Subunit c is the H+-translocating component of the F1F0 ATP synthase complex. H+ transport is coupled to conformational changes that ultimately lead to ATP synthesis by the enzyme. The properties of the monomeric subunit in a single-phase solution of chloroform-methanol-water (4:4:1) have been shown to mimic those of the protein in the native complex. Triple resonance NMR experiments were used to determine the complete structure of monomeric subunit c in this solvent mixture. The structure of the protein was defined by >2000 interproton distances, 64 (3)JN alpha, and 43 hydrogen-bonding NMR-derived restraints. The root mean squared deviation for the backbone atoms of the two transmembrane helices was 0.63 A. The protein folds as a hairpin of two antiparallel helical segments, connected by a short structured loop. The conserved Arg41-Gln42-Pro43 form the top of this loop. The essential H+-transporting Asp61 residue is located at a slight break in the middle of the C-terminal helix, just prior to Pro64. The C-terminal helix changes direction by 30 +/- 5 degrees at the conserved Pro64. In its protonated form, the Asp61 lies in a cavity created by the absence of side chains at Gly23 and Gly27 in the N-terminal helix. The shape and charge distribution of the molecular surface of the monomeric protein suggest a packing arrangement for the oligomeric protein in the F0 complex, with the front face of one monomer packing favorably against the back face of a second monomer. The packing suggests that the proton (cation) binding site lies between packed pairs of adjacent subunit c.

The stoichiometry of c subunits in the H ؉ -transporting Fo rotary motor of ATP synthase is uncertain, the most recent suggestions varying from 10 to 14. The stoichiometry will determine the number of H ؉ transported per ATP synthesized and will directly relate to the P͞O ratio of oxidative phosphorylation. The experiments described here show that the number of c subunits in functional complexes of F oF1 ATP synthase from Escherichia coli can be manipulated, but that the preferred number is 10. Mixtures of genetically fused cysteine-substituted trimers (c 3) and tetramers (c 4) of subunit c were coexpressed and the c subunits crosslinked in the plasma membrane. Prominent products corresponding to oligomers of c 7 and c10 were observed in the membrane and purified F oF1 complex, indicating that the c10 oligomer formed naturally. Oligomers larger than c 10 were also observed in the membrane fraction of cells expressing c3 or c4 individually, or in cells coexpressing c 3 and c4 together, but these larger oligomers did not copurify with the functional F oF1 complex and were concluded to be aberrant products of assembly in the membrane. The ATP made during oxidative and photo phosphorylation is synthesized by closely related enzymes located in the inner membrane of mitochondria, the thylakoid membrane of chloroplasts, and the plasma membrane of eubacteria (1). Recent evidence supports a rotary mechanism for ATP synthesis in which proton-transport-coupled rotation of an oligomeric ring of c subunits in the membrane is coupled to rotary movement of subunit ␥ between alternating catalytic sites in the ␣ 3 ␤ 3 sector of the enzyme (refs. 2-8; Fig. 1). The proton-motive force is thought to drive rotation of the c-ring by a transport mechanism by using half channels that connect the aspartyl-61 protonbinding site of subunit c in the middle of the membrane to the aqueous compartments on either side of the membrane. After H ϩ binding from the entrance half channel at the a 1 b 2 stator, the c subunit carrying the proton would rotate nearly 360°before encountering the H ϩ exit channel on the opposite side of the membrane. In such a mechanism, the number of H ϩ transported per revolution of the c oligomer would equal the number of subunit c in the ring. The number of subunit c would also determine the H ϩ ͞ATP stoichiometry (i.e., H ϩ transported per ATP synthesized) and directly relate to the P͞O ratio for oxidative phosphorylation, a fundamental parameter of biology (9).The number of c subunits in F o is controversial. Direct measurements of subunit ratios in Escherichia coli F o F 1 , after growth of cells on radioactive amino acids, indicated a range of 10 Ϯ 1 c per 3 ␣␤ pairs in the purified F o F 1 complex or 12 c per 3 ␣␤ in the membrane fraction in which F o F 1 was overproduced (10). More recently, genetically fused subunit c were generated by insertion of a loop between the C-terminal residue of transmembrane helix-2 (TMH-2) of the first subunit and the N-terminal residue of TMH-1 of the next subunit (11). The geneti...

F(1)F(o)-ATP synthase is a ubiquitous membrane protein complex that efficiently converts a cell's transmembrane proton gradient into chemical energy stored as ATP. The protein is made of two molecular motors, F(o) and F(1), which are coupled by a central stalk. The membrane unit, F(o), converts the transmembrane electrochemical potential into mechanical rotation of a rotor in F(o) and the physically connected central stalk. Based on available data of individual components, we have built an all-atom model of F(o) and investigated through molecular dynamics simulations and mathematical modeling the mechanism of torque generation in F(o). The mechanism that emerged generates the torque at the interface of the a- and c-subunits of F(o) through side groups aSer-206, aArg-210, and aAsn-214 of the a-subunit and side groups cAsp-61 of the c-subunits. The mechanism couples protonation/deprotonation of two cAsp-61 side groups, juxtaposed to the a-subunit at any moment in time, to rotations of individual c-subunit helices as well as rotation of the entire c-subunit. The aArg-210 side group orients the cAsp-61 side groups and, thereby, establishes proton transfer via aSer-206 and aAsn-214 to proton half-channels, while preventing direct proton transfer between the half-channels. A mathematical model proves the feasibility of torque generation by the stated mechanism against loads typical during ATP synthesis; the essential model characteristics, e.g., helix and subunit rotation and associated friction constants, have been tested and furnished by steered molecular dynamics simulations.

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Subunit a is the least understood of the three subunits that compose the F 0 sector in the Escherichia coli F 0 F 1 ATP synthase. In this study, we have substituted Cys into predicted extramembranous loops of the protein and used chemical modification to obtain topographical information on the folding of subunit a. The extent of labeling of the substituted Cys residues by fluorescein-5-maleimide was determined. The localization of reactive Cys residues was inferred from differences in the extent of labeling in inside out and right side out membrane vesicles. The NH 2 -terminal segment of subunit a was localized to the outside (periplasmic) surface and the COOH terminus to the cytoplasmic surface by these procedures. Loop residues in two periplasmic extramembranous loops and in two cytoplasmic extramembranous loops were also localized. The localization of two cytoplasmic Cys residues was confirmed by using 4-acetamido-4-maleimidylstilbene-2,2-disulfonic acid to block fluorescein-5-maleimide labeling. From the localization of the Cys residues, a model for the topography is proposed that consists of five transmembrane segments with the NH 2 terminus periplasmic and the COOH terminus cytoplasmic. The positions of second site suppressors, including several isolated here to the nonfunctional E219C and H245C substitutions, provide support for the topographical model proposed.

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F 1 F 0 ATP synthases generate ATP by a rotary catalytic mechanism in which H + transport is coupled to rotation of an oligomeric ring of c subunits extending through the membrane. Protons bind to and then are released from the aspartyl-61 residue of subunit c at the center of the membrane. Subunit a of the F 0 sector is thought to provide proton access channels to and from aspartyl-61. Here, we summarize new information on the structural organization of Escherichia coli subunit a and the mapping of aqueous-accessible residues in the second, fourth and ¢fth transmembrane helices (TMHs). Aqueous-accessible regions of these helices extend to both the cytoplasmic and periplasmic surface. We propose that aTMH4 rotates to alternately expose the periplasmic or cytoplasmic half-channels to aspartyl-61 of subunit c during the proton transport cycle. The concerted rotation of interacting helices in subunit a and subunit c is proposed to be the mechanical force driving rotation of the c-rotor, using a mechanism akin to meshed gears.

F 1 F 0 ATP synthases catalyze the formation of ATP utilizing the energy of a transmembrane H ϩ electrochemical gradient, generated by electron transport complexes and other ion pumping systems. Closely related ATP synthases are found in the plasma membrane of eubacteria, the inner membrane of mitochondria, and the thylakoid membrane of chloroplasts. The enzyme is a multisubunit complex with distinct extramembranous and transmembrane domains, termed F 1 and F 0 , respectively. Ion movement through F 0 is coupled to ATP synthesis/ hydrolysis at sites in F 1 (1, 2). The simplest F 1 sectors, as found in Escherichia coli, consist of five subunits in an ␣ 3 ␤ 3 ␥␦⑀ stoichiometry. Homologous subunits are found in mitochondria and chloroplasts. A high resolution structure of a substantial part of bovine F 1 shows the three ␣-and three ␤-subunits to alternate around a central core through which subunit ␥ extends and protrudes (3). The structure fits well with the binding change mechanism proposed by Boyer and co-workers (4), where each of the three ␤-subunits alternates between the loose binding of ADP plus P i , tight ADP plus P i binding and ATP synthesis, and ATP release during catalytic turnover.