A Na⁺ A₁ A_O ATP synthase with a V-type c subunit in a mesophilic bacterium

A₁ A_O 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 A₁ A_O ATP synthase with a V-type c subunit in a mesophilic organism revealed an A₁ A_O 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 A₁ A_O ATP synthase into liposomes, ATP-dependent primary transport of ²² Na⁺ as well as ΔµNa⁺ -driven ATP synthesis could be demonstrated. The Na⁺ A₁ A_O 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.