The stimulating role of subunit F in ATPase activity inside the A₁-complex of the Methanosarcina mazei Gö1 A₁A_O ATP synthase

A₁A_O ATP synthases couple ion-transport of the A_O sector and ATP synthesis/hydrolysis of the A₃B₃-headpiece via their stalk subunits D and F. Here, we produced and purified stable A₃B₃D- and A₃B₃DF-complexes of the Methanosarcina mazei Gö1 A-ATP synthase as confirmed by electron microscopy. Enzymatic studies with these complexes showed that the M. mazei Gö1 A-ATP synthase subunit F is an ATPase activating subunit. The maximum ATP hydrolysis rates (V_max) of A₃B₃D and A₃B₃DF were determined by substrate-dependent ATP hydrolysis experiments resulting in a V_max of 7.9 s^{- 1} and 30.4 s^{- 1}, respectively, while the K_M is the same for both. Deletions of the N- or C-termini of subunit F abolished the effect of ATP hydrolysis activation. We generated subunit F mutant proteins with single amino acid substitutions and demonstrated that the subunit F residues S84 and R88 are important in stimulating ATP hydrolysis. Hybrid formation of the A₃B₃D-complex with subunit F of the related eukaryotic V-ATPase of Saccharomyces cerevisiae or subunit ε of the F-ATP synthase from Mycobacterium tuberculosis showed that subunit F of the archaea and eukaryotic enzymes are important in ATP hydrolysis.