Localized energy coupling during photophosphorylation by chromatophores ofRhodopseudomonas capsulata N22

The principle of the dual inhibitor titration method for testing models of electron-transport phosphorylation is outlined, and the method is applied to the study of photophosphorylation in bacterial chromatophores. It is concluded that energy coupling is strictly localized in nature in this system, in the sense that free energy released by a particular electron-transport chain may be used only by a particular H⁺-ATP synthase. Dual inhibitor titrations using the uncoupler SF 6847 and the H⁺-ATP synthase inhibitor oligomycin indicate that uncouplers act by shuttling rapidly between the localized energy-coupling sites.     

Inhibition of energy conservation reactions in chromatophores of Rhodospirillum rubrum by antibiotics

The antibiotics efrapeptin and leucinostatin inhibited photosynthetic and oxidative phosphorylation and related reactions such as the dark and light ATP-P_i exchange reactions and the Mg-ATPase in Rhodospirillum rubrum chromatophores. Higher concentrations of leucinostatin were required for inhibition of the phenazine methosulfate-catalyzed photophosphorylation and light ATP-P_i exchange reaction than for the endogenous or succinate-induced photophosphorylation and dark ATP-P_i exchange reaction. Efrapeptin and leucinostatin inhibited the ATP-driven transhydrogenase while only the latter inhibited the light-driven transhydrogenase, proton gradient formation, and NAD⁺ reduction by succinate in chromatophores. Efrapeptin, but not leucinostatin, inhibited the soluble Ca-ATPase activity of the coupling factor obtained from chromatophores. The inhibition was competitive with ATP. It is concluded that efrapeptin is an effective energy transfer inhibitor whose site of action may be localized in the soluble coupling factor, while the effects of leucinostatin are more complex and cannot be explained as a simple uncoupling.     

The features of activation of free oxidation by α,ω-tetradecanedioic acid in liver mitochondria

It was found that α,ω-tetradecanedioic acid (TDA) at the concentration of 0–500 μM doubles the rate of nonphosphorylating respiration (free oxidation) of liver mitochondria in a dose-dependent manner. This effect of TDA is observed in the presence of the excess of EGTA, which eliminates the induction of the Ca²⁺-dependent nonspecific permeability of the mitochondrial inner membrane (pore opening). An unusually high concentration of cyclosporin A (10 mM) completely eliminates this effect when added to the mitochondria before or after TDA. The stimulatory effect of TDA is not accompanied by inhibition of oxidative ATP synthesis and decrease in the ADP/O ratio, in contrast to the effects of other activators of free oxidation, such as protonophore uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone and palmitic acid. It was shown that neither oligomycin, an inhibitor of H⁺-ATP synthase, nor ADP, ATP and P_i affected the activity of TDA. This is seen as an evidence that the effect of TDA is not associated with the influence on H⁺-ATP synthase and it differs from the action of membranotropic uncouplers. In the presence of the lipophilic cation tetraphenylphosphonium (TPP⁺) cyclosporin A does not affect the TDA-stimulated respiration of mitochondria, but carboxyatractylate and glutamate added after TDA do inhibit the respiration. In addition, under these conditions TDA decreases the rate of oxidative ATP synthesis and reduces the ADP/O ratio. It is assumed that the mechanism of the TDA-induced activation of free oxidation in liver mitochondria in the absence of TPP⁺ is similar to that of the so-called decouplers and is associated with the switching of the respiratory chain complexes to the idle mode (inner uncoupling).     

Melittin inhibition and uncoupling of spinach thylakoids

Melittin has been found to inhibit a photosystem I reaction (diaminodurene to methylviologen) in much the same way that it inhibits sequential electron transport through both photosystems (water to methylviologen). At much lower concentrations melittin uncouples ATP synthesis. Melittin inhibition and uncoupling are found to be irreversible indicating very tight association between melittin and the membrane. Melittin inhibits the light-induced proton pump and the light-induced thylakoid Mg⁺²-ATPase activity as well as the Ca⁺²-ATPase activity of isolated coupling factor. The results are consistent with both a conventional model where the uncoupling by melittin is related to its lytic properties and a model wherein melittin interacts directly with coupling factor causing an uncoupling condition.     

Photophosphorylation capacity of stable spheroplast preparations of anabaena

Spheroplasts from Anabaena 7119 (formerly designated Nostoc muscorum) were prepared in the presence of serum albumin in 0.5 molar sucrose. Electron transport and photophosphorylation were preserved (> 70% of the maximum rate for 1 week). The pH profile of electron transport and photophosphorylation in the reactions H₂O → NADP, H₂O → methyl viologen, and H₂O → ferricyanide shows that uncoupling by ammonia is small throughout and increases slightly with higher pH. ADP + Pi increased NADP reduction from H₂O by 2.5-fold. The ratios of ATP formed per electron pair transported ranged from 0.9 to 1.5. Effects of catalase and superoxide dismutase on the overall O₂ balance implicate pseudocyclic electron transport and phosphorylation. The quenching of 9-aminoacridine fluorescence indicates the formation of a Δ pH from 2 to 2.6 during illumination. This pH gradient is abolished by uncouplers; however, complete uncoupling is achieved only by 3-chlorocarbonyl cyanide phenylhydrazone or valinomycin + NH₄⁺. In the presence of NH₄⁺ alone, the membrane potential may act as the driving force for photophosphorylation.     

Generation of membrane potential during photosynthetic electron flow in chromatophores from Rhodopseudomonas capsulata

1. When cytochrome c₂ is available for oxidation by the photosynthetic reaction centre, the decay of the carotenoid absorption band shift generated by a short flash excitation of Rhodopseudomonas capsulata chromatophores is very slow (half-time approximately 10 s). Otherwise the decay is fast (half-time approximately 1 s in the absence and 0.05 s in the presence of 1,10-ortho-phenanthroline) and coincides with the photosynthetic back reaction.2. In each of these situations the carotenoid shift decay, but not electron transport, may be accelerated by ioniophores. The ionophore concentration dependence suggests that in each case the carotenoid response is due to a delocalised membrane potential which may be dissipated either by the electronic back reaction or by electrophoretic ion flux.3. At high redox potentials, where cytochrome c₂ is unavailable for photo-oxidation, electron transport is believed to proceed only across part of the membrane dielectric. Under such conditions it is shown that the driving force for carbonyl cyanide trifluoromethoxyphenyl hydrazone-mediated H⁺ efflux is nevertheless decreased by valinomycin/K⁺; demonstrating that the [BChl]₂ → Q electron transfer generates a delocalised membrane potential.     

The relation between membrane ionic current and ATP synthesis in chromatophores from Rhodopseudomonas capsulata

(1) Under conditions in which membrane potential (Δψ) was the sole contributor to the proton-motive force, the steady-state rate of ATP synthesis in chromatophores increased disproportionately when Δψ was increased: the rate had an approximately sixth-power dependence on Δψ. (2) Simultaneous measurements showed that the dissipative ionic current (J_DIS) across the chromatophore membrane had a related dependence on Δψ, i.e., the membrane conductance increased markedly as Δψ increased. (3) For comparable Δψ values, J_DIS was greater in phosphorylating than in non-phosphorylating chromatophores. For comparable actinic light intensities, Δψ was smaller in phosphorylating than in non-phosphorylating chromatophores. (4) At either low pH or in the presence of venturicidin, oligomycin or dicyclohexylcarbodiimide to inhibit ATP synthesis, J_DIS was substantially depressed, particularly at high Δψ. Even under these conditions the membrane conductance was dependent on Δψ. (5) Also in intact cells, J_DIS was depressed in the presence of venturicidin. Points 1–5 are interpreted in terms of a Δψ -driven H⁺ flux through the F₀ channel of the ATPase synthase. The high-power dependence of the F₀ conductance on Δψ determines the dependence of the rate of ATP synthesis on Δψ. The Δψ -dependent conductance of F₀ dominates the electrical properties of the membrane. In chromatophores the ionic current accompanying ATP synthesis was more than 50% of the total membrane ionic current at maximal Δψ. (6) The rate of cyclic electron transport was calculated from J_DIS. This led to an estimate of 0.77 ± 0.22 for the $\text{ATP}\text{2}\text{e}{}^{\mathrm{?}}$ ratio and of 3.5 ± 1.3 for the $\text{H}{}^{\mathrm{+}}\text{ATP}$ ratio. (7) Severe inhibition of the electron-transport rate by decreasing the light intensity led to an almost proportionate decrease in the rate of ATP synthesis. The chromatophores were able to maintain proportionality by confining electron-transport phosphorylation to a narrow range of Δψ. This is a consequence of the remarkable conductance properties of the membrane.     

Uncouplers can shuttle between localized energy-coupling sites during photophosphorylation by chromatophores of Rhodopseudomonas capsulata N22.

Two models of the action of uncoupler molecules in inhibiting photophosphorylation in bacterial chromatophores are considered: either uncoupler molecules shuttle rapidly between energy-coupling sites, or uncoupler molecules that are bound to particular sites in the chromatophores for a time that is comparable with the turnover time of the photophosphorylation apparatus may uncouple by a co-operative "substoichiometric' mechanism. It is found that the titre of uncoupler necessary to cause complete uncoupling is lowered if the rate of photophosphorylation is initially decreased by partially restricting electron flow with an appropriate titre of antimycin A. This result indicates that uncoupler molecules shuttle rapidly between energy coupling in which the energized intermediate between electron transport and phosphorylation is delocalized over the entire chromatophore membrane and those in which it is not. If the rate of photophosphorylation is partially restricted with the covalent H+-translocating ATP synthase inhibitor dicyclohexylcarbodi-imide, the titre of uncoupler necessary to effect complete inhibition of photophosphorylation is also decreased relative to that in which the covalent H+-ATP synthase inhibitor is absent. This important result appears to be inconsistent with models of electron-transport phosphorylation in which the "energized state' of the chromatophore membrane that is set up by electron transport and utilized in photophosphorylation is delocalized over the entire chromatophore membrane.     

Inactivation of Rhodospirillum rubrum coupling factor by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Modification of a tyrosine protected by phosphate

Chemical modification of Rhodospirillum rubrum chromatophores by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) results in inactivation of photophosphorylation, Mg²⁺-ATPase, oxidative phosphorylation and ATP-driven transhydrogenase, with apparent first-order kinetics. Other energy-linked reactions such as light-driven transhydrogenase and light-dependent proton uptake were insensitive to NBD-Cl. The Ca²⁺-ATPase activity of the soluble coupling factor from chromatophores (R. rubrum F₁) was inactivated by NBD-Cl with kinetics resembling those described for Mg²⁺-ATPase and photophosphorylation activities of chromatophores. Both NBD-chromatophores and NBD-R. rubrum F₁ fully recovered their activities when subjected to thiolysis by dithioerythritol. Phosphoryl transfer reactions of chromatophores and Ca²⁺-ATPase activity of R. rubrum F₁ were fully protected by 5 mM P_i against modification by NBD-Cl. ADP or ATP afforded partial protection. Analysis of the protection of Ca²⁺-ATPase activity by P_i indicated that NBD-Cl and P_i are mutually exclusive ligands. Spectroscopic studies revealed that tyrosine and sulfhydryl residues in R. rubrum F₁ underwent modification by NBD-Cl. However, the inactivation was only related to the modification of tyrosine groups.     

Interaction of a coupling factor from Rhodospirillum rubrum with coupling factor deficient chromatophores

A coupling factor necessary for the photophosphorylation and Mg²⁺-ATPase activities of Rhodospirillum rubrum chromatophores has been separated from these particles. Although the redox potential of coupling factor deficient chromatophores is slightly more oxidized than of the control, the addition of the coupling factor for reconstitution does not alter the redox potential. Phenazine methosulfate cannot restore or significantly enhance the photophosphorylation activities of uncoupled or reconstituted chromatophores compared to the control. The coupling factor can bind to coupling factor deficient membranes without addition of magnesium ions and thus restore the photophosphorylation and Mg²⁺-ATPase activities of these vesicles. The Ca²⁺-ATPase in the coupling factor preparation shows binding characteristics similar to those of the coupling factor.     

Na+-transporting ATPase in the plasma membrane of halotolerant microalga Dunaliella maritima operates as a Na+ uniporter

A fraction of inside-out membrane vesicles enriched in plasma membranes (PM) was isolated from Dunaliella maritima cells. Attempts were made to reveal ATP-driven Na⁺-dependent H⁺ efflux from the PM vesicles to external medium, as detected by alkalization of the vesicle lumen. In parallel experiments, ATP-dependent Na⁺ uptake and electric potential generation in PM vesicles were investigated. The alkalization of the vesicle lumen was monitored with an impermeant pH-sensitive optical probe pyranine (8-hydroxy-1,3,6-pyrenetrisulfonic acid), which was loaded into vesicles during the isolation procedure. Sodium uptake was measured with ²²Na⁺ radioactive label. The generation of electric potential in PM vesicles (positive inside) was recorded with a voltage-sensitive probe oxonol VI. Appreciable Na⁺-and ATP-dependent alkalization of vesicle lumen was only observed in the presence of a protonophore CCCP (carbonyl cyanide-chlorophenylhydrazone). In parallel experiments, CCCP accelerated the ATP-dependent ²²Na⁺ uptake and abolished the electric potential generated by the Na⁺-ATPase at the vesicle membrane. A permeant anion NO^? ₃ accelerated ATP-dependent ²²Na⁺ uptake and promoted dissipation of the electric potential like CCCP did. At the same time, NO^? ₃ inhibited the ATP-and Na⁺-dependent alkalization of the vesicle lumen. The results clearly show that the ATP-and Na⁺-dependent H⁺ efflux from PM vesicles of D. maritima is driven by the electric potential generated at the vesicle membrane by the Na⁺-ATPase. Hence, the Na⁺-transporting ATPase of D. maritima carries only one ion species, i.e., Na⁺. Proton is not involved as a counter-ion in the catalytic cycle of this enzyme.     

Correlation between ATP synthesis and the decay of the carotenoid band shift after single flash activation of chromatophores from Rhodopseudomonas capsulata

ATP synthesis and the acceleration of the decay of the carotenoid absorption band shift after single flash excitation of Rhodopseudomonas capsulata chromatophores were compared. The two processes behave similarly with respect to: (1) ADP and P_i concentration; (2) inhibition by efrapeptin and venturicidin, and (3) inhibition by valinomycin/K⁺ and by ionophores.Taken together with earlier evidence for the electrochromic nature of the carotenoid band shift the data support the contention that positive charge moves outwards across the chromatophore membrane during ATP synthesis and justify the method for determination of the H⁺/ATP ratio (Petty, K.M. and Jackson, J.B. (1979) FEBS Lett. 97, 367–372).The ability of nucleotide diphosphates in the presence of P_i and Mg²⁺ to give rise to the acceleration of the carotenoid shift decay closely correlates with the rate of phosphorylation of the nucleotides in steady-state light. Nucleotide triphosphates enhance the decay in parallel with their rate of hydrolysis.Adenylyl imidodiphosphate is itself without effect on the decay of the carotenoid shift and it does not prevent the ADP-induced acceleration. The analogue does prevent the ATP effect but only after repeated flashes.     

Ion transport and energy conservation in submitochondrial particles

Summary The combination of valinomycin and nigericin in the presence of K⁺ uncouples submitochondrial particles (SMP) as evidenced by: 1) loss and release of the oligomycin-induced respiratory control; 2) inhibition of the P/0 ratio; 3) inhibition of three energy-linked reactions — pyridine-nucleotide transhydrogenation, reversal of electron-transfer, and bromthymol blue and 8-anilino-1-naphtalenesulfonate responses; and 4) change of redox state of cytochromes to the same extent obtained with conventional uncouplers. Neither antibiotic alone, in the presence of K⁺, markedly affected the energized state of the system. Direct measurements of K⁺ and H⁺ movements showed that SMP did indeed translocate these ions in a predictable manner, i. e., a nigericin-stimulated influx of K⁺ to SMP, followed by a valinomycin-mediated efflux of the K⁺ taken up. The NH ₄ ⁺ -dependent uncoupling is demonstrated to be associated with the uptake of NH ₄ ⁺ by SMP with a consequent collapse of the pH gradient established during respiration, followed by a valinomycin-mediated efflux of the NH ₄ ⁺ taken up. The effects of cations and antibiotics can be mimicked by suitable combinations of cations and anions, suggesting that the valinomycin-mediated efflux of cations from SMP is electrophoretic in nature and can be replaced by an electrophoretic influx of appropriate anions. Analogies are drawn with observations reported on bacterial chromatophores and chloroplasts, and a general scheme is suggested. The implications of these results are discussed in terms of the current hypotheses of energy coupling in oxidative and photosynthetic phosphorylation.This investigation constitutes a portion of the work to be submitted to the Graduate School of Arts and Sciences, University of Pennsylvania, in partial fulfillment of the requirements for the degree of Doctor of Philosophy.     

Synergism of ammonium and palmitic acid in uncoupling of electron transfer and ATP synthesis in chloroplasts

Uncoupling by ammonium of electron transfer and ATP synthesis during linear transfer of electrons from water to photosystem 1 acceptors was studied in pea chloroplasts. It was shown that 40 μM palmitic acid decreased several-fold the ammonium concentrations necessary for 50% inhibition of ATP synthesis. The protonophore carbonyl cyanide m-chlorophenylhydrazone has no such property. The enhancement by palmitate of ammonium-induced uncoupling is accompanied by acceleration of basal electron transfer and decrease in the photoinduced uptake of hydrogen ions (H⁺). In the absence of ammonium, palmitate has no effect on basal transport and stimulates uptake of hydrogen ions. This means that in the case of combined action of palmitate and ammonium an additional leakage of H⁺ takes place, resulting in dissipation of the pH gradient. Synergic action of two metabolites, free fatty acid and ammonium, is supposed to provide for functioning of a system of mild regulation of energy coupling processes in native plant cell chloroplasts. Possible mechanisms of synergism are discussed.     

The kinetics of carotenoid absorption changes in intact cells of photosynthetic bacteria

The kinetics of carotenoid absorption changes have been measured in intact cells of Rhodopseudomonas capsulata after short flash excitation. The observed changes were consistent with the thesis that they indicate the development and dissipation of membrane potential. In the generation of the absorption changes in anaerobic cells, fast (complete in 0.5 ms) and slow (half-time 3 ms) components can be distinguished. The slow component corresponds kinetically to the rate of cytochrome c re-reduction and is similarly antimycin sensitive. These data are similar to those observed in isolated chromatophores which have been artifically poised with redox mediators. In aerobic intact cells the kinetic profile is altered, mainly because the decay of the carotenoid change is much faster. Inhibition of respiration with KCN leads to flash-induced changes similar to those in anaerobic cells. At least two components can be distinguished in the decay of the carotenoid absorption changes in anaerobic intact cells. Only the faster decay component was inhibited by venturicidin which suggests that it corresponds to H⁺ flux through the F₀F₁-ATPase during ATP synthesis. The contribution of the venturicidin-sensitive decay to the total decay was dependent upon the initial amplitude of the carotenoid absorption change produced by the flash group. This suggests that there is an apparent threshold of membrane potential for ATP synthesis. Supporting evidence was provided by the finding that venturicidin stimulated the steady-state light-induced carotenoid absorption change at high but not at low light intensities. The entire decay of the carotenoid absorption changes was stimulated by carbonyl cyanide p-trifluoromethoxyphenylhydrazone in a manner that can be interpreted as an ionophore catalysing the dissipation of membrane potential.     

Ratiometric fluorescence measurements of membrane potential generated by yeast plasma membrane H-ATPase reconstituted into vesicles

Potential-sensitive fluorescent probes oxonol V and oxonol VI were employed for monitoring membrane potential (Δψ) generated by the Schizosaccharomyces pombe plasma membrane H⁺-ATPase reconstituted into vesicles. Oxonol VI was used for quantitative measurements of the Δψ because its response to membrane potential changes can be easily calibrated, which is not possible with oxonol V. However, oxonol V has a superior sensitivity to Δψ at very low concentration of reconstituted vesicles, and thus it is useful for testing quality of the reconstitution. Oxonol VI was found to be a good emission-ratiometric probe. We have shown that the reconstituted H⁺-ATPase generates Δψ of about 160 mV on the vesicle membrane. The generated Δψ was stable at least over tens of minutes. An influence of the H⁺ membrane permeability on the Δψ buildup was demonstrated by manipulating the H⁺ permeability with the protonophore CCCP. Ratiometric measurements with oxonol VI thus offer a promising tool for studying processes accompanying the yeast plasma membrane H⁺-ATPase-mediated Δψ buildup.     

Two regimens of electrogenic cyclic redox chain operation in chromatophores of non-sulfur purple bacteria A study using antimycin A

Antimycin A causes a biphasic suppression of the light-induced membrane potential generation in Rhodospirillum rubrum and Rhodopseudomonas sphaeroides chromatophores incubated anaerobically. The first phase is observed at low antibiotic concentrations and is apparently due to its action as a cyclic electron transfer inhibitor. The second phase is manifested at concentrations which are greater than 1–2 μM and is due to uncoupling that may be connected with an antibiotic-induced dissipation of the electrochemical H⁺ gradient across the chromatophore membrane. The inhibitory effect of anti-mycin added at low concentrations under aerobic conditions is removed by succinate to a large extent. It is expected that the electrogenic cyclic redox chain in the bacterial chromatophores incubated under conditions of continuous illumination may function at two regimes: (1) as a complete chain involving all the redox components, and (2) as a shortened chain involving only the P-870 photoreaction center, ubiquinone and cytochrome c₂.     

Photophosphorylation in cell envelope vesicles from Halobacteriumhalobium

We have prepared vesicles from cell envelope membranes of $\text{Halobacterium}\text{halobium}$ strains R₁ and ET-15 which are able to synthesize ATP in response to illumination. This photophosphorylation is inhibited by dicyclohexylcarbodiimide (DCCD) and by phloretin. ATP synthesis in L vesicles from the R₁ strain (which contain bacteriorhodopsin) is inhibited by the protonophore 1799 but not by valinomycin. In M vesicles from the R₁ strain and in ET-15 vesicles (both contain halorhodopsin) photophosphorylation is inhibited by both 1799 and valinomycin. These data are consistent with the idea that light-driven ATP synthesis can be coupled to the electrochemical H⁺ gradient generated by bacteriorhodopsin or by halorhodopsin through the membrane potential component of protonmotive force.     

Freezing injury and uncoupling of phosphorylation from electron transport in chloroplasts

Freezing of chloroplast membranes uncouples photophosphorylation from electron transport and inactivates the light-dependent and thiol-requiring ATPase, conformational changes and the light-dependent proton uptake. All of these energy requiring activities can be protected against inactivation by addition of sucrose prior to freezing. The direct relation to photophosphorylation is demonstrated by the quantitatively similar response of photophosphorylation and the other activities to sucrose protection. Salts interfere with the protection afforded by sucrose.

In contrast to the light-dependent ATPase, the ATPase activities which are unmasked by digestion with trypsin show no significant response to freezing. Similarly, the chloroplast coupling factor, which is released from the membranes by ethylenediamine tetraacetic acid treatment, survives freezing. The membranes, which are depleted of the factor, are damaged by freezing.

The results suggest that uncoupling of phosphorylation from electron transport is caused by interference of freezing with a structure involved in the formation of a non-phosphorylated high energy state of chloroplasts. They are best explained on the basis of Mitchell's theory of phosphorylation. Since freezing alters the permeability properties of chloroplast membranes—frozen membrane vesicles no longer function as osmometers—it may be assumed that freezing uncouples phosphorylation from electron transport by preventing the formation of a pH gradient across the vesicle membranes owing to proton leakage through the membranes. From the results, the basic injury caused by freezing appears to consist in the alteration of permeability properties of biological membranes due to the dehydration which accompanies freezing.

     

Na+ Transport by the A1AO-ATP Synthase Purified from Thermococcus onnurineus and Reconstituted into Liposomes

The ATP synthase of many archaea has the conserved sodium ion binding motif in its rotor subunit, implying that these A₁A_O-ATP synthases use Na⁺ as coupling ion. However, this has never been experimentally verified with a purified system. To experimentally address the nature of the coupling ion, we have purified the A₁A_O-ATP synthase from T. onnurineus. It contains nine subunits that are functionally coupled. The enzyme hydrolyzed ATP, CTP, GTP, UTP, and ITP with nearly identical activities of around 40 units/mg of protein and was active over a wide pH range with maximal activity at pH 7. Noteworthy was the temperature profile. ATP hydrolysis was maximal at 80 °C and still retained an activity of 2.5 units/mg of protein at 45 °C. The high activity of the enzyme at 45 °C opened, for the first time, a way to directly measure ion transport in an A₁A_O-ATP synthase. Therefore, the enzyme was reconstituted into liposomes generated from Escherichia coli lipids. These proteoliposomes were still active at 45 °C and coupled ATP hydrolysis to primary and electrogenic Na⁺ transport. This is the first proof of Na⁺ transport by an A₁A_O-ATP synthase and these findings are discussed in light of the distribution of the sodium ion binding motif in archaea and the role of Na⁺ in the bioenergetics of archaea.