F-ATPase: Forced full rotation of the rotor despite covalent cross-link with the stator

Abstract

In ATP synthase (FoF1-ATPase) ion flow through the membrane-intrinsic portion, Fo, drives the central "rotor", subunits c10εγ, relative to the "stator" ab2δ(αβ)3. This converts ADP and Pi into ATP. Vice versa, ATP hydrolysis drives the rotation backwards. Covalent cross-links between rotor and stator subunits have been shown to inhibit these activities. Aiming at the rotary compliance of subunit γ we introduced disulfide bridges between γ (rotor) and α or β (stator). We engineered cysteine residues into positions located roughly at the "top," "center," and "bottom" parts of the coiled-coil portion of γ and suitable residues on α or β. This part of γ is located at the center of the (αβ)3 domain with its C-terminal part at the top of F1 and the bottom part close to the Fo complex. Disulfide bridge formation under oxidizing conditions was quantitative as shown by SDS-polyacrylamide gel electrophoresis and immunoblotting. As expected both the ATPase activities and the yield of rotating subunits γ dropped to zero when the cross-link was formed at the center (γL262C → αA334C) and bottom (γCys87 ↔ βD380C) positions. But much to our surprise disulfide bridging impaired neither ATP hydrolysis activity nor the full rotation of γ and the enzyme-generated torque of oxidized F1, which had been engineered at the top position (γA285C ↔ αP280C). Apparently the high torque of this rotary engine uncoiled the α-helix and forced amino acids at the C-terminal portion of γ into full rotation around their dihedral (Ramachandran) angles. This conclusion was supported by molecular dynamics simulations: If γCys285-Val286 are attached covalently to (αβ)3 and γAla1-Ser281 is forced to rotate, γGly282-Ala284 can serve as cardan shaft. © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.