrolysis. In this study, a new reaction intermediate of F(1)-ATPase was discovered at a temperature below 4 degrees C, which makes a pause at the same angle in its rotation as when ATP binds. The rate constant of the intermediate reaction was strongly dependent on temperature with a Q(10) factor of 19, implying that the intermediate reaction accompanies a large conformational change. Kinetic analyses showed that the intermediate state does not correspond to ATP binding or hydrolysis. The addition of ADP to the reaction mixture did not alter the angular position of the intermediate state, but specifically lengthened the time constant of this state. Conversely, the addition of inorganic phosphate caused a pause at an angle of +80 degrees from that of the intermediate state. These observations strongly suggest that the newly found reaction intermediate is an ADP-releasing step.     

Principal role of the arginine finger in rotary catalysis of F1-ATPase

F(1)-ATPase (F(1)) is an ATP-driven rotary motor wherein the γ subunit rotates against the surrounding α(3)β(3) stator ring. The 3 catalytic sites of F(1) reside on the interface of the α and β subunits of the α(3)β(3) ring. While the catalytic residues predominantly reside on the β subunit, the α subunit has 1 catalytically critical arginine, termed the arginine finger, with stereogeometric similarities with the arginine finger of G-protein-activating proteins. However, the principal role of the arginine finger of F(1) remains controversial. We studied the role of the arginine finger by analyzing the rotation of a mutant F(1) with a lysine substitution of the arginine finger. The mutant showed a 350-fold longer catalytic pause than the wild-type; this pause was further lengthened by the slowly hydrolyzed ATP analog ATPγS. On the other hand, the mutant F(1) showed highly unidirectional rotation with a coupling ratio of 3 ATPs/turn, the same as wild-type, suggesting that cooperative torque generation by the 3 β subunits was not impaired. The hybrid F(1) carrying a single copy of the α mutant revealed that the reaction step slowed by the mutation occurs at +200° from the binding angle of the mutant subunit. Thus, the principal role of the arginine finger is not to mediate cooperativity among the catalytic sites, but to enhance the rate of the ATP cleavage by stabilizing the transition state of ATP hydrolysis. Lysine substitution also caused frequent pauses because of severe ADP inhibition, and a slight decrease in ATP-binding rate.     

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.     

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.     

Torque Generation Mechanism of F1-ATPase upon NTP Binding

Molecular machines fueled by NTP play pivotal roles in a wide range of cellular activities. One common feature among NTP-driven molecular machines is that NTP binding is a major force-generating step among the elementary reaction steps comprising NTP hydrolysis. To understand the mechanism in detail,in this study, we conducted a single-molecule rotation assay of the ATP-driven rotary motor protein F₁-ATPase using uridine triphosphate (UTP) and a base-free nucleotide (ribose triphosphate) to investigate the impact of a pyrimidine base or base depletion on kinetics and force generation. Although the binding rates of UTP and ribose triphosphate were 10³ and 10⁶ times, respectively, slower than that of ATP, they supported rotation, generating torque comparable to that generated by ATP. Affinity change of F₁ to UTP coupled with rotation was determined, and the results again were comparable to those for ATP, suggesting that F₁ exerts torque upon the affinity change to UTP via rotation similar to ATP-driven rotation. Thus, the adenine-ring significantly enhances the binding rate, although it is not directly involved in force generation. Taking into account the findings from another study on F₁ with mutated phosphate-binding residues, it was proposed that progressive bond formation between the phosphate region and catalytic residues is responsible for the rotation-coupled change in affinity.     

Torque Generation Mechanism of F1-ATPase upon NTP Binding

Molecular machines fueled by NTP play pivotal roles in a wide range of cellular activities. One common feature among NTP-driven molecular machines is that NTP binding is a major force-generating step among the elementary reaction steps comprising NTP hydrolysis. To understand the mechanism in detail,in this study, we conducted a single-molecule rotation assay of the ATP-driven rotary motor protein F₁-ATPase using uridine triphosphate (UTP) and a base-free nucleotide (ribose triphosphate) to investigate the impact of a pyrimidine base or base depletion on kinetics and force generation. Although the binding rates of UTP and ribose triphosphate were 10³ and 10⁶ times, respectively, slower than that of ATP, they supported rotation, generating torque comparable to that generated by ATP. Affinity change of F₁ to UTP coupled with rotation was determined, and the results again were comparable to those for ATP, suggesting that F₁ exerts torque upon the affinity change to UTP via rotation similar to ATP-driven rotation. Thus, the adenine-ring significantly enhances the binding rate, although it is not directly involved in force generation. Taking into account the findings from another study on F₁ with mutated phosphate-binding residues, it was proposed that progressive bond formation between the phosphate region and catalytic residues is responsible for the rotation-coupled change in affinity.     

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.     

Electrostatic interactions in catalytic centers of F1-ATPase

The first part of this paper is a brief review of works concerned with the mechanisms of functioning of F0F1-ATP synthases. F0F1-ATP syntheses operate as rotating molecular machines that provide the synthesis of ATP from ADP and inorganic phosphate (Pi) in mitochondria, chloroplasts, and bacteria at the expense of the energy of electrochemical gradient of hydrogen ions generated across energy-transducing mitochondrial, chloroplast or, bacterial membranes. A distinguishing feature of these enzymes is that they operate as rotary molecular motors. In the second part of the work, we calculated the contribution of electrostatic interactions between charged groups of a substrate (MgATP), reaction products (MgADP and Pi), and charged amino acid residues of the F1-ATPase molecule to energy changes associated with the binding of ATP and its chemical transformations in the catalytic centers located at the interface of the alpha- and beta-subunits of the enzyme (oligomer complex alpha 3 beta 3 gamma of bovine mitochondrial ATPase). The catalytic cycle of ATP hydrolysis considered in the work includes conformational changes of alpha- and beta-subunits caused by unidirectional rotations of the central gamma-subunit. The results of our calculations are consistent with the idea that the energetically favorable process of ATP binding to the "open" catalytic center of F1-ATPase initiates the rotation of the gamma-subunit followed by ATP hydrolysis in another ("closed") catalytic center of the enzyme.     

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.     

Purine but not pyrimidine nucleotides support rotation of F(1)-ATPase

The binding change model for the F(1)-ATPase predicts that its rotation is intimately correlated with the changes in the affinities of the three catalytic sites for nucleotides. If so, subtle differences in the nucleotide structure may have pronounced effects on rotation. Here we show by single-molecule imaging that purine nucleotides ATP, GTP, and ITP support rotation but pyrimidine nucleotides UTP and CTP do not, suggesting that the extra ring in purine is indispensable for proper operation of this molecular motor. Although the three purine nucleotides were bound to the enzyme at different rates, all showed similar rotational characteristics: counterclockwise rotation, 120 degrees steps each driven by hydrolysis of one nucleotide molecule, occasional back steps, rotary torque of approximately 40 piconewtons (pN).nm, and mechanical work done in a step of approximately 80 pN.nm. These latter characteristics are likely to be determined by the rotational mechanism built in the protein structure, which purine nucleotides can energize. With ATP and GTP, rotation was observed even when the free energy of hydrolysis was -80 pN.nm/molecule, indicating approximately 100% efficiency. Reconstituted F(o)F(1)-ATPase actively translocated protons by hydrolyzing ATP, GTP, and ITP, but CTP and UTP were not even hydrolyzed. Isolated F(1) very slowly hydrolyzed UTP (but not CTP), suggesting possible uncoupling from rotation.     

Reaction mechanism of the membrane-bound ATPase of submitochondrial particles from beef heart

Submitochondrial particles from beef heart, washed with dilute solutions of KCl so as to activate the latent, membrane-bound ATPase, F1, may be used to study single site catalysis by the enzyme. [gamma-32P]ATP, incubated with a molar excess of catalytic sites, a condition which favors binding of substrate in only a single catalytic site on the enzyme, is hydrolyzed via a four-step reaction mechanism. The mechanism includes binding in a high affinity catalytic site, Ka = 10(12)M-1, a hydrolytic step for which the equilibrium constant is near unity, and two product release steps in which Pi dissociates from catalytic sites about 10 times more rapidly than ADP. Catalysis by the membrane-bound ATPase also is characterized by a 10(6)-fold acceleration in the rate of net hydrolysis of [gamma-32P]ATP, bound in the high affinity catalytic site, that occurs when substrate is made available to additional catalytic sites on the enzyme. These aspects of the reaction mechanism of the ATPase of submitochondrial particles closely parallel the reaction mechanism determined for solubilized, homogeneous F1 (Grubmeyer, C., Cross, R. L., and Penefsky, H. S. (1982) J. Biol. Chem. 257, 12092-12100). The finding that removal of the enzyme from the membrane does not significantly alter the properties of single site catalysis lends support to models of ATP synthesis in oxidative phosphorylation, catalyzed by membrane-bound F1, that have been based on the study of the soluble enzyme.     

Direct identification of the rotary angle of ATP cleavage in F1-ATPase from Bacillus PS3

F₁-ATPase is the world’s smallest biological rotary motor driven by ATP hydrolysis at three catalytic β subunits. The 120° rotational step of the central shaft γ consists of 80° substep driven by ATP binding and a subsequent 40° substep. In order to correlate timing of ATP cleavage at a specific catalytic site with a rotary angle, we designed a new F₁-ATPase (F₁) from thermophilic Bacillus PS3 carrying β(E190D/F414E/F420E) mutations, which cause extremely slow rates of both ATP cleavage and ATP binding. We produced an F₁ molecule that consists of one mutant β and two wild-type βs (hybrid F₁). As a result, the new hybrid F₁ showed two pausing angles that are separated by 200°. They are attributable to two slowed reaction steps in the mutated β, thus providing the direct evidence that ATP cleavage occurs at 200° rather than 80° subsequent to ATP binding at 0°. This scenario resolves the long-standing unclarified issue in the chemomechanical coupling scheme and gives insights into the mechanism of driving unidirectional rotation.     

Nucleotide exchange from the high-affinity ATP-binding site in SecA is the rate-limiting step in the ATPase cycle of the soluble enzyme and occurs through a specialized conformational state

We have characterized the kinetic and thermodynamic consequences of adenine nucleotide interaction with the low-affinity and high-affinity nucleotide-binding sites in free SecA. ATP binds to the hydrolytically active high-affinity site approximately 3-fold more slowly than ADP when SecA is in its conformational ground state, suggesting that ATP binding probably occurs when the enzyme is in another conformational state during the productive ATPase/transport cycle. The steady-state ATP hydrolysis rate is equivalent to the rate of ADP release from the high-affinity site under a number of conditions, indicating that this process is the rate-limiting step in the ATPase cycle of the free enzyme. Because efficient protein translocation requires at least a 100-fold acceleration in the ATPase rate, the rate-limiting process of ADP release from the high-affinity site is likely to play a controlling role in the conformational reaction cycle of SecA. This release process involves a large enthalpy of activation, suggesting that it involves a protein conformational change, and two observations indicate that this conformational change is different from the well-characterized endothermic conformational transition believed to gate the binding of SecA to SecYEG. First, nucleotide binding to the low-affinity site strongly inhibits the endothermic transition but does not reduce the rate of ADP release. Second, removal of Mg(2+) from an allosteric binding site on SecA does not perturb the endothermic transition but produces a 10-fold acceleration in the rate of ADP release. These divergent effects suggest that a specialized conformational transition mediates the rate-limiting ADP-release process in SecA. Finally, ADP, 2'-O-(N-methylanthraniloyl)-adenosine-5'-diphosphate (MANT-ADP), and adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S) bind with similar affinities to the high-affinity site and also to the low-affinity site as inferred from their consistent effects in inhibiting the endothermic transition. In contrast, adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP) shows 100-fold weaker affinity than ADP for the high-affinity site and no detectable interaction with the low-affinity site at concentrations up to 1 mM, suggesting that this nonhydrolyzable analogue may not be a faithful mimic of ATP in its interactions with SecA.     

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.     

Single-molecule FRET reveals nucleotide-driven conformational changes in molecular machines and their link to RNA unwinding and DNA supercoiling

Many complex cellular processes in the cell are catalysed at the expense of ATP hydrolysis. The enzymes involved bind and hydrolyse ATP and couple ATP hydrolysis to the catalysed process via cycles of nucleotide-driven conformational changes. In this review, I illustrate how smFRET (single-molecule fluorescence resonance energy transfer) can define the underlying conformational changes that drive ATP-dependent molecular machines. The first example is a DEAD-box helicase that alternates between two different conformations in its catalytic cycle during RNA unwinding, and the second is DNA gyrase, a topoisomerase that undergoes a set of concerted conformational changes during negative supercoiling of DNA.     

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.