Redox regulation of the rotation of F(1)-ATP synthase

In F(1)-ATPase, the smallest known motor enzyme, unidirectional rotation of the central axis subunit gamma is coupled to ATP hydrolysis. In the present study, we report the redox switching of the rotation of this enzyme. For this purpose, the switch region from the gamma subunit of the redox-sensitive chloroplast F(1)-ATPase was introduced into the bacterial F(1)-ATPase. The ATPase activity of the obtained complex was increased up to 3-fold upon reduction (Bald, D., Noji, H., Stumpp, M. T., Yoshida, M. & Hisabori, T. (2000) J. Biol. Chem. 275, 12757-12762). Here, we successfully observed the modulation of rotation of gamma in this chimeric complex by changes in the redox conditions. In addition we revealed that the suppressed enzymatic activity of the oxidized F(1)-ATPase complex was characterized by more frequent long pauses in the rotation of the gamma subunit. These findings obtained by the single molecule analysis therefore provide new insights into the mechanisms of enzyme regulation.     

Inverse regulation of rotation of F1-ATPase by the mutation at the regulatory region on the gamma subunit of chloroplast ATP synthase

In F1-ATPase, the rotation of the central axis subunit gamma relative to the surrounding alpha3beta3 subunits is coupled to ATP hydrolysis. We previously reported that the introduced regulatory region of the gamma subunit of chloroplast F1-ATPase can modulate rotation of the gamma subunit of the thermophilic bacterial F1-ATPase (Bald, D., Noji, H., Yoshida, M., Hirono-Hara, Y., and Hisabori, T. (2001) J. Biol. Chem. 276, 39505-39507). The attenuated enzyme activity of this chimeric enzyme under oxidizing conditions was characterized by frequent and long pauses of rotation of gamma. In this study, we report an inverse regulation of the gamma subunit rotation in the newly engineered F1-chimeric complex whose three negatively charged residues Glu210-Asp211-Glu212 adjacent to two cysteine residues of the regulatory region derived from chloroplast F1-ATPase gamma were deleted. ATP hydrolysis activity of the mutant complex was stimulated up to 2-fold by the formation of the disulfide bond at the regulatory region by oxidation. We successfully observed inverse redox switching of rotation of gamma using this mutant complex. The complex exhibited long and frequent pauses in its gamma rotation when reduced, but the rotation rates between pauses remained unaltered. Hence, the suppression or activation of the redox-sensitive F1-ATPase can be explained in terms of the change in the rotation behavior at a single molecule level. These results obtained by the single molecule analysis of the redox regulation provide further insights into the regulation mechanism of the rotary enzyme.     

Redox regulation of rotation of the cyanobacterial F1-ATPase containing thiol regulation switch

F(1)-ATP synthase (F(1)-ATPase) is equipped with a special mechanism that prevents the wasteful reverse reaction, ATP hydrolysis, when there is insufficient proton motive force to drive ATP synthesis. Chloroplast F(1)-ATPase is subject to redox regulation, whereby ATP hydrolysis activity is regulated by formation and reduction of the disulfide bond located on the γ subunit. To understand the molecular mechanism of this redox regulation, we constructed a chimeric F(1) complex (α(3)β(3)γ(redox)) using cyanobacterial F(1), which mimics the regulatory properties of the chloroplast F(1)-ATPase, allowing the study of its regulation at the single molecule level. The redox state of the γ subunit did not affect the ATP binding rate to the catalytic site(s) and the torque for rotation. However, the long pauses caused by ADP inhibition were frequently observed in the oxidized state. In addition, the duration of continuous rotation was relatively shorter in the oxidized α(3)β(3)γ(redox) complex. These findings lead us to conclude that redox regulation of CF(1)-ATPase is achieved by controlling the probability of ADP inhibition via the γ subunit inserted region, a sequence feature observed in both cyanobacterial and chloroplast ATPase γ subunits, which is important for ADP inhibition (Sunamura, E., Konno, H., Imashimizu-Kobayashi, M., Sugano, Y., and Hisabori, T. (2010) Plant Cell Physiol. 51, 855-865).     

Archaebacterial ATPases: relationship to other ion-translocating ATPase families examined in terms of immunological cross-reactivity

Immunological cross-reactivity among three types of H(+)-ATPases, that is, three archaebacterial ATPases, the F1-ATPase from thermophilic bacterium PS3 (TF1) and the vacuolar membrane ATPase from Saccharomyces cerevisiae, was examined by means of immunoblot analyses. The three archaebacterial ATPases were very similar in immunological cross-reactivity, suggesting that they belong to the same family of ATPases. Cross-reaction was also observed between the ATPase from Sulfolobus acidocaldarius, one of the three archaebacteria, and TF1. S. cerevisiae vacuolar ATPase reacted with the antibodies prepared against each of the three archaebacterial ATPases, but did not react with the antibody against TF1. Electron microscopic examination revealed that the oligomeric structure of Sulfolobus ATPase was very similar to that of F1-ATPase. These results, taken together, suggest that the archaebacterial ATPases share close structural similarities with the vacuolar ATPases, and, to a lesser degree, with the F0F1-ATPases.     

A new solution structure of ATP synthase subunit c from thermophilic Bacillus PS3, suggesting a local conformational change for H+-translocation

In F(o)F(1)-ATP synthase, an oligomer ring of F(o)c subunits acts as a rotary proton channel of the F(o)-proton motor. On the basis of the solution structure of the Escherichia coli F(o)c (EF(o)c) monomer, the rotation of the C-terminal helix coupled with the reorientation of the essential Asp61 side-chain on deprotonation was proposed to drive rotation of the whole c-ring. We have determined the NMR structure of F(o)c from thermophilic Bacillus PS3, TF(o)c, in an organic solvent mixture (chloroform/methanol (3:1, v/v)). Our results showed that, independent of pH, the carboxyl group of the essential Glu56 of TF(o)c protrudes toward the outside of the hairpin, a third orientation that differs from either of the two orientations in EF(o)c. Therefore, it would be inappropriate to draw conclusions about the mechanism of c-ring rotation on the basis of the conformations observed only for EF(o)c. The appearance of different hairpin structures shows that there are multiple energy minima for the hairpin structure in terms of helix rotation and axial displacement. The multiple energy minima may also provide a base for the different oligomeric states in the c-ring structure. A rotation mechanism of the F(o) motor coupled with H(+)-translocation is discussed on the basis of these results and the recently reported crystal structure of the c-ring from Ilyobacter tartaricus Na(+)-ATPase.     

An insight into the mechanism of inhibition and reactivation of the F(1)-ATPases by tentoxin

The mechanism of inhibition and reactivation of chloroplast ATP-synthase by the fungal cyclotetrapeptide tentoxin was investigated by photolabeling experiments, binding studies, and kinetic analysis using synthetic analogues of tentoxin. The alpha-subunit of chloroplast F(1)-ATPase (CF(1)) was specifically labeled by a photoactivatable tentoxin derivative, providing the first direct evidence of tentoxin binding to the alpha-subunit, and 3D homology modeling was used to locate tentoxin in its putative binding site at the alpha/beta interface. The non-photosynthetic F(1)-ATPase from thermophilic bacterium (TF(1)) proved to be also tentoxin-sensitive, and enzyme turnover dramatically increased the rate of tentoxin binding to its inhibitory site, contrary to what was previously observed with epsilon-depleted CF(1) [Santolini, J., Haraux, F., Sigalat, C., Moal, G., and André, F. (1999) J. Biol. Chem. 274, 849-858]. We propose that tentoxin preferentially binds to an ADP-loaded alpha beta pair, and mechanically blocks the catalytic cycle, perhaps by the impossibility of converting this alpha beta pair into an ATP-loaded alpha beta pair. Using (14)C-tentoxin and selected synthetic analogues, we found that toxin binding to the tight inhibitory site of CF(1) exerts some cooperative effect on the loose reactivatory site, but that no reciprocal effect exists. When the two tentoxin-binding sites are filled in reactivated F(1)-ATPase, they do not exchange their role during catalytic turnover, indicating an impairment between nucleotide occupancy and the shape of tentoxin-binding pocket. This analysis provides a mechanical interpretation of the inhibition of F(1)-ATPase by tentoxin and a clue for understanding the reactivation process.     

Microsecond time scale rotation measurements of single F1-ATPase molecules

A novel method for detecting F(1)-ATPase rotation in a manner sufficiently sensitive to achieve acquisition rates with a time resolution of 2.5 micros (equivalent to 400,000 fps) is reported. This is sufficient for resolving the rate at which the gamma-subunit travels from one dwell state to another (transition time). Rotation is detected via a gold nanorod attached to the rotating gamma-subunit of an immobilized F(1)-ATPase. Variations in scattered light intensity allow precise measurement of changes in the angular position of the rod below the diffraction limit of light. Using this approach, the transition time of Escherichia coli F(1)-ATPase gamma-subunit rotation was determined to be 7.62 +/- 0.15 (standard deviation) rad/ms. The average rate-limiting dwell time between rotation events observed at the saturating substrate concentration was 8.03 ms, comparable to the observed Mg(2+)-ATPase k(cat) of 130 s(-)(1) (7.7 ms). Histograms of scattered light intensity from ATP-dependent nanorod rotation as a function of polarization angle allowed the determination of the nanorod orientation with respect to the axis of rotation and plane of polarization. This information allowed the drag coefficient to be determined, which implied that the instantaneous torque generated by F(1) was 63.3 +/- 2.9 pN nm. The high temporal resolution of rotation allowed the measurement of the instantaneous torque of F(1), resulting in direct implications for its rotational mechanism.     

Single molecule behavior of inhibited and active states of Escherichia coli ATP synthase F1 rotation

ATP hydrolysis-dependent rotation of the F(1) sector of the ATP synthase is a successive cycle of catalytic dwells (～0.2 ms at 24 °C) and 120° rotation steps (～0.6 ms) when observed under V(max) conditions using a low viscous drag 60-nm bead attached to the γ subunit (Sekiya, M., Nakamoto, R. K., Al-Shawi, M. K., Nakanishi-Matsui, M., and Futai, M. (2009) J. Biol. Chem. 284, 22401-22410). During the normal course of observation, the γ subunit pauses in a stochastic manner to a catalytically inhibited state that averages ～1 s in duration. The rotation behavior with adenosine 5'-O-(3-thiotriphosphate) as the substrate or at a low ATP concentration (4 μM) indicates that the rotation is inhibited at the catalytic dwell when the bound ATP undergoes reversible hydrolysis/synthesis. The temperature dependence of rotation shows that F(1) requires ～2-fold higher activation energy for the transition from the active to the inhibited state compared with that for normal steady-state rotation during the active state. Addition of superstoichiometric ε subunit, the inhibitor of F(1)-ATPase, decreases the rotation rate and at the same time increases the duration time of the inhibited state. Arrhenius analysis shows that the ε subunit has little effect on the transition between active and inhibited states. Rather, the ε subunit confers lower activation energy of steady-state rotation. These results suggest that the ε subunit plays a role in guiding the enzyme through the proper and efficient catalytic and transport rotational pathway but does not influence the transition to the inhibited state.     

Stable structure of thermophilic proton ATPase beta subunit

F1-ATPase is the major enzyme for ATP synthesis in mitochondria, chloroplasts, and bacterial plasma membranes. F1-ATPase obtained from thermophilic bacterium PS3 (TF1) is the only ATPase which can be reconstituted from its primary structure. Its beta subunit constitutes the catalytic site, and is capable of forming hybrid F1's with E. coli alpha and gamma subunits. Since the stability of TF1 resides in its primary structure, we cloned a gene coding for TF1, and the primary structure of the beta subunit was deduced from the nucleotide sequence of the gene to compare the sequence with those of beta's of three major categories of F1's; prokaryotic membranes, chloroplasts, and mitochondria. The following results were obtained. Homology: The primary structure of the TF1 beta subunit (473 residues, Mr = 51,995.6) showed 89.3% homology with 270 residues which are identical in the beta subunits from human mitochondria, spinach chloroplasts, and E. coli. It contained regions homologous to several nucleotide-binding proteins. Secondary structure: The deduced alpha-helical (30.1%) and beta-sheet (22.3%) contents were consistent with those determined from the circular dichroism spectra. Residues forming reverse turns (Gly and Pro) were highly conserved among the F1 beta subunits. Substituted residues and stability of TF1: We compared the amino acid sequence of the TF1 beta subunit with those of the other F1 beta subunits mentioned above. The observed substitutions in the thermophilic subunit increased its propensities to form secondary structures, and its external polarity to form tertiary structure. Codon usage: The codon usage of the TF1 beta gene was found to be unique. The changes in codons that achieved these amino acid substitutions were much larger than those caused by minimal mutations, and the third letters of the optimal codons were either guanine or cytosine, except in codons for Gln, Lys, and Glu.     

pH dependence of H+ conduction through the membrane moiety of the H+-ATPase (F0 . F1) and effects of tyrosyl residue modification

A convenient and reliable method to measure passive H+-translocating activity (H+ conductivity) was developed; vesicles reconstituted from the membrane moiety (F0) of H+-ATPase (F0 . F1) and soybean phospholipids were loaded with KCl by a freeze-thaw-sonication procedure and the rate of H+ uptake caused by the K+ diffusion potential upon addition of valinomycin was followed with a pH meter. Of the methods tested, a dialysis method using cholate plus deoxycholate gave the best results for reconstitution. Using this method, H+ conductivity of the membrane moiety of H+-ATPase from a thermophilic bacterium PS3 (TF0) was analyzed. Dependence of H+ conductivity of TF0 on H+ concentration fitted a Michaelis-Menten equation showing a Vmax of 31.3 microgram ion/min . mg of TF0 and a Km of 0.095 microgram ion/liter. Upon modification of a tyrosyl residue of TF0 with iodine, the Km value shifted to 0.71 microgram ion/liter, while the Vmax remained constant. These results were interpreted as indicating that a single tyrosyl residue in N,N'-dicyclohexylcarbodiimide-binding proteolipid of TF0 plays an important role as an H+ donor in the the rate-limiting step of H+ permeation through TF0. TF1, the catalytic moiety of H+-ATPase from the thermophilic bacterium PS3, blocked H+ conduction through TF0. A 1:1 stoichiometry of TF1 and TF0 was found in ATP-dependent membrane potential generation as well as H+ conduction.     

Single site hydrolysis of 2',3'-O-(2,4,6-trinitrophenyl)-ATP by the F1-ATPase from thermophilic bacterium PS3 is accelerated by the chase-addition of excess ATP.

The interaction of 2',3'-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP) and TNP-ADP to F1-ATPase from a thermophilic bacterium PS3 (TF1) was investigated. When TNP-ADP or TNP-ATP was added to the isolated alpha or beta subunit of TF1, characteristic difference spectra were generated for each subunit. Difference spectra generated on addition of these analogs to TF1 resembled those observed for the beta subunit, indicating TNP analogs bind to the beta subunits in the molecule of TF1. Results of equilibrium dialysis showed that TNP-ADP binds to a single high affinity site on TF1 in the presence of Mg2+ with a dissociation constant of 2.2 nM. When TNP-ATP was added to TF1 in a substoichiometric molar ratio, it rapidly bound to TF1 and was slowly hydrolyzed. The hydrolysis proceeded nearly to completion without showing stable equilibrium between bound species of TNP-ATP and TNP-ADP. Similar to beef heart mitochondrial F1, this hydrolysis was greatly accelerated by the chase-addition of 100 microM ATP. However, the hydrolyzed product, TNP-ADP, remained bound on the beta subunit even after the chase.     

Single-site catalysis of F1-ATPase from thermophilic bacterium PS3 and its dominance in steady-state catalysis at low ATP concentration

Single-site catalysis by F1-ATPase from a thermophilic bacterium PS3 (TF1) was examined by incubating the enzyme with a submolar amount of radioactive ATP. The profile of single-site catalysis by TF1 at 23 degrees C was different from that of beef heart mitochondrial F1-ATPase (MF1). ATP hydrolysis on the enzyme and release of the products was rapid, and subsequent addition of non-radioactive ATP (cold chase) did not promote the hydrolysis of radioactive ATP, indicating that the rate-limiting step was not the step of product release but the step of ATP binding to the enzyme. Thus, the characteristic features of so-called uni-site catalysis were not observed. At 60 degrees C, whether in the presence or absence of phosphate ion, a small amount of bound [alpha, gamma-32P]ATP and cold chase promotion were observed. However, since bound 32P1 was not detected by centrifugal gel filtration, it is not yet certain whether TF1 has typical uni-site characteristics. Based on the hydrolytic turnover rate for single-site catalysis and analysis of the kinetics of steady-state catalysis, it is proposed that single-site catalysis is dominant even in steady-state catalysis at ATP concentrations of less than about 20 microM.     

Single-molecule Analysis of Inhibitory Pausing States of V1-ATPase

V(1)-ATPase, the hydrophilic V-ATPase domain, is a rotary motor fueled by ATP hydrolysis. Here, we found that Thermus thermophilus V(1)-ATPase shows two types of inhibitory pauses interrupting continuous rotation: a short pause (SP, 4.2 s) that occurred frequently during rotation, and a long inhibitory pause (LP, >30 min) that terminated all active rotations. Both pauses occurred at the same angle for ATP binding and hydrolysis. Kinetic analysis revealed that the time constants of inactivation into and activation from the SP were too short to represent biochemically predicted ADP inhibition, suggesting that SP is a newly identified inhibitory state of V(1)-ATPase. The time constant of inactivation into LP was 17 min, consistent with one of the two time constants governing the inactivation process observed in bulk ATPase assay. When forcibly rotated in the forward direction, V(1) in LP resumed active rotation. Solution ADP suppressed the probability of mechanical activation, suggesting that mechanical rotation enhanced inhibitory ADP release. These features were highly consistent with mechanical activation of ADP-inhibited F(1), suggesting that LP represents the ADP-inhibited state of V(1)-ATPase. Mechanical activation largely depended on the direction and angular displacement of forced rotation, implying that V(1)-ATPase rotation modulates the off rate of ADP.     

The ionic track in the F1-ATPase from the thermophilic Bacillus PS3

Only beta-beta cross-links form when the alpha(3)(betaE(395)C)(3)gammaK(36)C (MF(1) residue numbers) double mutant subcomplex of TF(1), the F(1)-ATPase from the thermophilic Bacillus PS3, is slowly inactivated with CuCl(2) in the presence or absence of MgATP. The same slow rate of inactivation and extent of beta-beta cross-linking occur upon treatment of the alpha(3)(betaE(395)C)(3)gamma single mutant subcomplex with CuCl(2) under the same conditions. In contrast, the alpha(3)(betaE(395)C)(3)gammaR(33)C and alpha(3)(betaE(395)C)(3)gammaR(75)C double mutant subcomplexes of TF(1) are rapidly inactivated by CuCl(2) under the same conditions that is accompanied by complete beta-gamma cross-linking. The ATPase activity of each mutant enzyme containing the betaE(395)C substitution is stimulated to a much greater extent by the nonionic detergent lauryldimethylamine oxide (LDAO) than wild-type enzyme, whereas the ATPase activities of the gammaR(33)C, gammaK(36)C, and gammaR(75)C single mutants are stimulated to about the same extent as wild-type enzyme by LDAO. This indicates that the E(395)C substitution in the (394)DELSEED(400) segment of beta subunits increases propensity of the enzyme to entrap inhibitory MgADP in a catalytic site during turnover. These results are discussed in perspective with (i) the ionic track predicted from molecular dynamics simulations to operate during energy-driven ATP synthesis by MF(1), the F(1)-ATPase from bovine heart mitochondria [Ma, J., Flynn, T. C., Cui, Q., Leslie, A. G. W., Walker, J. E., and Karplus, M. (2002) Structure 10, 921-931]; and (ii) the possibility that the betaE(395)C substitution might induce a global effect that alters affinity of noncatalytic sites for nucleotides or alters communication between noncatalytic sites and catalytic sites during ATP hydrolysis.     

Photoaffinity labeling of the TF1-ATPase from the thermophilic bacterium PS3 with 3'-O-(4-benzoyl)benzoyl ADP

3'-O-(4-Benzoyl)benzoyl ADP (BzADP) was used as a photoaffinity label for covalent binding of adenine nucleotide analogs to the nucleotide binding site(s) of the thermophilic bacterium PS3 ATPase (TF1). As with the CF1-ATPase (Bar-Zvi, D. and Shavit, N. (1984) Biochim. Biophys. Acta 765, 340-356) noncovalently bound BzADP is a reversible inhibitor of the TF1-ATPase. BzADP changes the kinetics of ATP hydrolysis from noncooperative to cooperative in the same way as ADP does, but, in contrast to the effect on the CF1-ATPase, it has no effect on the Vmax. In the absence of Mg2+ 1 mol BzADP binds noncovalently to TF1, while with Mg2+ 3 mol are bound. Photoactivation of BzADP results in the covalent binding of the analog to the nucleotide binding site(s) on TF1 and correlates with the inactivation of the ATPase. Complete inactivation of the TF1-ATPase occurs after covalent binding of 2 mol BzADP/mol TF1. Photoinactivation of TF1 by BzADP is prevented if excess of either ADP or ATP is present during irradiation. Analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the Bz[3H]ADP-labeled TF1-ATPase shows that all the radioactivity is incorporated into the beta subunit.     

Complete inhibition and partial Re-activation of single F1-ATPase molecules by tentoxin: new properties of the re-activated enzyme

During hydrolysis of ATP, the gamma subunit of the rotary motor protein F(1)-ATPase rotates within a ring of alpha(3)beta(3) subunits. Tentoxin is a phyto-pathogenic cyclic tetrapeptide, which influences F(1)-ATPase activity of sensitive species. At low concentrations, tentoxin inhibits ATP hydrolysis of ensembles of F(1) molecules in solution. At higher concentrations, however, ATP hydrolysis recovers. Here we have examined how tentoxin acts on individual molecules of engineered F(1)-ATPase from the thermophilic Bacillus PS3 (Groth, G., Hisabori, T., Lill, H., and Bald, D. (2002) J. Biol. Chem. 277, 20117-20119). We found that inhibition by tentoxin caused a virtually complete stop of rotation, which was partially relieved at higher tentoxin concentrations. Re-activation, however, was not simply a reversal of inhibition; while the torque appears unaffected as compared with the situation without tentoxin, F(1) under re-activating conditions was less susceptible to inhibitory ADP binding but displayed a large number of short pauses, indicating infringed energy conversion.     

Rotation of the proteolipid ring in the V-ATPase

V0V1-ATPase is a proton-translocating ATPase responsible for acidification of eukaryotic intracellular compartments and for ATP synthesis in archaea and some eubacteria. We demonstrated recently the rotation of the central stalk subunits in V1, a catalytic sector of V0V1-ATPase (Imamura, H., Nakano, M., Noji, H., Muneyuki, E., Ohkuma, S., Yoshida, M., and Yokoyama, K. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2312-2315), but the rotation of the proteolipid ring, a predicted counterpart rotor in the membrane V0 sector, has remained to be proven. V0V1-ATPase that retained sensitivity to N',N'-dicyclohexylcarbodiimide was isolated from Thermus thermophilus, immobilized onto a glass surface through the N termini of the A subunits of V1, and decorated with a bead attached to a proteolipid subunit of V0. Rotation of beads was observed in the presence of ATP, and direction of rotation was always counterclockwise viewed from the membrane side. The rotation proceeded at approximately 3.0 rev/s in average at 4 mm ATP and was abolished by N',N'-dicyclohexylcarbodiimide treatment. Thus, the rotation of the central stalk in V1 accompanies rotation of a proteolipid ring of V0 in the functioning V0V1-ATPase.     

Further investigations on the inorganic phosphate binding site of beef heart mitochondrial F1-ATPase

The possibility that 4-azido-2-nitrophenyl phosphate (ANPP), a photoreactive derivative of inorganic phosphate (Pi) [Lauquin, G., Pougeois, R., & Vignais, P. V. (1980) Biochemistry 19, 4620-4626], could mimic ATP was investigated. ANPP was hydrolyzed in the dark by sarcoplasmic reticulum Ca2+-ATPase in the presence of Ca2+ but not in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. ANPP was not hydrolyzed by purified mitochondrial F1-ATPase; however, ADP and ATP protected F1-ATPase against ANPP photoinactivation. On the other hand, the trinitrophenyl nucleotide analogues (TNP-ADP, TNP-ATP, and TNP-AMP-PNP), which bind specifically at the two catalytic sites of F1-ATPase [Grubmeyer, C., & Penefsky, H. (1981) J. Biol. Chem. 256, 3718-3727], abolished Pi binding on F1-ATPase; they do not protect F1-ATPase against ANPP photoinactivation. Furthermore, ANPP-photoinactivated F1-ATPase binds the TNP analogues in the same way as the native enzyme. The Pi binding site of F1-ATPase, which is shown to be photolabeled by ANPP, does not appear to be at the gamma-phosphate position of the catalytic sites.     

Complex cooperativity of ATP hydrolysis in the F(1)-ATPase molecular motor

F(1)-ATPase catalyses ATP hydrolysis and converts the cellular chemical energy into mechanical rotation. The hydrolysis reaction in F(1)-ATPase does not follow the widely believed Michaelis-Menten mechanism. Instead, the hydrolysis mechanism behaves in an ATP-dependent manner. We develop a model for enzyme kinetics and hydrolysis cooperativity of F(1)-ATPase which involves the binding-state changes to the coupling catalytic reactions. The quantitative analysis and modeling suggest the existence of complex cooperative hydrolysis between three different catalysis sites of F(1)-ATPase. This complexity may be taken into account to resolve the arguments on the binding change mechanism in F(1)-ATPase.