The stimulating role of subunit F in ATPase activity inside the A1-complex of the Methanosarcina mazei Gö1 A1AO ATP synthase.

Abstract A 1 A O ATP synthases couple ion-transport of the A O sector and ATP synthesis/hydrolysis of the A 3 B 3 -headpiece via their stalk subunits D and F. Here, we produced and purified stable A 3 B 3 D- and A 3 B 3 DF-complexes of the Methanosarcina mazei Go1 A-ATP synthase as confirmed by electron microscopy. Enzymatic studies with these complexes showed that the M. mazei Go1 A-ATP synthase subunit F is an ATPase activating subunit. The maximum ATP hydrolysis rates ( V max ) of A 3 B 3 D and A 3 B 3 DF 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 A 3 B 3 D-complex with subunit F of the related eukaryotic V-ATPase of Saccharomyces cerevisiae or subunit e of the F-ATP synthase from Mycobacterium tuberculosis showed that subunit F of the archaea and eukaryotic enzymes are important in ATP hydrolysis.