To establish a system to analyze ATP synthesis by the archaeal A(1)A(o) ATP synthase and to address the nature of the coupling ion, the operon encoding the A(1)A(o) ATP synthase from the mesophile Methanosarcina mazei Gö1 was cloned in an expression vector and it was expressed in the F(1)F(o) ATP synthase-negative mutant Escherichia coli DK8. Western blot analyses revealed that each of the subunits was produced, and the subunits assembled to a functional, membrane-embedded ATP synthase/ATPase. ATP hydrolysis was inhibited by dicyclohexylcarbodiimide but also by tributyltin, which turned out to be the most efficient inhibitor of the A(o) domain of A(1)A(o) ATP synthase known to date. ATP hydrolysis was not dependent on the Na(+) concentration of the medium, and inhibition of the enzyme by dicyclohexylcarbodiimide could not be relieved by Na(+). The enzyme present in the cytoplasmic membrane of E. coli catalyzed ATP synthesis driven by an artificial DeltapH but not by DeltapNa or DeltamuNa(+).

Archaea are a heterogenous group of microorganisms that often thrive under harsh environmental conditions such as high temperatures, extreme pHs and high salinity. As other living cells, they use chemiosmotic mechanisms along with substrate level phosphorylation to conserve energy in form of ATP. Because some archaea are rooted close to the origin in the tree of life, these unusual mechanisms are considered to have developed very early in the history of life and, therefore, may represent first energy-conserving mechanisms. A key component in cellular bioenergetics is the ATP synthase. The enzyme from archaea represents a new class of ATPases, the A₁A₀ ATP synthases. They are composed of two domains that function as a pair of rotary motors connected by a central and peripheral stalk(s). The structure of the chemically-driven motor (A₁) was solved by small-angle X-ray scattering in solution, and the structure of the first A₁A₀ ATP synthases was obtained recently by single particle analyses. These studies revealed novel structural features such as a second peripheral stalk and a collar-like structure. In addition, the membrane-embedded electrically-driven motor (A₀) is very different in archaea with sometimes novel, exceptional subunit composition and coupling stoichiometries that may reflect the differences in energy-conserving mechanisms as well as adaptation to temperatures at or above 100°C.