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

In ATP synthase (F0F1-ATPase) ion flow through the membrane-intrinsic porti on, Fo, drives the central "rotor", subunits c(10)epsilon gamma, relative t o the "stator" ab(2)delta(alpha beta)(3). This converts ADP and P-i into AT P. Vice versa, ATP hydrolysis drives the rotation backwards. Covalent cross -links between rotor and stator subunits have been shown to inhibit these a ctivities. Aiming at the rotary compliance of subunit gamma we introduced d isulfide bridges between gamma (rotor) and a or beta (stator). We engineere d cysteine residues into positions located roughly at the "top," "center," and "bottom" parts of the coiled-coil portion of gamma and suitable residue s on alpha or beta. This part of gamma is located at the center of the (alp ha beta)(3) domain with its C-terminal part at the top of F-1 and the botto m part close to the F-0 complex. Disulfide bridge formation under oxidizing conditions was quantitative as shown by SDS-polyacrylamide gel electrophor esis and immunoblotting. As expected both the ATPase activities and the yie ld of rotating subunits gamma dropped to zero when the cross-link was forme d 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 torq ue 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 (Rama chandran) angles. This conclusion was supported by molecular dynamics simul ations: 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) ca n serve as cardan shaft.