Reconstitution of ATP-32-Pi exchange by phospholipid addition to the purified oligomycin-sensitive ATPase from yeast mitochondria.

A purified preparation of the oligomycin-sensitive ATPase from yeast mitochondria has been shown to elicit ATP-³²P_i exchange when combined with phospholipids. The reconstitution was normally carried out by dialysis of an ATPase-phospholipid-bile detergent mixture, but could also be achieved by direct addition of the lipid. Vesicle structures with diameters between 200 and 1500 Å were seen by electron microscopy.The ATP-³²P_i exchange was independent of electron transport but sensitive to uncouplers and energy-transfer inhibitors. As in mitochondria, ATPase activity in the reconstituted system was stimulated by a range of uncouplers which inhibited ATP-³²P_i exchange. Taken together, the results raise the possibility that the terminal coupling mechanism might still be intact within the ATPase complex.     

Studies of the oligomycin-sensitive ATPase from yeast mitochondria. Reconstitution of ATP-32Pi exchange in the presence of phospholipids

A purified preparation of the oligomycin-sensitive ATPase from yeast mitochondria has been shown to elicit an oligomycin- and uncoupler-sensitive ATP-³²P_i exchange in the presence of phospholipids. Reconstitution was normally achieved by dialysis of an ATPase-phospholipid-cholate mixture. Following this procedure, vesicles with diameters between 200 and 1,500 Å were seen by electron microscopy. As in mitochondria, ATPase activity in the reconstituted system was stimulated by a range of uncouplers which inhibited ATP-³²P_i exchange. These and other findings suggest that the coupling mechanism may still be intact within the ATPase complex.     

Purification and reconstitution of the 32Pi-ATP exchange activity of bovine chromaffin granule membrane

Ghosts derived from bovine chromaffin granules have a ³²P_i-ATP exchange activity which is associated with the H⁺ pump of that membrane. This activity was low when compared to bacteria, chloroplasts or submitochondrial particles, but had similar properties (K_m for ATP and P_i, ATP/Mg²⁺ ratio, pH profile, inhibition by dicyclohexylcarbodiimide and tributyltin) to the ATPase from above membranes. The ³²P_i-ATP exchange activity was solubilized by cholate/octylglucoside mixtures. The soluble extract was lipid depleted by ammonium sulfate fractionation and partially purified by sucrose gradient centrifugation. The purified preparation was reconstituted with phospholipids by freeze-thawing. The reconstituted vesicles had a ³²P_i-ATP exchange sensitive to dicyclohexylcarbodiimide and trybutyltin and an ATPase with a sensitivity to the inhibitors which varied with the reconstitution conditions. The α- and β-subunits of F₁-ATPase were major components of the preparation.     

On the mechanism of H+ translocation by mitochondrial H+-ATPase. Studies with chemical modifier of tyrosine residues

In this paper a detailed study of the effect of nitration of tyrosine residues by tetranitromethane on H⁺ conduction and other reactions catalyzed by the H⁺-ATPase complex in phosphorylating submitochondrial particles, uncoupled particles, and the purified complex is presented. Tetranitromethane treatment of submitochondrial particles results in marked inhibition of ATP hydrolysis, ATP-³³P_i exchange, and proton conduction by the H⁺-ATPase complex. These effects are caused by nitration of tyrosine residues of H⁺-ATPase complex as shown by the appearance of the absorption peak at 360 nm (specific for nitrotyrosine formation) and inhibition of ATP hydrolysis and ATP-³³P_i exchange in the complex purified from tetranitromethane-treated particles. H⁺ conduction in phospholipid vesicles inlaid with F₀ is also inhibited by tetranitromethane treatment. These observations indicate that tyrosine residue(s) of F₀ are critically involved in energy-linked proton translocation in the ATP-ase complex.     

ATP synthesis catalyzed by the ATPase complex from Rhodospirillum rubrum reconstituted into phospholipid vesicles together with bacteriorhodopsin

The coupling factor ATPase complex extracted by Triton X-100 from the photosynthetic bacterium Rhodospirillum rubrum could be incorporated into phospholipid vesicles after removal of the Triton. Vesicles reconstituted with this F₀ · F₁-type ATPase together with bacteriorhodopsin were found to catalyze, in the light, net ATP synthesis which was inhibited by the energy transfer inhibitors oligomycin and N,N-dicyclohexylcarbodiimide as well as by uncouplers. In vesicles reconstituted with the crude ATPase up to 50% of the observed rate of phosphorylation was independent on light and bacteriorhodopsin and insensitive to the above-listed inhibitors. This dark activity was, however, completely blocked by the adenylate kinase inhibitor, p¹,p⁵-di(adenosine-5′)pentaphosphate, which did not affect at all the net light-dependent phosphorylation nor the ATP-³²P_i exchange reaction. Vesicles reconstituted with the purified ATPase catalyzed only the light- and bacteriorhodopsin-dependent diadenosine pentaphosphate-insensitive phosphorylation. The rate of this photophosphorylation was found to be proportional to the amount of ATPase and bacteriorhodopsin, and linear for at least 20 min of illumination. These results indicate that the purified ATPase contains the complete assembly of subunits required to transduce electrochemical gradient energy into chemical energy.     

Effect of lysolecithin treatment on the structure and functions of the mitochondrial inner membrane

Lysolecithin treatment of electron transport particles (ETP) generated membrane fragments capable of catalyzing ATP-³²P_i exchange, which was resistant to the uncoupling action of Valinomycin plus Nigericin or Valinomycin plus Monensin A in the presence of K⁺. Electron micrographs of ultrathin, positively stained sections of lysolecithin treated ETP were virtually devoid of circular patterns characteristic of closed vesicles. The results suggest that the closed vesicular structure of the mitochondrial inner membrane demanded by the chemiosmotic hypothesis of energy transduction (1) may not be essential for the ATP-³²P_i exchange reaction.     

Formation of ATP by the adenosine triphosphatase complex from spinach chloroplasts reconstituted together with bacteriorhodopsin

The energy-linked ATPase complex has been isolated from spinach chloroplasts. This protein complex contained all the subunits of the chloroplast coupling factor (CF₁) as well as several hydrophobic components. When the activated complex was reconstituted with added soybean phospholipids, it catalyzed the exchange of radioactive inorganic phosphate with ATP. Sonication of the complex into proteoliposomes together with bacteriorhodopsin yielded vesicles that catalyzed light-dependent ATP formation. Both the ³²P_i-ATP exchange reactions and ATP formation were sensitive to uncouplers such as 3-tert-butyl-5,2′-dichloro-4′-nitrosalicylanilide, bis-(hexafluoroacetonyl)acetone and carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone, that act to dissipate a proton gradient. The energy transfer inhibitors dicyclohexylcarbodiimide, triphenyltin chloride and 2-β-d-glucopyranosyl-4,6′-dihydroxydihydrochalcone were also effective inhibitors of both reactions.     

Characterization of the tonoplast proton pumps in Cucumis sativus L. root cells

Large-scale preparation of highly purified tonoplast from cucumber (Cucumis sativus L.) roots was obtained after centrifugation of microsome pellet (10,000 – 80,000 g) on discontinuous sucrose density gradient (20, 28, 32 and 42 %). Lack of PEP carboxylase (cytosol marker) and cytochrome c oxidase (mitochondrial marker) together with a slight activity of VO₄-ATPase (plasma membrane marker) and NADH-cytochrome c reductase (ER marker) in tonoplast preparation confirmed its high purity. Using latency of nitrate-inhibited ATPase and H⁺ pumping as criteria it was established that the majority of tonoplast vesicles were sealed and oriented right(cytoplasmic)-side-out. Strong acidification of the interior of vesicles observed at the presence of both, ATP and PP_i, confirmed that obtained tonoplast contains two classes of proton pumps: V-ATPase and H⁺PP_iase. To examine and characterise of proton-transport systems in tonoplast, the effect of various inhibitors on H⁺ pumping and hydrolytic activities of ATPase and PP_iase were measured. ATP-dependent activities (H⁺ flux and ATP hydrolysis) were specifically decreased by nitrate and bafilomycin A₁, whereas the PP_iase activities were reduced in the presence of fluoride and Na⁺ ions. Both enzymes showed a similar sensitivity to DCCD and DES. The results of experiments with KCl and NaCl suggested that the vacuolar ATPase was stimulated by Cl⁻, whereas the vacuolar Pp_iase requires K⁺ ions for its activity.     

Partial Purification and Characterization of the Phosphate Transporter from Bovine Heart Mitochondria

A highly active phosphate transporter was extracted with octylglucoside from bovine heart submitochondrial particles that were first partially depleted of other membrane components. It was then partially purified by ammonium sulfate fractionation. After reconstitution of the transporter into liposomes prepared with a crude mixture of soybean phospholipids, the P_i/OH exchange, but not the P_i/P_i exchange, was stimulated three- to fourfold by valinomycin and nigericin in the presence of K⁺. Both P_i/OH and P_i/P_i exchange activities were sensitive to mercurials and other SH reagents. The rutamycin-sensitive ATPase complex from mitochondria was reconstituted together with the phosphate transporter and adenine nucleotide transporter into liposomes. After inhibition of externally located ATPase, the hydrolysis of ATP was sensitive to atractyloside and mersalyl.     

ATP synthesis catalyzed by a V-ATPase: an alternative pathway for energy conservation operating in plant vacuoles?

The electrochemical H⁺ gradient generated in tonoplast vesicles isolated from maize seeds was found to be able to drive the reversal of the catalytic cycle of both vacuolar H⁺-pumps (Façanha and de Meis, 1998). Here we describe the reversibility of the vacuolar V-type H⁺-ATPase (V-ATPase) even in the absence of the H⁺ gradient in a water-Me₂SO co-solvent mixture, resulting in net synthesis of [γ-³²P]ATP from [³²P]P_i and ADP. The water-Me₂SO (5 to 20 %) media promoted inhibition of both PP_i hydrolysis and synthesis reactions whereas it slightly affected the ATP hydrolysis and clearly stimulated the ATP synthesis, which was unaffected by uncoupling agents (FCCP, Triton X-100 or NH₄⁺). This effect of Me₂SO on the ATP⇔³²P exchange reaction seems to be related to a decrease of the apparent K_m of the V-ATPase for P_i. The results are in accordance to the concept that the energetics of ATP synthesis catalysis depends on the solvation energies interacting in the enzyme microenvironment. A possible physiological significance of this phenomenon for the metabolism of desiccation-tolerant plant cells is discussed.Key words: bind energy, proton pumps, proton gradient, DMSO, corn seeds, V₁V₀-ATPase, membrane bound H⁺-pyrophosphatase     

Mycochromone decreases ATP-driven proton translocation in plant plasma membrane- and tonoplast-enriched vesicles

Abstract Mycochromone, a metabolite produced by Mycosphaerella rosigena, inhibits the ATP-dependent proton translocation and the ATP-generated electrical potential in pea stem tonoplast-enriched vesicles, without affecting the H⁺/K⁺ exchange induced by nigericin or an artificially imposed proton gradient. The inhibition is dependent on the time of pre-incubation and mycochromone concentration. In addition, mycochromone inhibits the ATP-dependent proton translocation in radish plasma membrane-enriched vesicles, though it does not alter ATPase activity (evaluated by hydrolysis of ATP) in either type of plant vesicle. Mycochromone seems to act on the H⁺ channels for proton translocation of the H⁺-pumping ATPase localized on plasmalemma and tonoplast, without affecting the catalytic site of ATP hydrolysis.     

The yeast mitochondrial adenosine triphosphatase complex. Purification, subunit composition, and some effects of protease inhibitors.

(i) The method of preparing the oligomycin-insensitive F₁-ATPase by chloroform treatment of mitochondrial membranes (Beechey et al., 1975, Biochem. J.148, 533–537) has been modified such that a five-subunit protein is obtained from yeast with an activity of 140 μmol of ATP hydrolyzed/min/mg of protein. Repetition of this procedure in the presence of protease inhibitors (in particular, p-aminobenzamidine) allows isolation of a four-subunit protein with an activity of 243 μmol of ATP hydrolyzed/min/ mg of protein, (ii) A modified procedure is described for the preparation of the yeast oligomycin-sensitive F₁-F₀ ATPase complex, making use of protease inhibitors throughout and solubilization of the ATPase from mitochondrial membranes using Triton X-100 and sodium deoxycholate simultaneously. Two polypeptides Of 42,000 and 29,000 molecular weight are eliminated, the largest corresponding to the missing band of the F₁ sector. The complex retains oligomycin- and uncoupler-sensitive ATP-³²P_i exchange and ATP-driven proton uptake, indicating the retention of a complete coupling mechanism. (iii) F₁-ATPase is released from the F₁-F₀ complex by brief heating at 50 °C in the presence of ATP. The remaining hydrophobic polypeptides aggregate and are isolated by centrifugation. The F₁ sector can be isolated containing either four or five subunits depending on whether the starting F₁-F₀ complex contained the 42,000 and 29,000 molecular weight polypeptides. (iv) Sensitivity of the F₁-F₀ ATPase complex to oligomycin and dicyclohexylcarbodiimide varies considerably depending on the activity measured and whether the complex was first reconstituted with phospholipids. The degree of inhibitor sensitivity is considered a poor guide to intactness of the complex.     

Potassium Stimulation of Corn Root Plasmalemma ATPase : II. H-Pumping in Native and Reconstituted Vesicles with Purified ATPase

The stimulation by K⁺ of the initial rate of H⁺-pumping by ATPase was studied in native plasmalemma (Zea mays L. var Mona) vesicles and in reconstituted vesicles with enzyme purified on a glycerol gradient. In reconstituted vesicles, a very high H⁺-pumping rate (200,000% quenching per minute per milligram protein) was obtained with 9-amino-6-chloro-2-methoxyacridine provided that the pump was short-circuited by K⁺-valinomycin. A constant ionic strength was used to prevent indirect stimulation by the electrostatic effects of K⁺ salts. Indirect stimulation of H⁺-pumping by the short-circuiting effect of internal K⁺, could be abolished by using the permeant anions NO₃⁻ and Br⁻ in native, but not in reconstituted vesicles. In both materials, half-stimulation of the H⁺-pumping by K⁺ was observed at about 5 millimolar. The same stimulation was obtained when K⁺ was present only in the external solution or when it was present both outside and inside the vesicles. It was concluded that the stimulating effect of K⁺ on the H⁺-pumping evidenced in these experiments on both native and reconstituted vesicles was due to a direct effect of the cation on the cytoplasmic face of the ATPase. These results are discussed within the context of the hypothesis of an active K⁺ transport driven by the ATPase through a direct H⁺/K⁺ exchange mechanism.     

Exchange between inorganic phosphate and adenosine triphosphate in (Na+,K+)-ATPase

(Na⁺,K⁺)-ATPase is able to catalyze a continuous ATP?P_i exchange in the presence of Na⁺ and in the absence of a transmembrane ionic gradient. At pH 7.6 the Na⁺ concentration required for half-maximal activity is 85 mM and at pH 5.1 it is 340 mM. In the presence of optimal Na⁺ concentration, the rate of exchange is maximal at pH 6.0 and varies with ADP and P_i concentration in the assay medium. ATP?P_i exchange is inhibited by K⁺ and by ouabain.     

Asymmetrical distribution of thiol groups involved in ATP-32Pi exchange on mitochondrial membranes.

The use of mitoplasts, that is mitochondria devoid of outer membrane oriented as normal mitochondria, and of sonicated vesicles, the membrane of which is inside-out has shown that the thiol groups involved in the process of ATP synthesis are on the matrix face of the mitochondrial membrane: carboxypyridine disulfide (CPDS) a thiol reagent that cannot penetrate across hydrophobic membranes does not inhibit the ATP-³²P_i exchange catalyzed by mitoplasts, while 5,5′-dithio-$\text{bis}$-(2-nitrobenzoate), which penetrates more readily, can completely inhibit this exchange. In contrast, both reagents react similarly with inside-out vesicles. The nature of the component of the ATPase-ATP synthase complex to which this thiol group may belong is discussed.     

Inhibition of the (Na+ + K+)-dependent ATPase by inorganic phosphate

Inhibition of the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-dependent}$ ATPase by inorganic phosphate, P_i, was examined in terms of product inhibition of the various activities catalyzed by an enzyme preparation from rat brain, and considered in terms of the specific transport processes of the membrane Na⁺,K⁺-pump that these activities reflect. The K⁺-dependent phosphatase activity of the enzyme was most sensitive to P_i, and inhibition was competitive toward the substrate, nitrophenyl phosphate, as would be expected if P_i were released from the same enzyme form that bound substrate. However, this enzymatic activity does not seem to represent a transport process, and thus a cyclical discharge of K⁺ may not be involved. The Na⁺-dependent exchange activity was unaffected by P_i, in accord with the absence of P_i release in the reaction sequence. For the corresponding Na⁺/Na⁺ exchange function of the pump, which reportedly does not involve ATP hydrolysis either, prior release of P_i obviously cannot be required for Na⁺ discharge. With the Na⁺-dependent ATPase activity, measured using micromolar concentrations of ATP, P_i inhibited, but far less than with the phosphatase activity, and inhibition was not competitive toward ATP. Moreover, inhibition decreased as the Na⁺ concentration was raised from 10 to 100 mM. This elevated concentration of Na⁺ also led to substrate inhibition. For this ATPase activity, and the corresponding transport process, uncoupled Na⁺ efflux, the findings suggest that Na⁺ discharge follows P_i release, in contrast to Na⁺/Na⁺ exchange. The $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-dependent}$ ATPase activity, measured with millimolar concentrations of ATP and reflecting the coupled Na⁺,K⁺-transport function, was similarly sensitive to P_i, and again inhibition was not competitive toward ATP. However, in this case inhibition did not increase as the Na⁺ concentration was lowered. For this activity, and the associated transport process, the site of Na⁺ discharge in the overall reaction sequence remains unresolved.     

Properties of a plasmalemma ATPase of the maize scutellum

Properties of a plasmalemma phosphatase of the maize scutellum, tentatively identified as an ATPase in a previous paper, were investigated. Fresh and frozen-thawed scutellum slices, that had been treated with 10 mM HCl to destroy acid phosphatases, were used as a source of enzyme. With the exceptions of the Na⁺, K⁺ and dinitrophenol experiments, the two kinds of slices gave similar results. ATP and CTP were the best substrates for the enzyme followed by TTP, UTP, CDP, ADP and GTP. UDP, nucleoside monophosphates, sugar phosphates, inorganic pyrophosphate and p-nitrophenyl phosphate were relatively ineffective as substrates. The K_m's for ATP and ADP were 0.65 and 5 mM, respectively, but the two substrates gave the same V_max (49.8 μmol P_i/hr/g slices). Previously, it was shown that the products of ATP hydrolysis are ADP, AMP and P_i. Using these previous results and from the time courses of ATP disappearance from the bathing solution and the appearance of P_i and ADP, it was concluded that ATP and ADP were hydrolysed by the same enzyme. The ATPase was not inhibited by oligomycin. N-N′-Dicyclohexylcarbodiimide (DCCD) was a poor inhibitor, and a water soluble analog of DCCD, 1-ethyl-3 (3 dimethyl-aminopropyl)-carbodiimide, gave only 33% inhibition. The relative effectiveness of divalent cations for stimulating ATPase activity was Mn²⁺ > Mg²⁺ ? Ca²⁺ > Co²⁺ · Na⁺ and K⁺ gave a small additional stimulation in the presence of Mg²⁺. However, Na⁺ and K⁺ gave a much greater stimulation when no divalent cation was added, and this occurred only when fresh slices were used. Dinitrophenol also increased ATPase activity only when fresh slices were used. Since it is likely that both the uptake of Na⁺ and K⁺ and the action of dinitrophenol would lower the electrochemical gradient of protons across the plasmalemma, the different results obtained with fresh slices indicate that the ATPase in these slices was under the constraint of a proton gradient.     

Proton Transport Activity of the Purified Vacuolar H-ATPase from Oats : Direct Stimulation by Cl

To determine whether the detergent-solubilized and purified vacuolar H⁺-ATPase from plants was active in H⁺ transport, we reconstituted the purified vacuolar ATPase from oat roots (Avena sativa var Lang). Triton-solubilized ATPase activity was purified by gel filtration and ion exchange chromatography. Incorporation of the vacuolar ATPase into liposomes formed from Escherichia coli phospholipids was accomplished by removing Triton X-100 with SM-2 Bio-beads. ATP hydrolysis activity of the reconstituted ATPase was stimulated twofold by gramicidin, suggesting that the enzyme was incorporated into sealed proteoliposomes. Acidification of K⁺-loaded proteoliposomes, monitored by the quenching of acridine orange fluorescence, was stimulated by valinomycin. Because the presence of K⁺ and valinomycin dissipates a transmembrane electrical potential, the results indicate that ATP-dependent H⁺ pumping was electrogenic. Both H⁺ pumping and ATP hydrolysis activity of reconstituted preparations were completely inhibited by <50 nanomolar bafilomycin A₁, a specific vacuolar type ATPase inhibitor. The reconstituted H⁺ pump was also inhibited by N,N′-dicyclohexylcarbodiimide or NO₃⁻ but not by azide or vanadate. Chloride stimulated both ATP hydrolysis by the purified ATPase and H⁺ pumping by the reconstituted ATPase in the presence of K⁺ and valinomycin. Hence, our results support the idea that the vacuolar H⁺-pumping ATPase from oat, unlike some animal vacuolar ATPases, could be regulated directly by cytoplasmic Cl⁻ concentration. The purified and reconstituted H⁺-ATPase was composed of 10 polypeptides of 70, 60, 44, 42, 36, 32, 29, 16, 13, and 12 kilodaltons. These results demonstrate conclusively that the purified vacuolar ATPase is a functional electrogenic H⁺ pump and that a set of 10 polypeptides is sufficient for coupled ATP hydrolysis and H⁺ translocation.     

Characterization of a plasma membrane H+-ATPase from the extremely acidophilic algaDunaliella acidophila

Summary Dunaliella acidophila is an unicellular green alga which grows optimally at pH 0–1 while maintaining neutral internal pH. A plasma membrane preparation of this algae has been purified on sucrose density gradients. The preparation exhibits vanadatesensitive ATPase activity of 2 mol P_i/mg protein/min, an activity 15 to 30-fold higher than that in the related neutrophilic speciesD. salina. The following properties suggest that the ATPase is an electrogenic plasma membrane H⁺ pump. (i) ATP induces proton uptake and generates a positive-inside membrane potential as demonstrated with optical probes. (ii) ATP hydrolysis and proton uptake are inhibited by vanadate, diethylstilbestrol, dicyclohexylcarbodiimide and erythrosine but not by molybdate, azide or nitrate. (iii) ATP hydrolysis and proton uptake are stimulated by fussicoccin in a pH-dependent manner as found for plants plasma membrane H⁺-ATPase. Unusual properties of this enzyme are: (i) theK _m for ATP is around 60 M, considerably lower than in other plasma membrane H⁺-ATPases, and (ii) the ATPase activity and proton uptake are stimulated three to fourfold by K⁺ and to a smaller extent by other monovalent cations. These results suggest thatD. acidophila possesses a vanadate-sensitive H⁺-ATPase with unusual features enabling it to maintain the large transmembrane pH gradient.     

Factors defining the functional coupling of bacteriorhodopsin and ATP synthase in liposomes

Optimal conditions for the co-reconstitution of bacteriorhodopsin and yeast mitochondria ATP synthase were determined. Reconstitution was achieved with a quick two-step procedure. Preparations obtained by this method displayed in optimal cases 2–3-times higher activities (up to 500 nmol ATP/min per mg protein) compared with maximal values reported in the literature, when light-driven ATP synthesis was measured under similar conditions. The final activities depended on the purification method used for the ATP synthase, and it is shown that the oligomycin-sensitive ATP hydrolysis activity was not a good measure for the ability of the ATP synthase preparations to perform ATP synthesis after co-reconstitution. Light-driven ATP synthesis activities depended also on the type of phospholipid used, soybean phospholipid giving the best results. A close relation to the bacteriorhodopsin proton pump activity was found. Using different phospholipids, different $\text{H}{}^{\mathrm{+}}\text{ATP}$ ratios were found, calculated from ATP synthesis activities and initial and steady-state light-driven proton pump activities. From this, together with the findings that the ATP synthase displayed the same ATP hydrolysis and ATP-³²P_i exchange activities with these different phospholipids used, it is concluded that the protein distribution for the two proteins among the liposomes is different relative to each other for the different phospholipids. The light-driven ATP synthesis activity did not correlate with the variation in leakiness of the membrane for protons when different phospholipids were used. An explanation is given by the finding that at high light intensities, the ATP synthesis became independent of the presence of protonophore.