ATP Synthesis by F0F1-ATP Synthase Independent of Noncatalytic Nucleotide Binding Sites and Insensitive to Azide Inhibition*

Dirk BaldDagger , Toyoki Amano, Eiro Muneyuki, Bruno Pitard§, Jean-Louis Rigaud§, Jochen Kruip, Toru Hisabori, Masasuke Yoshidapar , and Masaki Shibata

From the Research Laboratory of Resources Utilization, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226, Japan and the § Institute Curie, Section de recherche, UMR 168 and LCR-CEA 8, 11 rue Pierre et Marie Curie, 75231 Paris, France

    ABSTRACT
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Procedures
Results
Discussion
References

ATP hydrolyzing activity of a mutant alpha 3beta 3gamma subcomplex of F0F1-ATP synthase (Delta NC) 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, Delta NC F0F1-ATP synthase catalyzed continuous turnover of ATP synthesis while it could not promote ATP-driven proton translocation. ATP synthesis by Delta NC 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 Delta NC 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.

    INTRODUCTION
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Abstract
Introduction
Procedures
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References

The F0F1-ATP synthase is a ubiquitous enzyme in plasma membranes of bacteria, inner membranes of mitochondria, and thylakoid membranes of chloroplasts which utilizes a transmembrane electrochemical potential difference of protons (Delta µH+)1 for ATP synthesis (1, 2). It can be reversibly separated into a hydrophilic, water-soluble F1 part and a hydrophobic, membrane-embedded F0 part. F0 conducts protons across the membrane. F1, also called F1-ATPase, has a subunit composition of alpha 3beta 3gamma delta epsilon and shows strong activity of ATP hydrolysis. According to the crystal structure of the major part of beef heart mitochondrial F1-ATPase (3), alpha  and beta  subunits are alternatively arranged to form a hexagonal cylinder with a central cavity through which coiled-coil alpha  helices of the gamma  subunit penetrate. F1-ATPase was recently proven to be a "rotary motor enzyme", the first ever found in the biological world; the gamma  subunit rotates within an alpha 3beta 3 hexagon during ATP hydrolysis (4-6).

The F1-ATPase has six nucleotide binding sites. Three of them are catalytic and located mainly on the beta  subunits. The other three, called noncatalytic sites, are mainly located on the alpha subunits. The function of the noncatalytic site had been obscure but recent studies indicated its regulatory role as follows. During multiple turnover of ATP hydrolysis, MgADP is prone to be entrapped in a catalytic site producing the MgADP inhibited form of the enzyme, which is inactive in ATP hydrolysis. ATP binding to the noncatalytic sites causes release of this inhibitory MgADP from the affected catalytic site and relieves the enzyme from the MgADP inhibited form (7-14). In steady-state catalysis of ATP hydrolysis, the relative population of the MgADP inhibited form of F1-ATPase is in an equilibrium determined between entrapping inhibitory MgADP in a catalytic site and releasing inhibitory MgADP by ATP binding to noncatalytic sites. Azide inhibits ATPase activity of F1-ATPase by stabilizing the MgADP inhibited form (7, 8, 15).

Results obtained for a mutant (Delta NC) alpha 3beta 3gamma subcomplex from the thermophilic Bacillus PS3 provided strong evidence for the proposed role of noncatalytic sites (16). The Delta NC enzyme is a quadruple mutant (alpha K175A/alpha T176A/alpha D261A/alpha D262A) and completely lacks the ability to bind ATP (and ADP) to the noncatalytic sites. One may predict that, with the noncatalytic sites being empty, this mutant cannot release entrapped inhibitory MgADP from a catalytic site, and all enzyme molecules fall into the MgADP inhibited form rapidly during catalytic turnover. Indeed, the Delta NC enzyme hydrolyzed ATP at an initial rate similar to that of the wild-type enzyme, but it decayed rapidly to an inactivated form. In the presence of ATP, the Delta NC enzyme did not release preloaded inhibitory [3H]ADP, whereas the wild-type enzyme did (16). Thus, binding of nucleotide to noncatalytic sites seems to be essential for continuous catalytic turnover of ATP hydrolysis by F1-ATPase.

The occurrence of the MgADP inhibited form during ATP hydrolysis reaction has been taken as established but the occurrence of it during ATP synthesis reaction has been questioned by Syroeshkin et al. (17). Using beef heart submitochondrial particles in the presence of substoichiometric amounts of oligomycin, they tested the effect of azide on ATP synthesis and ATP hydrolysis. Interestingly, ATP synthesis was not inhibited by azide, whereas ATP hydrolysis was strongly inhibited. Different azide sensitivity of ATP synthesis and ATP hydrolysis was also reported for chloroplast thylakoid membranes (18). This indicates that generation of the MgADP inhibited form is avoided in ATP synthesis reaction, and Syroeshkin et al. (17) have suggested a possibility that ATP synthesis catalyzed by submitochondrial particles is not a simple reversal of ATP hydrolysis. Since they have used submitochondrial particles and monitored ATP hydrolysis and synthesis indirectly by pH change, questions have remained, such as is the reaction kinetics of azide-insensitive ATP synthesis the same as that in the absence of azide, are there special protein components that are responsible for the azide-insensitive ATP synthesis, and is this a phenomenon universal in any F0F1-ATP synthases? A direct measurement with purified enzyme is expected to lead to a more definite conclusion.

Continuous turnover of ATP synthesis by the purified F0F1-ATP synthase was measured with reconstituted proteoliposomes (19). In that experiment, thermophilic F0F1-ATP synthase was incorporated into membranes of liposomes together with bacteriorhodopsin. Under illumination, bacteriorhodopsin pumped protons into proteoliposomes and established a stable Delta µH+, and the F0F1-ATP synthase catalyzed continuous synthesis of ATP for several hours. Considerable improvement of this assay system was made recently (20, 21), and a biphasic time course of ATP synthesis by thermophilic F0F1-ATP synthase under constant Delta µH+ was found; a first phase with a basic reaction rate accelerated to a second phase with a maximum rate (22). Labeling experiments with 2-N3-[alpha -32P]ATP revealed that ATP binding to a noncatalytic site was responsible for the shift from the first phase to the second phase during catalysis (22). Here, we have further extended the versatility of the assay system to be applicable for the F0F1-ATP synthase assembled from subcomplexes and subunits. We assembled the Delta NC alpha 3beta 3gamma subcomplex into F0F1-ATP synthase and used it for the preparation of bacteriorhodopsin-F0F1-ATP synthase proteoliposomes (bR-F0F1 liposomes). If the ATP synthesis reaction by F0F1-ATP synthase is just the reverse reaction of ATP hydrolysis, the Delta NC F0F1-ATP synthase cannot catalyze continuous ATP synthesis because of rapid accumulation of the MgADP inhibited form due to defective noncatalytic sites. However, the result showed that the Delta NC F0F1-ATP synthase was capable of ATP synthesis, whereas it could not mediate ATP-driven proton translocation. ATP synthesis, either by the wild-type or the Delta NC F0F1-ATP synthases, proceeded in the presence of azide. These results indicate that the MgADP inhibited form is not generated during multiple turnover of ATP synthesis, and in this sense, the reaction pathway of ATP synthesis is not just the reversal of that of ATP hydrolysis.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Chemicals-- Phosphatidylcholine and phosphatidic acid were purchased from Avanti Polar Lipids, beta -octylglucopyranoside was from Nacalai, Bio-Beads SM-2 were from Bio-Rad, and luciferin/luciferase kit was from Boehringer Mannheim.

Preparation of Enzymes-- Authentic F0F1-ATP synthase was purified from the thermophilic Bacillus PS3 as described previously (23). F0 was obtained from urea-treated F0F1-ATP synthase by centrifugation (24). The wild-type (specific activity, about 1.8 µmol of ATP hydrolyzed/mg/min at 40 °C at pH 7.3) and the Delta NC alpha 3beta 3gamma complexes were purified from overexpressed Escherichia coli cells strain JM103 (uncB-uncD) (25) carrying the plasmids pKABG1 (16) and pKABG1alpha k175A/alpha T176A/alpha D261A/alpha D262A, respectively (16). Over-expressed delta  and epsilon  subunits of Bacillus PS3 F1-ATPase were isolated from E. coli strain JM109 carrying the pKD2 plasmid and strain BL23 carrying the pTE2 plasmid, respectively (26). Purified proteins were stored as ammonium sulfate precipitates at 4 °C. F1-ATPase was assembled from the wild-type or Delta NC alpha 3beta 3gamma complex and the isolated delta  and epsilon  subunits, and excess free subunits were removed by centrifugal filtration (26). To assemble F0F1-ATP synthase further, 85 µg of F1-ATPase (a 2.5-fold molar excess to F0 assuming molecular mass ratio of F1 and F0, 2.7:1) was mixed with 12.5 µg of F0 suspended in 10 mM Tricine/NaOH (pH 8.0) and incubated on ice for 1 h. This mixture was used directly for reconstitution into liposomes. In the case of authentic F0F1-ATP synthase, 45 µg of F0F1-ATP synthase, an amount equivalent to 12.5 µg of F0, was used for proteoliposome reconstitution. Protein concentration was determined by the bischinchonic acid (BCA) method (27).

Assay of ATP Synthesis-- F0F1-ATP synthase and bacteriorhodopsin were incorporated into membranes of liposomes according to the method described previously (20, 21) with minor modifications. Briefly, liposomes consisting of 90% phosphatidylcholine and 10% phosphatidic acid were formed by reversed phase evaporation. Purple membranes from Halobacterium halobium were solubilized by overnight incubation with 2% Triton X-100. For reconstitution of proteoliposomes, 4 mg of liposomes (250 µl) were mixed with 600 µl of the reaction buffer (25 mM potassium phosphate buffer, pH 7.3, 50 mM Na2SO4, 50 mM K2SO4), 40 µl of 20% (w/v) Triton X-100, 50 µl of bacteriorhodopsin solution (4 mg/ml), and 15 µl of the authentic F0F1-ATP synthase (3 mg/ml) in 20 mM Tris/SO4, pH 8.0, 1.5% (w/v) beta -octylglucopyranoside or an equivalent amount of assembled F0F1-ATP synthase. The resultant clear solution was incubated at room temperature for 5 min, and then 40 µl of 14.8% (w/v) beta -octylglucopyranoside was added. After a 2-min incubation, 10 µl of 20 mM pyranine solution in ethanol and wet Bio-Beads (80 mg) were added and incubated for 1 h at room temperature while stirring. Addition of Bio-Beads and the 1-h incubation with Bio-Beads was repeated three times. The proteoliposomes were washed twice with the reaction buffer by centrifugation (30 min, 150,000 × g), and the pellet was suspended in 1 ml of the reaction buffer. A 200-µl aliquot of the prepared proteoliposomes (bR-F0F1 liposomes) was mixed with 600 µl of the reaction buffer, and 2 mM (final concentration) ADP was added. The reaction mixture was preilluminated by three 300 W slide projectors for 15 min on a magnetic stirrer at 40 °C. When indicated, different concentrations of ADP and potassium phosphate buffer were used. The reaction was started by addition of 2 mM (final concentration) MgSO4. At 15-min intervals, 50 µl-aliquots were taken, and the reaction was stopped by addition of the same volume of 4% trichloroacetic acid. The amount of ATP was determined by the luciferin-luciferase assay method using a luminescence reader BLR201 (Aloka). When the effect of azide was tested, bR-F0F1 liposomes were incubated with 5 mM (final concentration) sodium azide for 20 min at 40 °C prior to initiation of pre-illumination. When indicated, carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) (final concentration, 5 µM) was added directly before initiation of the ATP synthesis assay. Activity of ATP synthesis was expressed as amounts of synthesized ATP per mg of F0F1-ATP synthase per min. When the dependence of ATP synthesis activity on Pi and ADP concentrations was analyzed, Km and Vmax values were calculated from a Hanes-type plot.

Assay for ATP-driven Proton Translocation-- ATP-driven proton translocation into bR-F0F1 liposomes was detected by decrease of pyranine fluorescence (20, 22) with an FP 777 fluorometer (Jasco) equipped with a stirring facility. An aliquot of 150 µl of bR-F0F1 liposomes (about 5 µg of F0F1-ATP synthase) was preincubated at 40 °C in 2 ml of the reaction buffer supplemented with 7 mM MgCl2, and a base line was monitored for 2-3 min. The reaction was started by adding 5 mM ATP. Excitation and emission wavelengths were 460 and 510 nm, respectively. Excess fluorescence from pyranine molecules not entrapped into bR-F0F1 liposomes was quenched by addition of 20 mM p-xylene-bispyridinium bromide. When the effect of azide was tested, bR-F0F1 liposomes were preincubated at 40 °C for 20 min with 5 mM sodium azide.

    RESULTS
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Procedures
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ATP Synthesis by Assembled F0F1-ATP Synthase-- Since an assay for ATP synthesis by F0F1-ATP synthase assembled from F0- and F1-ATPase has not been reported previously, we have optimized the assay procedures. An attempt to reconstitute bR-F0F1 liposomes by adding F1-ATPase to bacteriorhodopsin-F0 proteoliposomes did not give optimal results probably because of incomplete binding of F1-ATPase to membrane-embedded F0. We mixed F0 and F1-ATPase and then reconstituted proteoliposomes as described under "Experimental Procedures." As shown in Fig. 1, the assembled wild-type F0F1-ATP synthase catalyzed ATP synthesis efficiently, a rate of ~90% of the authentic enzyme. ATP synthesis by the authentic F0F1-ATP synthase proceeded in two phases (Fig. 1, closed circles) (22), and the assembled wild-type F0F1-ATP synthase also showed a biphasic kinetic (Fig. 1, open circles). A first phase with a basic rate in the initial 20 min was accelerated 1.7-1.8-fold to a second phase with a maximum rate, and it continued for 60 min without deceleration. In agreement with the reported values (22), the activities of ATP synthesis of the authentic F0F1-ATP synthase were 130 nmol of ATP/mg/min (first phase, 0-5 min) and 230 nmol of ATP/mg/min (second phase, 40-60 min). The activity of the assembled F0F1-ATP synthase was 110 nmol of ATP/mg/min (first phase) and 190 nmol of ATP/mg/min (second phase). These values (turnover rate, 1 to ~2 ATP/sec/enzyme) were about 10-20% of the ATP hydrolysis activity by the same F0F1-ATP synthase in bR-F0F1-liposomes (1.5-2.5 µmol of ATP hydrolyzed/mg/min). This relatively low activity of ATP synthesis is attributable to the limited Delta µH+ attainable with bacteriorhodopsin as analyzed in a previous paper (20). ATP synthesis by the assembled Delta NC (Fig. 1) and the assembled wild-type F0F1-ATP synthase (Fig. 2B) was completely suppressed by addition of the uncoupler FCCP. Hereafter, we describe only the properties of the assembled enzyme.


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Fig. 1.   ATP synthesis by authentic wild-type (WT), assembled wild-type (WT), and Delta NC F0F1-ATP synthase. bR-F0F1 liposomes were illuminated, and synthesized ATP was measured at indicated times. Three kinds of F0F1-ATP synthases were used to prepare bR-F0F1 liposomes: wild-type FoF1-ATP synthase isolated from Bacillus PS3 (authentic WT F0F1), F0F1-ATP synthase assembled from wild-type F1-ATPase and F0 (assembled WT F0F1), and F0F1-ATP synthase assembled from Delta NC F1-ATPase and F0 (Delta NC F0F1). When indicated, 5 µM FCCP was added. Other experimental details are described under "Experimental Procedures."


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Fig. 2.   Effect of azide on ATP synthesis. A, ATP synthesis by Delta NC F0F1-ATP synthase in the presence or absence of 5 mM sodium azide. B, ATP synthesis by wild-type (WT) F0F1-ATP synthase in the presence and absence of 5 mM sodium azide. bR-F0F1 liposomes were preincubated at 40 °C for 20 min in the presence or absence of 5 mM sodium azide before preillumination. When indicated, 5 µM FCCP was added. Other experimental details are described under "Experimental Procedures."

To our surprise, the Delta NC F0F1-ATP synthase could catalyze ATP synthesis at a rate (90 nmol of ATP/mg/min) only slightly slower than the rate of the first phase of the wild-type F0F1-ATP synthase (Fig. 1, open squares). Therefore, occupation of a noncatalytic site by ATP (or ADP) is not a prerequisite for catalytic turnover of ATP synthesis. Interestingly, the Delta NC F0F1-ATP synthase showed a monophasic, linear time course. Richard et al. (22) reported that, when ATP synthesis by the authentic F0F1-ATP synthase was initiated by addition of 2 mM ADP together with 10 µM ATP, it started at a maximum velocity from the beginning without the first phase, and kinetics apparently turned to be monophasic. The monophasic kinetic of ATP synthesis by the Delta NC F0F1-ATP synthase did not change when 10 µM ATP (and 2 mM ADP) was added to the reaction mixture before the ATP synthesis reaction was started (data not shown).

ATP Synthesis in the Presence of Azide-- The Delta NC alpha 3beta 3gamma subcomplex is unable to catalyze steady-state ATP hydrolysis due to rapid accumulation of the MgADP inhibited form during catalytic turnover (16). Nonetheless, the Delta NC F0F1-ATP synthase can catalyze continuous ATP synthesis as described above, indicating that the MgADP inhibited form may not be generated during ATP synthesis. Then, it is interesting to test the effect of azide on ATP synthesis. As shown in Fig. 2A (closed squares), ATP synthesis by the Delta NC F0F1-ATP synthase was hardly inhibited by 5 mM sodium azide (85-90 nmol of ATP/mg/min). The wild-type F0F1-ATP synthase also synthesized ATP in the presence of 5 mM azide (Fig. 2B, open triangles). The initial rate of ATP synthesis is nearly the same as the value obtained for the enzyme in the absence of azide (100 nmol of ATP/mg/min). However, ATP synthesis in the presence of 5 mM azide did not show the typical second phase, and the acceleration was only 1.2-fold. Higher concentrations of azide up to 10 mM did not change the kinetics and rate of ATP synthesis (data not shown).

Km for ADP and Pi-- The dependence of rates of ATP synthesis on the concentration of ADP and Pi by the Delta NC F0F1-ATP synthase was almost the same as that of the first phase of the wild-type F0F1-ATP synthase. They show a Michaelis-Menten type kinetics with apparent Km(Pi), 8.9 mM (Delta NC) and 6.3 mM (wild-type) (Fig. 3A), and apparent Km(ADP), 0.30 mM (Delta NC) and 0.41 mM (wild-type) (Fig. 3B). Similar dependence of the ATP synthesis rates on the concentrations of ADP and Pi was observed for the catalysis of the second maximum phase by the wild-type F0F1-ATP synthase except that the apparent Vmax value was 1.7-fold of that of the first phase. Furthermore, the first phase of ATP synthesis in the presence of 5 mM azide by the wild-type F0F1-ATP synthase was characterized by similar kinetic parameters, apparent Km(Pi), 7.5 mM and apparent Km(ADP), 0.31 mM (Fig. 3, A and B).


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Fig. 3.   Effect of concentrations of (A) Pi and (B) ADP on the rates of ATP synthesis. For wild-type (WT) F0F1-ATP synthase, rates at the first phase and the second phase were separately analyzed. ADP concentration of panel A was fixed at 2 mM, and Pi concentration of panel B was 25 mM. Lines were theoretical ones obtained from calculated Km and Vmax values. Other experimental details are described under "Experimental Procedures."

ATP-driven Proton Translocation-- The same preparation of bR-F0F1 liposomes used for ATP synthesis assay of the Delta NC F0F1-ATP synthase could not promote ATP-driven proton translocation (Fig. 4), as expected from the fact that the Delta NC alpha 3beta 3gamma subcomplex could not catalyze steady-state ATP hydrolysis (16). The bR-F0F1 liposomes prepared with the same procedures using the wild-type F0F1-ATP synthase could translocate protons driven by ATP hydrolysis. In the presence of 5 mM azide, proton translocation by the wild-type F0F1-ATP synthase started at a slow initial rate, and it was stopped completely in a couple of min. Since it has been known that ATP hydrolyzing activity of the wild-type F1-ATPase in the presence of azide is also subjected to turnover-dependent inactivation (8, 15), this observation is reasonably interpreted as the result of azide-facilitated accumulation of the MgADP inhibited form during turnover of ATP hydrolysis.


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Fig. 4.   ATP-driven proton translocation. bR-F0F1 liposomes containing wild-type (WT) or Delta NC F0F1-ATP synthase were used for experiments. Translocation of protons into bR-F0F1 liposomes was monitored by decrease of pyranine fluorescence. Reactions were started by addition of 5 mM ATP. Other experimental details are described under "Experimental Procedures."

    DISCUSSION
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Noncatalytic Sites Are Not Essential for ATP Synthesis-- The mutant Delta NC F0F1-ATP synthase, which lacks the ability to bind adenine nucleotide to noncatalytic nucleotide binding sites, can still synthesize ATP at a rate comparable with that of the wild-type enzyme (Fig. 1). Thus, nucleotide binding to noncatalytic sites is not a prerequisite for ATP synthesis. Related to this conclusion, Weber et al. (29) observed that E. coli cells carrying mutant F0F1-ATP synthase (alpha D261N/alpha R365W) with impaired noncatalytic sites grew well on succinate plates. Although their mutant showed normal ATP hydrolyzing activity and hence the growth on succinate was not surprising, their observation is consistent with our conclusion. An optional role of noncatalytic sites in ATP synthesis could be related to the rigid structure of the alpha  subunit. In the crystal structure of mitochondrial F1-ATPase (3), noncatalytic sites on the three alpha  subunits are all occupied by AMP-PNP while they are all empty in the crystal structure of the alpha 3beta 3 subcomplex of thermophilic F1-ATPase (30). Nonetheless, the structure of the alpha  subunit of the former is very similar to that of the alpha 3beta 3 subcomplex, indicating that the structure of the alpha  subunit is not changed significantly by the binding of adenine nucleotide, which is always in the "closed" structure (3). This is in sharp contrast to the structure of beta  subunits which changes from "open" to "closed" form by nucleotide binding.

ATP Synthesis Is Not a Simple Reversal of ATP Hydrolysis-- One may raise questions from observations reported here. Why can ATPase-inactive Delta NC F0F1-ATP synthase be active in ATP synthesis, and why can F0F1-ATP synthase synthesize ATP in the presence of azide, an inhibitor of ATPase activity? In both instances, rapid accumulation of the MgADP inhibited form during turnover of ATP hydrolysis is responsible for the absence of steady-state ATPase activities (8, 16). But, in the ATP synthesis reaction, generation of the MgADP inhibited form is apparently avoided, and therefore, the reaction kinetics are obviously different from those in ATP hydrolysis. Continuous ATP synthesis is measurable only in the presence of Delta µH+, whereas ATP hydrolysis is usually assayed in the absence of Delta µH+. The straightforward explanation of our observation is that Delta µH+ somehow directly blocks the generation of the MgADP inhibited form. Another possibility is that Delta µH+ increases binding affinity of Pi to F0F1-ATP synthase. This assumption has some experimental support (31-33) and constitutes a part of the binding change mechanism (1). Because the binding order of ADP and Pi to F0F1-ATP synthase is random (34), there are two pathways between F0F1(MgADP·Pi) and F0F1. In the absence of Delta µH+ (Fig. 5A), a significant fraction of the ATP hydrolysis reaction proceeds through the pathway F0F1(MgADP·Pi) right-arrow F0F1(MgADP) right-arrow F0F1, and the MgADP inhibited form (F0F1*(MgADP)) is produced from F0F1(MgADP). With increased affinity of Pi to F0F1 in the presence of Delta µH+ (Fig. 5B), the reaction pathway F0F1 right-arrow F0F1(Pi) right-arrow F0F1(MgADP·Pi) becomes dominant, and the relative population of F0F1(MgADP) decreases. In addition, binding affinity of Pi to F0F1(MgADP) to form F0F1(MgADP·Pi) may also be increased, and the population of F0F1(MgADP) further decreases. As a consequence, generation of F0F1*(MgADP) is suppressed.


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Fig. 5.   Scheme of possible reaction pathways of F0F1-ATP synthase (A) in the presence and in the absence (B) of Delta µH+. Thick arrows indicate favorable reaction steps. F0F1*(MgADP) represents the MgADP inhibited form. Azide facilitates formation of F0F1*(MgADP) while ATP binding to noncatalytic site stimulates returning of the enzyme from F0F1*(MgADP) to a productive intermediate F0F1(MgADP). This scheme represents an extension of a previous one proposed by Muneyuki et al. (15). For details, see "Discussion."

The azide-insensitive ATP synthesis by submitochondrial particles has been analyzed by Syroeshkin et al. (17). They proposed that Delta µH+-driven ATP synthesis is not a simple reversal of ATP hydrolysis and assumed the existence of "synthase" and "hydrolase" states of F0F1-ATP synthase. Our results provide strong evidence for their proposal. In addition, it is now clear that endogenously bound nucleotide at the noncatalytic site is not directly related to the difference between synthase and hydrolase, that purified F0F1-ATP synthase made up from eight kinds of subunits is by itself responsible for the azide-insensitive ATP synthesis, and that this phenomenon is probably universal in any F0F1-ATP synthases. According to our explanation as described above, the synthase state may be the state of F0F1-ATP synthase in which the generation of the MgADP inhibited form is blocked or the affinity of Pi binding to the enzyme is greatly enhanced by the imposed Delta µH+.

Acceleration of ATP Synthesis by ATP Binding to Noncatalytic Sites Is Prevented by Azide-- The Delta NC F0F1-ATP synthase synthesized ATP with a linear time course at a rate corresponding to the first phase of ATP synthesis by the wild-type F0F1-ATP synthase (Fig. 1). This observation means that nucleotide binding to noncatalytic sites is necessary for the shift from the first phase to the second maximum phase of ATP synthesis and provides other evidence for the previous conclusion made by Richard et al. (22) that the shift from the first to the second maximum phase is triggered by the ATP binding to noncatalytic sites. ATP synthesis by wild-type F0F1-ATP synthase in the presence of azide obeyed a nearly monophasic time course similar to that by the Delta NC F0F1-ATP synthase (Fig. 2B). Addition of azide to the Delta NC F0F1-ATP synthase did not change the kinetics, that is the effect of azide and that of deficient noncatalytic sites are not additive. This implies that azide blocks a step in sequential events from ATP binding to the noncatalytic site to propagation of rate acceleration. Based on a labeling experiment during preincubation with 2-azido-[alpha -32P]ADP, Richard et al. (22) suggested that the rate acceleration of ATP synthesis by ATP binding to noncatalytic site is not related to the release of inhibitory MgADP. Therefore, azide possibly inhibits the shift from the first phase to the second maximum phase in a manner that is probably different from its previously known mechanism, stabilization of the MgADP inhibited form. Actually, because Km values of the catalysis of the second maximum phase of the wild-type F0F1-ATP synthase are almost identical to that of the first phase and only Vmax was increased (Fig. 3), rate acceleration may occur at the step F0F1(MgADP·Pi) right-arrow F0F1(MgATP).

    ACKNOWLEDGEMENT

We are indebted to H. Noji and Y. Kato for productive discussion.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a post-doctoral fellowship from the Japan Society for the Promotion of Science.

Recipient of a fellowship from the European Union/Japan Society for the Promotion of Science.

par To whom correspondence should be addressed: Research Laboratory of Resources Utilization, R-1, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226, Japan. Tel.: 81-45-924-5233; Fax: 81-45-924-5277; E-mail: myoshida{at}res.titech.ac.jp.

1 The abbreviations used are: Delta µH+, transmembrane electrochemical potential of protons; bR-F0F1 liposomes, proteoliposomes reconstituted from bacteriorhodopsin and F0F1-ATP synthase; FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone; Delta NC, F0F1-ATP synthase or its subcomplex containing the mutant alpha  subunits with the four point mutations alpha K175A/alpha T176A/alpha D261A/alpha D262A; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

  1. Boyer, P. D. (1997) Annu. Rev. Biochem. 66, 717-749[CrossRef][Medline] [Order article via Infotrieve]
  2. Weber, J., and Senior, A. E. (1997) Biochim. Biophys. Acta 1319, 19-58[Medline] [Order article via Infotrieve]
  3. Abrahams, J. P., Leslie, A., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628[CrossRef][Medline] [Order article via Infotrieve]
  4. Duncan, T. M., Bulygin, V. V., Zhou, Y., Hutcheon, M. L., Cross, R. L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10964-10968[Abstract]
  5. Sabbert, D., Engelbrecht, S., and Junge, W. (1996) Nature 381, 623-625[CrossRef][Medline] [Order article via Infotrieve]
  6. Noji, H., Yasuda, R., Yoshida, M., and Kinosita, K. (1997) Nature 386, 299-302[CrossRef][Medline] [Order article via Infotrieve]
  7. Vasilyeva, E. A., Minkov, I. B., Fitin, A. F., Vinogradov, A. D. (1982) Biochem. J. 202, 15-23[Medline] [Order article via Infotrieve]
  8. Fitin, A. F., Vasilyeva, E. A., and Vinogradov, A. D. (1979) Biochem. Biophys. Res. Commun. 86, 434-439[Medline] [Order article via Infotrieve]
  9. Milgrom, Y., Ehler, L. L., and Boyer, P. D. (1990) J. Biol. Chem. 265, 18725-18728[Abstract/Free Full Text]
  10. Milgrom, Y. M., Ehler, L. L., and Boyer, P. D. (1991) J. Biol. Chem. 266, 11551-11558[Abstract/Free Full Text]
  11. Murataliev, M. B., and Boyer, P. D. (1992) Eur. J. Biochem. 209, 681-687[Abstract]
  12. Jault, J. M., and Allison, W. S. (1993) J. Biol. Chem. 268, 1558-1566[Abstract/Free Full Text]
  13. Paik, S. R., Jault, J. M., and Allison, W. S. (1994) Biochemistry 33, 126-133[Medline] [Order article via Infotrieve]
  14. Hyndman, D. J., Milgrom, Y. M., Bramhall, E. A., Cross, R. L. (1994) J. Biol. Chem. 269, 28871-28877[Abstract/Free Full Text]
  15. Muneyuki, E., Makino, M., Kamata, H., Kagawa, Y., Yoshida, M., and Hirata, H. (1993) Biochim. Biophys. Acta 1144, 62-68[Medline] [Order article via Infotrieve]
  16. Matsui, T., Muneyuki, E., Honda, M., Allison, W. S., Dou, C., Yoshida, M. (1997) J. Biol. Chem. 272, 8215-8221[Abstract/Free Full Text]
  17. Syroeshkin, A. V., Vasilyeva, E. A., Vinogradov, A. D. (1995) FEBS Lett. 366, 29-32[CrossRef][Medline] [Order article via Infotrieve]
  18. Wei, J., Howlett, B., and Jagendorf, A. T. (1988) Biochim. Biophys. Acta 934, 72-79
  19. Yoshida, M., Sone, N., Hirata, H., and Kagawa, Y. (1975) Biochem. Biophys. Res. Commun. 67, 1295-1300[Medline] [Order article via Infotrieve]
  20. Pitard, B., Richard, P., Dunach, M., Girault, G., and Rigaud, J. L. (1996) Eur. J. Biochem. 235, 769-778[Abstract]
  21. Pitard, B., Richard, P., Dunach, M., and Rigaud, J. L. (1996) Eur. J. Biochem. 235, 779-788[Abstract]
  22. Richard, P., Pitard, B., and Rigaud, J. L. (1995) J. Biol. Chem. 270, 21571-21578[Abstract/Free Full Text]
  23. Sone, N., Yoshida, M., Hirata, H., and Kagawa, Y. (1975) J. Biol. Chem. 250, 7917-7923[Abstract]
  24. Okamoto, H., Sone, N., Hirata, H., Yoshida, M., and Kagawa, Y. (1977) J. Biol. Chem. 252, 6125-6131[Abstract]
  25. Monticello, R. A., Angov, E., and Brusilov, W. (1992) J. Bacteriol. 174, 3370-3376[Abstract]
  26. Kato, Y., Matsui, T., Tanaka, N., Muneyuki, E., Hisabori, T., and Yoshida, M. (1997) J. Biol. Chem. 272, 24906-24912[Abstract/Free Full Text]
  27. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., Klenk, D. C. (1985) Anal. Biochem. 150, 76-85[Medline] [Order article via Infotrieve]
  28. Deleted in proof
  29. Weber, J., Bowman, C., Wilke-Mounts, S., and Senior, A. E. (1995) J. Biol. Chem. 270, 21045-21049[Abstract/Free Full Text]
  30. Shirakihara, Y., Leslie, A. G., Abrahams, J. P., Walker, J. E., Ueda, T., Sekimoto, Y., Kambara, M., Saika, M., Kagawa, Y., Yoshida, M. (1997) Structure 6, 825-836
  31. Rosing, J., Kayalar, C., and Boyer, P. D. (1977) J. Biol. Chem. 252, 2478-2485[Abstract]
  32. Choate, G. L., Hutton, R. L., and Boyer, P. D. (1979) J. Biol. Chem. 254, 286-290[Abstract]
  33. Feldman, R., and Sigman, D. S. (1983) J. Biol. Chem. 258, 12178-12183[Abstract/Free Full Text]
  34. Perez, J. A., and Ferguson, S. J. (1990) Biochemistry 29, 10503-10518[Medline] [Order article via Infotrieve]


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