F 1 -ATPase, the C-terminal End of Subunit  Is Not Required for ATP Hydrolysis-driven Rotation* Received for publication, February 28, 2002, and in revised form, April 9, 2002 Published, JBC Papers in Press, April 18, 2002, DOI 10.1074/jbc.M201998200 Martin Mu ¨ ller, Oliver Pa ¨ nke, Wolfgang Junge, and Siegfried Engelbrecht‡ From the Universita ¨ t Osnabru ¨ ck, FB Biologie, Abt. Biophysik, Barbarastrasse 11, 49076 Osnabru ¨ ck, Germany ATP hydrolysis by the isolated F 1 -ATPase drives the rotation of the central shaft, subunit , which is located within a hexagon formed by subunits () 3 . The C-ter- minal end of  forms an -helix which properly fits into the “hydrophobic bearing” provided by loops of sub- units  and . 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 F 1 -ATPase). The ATP hydrolysis activity of a load- free ensemble of F 1 with 12 residues deleted decreased to 24% of the control. EF 1 with deletions of 15 or 18 resi- dues was inactive, probably because it failed to assem- ble. The average torque generated by a single molecule of EF 1 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 dur- ing 60  19% of the observation time). Activation energy analysis with the ensemble revealed a moderate de- crease from 54 kJ/mol for EF 1 (full-length ) to 34 kJ/mol for EF 1 (-12). These observations imply that the intact- ness of the C terminus of subunit  provides structural stability and/or routing during assembly of the enzyme, but that it is not required for the rotary action under load, proper. ATP is the universal free energy currency of prokaryotic and eukaryotic cells. It is synthesized in mitochondria, chloro- plasts, and the cytoplasm of prokaryotic cells by F 0 F 1 -ATP synthase (cf. Refs. 1– 6 for recent reviews). The enzyme works like a (reversible) rotary molecular machine with two motors/ generators mounted on a common shaft and hold together by an eccentric stator (7–11). In ATP synthesis mode the F 0 part translocates protons, thereby converting protonmotive force into the mechanical energy of rotary motion. Rotation is for- warded through the shaft into the F 1 part where it drives ATP synthesis. In ATP hydrolysis mode the rotation is reversed, and ions are pumped through F 0 in the opposite direction. The Escherichia coli enzyme (EF 1 ), 1 has the simplest subunit com- position. It consists of eight different subunits, five in the peripheral F 1 portion and three in the membrane-intrinsic F 0 , with stoichiometries of () 3  for F 1 and probably ab 2 c 10 for F 0 (12). In view of the rotary mechanism they also can be organized into “rotor” (c) and “stator” (ab). According to the crystal structure of bovine heart mitochondrial F 1 (13) the C-terminal region of subunit  properly fits into a supposed “hydrophobic bearing” formed by loops in the upper portion of the hexagon of subunits () 3 . Multiple sequence alignments showed that this region of  is more conserved than the remain- der (14, 15). One would expect therefore that truncations, point mutations, and covalent cross-links between the “bearing” and the rotor should inhibit the activity. But this expectation was not always met. 1) EF 1 with truncated  (lacking 10 C-terminal residues) was still active (15). 2) The ATPase activity of the homologous enzyme from chloroplasts (CF 1 ) tolerated trunca- tions of  up to 20 C-terminal deletions, 10 –16 residue trunca- tions even resulted in activation of the ATP hydrolysis activity (16). 3) Point mutations in the C-terminal region of E. coli  were tolerated in many cases, including some that changed polar residues into hydrophobic ones or even caused a charge reversal (15). 4) A number of second site mutations were iden- tified within the region of residues 269 –280 in E. coli , which restored energy coupling (17) in the significantly impaired mu- tants M23R or M23K. These constructs, however, were not able to build up protonmotive force to the extent of wild type enzyme despite comparable levels of ATPase activity (18) and despite generation of the same apparent torque (19). These restoring second site point mutations often resulted from the substitution of bulky residues with smaller ones, but in one case Ala was substituted with Val, thus increasing the occupied volume of the side chain significantly (17). Later, segments were identified in  by suppressor mutagenesis and second site mutagenesis, which are separated in the three-dimensional structure but still restored energy coupling if combined (20). 5) The effects of a deleterious frameshift in E. coli  could be mended by point mutations in subunit , at quite a distance from the frameshift region within  (Thr 277 –Val 286 (21)). 6) Most surprisingly, a covalent link between the penultimate C-terminal residue of EF 1 - and a nearby residue of  (A285C 7 P280C (22)) neither inhibited ATP hydrolysis nor the ro- tation of subunit  relative to () 3 and the torque generation under load. It would appear that the C-terminal part of  does play an important role in ATP synthase, but according to the foregoing not to the extent of certain residues being absolutely required. The situation is reminiscent of the “DELSEED” se- quence in subunit , which, despite conservation among many species, still tolerated not only one single point mutation (7) but even complete substitution of the acidic residues by alanines (23). The pronounced interplay of the rotor subunit  with its partners  and  is underlined by the fact that revertants map to distant regions not only located on the defective  itself, but also on . In the above cited work with truncated subunit  the activity of the enzyme constructs has been measured by ATP hydrolysis * This work was supported by grants from the Deutsch Forschungs- gemeinschaft (SFB 431/D1) (to W. J. and S. E.), by the Human Science Frontiers Project (to W. J.), and by the Fonds der chemischen Industrie (to W. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ To whom correspondence should be addressed. Fax: 49-541-969- 2870; E-mail: engel@uos.de. 1 The abbreviations used are: EF 1 , E. coli F 1 -ATPase; EF 1 (-x), EF 1 with subunit  lacking x C-terminal amino acid residues (x = 3, 6, 9, 12, 15, 18); CF 1 , chloroplast F 1 -ATPase; Ni-NTA, nickel-nitrilotriacetic acid; MOPS, 4-morpholinepropanesulfonic acid. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 26, Issue of June 28, pp. 23308 –23313, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. This paper is available on line at http://www.jbc.org 23308 by guest on February 5, 2016 http://www.jbc.org/ Downloaded from