F1-ATPase, the C-terminal end of subunit γ is not required for ATP hydrolysis-driven rotation

Abstract

ATP hydrolysis by the isolated F1-ATPase drives the rotation of the central shaft, subunit γ, which is located within a hexagon formed by subunits (αβ)3. The C-terminal end of γ forms an α-helix which properly fits into the "hydrophobic bearing" provided by loops of subunits α and β. This "bearing" is expected to be essential for the rotary function. We checked the importance of this contact region by successive C-terminal deletions of 3, 6, 9, 12, 15, and 18 amino acid residues (Escherichia coli F1-ATPase). The ATP hydrolysis activity of a load-free ensemble of F1 with 12 residues deleted decreased to 24% of the control. EF1 with deletions of 15 or 18 residues was inactive, probably because it failed to assemble. The average torque generated by a single molecule of EF1 when loaded by a fluorescent actin filament was, however, unaffected by deletions of up to 12 residues, as was their rotational behavior (all samples rotated during 60 ± 19% of the observation time). Activation energy analysis with the ensemble revealed a moderate decrease from 54 kJ/mol for EF1 (full-length γ) to 34 kJ/mol for EF1(γ-12). These observations imply that the intactness of the C terminus of subunit γ provides structural stability and/or routing during assembly of the enzyme, but that it is not required for the rotary action under load, proper.