• A1AO ATP synthase;
  • Archaea;
  • hyperthermophile;
  • Na+;
  • Pyrococcus

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.