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The rotor subunit c of the A1AO ATP synthase of the hyperthermophilic archaeon Pyrococcus furiosus contains a conserved Na+-binding motif, indicating that Na+ is a coupling ion. To experimentally address the nature of the coupling ion, we isolated the enzyme by detergent solubilization from native membranes followed by chromatographic separation techniques. The entire membrane-embedded motor domain was present in the preparation. The rotor subunit c was found to form an SDS-resistant oligomer. Under the conditions tested, the enzyme had maximal activity at 100 °C, had a rather broad pH optimum between pH 5.5 and 8.0, and was inhibited by diethystilbestrol and derivatives thereof. ATP hydrolysis was strictly dependent on Na+, with a Km of 0.6 mm. Li+, but not K+, could substitute for Na+. The Na+ dependence was less pronounced at higher proton concentrations, indicating competition between Na+ and H+ for a common binding site. Moreover, inhibition of the ATPase by N′,N′-dicyclohexylcarbodiimide could be relieved by Na+. Taken together, these data demonstrate the use of Na+ as coupling ion for the A1AO ATP synthase of Pyrococcus furiosus, the first Na+ A1AO ATP synthase described.
Membrane-bound, multisubunit, ion-translocating ATP synthases/ATPases are present in every domain of life. They arose from a common ancestor, but evolved into three distinct classes of ATP synthases/ATPases: the F1FO ATP synthase present in bacteria, mitochondria and chloroplasts, the A1AO ATP synthase present in archae, and the V1VO ATPase associated with cytoplasmic organelles such as vacuoles in eukaryotes [1–4]. A common feature of ATP synthases/ATPases is their organization into two domains, a hydrophilic and a membrane-bound domain, that are connected by (at least) two stalks, one central and one to two peripheral [5–12]. The hydrophilic domain catalyzes ATP hydrolysis [13–16], and the membrane-bound domain translocates ions from one side of the membrane to the other against their electrochemical gradient [17–20].
ATP synthases/ATPases are rotary machines that work as a pair of coupled motors: a chemically driven motor (F1/A1/V1) that is attached to the membrane, and an ion gradient-driven membrane-embedded motor (FO/AO/VO) [21–27]. The membrane-embedded motor is composed of a stator and a rotor. The rotor is composed of multiple copies of subunit c that form an oligomeric ring of noncovalently linked subunits, and rotation of the c ring is obligatorily coupled to ion flow across the membrane [28–31]. Most F1FO ATP synthases and V1VO ATPases use the proton as a coupling ion, but some use sodium ions instead. The ion-binding site is located in subunit c. The bacterial 8 kDa c subunit monomer folds in the membrane like a hairpin, with two transmembrane helices connected by a cytoplasmic loop, and a single carboxylic acid (aspartate or glutamate [D61 in Escherichia coli; E65 in Ilyobacter tartaricus]) has been identified as the protonatable group in the rotor subunit [32–37]. The recently determined high-resolution structure of the rotor from I. tartaricus revealed that the coordination sphere for Na+ is formed by side-chain oxygens of Gln32 and Glu65 of one subunit, and the hydroxyl group of Ser66 and the backbone carbonyl oxygen of Val63 of the neighboring subunit . The motif is conserved in Na+ F1FO ATP synthases [39,40]. V1VO ATPases have a ≈ 16 kDa subunit c that arose by gene duplication and fusion, giving rise to a monomer with four transmembrane helices. The active carboxylate is conserved in helix four but not in helix two. The Na+ coordination sphere in the c ring of Enterococcus hirae is composed of helix two and four of one monomer. The side chains of T64, Q65, Q110 and E139, and in addition the backbone carbonyl of L61, form the Na+-binding pocket .
Na+-translocating ATPases have so far been found only in anaerobic prokaryotes such as Propionigenium modestum, I. tartaricus and Acetobacterium woodii (F1FO ATP synthase) and Caloramator fervidus, Clostridium paradoxum and E. hirae (annotated or described as ‘bacterial’ V1VO ATPases, but there is debate about the classification in the literature) [42–47]. They have been found to be advantageous in analyzing the mechanism of ion transport and the structure of the c ring. In contrast to F1FO ATP synthases and V1VO ATPases, Na+-dependent A1AO ATP synthases have not been described. Experimental confirmation of the use of Na+ as a coupling ion in A1AO ATP synthases has so far not been obtained, due to the lack of purified and coupled A1AO ATP synthases. All preparations but one, from Methanocaldococcus jannaschii, lacked the membrane-embedded motor . To experimentally address the nature of the coupling ion in the A1AO ATP synthase, we have enriched and studied the A1AO ATP synthase from the hyperthermophilic archaeon Pyrococcus furiosus. This preparation contains the membrane-embedded motor, and we will present evidence that it is an Na+-dependent enzyme. This is the first description of an Na+ A1AO ATP synthase.
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In anaerobic environments, microbes often grow on substrates that allow for the synthesis of only 1–4 mol of ATP , and in addition, the concentration of membrane-permeable organic acids such as acetate or succinate will counteract proton-based energetics, due to uncoupling effects [60,61]. It has long been known that anaerobic members of the third domain of life, the Archaea, such as the methanogens, couple metabolic activities to the generation of primary, transmembrane electrochemical Na+ gradients [13,62,63], but Na+-driven ATP synthesis could not be demonstrated unequivocally using whole cells . However, sequence comparisons indicated that the Na+-binding motif in subunit c of F1FO ATP synthases and V1VO ATPases is present in subunit c of some A1AO ATP synthases, i.e. those from the methanogens, pyrococci, Thermoplasmatales, Archaeoglobales and halobacteria . Unfortunately, a dependence on Na+ of ATP synthesis or ATP hydrolysis using purified enzymes could not be demonstrated, due to the lack of purified, intact A1AO ATP synthases .
Here, we have established a protocol to enrich the A1AO ATP synthase of P. furiosus. The enzyme contained the membrane-embedded motor, and the A1 and AO domains were functionally coupled, as is evident from the inhibition of ATP hydrolysis by N′,N′-dicyclohexylcarbodiimide and the Na+ dependence of ATP hydrolysis. The enzyme exhibited the highest ATPase activities at 100 °C, an outstanding feature of a membrane-embedded macromolecular transport machinery. The A1AO ATP synthase from P. furiosus is one of the most interesting members of the class of A1AO ATP synthases/ATPases. It has a V1VO-type c subunit that could contribute to the thermostability of the rotor. Furthermore, the c subunit has only one ion-binding site in four transmembrane helices, and this would argue for a function of the enyzme as an ATPase, not an ATP synthase. However, ATP synthesis was demonstrated in vesicle preparations , and as the A1AO ATP synthase genes are the only ATP synthase genes on the chromosome , this would argue for the A1AO ATP synthase catalyzing ATP synthesis in vivo, despite the V1VO-type c subunit. The structural basis for ATP synthesis in the P. furiosus enzyme is unknown, but should reside in the membrane-embedded motor . Unfortunately, the preparation described here contained thermosomes that interfered with structural analyses. Attempts to remove the thermosomes by pH changes, washing the membranes with NaCl, LiCl or CaCO3, anion exchange chromatography and several gel filtrations (Superose 6, S300, S400 and S1000) were unsuccessful.
Most important, we have established that the A1AO ATP synthase of P. furiosus uses Na+ as a coupling ion. This is based on the Na+ dependence of ATP hydrolysis, the protection from N′,N′-dicyclohexylcarbodiimide inhibition by Na+, and the presence of an Na+-binding motif in subunit c. The Na+-binding motif is identical to the motif established experimentally for the 16 kDa c subunit of E. hirae. Na+ is bound by only one subunit c. In contrast, the Na+-binding site in the F1FO ATP synthase from I. tartaricus is formed by two subunits, and this bridging of subunit c by Na+ is thought to be the structural basis for SDS resistance of the c ring of Na+ F1FO ATP synthases . Interestingly, we observed partial SDS resistance of the c ring of P. furiosus. As the analogy with the E. hirae c ring would argue against subunit bridging by Na+, a more general mechanism may enable SDS resistance in the P. furiosus c ring.
The presence of an Na+-dependent A1AO ATP synthase gives the first hint of Na+-based bioenergetics in P. furiosus. This hyperthermophile is known to grow by fermentation of sugars. During fermentation, the electrochemical ion gradient across the membrane is established by the A1AO ATP synthase. In addition, P. furiosus can also couple hydrogen oxidation to the synthesis of ATP by a chemiosmotic mechanism . A potential role of Na+ in the bioenergetics of P. furiosus was not addressed in previous studies, but this is not excluded. A hydrogenase of the Ech type has been discussed as a primary Na+ pump in some methanogens . A membrane-bound hydrogenase is present in P. furiosus, and could couple hydrogen oxidation to the generation of transmembrane primary Na+ potential. As outlined above, Na+-based bioenergetics are of advantage in alkaline , anaerobic  and hot environments , and may contribute to successful life at high temperatures.