A sodium ion-dependent A1AO ATP synthase from the hyperthermophilic archaeon Pyrococcus furiosus.

The rotor subunit c of the A(1)A(O) 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 degrees 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 K(m) 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 A(1)A(O) ATP synthase of Pyrococcus furiosus, the first Na(+) A(1)A(O) ATP synthase described.