A Na+ A1AO ATP synthase with a V‐type c subunit in a mesophilic bacterium.

A1AO ATP synthases with a V‐type c subunit have only been found in hyperthermophilic archaea which makes bioenergetic analyses impossible due to the instability of liposomes at high temperatures. A search for a potential archaeal A1AO ATP synthase with a V‐type c subunit in a mesophilic organism revealed an A1AO ATP synthase cluster in the anaerobic, acetogenic bacterium Eubacterium limosum KIST612. The enzyme was purified to apparent homogeneity from cells grown on methanol to a specific activity of 1.2 U·mg−1 with a yield of 12%. The enzyme contained subunits A, B, C, D, E, F, H, a, and c. Subunit c is predicted to be a typical V‐type c subunit with only one ion (Na+)‐binding site. Indeed, ATP hydrolysis was strictly Na+‐dependent. N,N′‐dicyclohexylcarbodiimide (DCCD) inhibited ATP hydrolysis, but inhibition was relieved by addition of Na+. Na+ was shown directly to abolish binding of the fluorescence DCCD derivative, NCD‐4, to subunit c, demonstrating a competition of Na+ and DCCD/NCD‐4 for a common binding site. After incorporation of the A1AO ATP synthase into liposomes, ATP‐dependent primary transport of 22Na+ as well as ΔµNa+‐driven ATP synthesis could be demonstrated. The Na+ A1AO ATP synthase from E. limosum is the first ATP synthase with a V‐type c subunit from a mesophilic organism. This will enable future bioenergetic analysis of these unique ATP synthases. [ABSTRACT FROM AUTHOR]