γM23K, γm232K, and γl77K single substitutions in the TF1-ATPase lower ATPase activity by disrupting a cluster of hydrophobic side chains

In crystal structures of the bovine F 1 -ATPase (MF 1 ), the side chains of γMet 2 3 , γMet 2 3 2 , and γLeu 7 7 interact in a cluster. Substitution of the corresponding residues in the α 3 β 3 γ subcomplex of TF 1 with lysine lowers the ATPase activity to 2.3, 11, and 15%, respectively, of that displayed by wild-type. In contrast, TF 1 subcomplexes containing the γM 2 3 C, γM 2 3 2 C, and γL 7 7 C substitutions display 36, 36, and 130%, respectively, of the wild-type ATPase activity. The ATPase activity of the γM 2 3 C/γM 2 3 2 C double mutant subcomplex is 36% that of the wild-type subcomplex before and after cross-linking the introduced cysteines, whereas the ATPase activity of the γM 2 3 C/L 7 7 C double mutant increased from 50 to 85% that of wild-type after cross-linking the introduced cysteines. Only β-β cross-links formed when the α 3 (βE 3 9 5 C) 3 γM 2 3 C double mutant was inactivated with CuCl 2 . The overall results suggest that the attenuated ATPase of the mutant subcomplexes containing the γM 2 3 K, γL 7 7 K, and γM 2 3 2 K substitutions is caused by disruption of the cluster of hydrophobic amino acid side chains and that the midregion of the coiled-coil comprised of the amino- and carboxyl-terminal a helices of the y subunit does not undergo unwinding or major displacement from the side chain of γLeu 7 7 during ATP-driven rotation of the y subunit.