Pause and rotation of F(1)-ATPase during catalysis.

F(1)-ATPase is a rotary motor enzyme in which a single ATP molecule drives a 120 degrees rotation of the central gamma subunit relative to the surrounding alpha(3)beta(3) ring. Here, we show that the rotation of F(1)-ATPase spontaneously lapses into long (approximately 30 s) pauses during steady-state catalysis. The effects of ADP-Mg and mutation on the pauses, as well as kinetic comparison with bulk-phase catalysis, strongly indicate that the paused enzyme corresponds to the inactive state of F(1)-ATPase previously known as the ADP-Mg inhibited form in which F(1)-ATPase fails to release ADP-Mg from catalytic sites. The pausing position of the gamma subunit deviates from the ATP-waiting position and is most likely the recently found intermediate 90 degrees position.

ATP synthase--a marvellous rotary engine of the cell.

ATP synthase can be thought of as a complex of two motors--the ATP-driven F1 motor and the proton-driven Fo motor--that rotate in opposite directions. The mechanisms by which rotation and catalysis are coupled in the working enzyme are now being unravelled on a molecular scale.

Alkane derivative-bacteriorhodopsin interaction: proton transport and protein structure.

The effects of alkane derivatives, 1-alcohols (ROH), aliphatic amine hydrochlorides (RNH(2).HCl) and sodium aliphatic carboxylates (ROONa), on the proton pumping activity of bacteriorhodopsin (bR) in a purple membrane have been examined. Photocurrents in bR in the purple membrane adsorbed onto polyester thin film were recorded before and after exposure to these test substances. The peak photocurrent in bR was reversibly suppressed by each substance. From the dose-response curve, the concentrations required to reduce the peak capacitive current by 50% were determined for each homolog and then the standard free energies per CH(2) for the adsorption of the alkane derivatives to the site of action were estimated: -3.13 kJ mol(-1) for ROH, -3.05 kJ mol(-1) for RNH(3)(+), and -2.95 kJ mol(-1) for ROO(-). The proton pumping activity of bR was mainly suppressed by the hydrophobic interaction with the additive. The relative potencies of the functional groups of the alkane derivatives were almost comparable between 1-octanol (C(8)OH) and octylamine hydrochloride (C(8)NH(3)(+)) and about 10 times less effective for sodium octanoate (C(8)OO(-)) than for others. The addition of C(8)OH or C(8)OO(-) changed the absorption spectra of bR with a maximum at 560 nm to the spectra of the intermediate state with a maximum at 480 nm, while the C(8)NH(3)(+) decreased the intensity of the 560 nm band only with no blue-shift by the 480 nm band. We conclude that the action of the alkane derivatives is nonspecific and directed to all organized purple membrane structures and that the binding sites of the ROH and ROO(-) are different from that of RNH(3)(+).

Properties of the stochastic energization-relaxation channel model for vectorial ion transport.

A model for the primary active transport by an ion pump protein is proposed. The model, the "energization-relaxation channel model," describes an ion pump as a multiion channel that undergoes stochastic transitions between two conformational states by external energy supply. When the potential profile along ion transport pathway is asymmetrical, a net ion flux is induced by the transitions. In this model, the coupling of the conformational change and ion transport is stochastic and loose. The model qualitatively reproduces known properties of active transport such as the effect of ion concentration gradient and membrane potential on the rate of transport and the inhibition of ion transport at high ion concentration. We further examined the effect of various parameters on the ion transport properties of this model. The efficiency of the coupling was almost 100% under some conditions.

Rotation of F(1)-ATPase and the hinge residues of the beta subunit.

Rotation of a motor protein, F(1)-ATPase, was demonstrated using a unique single-molecule observation system. This paper reviews what has been clarified by this system and then focuses on the role of residues at the hinge region of the beta subunit. We have visualised rotation of a single molecule of F(1)-ATPase by attaching a fluorescent actin filament to the top of the beta subunit in the immobilised F(1)-ATPase, thus settling a major controversy regarding the rotary catalysis. The rotation of the beta subunit was exclusively in one direction, as could be predicted by the crystal structure of bovine heart F(1)-ATPase. Rotation at low ATP concentrations revealed that one revolution consists of three 120 degrees steps, each fuelled by the binding of an ATP to the beta subunit. The mean work done by a 120 degrees step was approximately 80 pN nm, a value close to the free energy liberated by hydrolysis of one ATP molecule, implying nearly 100% efficiency of energy conversion. The torque is probably generated by the beta subunit, which undergoes large opening-closing domain motion upon binding of AT(D)P. We identified three hinge residues, betaHis179, betaGly180 and betaGly181, whose peptide bond dihedral angles are drastically changed during domain motion. Simultaneous substitution of these residues with alanine resulted in nearly complete loss (99%) of ATPase activity. Single or double substitution of the two Gly residues did not abolish the ATPase activity. However, reflecting the shift of the equilibrium between the open and closed forms of the beta subunit, single substitution caused changes in the propensity to generate the kinetically trapped Mg-ADP inhibited form: Gly180Ala enhanced the propensity and Gly181Ala abolished the propensity. In spite of these changes, the mean rotational torque was not changed significantly for any of the mutants.

alpha3beta3gamma complex of F1-ATPase from thermophilic Bacillus PS3 can maintain steady-state ATP hydrolysis activity depending on the number of non-catalytic sites.

Homogeneous preparations of alpha(3)beta(3)gamma complexes with one, two or three non-competent non-catalytic site(s) were performed as described [Amano, Hisabori, Muneyuki, and Yoshida (1996) J. Biol. Chem. 271, 18128-18133] and their properties were compared with those of the wild-type complex. The ATPase activity of the complex with three non-competent non-catalytic sites decayed rapidly to an inactivated state, as reported previously [Matsui, Muneyuki, Honda, Allison, Dou, and Yoshida (1997) J. Biol. Chem. 272, 8215-8221]. In contrast, the complex with one or two non-competent non-catalytic sites displayed a substantial steady-state phase activity depending on the number of non-competent non-catalytic sites in the complex. This result indicates that one competent non-catalytic site can maintain the continuous catalytic turnover of the enzyme and can potentially relieve all three catalytic sites from inhibition by MgADP(-). Furthermore, the results suggest that the interaction between three non-catalytic sites might not be as strong as that between catalytic sites, which are all strictly required for a continuous catalytic turnover.

The noncatalytic site-deficient alpha3beta3gamma subcomplex and FoF1-ATP synthase can continuously catalyse ATP hydrolysis when Pi is present.

We investigated ATP hydrolysis by a mutant (DeltaNC) alpha3beta3gamma subcomplex of F0F1-ATP synthase from the thermophilic Bacillus PS3 that is defective in the noncatalytic nucleotide binding sites. This mutant subcomplex was activated by inorganic phosphate ions (Pi) and did not show continuous ATP hydrolysis activity in the absence of Pi. Pi also activated the wild-type alpha3beta3gamma subcomplex in a similar manner. Sulphate activated wild-type alpha3beta3gamma but not DeltaNC alpha3beta3gamma, indicating that Pi activation did not involve noncatalytic sites but that sulphate activation did. Pi also activated ATP hydrolysis and coupled proton translocation by the wild-type and DeltaNC F0F1-ATP synthases reconstituted into vesicle membranes.

Chloride concentration dependency of the electrogenic activity of halorhodopsin.

The capacitive photoelectric current responses of the halorhodopsins from Halobacterium salinarum (shR) and from Natronobacterium pharaonis (phR) were studied using membrane fragments adsorbed onto a thin polyester film. The electric current of shR was not much affected by ionic strength or cations present in the medium (Na+, K+, Li+, Mg2+, or Ca2+), but was greatly influenced by the Cl- concentration. It increased biphasically as the Cl- concentration increased from 0 to 5 M, then decreased and almost vanished at around 10 or 12 M. Apparent Kd's of about 0.1 and 6 M were deduced for the Kd of Cl- uptake sites. We had to assume a sigmoidal increase of Cl- binding with a Hill coefficient of about 8 at the cytoplasmic, Cl- release site(s). The half-maximum Cl- concentration for the sigmoidal binding was about 7.5 M. The electric current of phR had a maximum around 30 mM Cl- and biphasically decreased at higher Cl- concentrations. The apparent Kd for the Cl- uptake site was 5 mM. The biphasic decrease in the transport activity was explained by assuming a sum of simple hyperbolic type binding (Kd = 0.2 M) and sigmoidally increasing binding with a Hill coefficient of 10 on the cytoplasmic side. The half-maximum concentration of the latter cooperative binding was 5.6 M. This great difference between the apparent affinity of the release site of shR and that of phR can explain the previously reported difference between the Cl- dependency of their photocycles. These results also suggest that there may be multiple Cl- binding sites in the Cl- transport pathway. A simple sequence of Cl- transport steps based on a multiion channel model is proposed.

Time-resolved measurements of photovoltage generation by bacteriorhodopsin and halorhodopsin adsorbed on a thin polymer film.

We constructed a time-resolved photovoltage measurement system and examined the photovoltage kinetics of wild-type bacteriorhodopsin, its D96N mutant, and halorhodopsins from Halobacterium salinarum and Natronobacterium pharaonis. Upon illumination with a laser flash, wild-type bacteriorhodopsin showed photovoltage generation with fast (10-100 micros range) and slow (ms range) components while D96N lacked the latter, as reported previously [Holz, M., Drachev, L.A., Mogi, T., Otto, H., Kaulen, A.D., Heyn, M.P., Skulachev, V.P., and Khorana, H.G. (1989) Proc. Natl. Acad. Sci. USA 86, 2167-2171]. In contrast, photovoltage generation in halorhodopsins from H. salinarum and N. pharaonis was significant only in the ms time range. On the basis of the photovoltage kinetics and photocycle, we conclude that major charge (chloride) movements within halorhodopsin occur during the formation and decay of the N intermediate in the ms range. These observations are discussed in terms of the "Energization-Relaxation Channel Model" [Muneyuki, E., Ikematsu, M., and Yoshida, M. (1996) J. Phys. Chem. 100, 19687-19691].

Cross-linking of two beta subunits in the closed conformation in F1-ATPase.

In the crystal structure of mitochondrial F1-ATPase, two beta subunits with a bound Mg-nucleotide are in "closed" conformations, whereas the third beta subunit without bound nucleotide is in an "open" conformation. In this "CCO" (beta-closed beta-closed beta-open) conformational state, Ile-390s of the two closed beta subunits, even though they are separated by an intervening alpha subunit, have a direct contact. We replaced the equivalent Ile of the alpha3beta3gamma subcomplex of thermophilic F1-ATPase with Cys and observed the formation of the beta-beta cross-link through a disulfide bond. The analysis of conditions required for the cross-link formation indicates that: (i) F1-ATPase takes the CCO conformation when two catalytic sites are filled with Mg-nucleotide, (ii) intermediate(s) with the CCO conformation are generated during catalytic cycle, (iii) the Mg-ADP inhibited form is in the CCO conformation, and (iv) F1-ATPase dwells in conformational state(s) other than CCO when only one (or none) of catalytic sites is filled by Mg-nucleotide or when catalytic sites are filled by Mg2+-free nucleotide. The alpha3beta3gamma subcomplex containing the beta-beta cross-link retained the activity of uni-site catalysis but lost that of multiple catalytic turnover, suggesting that open-closed transition of beta subunits is required for the rotation of gamma subunit but not for hydrolysis of a single ATP.

V-ATPase of Thermus thermophilus is inactivated during ATP hydrolysis but can synthesize ATP.

The ATP hydrolysis of the V1-ATPase of Thermus thermophilus have been investigated with an ATP-regenerating system at 25 degreesC. The ratio of ATPase activity to ATP concentration ranged from 40 to 4000 microM; from this, an apparent Km of 240 +/- 24 microM and a Vmax of 5.2 +/- 0.5 units/mg were deduced. An apparent negative cooperativity, which is frequently observed in case of F1-ATPases, was not observed for the V1-ATPase. Interestingly, the rate of hydrolysis decayed rapidly during ATP hydrolysis, and the ATP hydrolysis finally stopped. Furthermore, the inactivation of the V1-ATPase was attained by a prior incubation with ADP-Mg. The inactivated V1-ATPase contained 1.5 mol of ADP/mol of enzyme. Difference absorption spectra generated from addition of ATP-Mg to the isolated subunits revealed that the A subunit can bind ATP-Mg, whereas the B subunit cannot. The inability to bind ATP-Mg is consistent with the absence of Walker motifs in the B subunit. These results indicate that the inactivation of the V1-ATPase during ATP hydrolysis is caused by entrapping inhibitory ADP-Mg in a catalytic site. Light-driven ATP synthesis by bacteriorhodopsin-VoV1-ATPase proteoliposomes was observed, and the rate of ATP synthesis was approximately constant. ATP synthesis occurred in the presence of an ADP-Mg of which concentration was high enough to induce complete inactivation of ATP hydrolysis of VoV1-ATPase. This result indicates that the ADP-Mg-inhibited form is not produced in ATP synthesis reaction.

A new system for the measurement of electrogenicity produced by ion pumps using a thin polymer film: examination of wild type bacteriorhodopsin and the D96N mutant over a wide pH range.

We developed a new assay system for the measurement of capacitive electric currents generated by ion pumps using the thin polymer film 'Lumirror' (Toray Co., Japan). This system enables us to examine the electrogenicity of ion pumps over a wide range of experimental conditions with high reproducibility due to the mechanical and chemical stability, the high electric resistance and the high electric capacitance of the thin polymer film. Using this method, we examined the photoelectric response of wild type bacteriorhodopsin and its D96N mutant over a wide pH range (2.8-10.0). The results were explained in terms of the affinities of the proton binding sites for translocated protons. A possibility that the direction of the proton transfer from the Schiff base was influenced by the protonation/deprotonation state of the surrounding proton binding sites was suggested. We also found that this film can be used as a substrate for atomic force microscopy (AFM) samples and hence the active purple membrane was observed with AFM.

ATP synthesis by F0F1-ATP synthase independent of noncatalytic nucleotide binding sites and insensitive to azide inhibition.

ATP hydrolyzing activity of a mutant alpha3beta3gamma subcomplex of F0F1-ATP synthase (DeltaNC) from the thermophilic Bacillus PS3, which lacked noncatalytic nucleotide binding sites, was inactivated completely soon after starting the reaction (Matsui, T., Muneyuki, E. , Honda, M., Allison, W. S., Dou, C., and Yoshida, M. (1997) J. Biol. Chem. 272, 8215-8221). This inactivation is caused by rapid accumulation of the "MgADP inhibited form" which, in the case of wild-type enzyme, would be relieved by ATP binding to noncatalytic sites. We reconstituted F0F1-ATP synthase into liposomes together with bacteriorhodopsin and measured illumination-driven ATP synthesis. Remarkably, DeltaNC F0F1-ATP synthase catalyzed continuous turnover of ATP synthesis while it could not promote ATP-driven proton translocation. ATP synthesis by DeltaNC F0F1-ATP synthase, as well as wild-type enzyme, proceeded even in the presence of azide, an inhibitor of ATP hydrolysis that stabilizes the MgADP inhibited form. The time course of ATP synthesis by DeltaNC F0F1-ATP synthase was linear, and gradual acceleration to the maximal rate, which was observed for the wild-type enzyme, was not seen. Thus, ATP synthesis can proceed without nucleotide binding to noncatalytic sites even though the rate is sub-maximal. These results indicate that the MgADP inhibited form is not produced in ATP synthesis reaction, and in this regard, ATP synthesis may not be a simple reversal of ATP hydrolysis.

Thermophilic F1-ATPase is activated without dissociation of an endogenous inhibitor, epsilon subunit.

Subunit complexes (alpha3beta3gamma, alpha3beta3gammadelta, alpha3beta3gammaepsilon, and alpha3beta3gammadeltaepsilon) of thermophilic F1-ATPase were prepared, and their catalytic properties were compared to know the role of delta and epsilon subunits in catalysis. The presence of delta subunit in the complexes had slight inhibitory effect on the ATPase activity. The effect of epsilon subunit was more profound. The (-epsilon) complexes, alpha3beta3gamma and alpha3beta3gammadelta, initiated ATP hydrolysis without a lag. In contrast, the (+epsilon) complexes, alpha3beta3gammaepsilon and alpha3beta3gammadeltaepsilon, started hydrolysis of ATP (<700 microM) with a lag phase that was gradually activated during catalytic turnover. As ATP concentration increased, the lag phase of the (+epsilon) complexes became shorter, and it was not observed above 1 mM ATP. Analysis of binding and hydrolysis of the ATP analog, 2',3'-O-(2,4,6-trinitrophenyl)-ATP, suggested that the (+epsilon) complexes bound substrate only slowly. Differing from Escherichia coli F1-ATPase, the activation of the (+epsilon) complexes from the lag phase was not due to dissociation of epsilon subunit since the re-isolated activated complex retained epsilon subunit. This indicates that there are two alternative forms of the (+epsilon) complex, inhibited form and activated form, and the inhibited one is converted to the activated one during catalytic turnover.

A single mutation at the catalytic site of TF1-alpha3beta3gamma complex switches the kinetics of ATP hydrolysis from negative to positive cooperativity.

Previously, we reported the substitution of Tyr341 of the F1-ATPase beta subunit from a thermophilic Bacillus strain PS3 with leucine, cysteine, or alanine (M. Odaka et al. J. Biochem., 115 (1994) 789-796). These mutations resulted in a great decrease in the affinity of the isolated beta subunit for ATP-Mg and an increase in the apparent Km of the alpha3beta3gamma complex in ATP hydrolysis when examined above 0.1 mM ATP. Here, we examined the ATPase activity of the mutant complexes in a wide range of ATP concentration and found that the mutants exhibited apparent positive cooperativity in ATP hydrolysis. This is the first clear demonstration that a single mutation in the catalytic sites converts the kinetics from apparent negative cooperativity in the wild-type alpha3beta3gamma complex to apparent positive cooperativity. The conversion of apparent cooperativity could be explained in terms of a simple kinetic scheme based on the binding change model proposed by Boyer.

Catalytic activity of the alpha3beta3gamma complex of F1-ATPase without noncatalytic nucleotide binding site.

A mutant alpha3beta3gamma complex of F1-ATPase from thermophilic Bacillus PS3 was generated in which noncatalytic nucleotide binding sites lost their ability to bind nucleotides. It hydrolyzed ATP at an initial rate with cooperative kinetics (Km(1), 4 microM; Km(2), 135 microM) similar to the wild-type complex. However, the initial rate decayed rapidly to an inactivated form. Since the inactivated mutant complex contained 1.5 mol of ADP/mol of complex, this inactivation seemed to be caused by entrapping inhibitory MgADP in a catalytic site. Indeed, the mutant complex was nearly completely inactivated by a 10 min prior incubation with equimolar MgADP. Analysis of the progress of inactivation after initiation of ATP hydrolysis as a function of ATP concentration indicated that the inactivation was optimal at ATP concentrations in the range of Km(1). In the presence of ATP, the wild-type complex dissociated the inhibitory [3H]ADP preloaded onto a catalytic site whereas the mutant complex did not. Lauryl dimethylamineoxide promoted release of preloaded inhibitory [3H]ADP in an ATP-dependent manner and partly restored the activity of the inactivated mutant complex. Addition of ATP promoted single-site hydrolysis of 2',3'-O-(2,4,6-trinitrophenyl)-ATP preloaded at a single catalytic site of the mutant complex. These results indicate that intact noncatalytic sites are essential for continuous catalytic turnover of the F1-ATPase but are not essential for catalytic cooperativity of F1-ATPase observed at ATP concentrations below approximately 300 microM.

The heterogeneous interaction of substoichiometric TNP-ATP and F1-ATPase from Escherichia coli.

The interactions of substoichiometric TNP-ATP and F1-ATPase from Escherichia coli (EF1) were examined and compared with those in the case of mitochondrial F1-ATPase (MF1) and F1-ATPase from thermophilic Bacillus PS3 (TF1). EF1 hydrolyzed substoichiometric TNP-ATP faster than TF1 or MF1, although some 20% of the TNP-ATP remained unhydrolyzed even in the presence of excess chase ATP. The affinity of the catalytic site of EF1 for the product, TNP-ADP, was weaker than that of TF1 or MF1, and the TNP-ADP was readily released upon addition of excess ATP. The amplitude of the difference absorption spectrum induced by binding of TNP-AT(D)P to EF1 was smaller than that of MF1 or TF1 under similar experimental conditions. When an excess amount of TNP-ATP was added to EF1 and the change of the difference spectrum was measured, the shape of the difference spectrum of the ATP-replaceable fraction was very similar to that in the case of binding of TNP-ATP to the isolated beta subunit of TF1, indicating that the rapidly replaceable fraction of bound TNP-ATP was actually at the catalytic site and most of the non-replaceable portion was bound at noncatalytic sites. Weaker affinity of the catalytic site for TNP-ATP may account for the heterogeneous binding and hydrolysis under the conditions described in this paper.

Catalytic activities of alpha3beta3gamma complexes of F1-ATPase with 1, 2, or 3 incompetent catalytic sites.

In order to know how many functional catalytic sites are necessary for ATPase activity of F1-ATPase from a thermophilic Bacillus PS3, a new method of isolating homogeneous preparations of the alpha3beta3gamma complex with 1, 2, or 3 incompetent catalytic sites was developed. Ten glutamic acids (Glu.Tag) were linked to the C terminus of the catalytically incompetent beta(E190Q) subunit. The Glu.Tag itself did not affect ATPase activity of the complexes. Two kinds of alpha3beta3gamma complexes, one containing beta(wild-type) and the other Glu.Tag-linked beta(E190Q), were mixed, urea-denatured, and dialyzed, and alpha3beta3gamma complexes were reconstituted. Each of the complexes containing a different number of Glu.Tag-linked beta(E190Q) was separated by anion-exchange chromatography and analyzed. The results were as follows. 1) Normal steady-state ATPase activity requires three intact catalytic sites. 2) Chase-acceleration, a catalytic cooperativity, requires at least two intact catalytic sites. 3) Single-site catalysis can be mediated by a single intact catalytic site alone. Rescrambling of subunits between complexes could occur when the complex was aged under certain conditions, and this might be one of the reasons for previous contradictory results (Miwa, K., Ohtsubo, M., Denda, K., Hisabori, T., Date, T., and Yoshida, M.(1989) J. Biochem. (Tokyo) 106, 730-734).