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Abstract:
The ATP synthase of many archaea has the conserved sodium ion binding motif in its rotor subunit, implying that these A1AO-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 A1AO-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 A1AO-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 A1AO-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
