F-ATPase: Forced Full Rotation of the Rotor Despite Covalent Cross-link with the Stator

Abstract

In ATP synthase (F O F 1 -ATPase) ion flow through the membrane-intrinsic portion, F O , drives the central "rotor", subunits c 10 ⑀␥, relative to the "stator" ab 2 ␦(␣␤) 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 ␥ we introduced disulfide bridges between ␥ (rotor) and ␣ or ␤ (stator). We engineered cysteine residues into positions located roughly at the "top," "center," and "bottom" parts of the coiled-coil portion of ␥ and suitable residues on ␣ or ␤. This part of ␥ is located at the center of the (␣␤) 3 domain with its Cterminal part at the top of F 1 and the bottom part close to the F O complex. Disulfide bridge formation under oxidizing conditions was quantitative as shown by SDSpolyacrylamide gel electrophoresis and immunoblotting. As expected both the ATPase activities and the yield of rotating subunits ␥ dropped to zero when the cross-link was formed at the center (␥L262C 7 ␣A334C) and bottom (␥Cys 87 7 ␤D380C) positions. But much to our surprise disulfide bridging impaired neither ATP hydrolysis activity nor the full rotation of ␥ and the enzyme-generated torque of oxidized F 1 , which had been engineered at the top position (␥A285C 7 ␣P280C). Apparently the high torque of this rotary engine uncoiled the ␣-helix and forced amino acids at the C-terminal portion of ␥ into full rotation around their dihedral (Ramachandran) angles.

Preliminary Estimation of Rotary Torque Produced by Proton-Motive Force in Fully Functional F0F1-ATPase

“…1) [16]. Studies on cross-linking between and ( P280C/ A285C) revealed that the torque generated by ATP hydrolysis can overcome the artificial clamping of C-terminal portion of probably by unwinding the -helix to form a swivel joint [16,17]. But until now it is not known whether covalent disulfide-bridging between and ( P280C/ A285C) inhibits the proton-pumping activity during oxidative phosphorylation.…”

“…1). It has been reported that disulfide bridges can easily form between and when it was treated with CuCl 2 in these two mutants F 1 [16]. In the preparation of inner membranes, no DTT or CuCl 2 was added, so in the following experiment, we treated the vesicles with 200 μM CuCl 2 or 50 mM DTT to ensure of the complete oxidation or reduction.…”

“…Cross-linked products were analyzed by SDS-PAGE (15%), followed by immunoblotting for identification with polyclonal antibody against [16].The introduced cysteines (red and green dots) in MM10 and PP2 are at the center and top positions, respectively. (This picture is from [16], with the permission of Dr. S. Engelbrecht).…”

“…E. Senior [18]. The E. coli strain pPP2/DK8 ( A334C/ L262C ) and plasmid pMM10( P280C/ A285C) were kindly presented by Prof. Siegfried Engelbrecht [16]. The plasmid pMM10 was transformed into E. coli strain DK8 [19].…”

“…It has been reported that covalent disulfide-bridging between subunits and at positions P280C and A285C did not inhibit the ATPase activity of F 1 and the rotation of subunit relative to 3 3 hexagon (Fig. 1) [16]. Studies on cross-linking between and ( P280C/ A285C) revealed that the torque generated by ATP hydrolysis can overcome the artificial clamping of C-terminal portion of probably by unwinding the -helix to form a swivel joint [16,17].…”