ATP synthase is the universal enzyme that manufactures cellular ATP using the energy stored in a transmembrane ion gradient. This energy gradient has two components: the concentration difference (ΔpH or ΔpNa ϩ ) and the electrical potential difference ΔΨ, which are thermodynamically equivalent. However, they are not kinetically equivalent, as the mitochondrial and bacterial ATP synthases require a transmembrane potential, ΔΨ, but the chloroplast enzyme has appeared to operate on ΔpH alone. Here we show that, contrary to the accepted wisdom, the 'acid bath' procedure used to study the chloroplast enzyme develops not only a ΔpH but also a membrane potential, and that this potential is essential for ATP synthesis. Thus, for the chloroplast and other ATP synthases, the membrane potential is the fundamental driving force for their normal operation. We discuss the biochemical reasons for this phenomenon and a model that is consistent with these new experimental facts.

We describe here purification and biochemical characterization of the F 1 F o -ATP synthase from the thermoalkaliphilic organism Bacillus sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an F 1 F o -ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F 1 subunits ␣, ␤, ␥, ␦, and and F o subunits a, b, and c. The subunits were identified by N-terminal protein sequencing and mass spectroscopy. A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity. ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis >15-fold. This activation was the same for either the F 1 F o holoenzyme or the isolated F 1 moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F 1 . After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (⌬). A transmembrane proton gradient or sodium ion gradient in the absence of ⌬ was not sufficient to drive ATP synthesis. ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone, while the electroneutral Na ؉ /H ؉ antiporter monensin had no effect. Neither ATP synthesis nor ATP hydrolysis was stimulated by Na ؉ ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic Bacillus species. The ATP synthase was specifically modified at its c subunits by N,N-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis.

The conserved glutamate residue at position 65 of the Propionigenium modestum c subunit is directly involved in binding and translocation of Na+ across the membrane. The site-specific introduction of the cQ32I and cS66A substitutions in the putative vicinity to cE65 inhibited growth of the single-site mutants on succinate minimal agar, indicating that both amino acid residues are important for proper function of the oxidative phosphorylation system. This growth inhibition was abolished, however, if the cF84L/cL87V double mutation was additionally present in the P. modestum c subunit. The newly constructed Escherichia coli strain MPC848732I, harboring the cQ32I/cF84L/cL87V triple mutation, revealed a change in the coupling ion specificity from Na+ to H+. ATP hydrolysis by this enzyme was therefore not activated by NaCl, and ATP-driven H+ transport was not affected by this alkali salt. Both activities were influenced, however, by LiCl. These data demonstrate the loss of the Na+ binding site and retention of Li+ and H+ binding sites within this mutant ATPase. In the E. coli strain MPC848766A (cS66A/cF84L/cL87V), the specificity of the ATPase was further restricted to H+ as the exclusive coupling ion. Therefore, neither Na+ nor Li+ stimulated the ATPase activity, and no ATP-driven Li+ transport was observed. The ATPase of the E. coli mutant MPC32N (cQ32N) was activated by NaCl and LiCl. The mutant ATPase exhibited a 5-fold higher Km for NaCl but no change in the Km for LiCl in comparison to that of the parent strain. These results demonstrate that the binding of Na+ to the c subunit of P. modestum requires liganding groups provided by Q32, E65, and S66. For the coordination of Li+, two liganding partners, E65 and S66, are sufficient, and H+ translocation was mediated by E65 alone.

The mechanism by which ion-flux through the membrane-bound motor module (F0) induces rotational torque, driving the rotation of the gamma subunit, was probed with a Na+-translocating hybrid ATP synthase. The ATP-dependent occlusion of 1 (22)Na+ per ATP synthase persisted after modification of the c subunit ring with dicyclohexylcarbodiimide (DCCD), when 22Na+ was added first and ATP second, but not if the order of addition was reversed. These results support the model of ATP-driven rotation of the c subunit oligomer (rotor) versus subunit a (stator) that stops when either a 22Na+-loaded or a DCCD-modified rotor subunit reaches the Na+-impermeable stator. The ATP synthase with a Na+-permeable stator catalyzed 22Na+out/Na+in-exchange after reconstitution into proteoliposomes, which was not significantly affected by DCCD modification of the c subunit oligomer, but was abolished by the additional presence of ATP or by a membrane potential (DeltaPsi) of 90 mV. We propose that in the idling mode of the motor, Na+ ions are shuttled across the membrane by limited back and forth movements of the rotor against the stator. This motional flexibility is arrested if either ATP or DeltaPsi induces the switch from idling into a directed rotation. The Propionigenium modestum ATP synthase catalyzed ATP formation with DeltaPsi of 60-125 mV but not with DeltapNa+ of 195 mV. These results demonstrate that electric forces are essential for ATP synthesis and lead to a new concept of rotary-torque generation in the ATP synthase motor.

We have recently isolated a mutant (aK220R, aV264E, aI278N) of the Na-translocating Escherichia coli/ Propionigenium modestum ATPase hybrid with a Na-inhibited growth phenotype on succinate. ATP hydrolysis by the reconstituted mutant ATPase was inhibited by external (N side) NaCl but not by internal (P side) NaCl. In contrast, LiCl activated the ATPase from the N side and inhibited it from the P side. A similar pattern of activation and inhibition was observed with NaCl and the ATPase from the parent strain PEF42. We conclude from these results that the binding sites for the coupling ions on the c subunits are freely accessible from the N side. Upon occupation of these sites, the ATPase becomes more active, provided that the ions can be further translocated to the P side through a channel of the a subunit. If by mutation of the a subunit this channel becomes impermeable for Na , N side Na ions specifically inhibit the ATPase activity. These conclusions were corroborated by the observation that proton transport into proteoliposomes containing the mutant ATPase was abolished by N side but not by P side Na ions. In contrast, LiCl affected proton translocation from either side, similar to the sidedness effect of Na ions on H transport by the parent hybrid ATPase. If the ATPase carrying the mutated a subunit was incubated with 22 NaCl and ATP, 1 mol 22 Na /mol enzyme was occluded. With the parent hybrid ATPase, 22 Na occlusion was not observed. The occluded 22 Na could be removed from its tight binding site by 20 mM LiCl, while incubation with 20 mM NaCl was without effect. Li but not Na is therefore apparently able to pass through the mutated a subunit and make the entrapped Na ions accessible again to the aqueous environment. These results suggest an ion translocation mechanism through F 0 that in the ATP hydrolysis mode involves binding of the coupling ions from the cytoplasm to the multiple c subunits, ATP-driven rotation to bring a Na , Li , or H-loaded c subunit into a contact site with the a subunit and release of the coupling ions through the a subunit channel to the periplasmic surface of the membrane.