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

Abstract

In ATP synthase (F0F1-ATPase) ion flow through the membrane-intrinsic portion, Fo, drives the central ``rotor'', subunits c(10)epsilon gamma, relative to the ``stator'' ab(2)delta(alpha beta)(3). This converts ADP and P-i 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 gamma we introduced disulfide bridges between gamma (rotor) and a or beta (stator). We engineered cysteine residues into positions located roughly at the ``top,'' ``center,'' and ``bottom'' parts of the coiled-coil portion of gamma and suitable residues on alpha or beta. This part of gamma is located at the center of the (alpha beta)(3) domain with its C-terminal part at the top of F-1 and the bottom part close to the F-0 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 gamma dropped to zero when the cross-link was formed at the center (gamma L262C <----> alpha A334C) and bottom (,gamma Cys(87) <----> beta D380C) positions. But much to our surprise disulfide bridging impaired neither ATP hydrolysis activity nor the full rotation of gamma and the enzyme-generated torque of oxidized F-1, which had been engineered at the top position (gamma A285C <----> alpha P280C). Apparently the high torque of this rotary engine uncoiled the a-helix and forced amino acids at the C-terminal portion of gamma into full rotation around their dihedral (Ramachandran) angles. This conclusion was supported by molecular dynamics simulations: If gamma Cys(285)-Val(286) are attached covalently to (alpha beta )3 and gamma Ala(1)-Ser(281) is forced to rotate, gamma GlY(282)-Ala(284) can serve as cardan shaft.