ATP Synthesis by Oxidative Phosphorylation

TOC

ATP Synthesis by Oxidative Phosphorylation

doi: 10.1128/ecosal.3.2.3

Module 3.2.3

STEVEN B. VIK

[SECTION EDITOR: VALLEY STEWART]

Posted October 19, 2007

Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275-0376

Mailing address: Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275-0376. Phone: (214) 768-4228. Fax: (214) 768-3955. E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it .

INTRODUCTION

Oxidative phosphorylation can occur in Escherichia coli under both aerobic and anaerobic conditions. The oxidation of certain substrates generates a proton motive force across the inner membrane. This energy can be used by an enzyme called the F₁F_o ATP synthase (EC 3.6.1.34) for the net synthesis of adenosine 5'-triphosphate (ATP). In the absence of a terminal electron acceptor, or in vitro, the enzyme readily functions in reverse, such that ATP hydrolysis can drive the translocation of protons from the cytoplasmic side of the membrane to the periplasmic side. Since the 1970s, a great deal of research has been directed toward the understanding of this enzyme, in part, because it is a prototype for the mitochondrial ATP synthase found in animals. Several other bacterial enzymes have also served as model systems, including the thermophilic Bacillus PS3 and the Na⁺-translocating enzymes from Propionigenium modestum and Ilyobacter tartaricus.

The ATP synthase is a most remarkable enzyme because it functions as a rotary motor. During ATP synthesis electrical energy is converted to chemical energy through mechanically driven conformational changes. The visualization of this rotation through elegant single-molecule studies was a transforming event for many researchers. It also signals that new standards are evolving for understanding the mechanisms of large, macromolecular complexes.

The ATP synthase from E. coli is formed from eight different polypeptide subunits, in a stoichiometry of α₃β₃γδεab₂c₁₀ (see Fig. 1). Traditionally, these subunits have been divided into two groups that reflect the ability to isolate two separate subcomplexes in the laboratory. F₁ comprises α, β, γ, δ, and ε, and it can be isolated as a water-soluble ATP hydrolase. There are six nucleotide binding sites, corresponding to the six α and β subunits. Three of these, found on the β subunits, are catalytic sites. A few residues from α also participate. The other three are termed noncatalytic sites, and are found on α subunits. They are not considered regulatory sites, but rather are probably molecular relics. The other sector, called F_o, comprises a, b, and c subunits. These are integral membrane proteins that form a proton pore through the membrane in the absence of F₁. (The terminology of F₁ and F_o comes from coupling Factor 1 and coupling Factor sensitive to oligomycin.)

Fig. 1Model of the ATP synthase from E. coli. The three α subunits are red, and the three β subunits are yellow. The γ subunit is dark blue, and its central shaft is shown by a blue dotted line. ε is dark green. The 10 c subunits are alternating light blue and green. The a subunit is brown, the dimeric b subunits are violet, and δ is orange. Details and references are in the text. (Reproduced from Biochim. Biophys. Acta [475] with permission from Elsevier, © 2006.)

In terms of rotary function the subunits are now classified as rotor or stator subunits. The rotor consists of γ, ε, and the oligomeric c subunits. The ring of c subunits resides in the membrane, and its cytoplasmic surface interacts with the globular domains of γ and ε. The γ subunit also contains two long helices that form a shaft inside the hexamer of alternating α and β subunit. The ε subunit contains two much shorter helices that appear to be highly mobile. The stator consists of α, β, δ, a, and b. The α/β hexamer is held to the membrane through δ, which is located at the membrane distal end, or top. The δ subunit connects through the elongated dimeric b subunits, that extend all the way down to the membrane. The b subunits interact with the a subunit in and near the membrane surface. In the membrane, the rotor subunit c interacts with the stator subunit a. The rotor subunits γ and ε both interact with the stator subunits α and β.

During ATP synthesis protons enter F_o from the periplasm. The initial pathway is probably through subunit a. Near the center of the membrane, protons bind to an unprotonated carboxyl group in a c subunit. This triggers one step of rotation of the ring of c subunits. Each proton travels nearly one complete revolution of the ring of c subunits until it returns to the a:c interface, where it escapes to the cytoplasm along the a:c interface. During rotation of the c subunits, the γ and ε subunits also rotate. Due to its inherent asymmetry, the shaft of the γ subunit interacts differently with each of the β subunits, and so it influences each catalytic site differently, at any one moment. Therefore, during one revolution, each catalytic site will experience a cycle of events that includes binding of substrates ADP and inorganic phosphate (P_i), synthesis of ATP, and release of products. This is referred to as the binding-change mechanism, and there are several variants of it in practice. The mechanical nature of the coupling of the proton movement to the chemical steps especially comes into play because the protons generate ten steps of rotation per cycle, whereas the chemical synthesis occurs in three steps per cycle. There are known to be two substeps in the chemical reaction, but since some organisms have up to 15 c subunits per 3 β subunits, a more general mechanism for coordinating these numerical differences likely exists. And so, coupling has been proposed to be elastic, and there are two places where it is thought this might occur. As the c subunits rotate, the shaft of γ would resist rotation, and the part of γ between the shaft and the c subunits might adopt partially twisted conformations that can store that energy, until the requisite number of steps occur and the shaft finally turns. Likewise, the extended b subunit might store energy in twisted conformations in its polypeptide chain between its connections to δ and to subunit a.

This represents a general view of the mechanism of ATP synthesis. During this review other topics, including operon structure, expression, assembly, and purification of the ATP synthase will be discussed. Also, evidence for mechanistic features of the enzyme will be examined, not only from the E. coli system, but also from other organisms when appropriate. Numerous reviews of various aspects of the ATP synthase have been published, including on the F₁-ATPase (20, 83, 251, 480), oxidative phosphorylation (191, 401), the F_o sector (89, 207), and on the ATP synthase (52, 71, 97, 105, 118, 129, 144, 323, 402, 506).

GENE ORDER, POLYPEPTIDE COMPOSITION, AND PROMOTERS

Operon Structure

The atp operon was initially called the unc operon (for uncoupled), because mutants displayed a phenotype in which electron transport reactions, e.g., oxidation of succinate, were not coupled to ATP synthesis. These mutations were mapped to an operon encoding the ATP synthase, atpIBEFHAGDC, which is located at 84.4 min on the E. coli chromosome map near the origin of replication (38). It has also been located by the E. coli genome sequencing project at the complement of nucleotides 3913576 to 3920463 (http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=49175990) (43). In addition to the eight structural genes, there is a ninth gene, atpI, the function of which remains unknown, and it is discussed in the next section. The gene order and corresponding polypeptides (180) are shown in Fig. 2. Many other bacteria contain a homolog to atpI, or contain other unknown genes associated with atp genes. The organization of atp genes in other bacteria can diverge greatly from that of E. coli, and several representative ones are shown in Table 1. The genes are not always found in a single operon, but when they are found together, they generally preserve the order seen in E. coli. Cyanobacteria such as Anabaena (85, 293) have two distinct b subunits, b' and b, and often have two operons, one coding for β and ε, and one for all the rest. Rhodospirillum rubrum has two operons (125, 126), split according to F₁ and F_o. In Desulfotalea psychrophilia the operon is split yet another way (367). In Anaplasma phagocytophilum the operon is split into four clusters of atp genes (208). In Hahella chejuensis there appear to be two complete operons of similar genes (224), but one operon lacks δ and starts with the genes for β and ε. In Streptococcus there is a single operon, but the order of genes for a and c are reversed relative to other operons (261).

Fig. 2Operon structure. The atp operon of E. coli has nine genes, which code for the indicated subunits. Genes atpB-C code for the eight proteins of the ATP synthase. The i protein is expressed poorly and has unknown function.

Table 1Gene organization in several representative species

atpI and the i Protein

Since the discovery of the atpI gene, it has been of great interest to learn if the i protein has a role related to the ATP synthase. Early studies showed that the i protein could be expressed from a plasmid in minicells, and that it fractionated with the membrane (65). Similar studies showed that it was expressed in wild-type cells, and could be detected by antibodies in membranes or in preparations of F_o or F₁F_o (394). No clear phenotype could be detected in a knockout strain of atpI (152). Overproduction of the i protein led to a determination of its N terminus, Ser-Val-Ser-Leu, and a hint that its overproduction was deleterious under growth with succinate as a carbon source (394, 395). Currently, the i protein is not considered to be a component of the ATP synthase in E. coli, and its role, if any, in the expression, assembly, or function of the ATP synthase remains unknown. A recent study (203) of a Bacillus alkaliphilic species pseudofirmus OF4 indicated that atpI, and a novel gene atpZ, are important for Mg²⁺ uptake, and expression of the atp structural genes.

Promoter for the atp Operon

The promoter for the atp operon was sequenced and analyzed by several different groups in the early 1980s (153, 239, 329, 365, 463). The transcription start site was determined to be 73 nucleotides upstream from the predicted start codon for atpI (365). Evidence for several other weaker promoters in this region has been presented (239, 329, 365), but these are unlikely to be of significance. Little evidence has been found for transcriptional regulation of the atp operon. Kasimoglu et al. (243) have examined the expression of lacZ fusions to single or low copy atpI genes under a variety of cell growth condtions. The expression did not vary by more than a factor of three. In general, there was higher expression when the carbon source was nonfermentable, such as glycerol or succinate, and its expression was lower when grown on a rich medium.

POSTTRANSCRIPTIONAL REGULATION OF EXPRESSION

Stability of the Transcript

The eight different subunits of the ATP synthase, which vary 10-fold in their stoichiometry in the assembled enzyme, are expressed from a single operon, and so it is an interesting question as to how this is accomplished. It seemed unlikely that all would be synthesized at a similar rate, and the excess subunits degraded. Early experiments (64) in which synthesis of all subunits from a plasmid, both in vitro, and in minicells, showed that the rates of synthesis of the subunits varied in a way that resembled the stoichiometry of the assembled enzyme. Studies in subsequent years, reviewed in references 294 and 295, demonstrated that several different mechanisms accounted for the differential synthesis of the subunits. One issue is the role of mRNA stability. The full-length transcript should be about 7 kb, but this has seldom been seen (227, 356, 388), unless the strain lacks a functional RNase E (356). The 5' end of the transcript is least stable, corresponding to atpI and atpB (296, 388). The half-life of atpI is several times shorter than the rest of the operon, and that of atpB is intermediate (268, 397). Further studies showed that at least five discrete endonucleolytic sites exist near the 3' end of atpB (355). Consistent with this finding, it was shown that the atpB transcript was unstable, even after the gene had been transferred to an internal site in the operon ( 397). RNase E-dependent cleavage was also found at the 3' end of the atp transcript, in which most of the atpC transcript is released (356). This was thought not to be important for differential rates of synthesis, because the transcript of the upstream genes is also destabilized. This might be a consequence of the removal of a transcription termination sequence that is found following the atpC gene (354, 356, 514).

Translational Initiation

A second mechanism involves ribosome binding and the rate of translational initiation. The most striking example of this regulation was the discovery that a region approximately 20 to 40 bp upstream of the ribosome binding site for atpE contributed to the high rate of synthesis of subunit c (297). This segment could also enhance translation of unrelated genes (299). Ribosome binding sites in the atp transcript were studied by two groups (269, 388), who found that little or no ribosomes bound to atpG, indicative of translational coupling to the upstream gene, atpA. Various evidence has been presented for translational coupling by several research groups. Coupling between atpEF, atpFH, atpHA, and atpAG was shown by McCarthy and colleagues (155, 198, 373), between atpFH and atpAG by Brusilow and colleagues (27, 359), and between atpDC by Dunn and Dallmann (86, 111). In general, translational coupling might arise from two different mechanisms, as has been discussed previously (86, 373). In the first case, the 30S ribosome might remain bound to the mRNA after the upstream polypeptide has been released, and reinitiate without dissociation. In the second case, translation through an upstream region might open up the ribosome binding site for the coupled gene. This mechanism would likely involve secondary structure in the mRNA. The first mechanism is more likely to be involved when the rate of translation is decreased, e.g., atpDC, while the second mechanism is more likely if the rate is increased, e.g., atpHA. One study (255) analyzed the atpEF region by mutagenizing several bases in regions of predicted secondary structure, and then measuring the effect on synthesis of subunit b. Some mutations caused a threefold increase in the rate of synthesis, supporting the view that secondary structure can impede translation. Furthermore, atpF is the only gene in the operon with a GUG initiation codon. When this was changed to AUG, the rate of translation increased about twofold (255). No evidence exists that biased codon usage affects the differential translation seen in the atp operon (298).

Proteolysis

In summary, a variety of mechanisms exist that allow rapid synthesis of subunits that are needed in high amounts (c, α, and β), or that allow lower rates of synthesis of subunits that are needed in lower amounts (a, δ, γ, and ε), or that follow the highly expressed atpE (b subunit). Although proteolysis does not seem to be important for maintaining the appropriate level of each subunit, there is one exception to this generalization. A member of the AAA+ protease family, FtsH, has been shown to selectively target several membrane proteins, including subunit a (13). This could provide a mechanism to limit overexpression of subunit a. Several early studies had shown various consequences of overproduction of subunit a (238, 462), including morphological changes (124, 464). In general, the overproduction of membrane proteins tends to be deleterious to a cell, and this has been examined in particular for ATP synthase proteins (311). Subunit a might be especially problematic when overproduced because of its role in proton translocation. In addition to the FtsH protease, the level of subunit a appears to be limited by an additional mechanism. An internal ribosome binding site has been reported to allow translation of a polypeptide that is not in-frame with subunit a (292). Mutagenesis of this region increased the rate of synthesis of subunit a. In a study from 2003, the instability of the atpB transcript was found responsible for the difficulty of overexpression this subunit, and was shown to be due to a central region, coding for amino acids 92 to 171 (31). These authors have proposed that degradation of the mRNA and proteolysis of excess protein are part of a scheme to minimize the level of subunit a.

ASSEMBLY OF THE ATP SYNTHASE

Assembly of F₁

The assembly pathway of the ATP synthase has long been of interest to researchers (63), but as is true for most multipolypeptide complexes, no detailed picture yet exists for its assembly in the cell. Even so, progress has been made in terms of the interactions of some of the subunits. Initial studies focused on whether F₁ and F_o could assemble independently of each other. It was shown, using a plasmid-based expression system (254), that a functional F₁ could assemble in the absence of genes for F_o. These results were consistent with earlier in vitro experiments (112) in which a functional F₁ was assembled from purified individual subunits. Subunits α, β, and γ were sufficient to form an enzyme capable of ATP hydrolysis, and δ and ε were additionally necessary to bind the enzyme to F_o to reconstitute functions associated with oxidative phosphorylation (415, 428).

Assembly of F_o

Analysis of the assembly of F_o in vivo yielded a variety of results, and so, were initially difficult to interpret. Early reports indicated that a functional F_o would not assemble in the cell unless F₁ subunits, in particular α or β, were present (79, 252). Later experiments showed that a functional F_o could assemble in the absence of α and β subunits (32, 132). One role for the F₁ subunits was proposed to be protection of F_o from proteolysis, in particular subunit b, which can be significant if the cells are harvested relatively late. A role for the δ subunit was investigated by the labs of Brusilow and Altendorf (28, 312, 313, 358). The coexpression of δ with a, b, and c increased the proton permeability of the membranes. In light of current structural information, not known at the time of those studies, it can now be seen how the presence of δ, and F₁, could stabilize the dimeric form of subunit b, and therefore increase assembly and functionality of F_o in vivo. Elegant studies (391, 393) performed with purified subunits a, b, and c showed that these three subunits alone are capable of forming a functional F_o. It is likely that results of studies of assembly in vivo are sensitive to the amounts and ratios of the various subunits, and so the outcomes will depend on the host strain and details of the expression systems for the various subunits. The assembly of F_o was addressed by Hermolin and Fillingame (200), who probed membranes from strains that lacked expression of each of the three subunits because of chromosomal nonsense mutations. They found that, in strains that did not express subunits b or c, the membranes also lacked subunit a, as determined by immunoblotting. These strains also lacked membrane-bound ATPase activity. However, in strains that did not express subunit a, the membranes did contain subunits b and c, and had substantial levels of membrane-bound ATPase activity, as if assembly had proceeded. Earlier work (459) had shown that from a strain with a mutation in atpF (bG9D), an intact F₁F_o complex could be immunoprecipitated that lacked only subunit a. In a different bacterial system (thermophilic Bacillus PS3) it was shown in 2004 (345) that an F₁F_o complex could be purified in the absence of subunit a, and that partial recovery of function was achieved by adding the purified subunit a to the preparation. Although it was suggested that subunit a might enter the complex last, it seems more likely that subunit a would associate immediately with dimeric b, and/or with the oligomeric ring of c, and then larger complexes might form. An inherent difficulty of studying assembly under conditions in which one or more genes have been deleted is that different pathways might be taken in the absence of one subunit. In that case, intermediates detected are not necessarily on the normal pathway.

Interactions between F₁ and F_o

Other insights into the assembly pathway were gained from studies of the purified subunits. Studies have shown that δ binds only to dimeric subunit b (110, 376, 418) and that δ binds only to oligomeric α (407). This would seem to be a mechanism that favors the interaction of fully assembled α₃β₃γε and ab₂c₁₀ units. These results also argue against an integrated assembly pathway of F₁ and F_o subunits, such as was first proposed in 1981 (79).

Assembly Factors for F_o Polypeptides

No folding or assembly factors are known to interact specifically with F₁ subunits of the E. coli enzyme, but that does not rule out their existence. Turning to F_o subunits, recent studies (450, 451, 505) have indicated that subunit c requires the yidC pathway for assembly into membranes. To distinguish between possible roles in membrane insertion or oligomerization, Kol et al. (259) analyzed two subunit c mutants that fail to form oligomers. Both were found to be inserted into the membrane in a YidC-dependent way, but both failed to oligomerize in the presence of YidC. Therefore, YidC is not strictly involved in the oligomerizaion of membrane proteins, but is more likely a membrane insertase. Subunit a was also shown to require YidC for membrane insertion (505), but that could be, in part, a consequence of its need for subunit c to become stabilized in the membrane (200). Furthermore, it was found that the N-terminal region of subunit a requires proton motive force to be transported to the periplasm, while the first periplasmic loop (residues approximately 125 to 140) does not. Note, however, that these experiments were carried out with a genetically fused polypeptide at the C terminus of subunit a, and this might have affected its interactions with other F_o proteins or otherwise disrupted its normal folding.

PURIFICATION AND EXPRESSION SYSTEMS

Purifications of F₁

Purification of the ATP synthase from E. coli has been actively pursued since about 1970. Early work on prokaryotic ATPases was reviewed in 1979 (105). A variety of purification schemes for the F₁ sector were described by Evans (121, 122), Kobayashi and Anraku (257), Bragg and Hou (57), Hanson and Kennedy (181), Futai et al. (145), and Nelson et al. (327). These schemes typically involved washing E. coli membranes with EDTA in low-ionic-strength buffers, which selectively extracts the F₁ complex. Most procedures of that time lacked the δ subunit, which prevents functional reconstitution with stripped membranes. Notable exceptions were the procedures of Bragg and Hou (57) and Futai et al. (145) that did contain the δ subunit. Vogel and Steinhart (461) drew upon the earlier procedures and introduced polyethylene glycol precipitation and gel filtration chromatography, which also generated a five-subunit F₁ preparation. Senior et al. (404, 405) incorporated features of the procedures from these latter preparations. Further modifications have been made of that procedure (18, 493).

Purifications of F₁F_o

Purification of F₁F_o complexes requires the use of detergents to extract the enzyme from the membrane. Two of the earliest successful procedures used Triton X-100 (60) or deoxycholate (183) for extraction, and both yielded only partially pure preparations. (See also reference 330.) The activities of both preparations were stimulated by addition of phospholipids, and both were sensitive to inhibition of ATP hydrolysis by dicyclohexylcardodiimide, a signature of normal interactions between F₁ and F_o (36, 37). Key findings from the Gibson lab were that p-aminobenzamidine inhibited the loss of F₁ from membranes, and that ε-aminocaproic acid, a protease inhibitor, also improved preparations (78, 106). Friedl et al. (142) incorporated these ingredients into a preparation that used a new detergent, acyl (C11–C17) amidopropyldimethylaminoxide (Aminoxid WS 35), and DEAE ion-exchange chromatography. Foster and Fillingame (136) modified an earlier procedure (183) by increasing the concentration of deoxycholate, adding an ammonium sulfate fractionation step, and adding phospholipids to the sucrose gradient. With these modifications, the two procedures yielded substantially pure 8-subunit preparations. The use of a strain deficient in cytochrome bo was found to improve the purity of both F₁ and F₁F_o preparations (176). The detergent n-octyl-β-d-glucopyranoside was introduced for purification of F_o (393) and has been incorporated into procedures for several F₁F_o preparations (135, 215, 265).

Expression Systems

For achieving high levels of expression, an inducible lambda system was used by several research groups (242, 316, 393). Specialized expression vectors and genetically fused tags have been used to facilitate purification and mutagenesis of the ATP synthase. Moriyama et al. (315) introduced the vector pBWU13, which encoded the entire atp operon, lacked the rop gene, and led to high expression of the ATP synthase. By using it in the background of an atp operon deletion strain, DK8 (253), a high-purity preparation could be achieved with a simple glycerol gradient procedure. Kuo et al. (265) introduced the cysteine-less ATP synthase, encoded on a plasmid to facilitate studies that required unique cysteine residues. They also introduced a FLAG epitope tag at the N terminus of the β subunit, for purification and immunodetection, similar to the construct of Zhou et al. (512). A hexahistidine tag introduced at the N terminus of β (352) has also been used for purification of the ATP synthase by nickel-nitrilotriacetic acid (Ni-NTA) chromatography (215). Similarly, a histidine tag at the N terminus of α is compatible with a functional enzyme (382). Histidine and other types of tags have been placed at the termini of many subunits. A hexahistidine tag at the N terminus of subunits a or b has little or no effect on their expression and can be used to purify ab₂ complexes (423). However, a histidine tag at the C terminus of subunit a was found to be detrimental to expression of subunit a (423), but not if an hemagglutinin (HA) epitope tag is inserted between the terminus and the histidine tag (465). With the c subunit, it was found that a hexahistidine tag at the N terminus resulted in an inactive enzyme, while at the C terminus a functional F₁F_o complex could be isolated after extraction with CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate} detergent (437). Others (436) were able to incorporate a histidine tag near the N terminus of subunit c without effect on function. Clearly, the details of the construct are often important. Tags have also been attached to both the C terminus and the N terminus of γ without impairing activity (116). Glutathione S-transferase has been fused to the N termini of δ and ε, and it did not interfere with the assembly of a functional F₁ complex (411).

Subunit Composition and Stoichiometry

The subunit composition and stoichiometry of the E. coli ATP synthase were largely resolved by the early 1980s. DNA sequence determination of the atp operon was carried out by three research groups, specifically, Walker and colleagues (153, 154, 384), Kanazawa, Futai, and colleagues (236, 237, 239, 240, 241, 289), and Friedl, von Meyenburg, and colleagues (328); their results were published in 1981 to 1982. The polypeptide chain of subunit c had been sequenced earlier (206, 399). Walker et al. (467) published an annotated version of the DNA sequence of the atp operon in 1984, and no changes in this sequence were necessary after completion of the E. coli genome-sequencing project (43). Walker et al. (466), also published an examination of the DNA sequence that neighbors the atp operon, encompassing about 17 kilobases. The sizes of the subunits are shown in Table 2. The stoichiometry of the subunits, α₃β₃γδεab₂c₁₀, was correctly determined by two groups using isotopic labeling of E. coli cultures: F₁ by Bragg and Hou (61), and F₁F_o by Foster and Fillingame (137). The stoichiometry of the c subunit was given as 10 ± 1, and this value was not widely accepted as reasonable until 1999, when the crystal structure of the yeast enzyme showed 10 c subunits (430). Until that time many had expected the number of c subunits to be a multiple of three. Studies of gene fusions of 2 and 3 c subunits had shown that both constructs supported an active enzyme, and disulfide cross-linking generated c₁₂ oligomers (228). Later, a reexamination of this question (226) used fusions of 3 and 4 c subunits. After disulfide cross-linking, oligomers of 10 and larger were seen, but only those of 10 copurified with a functional F₁F_o complex. It was concluded that 10 is the preferred stoichiometry.

Table 2Features of the subunits of the ATP synthase from *E. coli*

Variation in the Number of c Subunits

In other species, the number of c subunits in the oligomeric ring has been found to be 11 (422), 14 (400), and 15 (364). If 10 c subunits are favored in a ring, it is possible that other numbers of protomers, such as 9, 11, or 12, might form a very similar structure, because the packing interactions between the subunits would be nearly identical. Interactions with other subunits might be altered to a greater extent, such that assembly would not occur. The stoichiometry of c subunits is an important issue since it determines the number of protons transported per ATP. It has been argued that if two different stoichiometries coexisted in the cell, it might allow for a situation in which one type of enzyme would synthesize ATP while the other type would hydrolyze it (226). Evidence has been presented that the stoichiometry of c subunits in an F₁F_o complex might vary according to the rate of translation (389), or be controlled by the growth medium (390). Others (459) have also reported higher-than-normal stoichiometric ratios of c:b following anti-F₁ immunoprecipitations of F₁F_o complexes. There are two possible explanations. (i) Self-assembled rings of c subunits, which are known to occur (30), might associate with normal F₁F_o complexes. (ii) Some complexes that were immunoprecipitated might lack the peripheral stalk, as was seen in the yeast F₁F_o crystal structure (430), thereby elevating the c:b ratio. The ability to resolve complexes of F₁F_o with different subunit compositions and to determine those stoichiometries accurately will be necessary to resolve this question.

HIGH-RESOLUTION STRUCTURES

Structures of α₃β₃γ

No high-resolution structural model of the intact ATP synthase is available from E. coli or from any other source. However, a rather detailed model can be pieced together from structural data from several different organisms, as was first done in 1997 (118). This includes both low- and high-resolution structures of subcomplexes of F₁F_o from various sources, electron cryomicroscopy of F₁ and F₁F_o from E. coli, high-resolution structures of individual subunits from the E. coli enzyme, and numerous other kinds of structural studies.

The structure of F₁ from bovine mitochondria has been solved in the presence of numerous different inhibitors and nucleotides at resolutions down to 1.9 Å (1, 2, 49, 50, 62, 156, 308, 348, 452). F₁ has also been crystallized from other organisms, including E. coli (194, 195), Bacillus PS3 (412), rat liver mitochondria (39, 74), spinach chloroplast (171, 172), and yeast mitochondria (234), although the E. coli structures were solved only to 4.4-Å resolution. See Fig. 3A and B for a view of the α, β, and γ subunits, which appear to be structurally very similar when comparing the E. coli and bovine forms of F₁. The three α and β subunits alternate in a sixfold pseudosymmetrical hexamer. The α and β subunits are homologous, and about 20% identical in sequence. Each is composed of three domains. The N-terminal domains consist entirely of beta-strands, including a six-stranded beta-barrel. The central domains contain a Rossmann fold, a nucleotide binding domain that consists of alternating alpha-helices and beta-strands. The C-terminal domain is formed by a bundle of alpha-helices, 7 in the α subunits and 6 in the β, accounting for most of the size difference between these subunits. The subunits are arranged with the C-terminal domains proximal to the membrane surface, and the N-terminal domains distal. The strongest interactions of the α and β subunits appear to occur at the "crown" through the N-terminal domains, which form a continuous 24-stranded circular beta-sheet. These interactions appear to remain intact, while other parts of the protein undergo major conformational changes. The conformations of the β subunits have been termed β_E, (empty of nucleotides), β_DP (with ADP bound), and β_TP (with adenylyl imidodiphosphate, AMP-PNP, bound), as found in the first crystal structure (2). It was recently determined (49) that by omitting azide in the crystallization medium, AMP-PNP also binds to the β_DP site, and the conformation of the subunit is slightly different.

Fig. 3AStructure of α₃β₃γ from the bovine enzyme. The three α subunits are red, the three β subunits are yellow, and γ is blue. The two long helices of γ can be seen to fill the central cavity formed by the α-β hexamer. The β subunit in the front has a bound AMP-PNP, shown in magenta. The Protein Data Bank ID code is 2jdi (49).

Fig. 3BStructure of α₃β₃γ from the bovine enzyme. A top view of the same structure in Fig. 3A, now shown in space-filling. The Protein Data Bank ID code is 2jdi (49).

The γ subunit is composed of two domains. The N and C termini are both alpha-helical, extending from residues 1 to 66 and 211 to 286 in the E. coli enzyme (194). These helices form an antiparallel, left-handed coiled coil that occupies the central channel of the α-β hexamer. This coiled coil forms an aymmetric shaft that interacts differently with each of the α and β subunits, at any moment. Two prominent regions of interaction have been termed "catches" (2) and are illustrated in Fig. 4A and B. The so-called DELSEED sequence (residues 380 to 386 in E. coli numbering) of the C-terminal domain of β in the β_TP conformation is in contact with residues of γ. A second catch occurs between the nucleotide binding domain of β in the β_E conformation and the C terminus of γ. These are key elements of the mechanics of coupling. The central region of γ is composed of a five-stranded beta-sheet, flanked by six alpha-helices. This globular domain of γ is exposed at the end of F₁ that is proximal to the membrane and is where the ε subunit binds.

Fig. 4AInteractions of γ and β in the central cavity. The lower "catch" is indicated with a black asterisk. The β subunit has a bound AMP-PNP. It is in the β_TP conformation. The Protein Data Bank ID code is 2jdi (49).

Fig. 4BInteractions of γ and β in the central cavity. The upper "catch" is shown by a black asterisk. The β subunit has no nucleotides bound. It is in the β_E conformation. The Protein Data Bank ID code is 2jdi (49).

Structures of δ and ε

The structure of the isolated ε subunit of E. coli was solved by both X-ray crystallography (446) and nuclear magnetic resonance spectroscopy (489, 490). Both methods revealed a two-domain protein, with an N-terminal 10-stranded beta-barrel and a C-terminal alpha-helical coiled coil. Interestingly, when ε was cocrystallized with the globular fragment of γ (375), the two alpha-helices moved away from the beta-barrel, and became more extended. See Fig. 5A and B for views of these two conformations of the ε subunit. Evidence for such a rearrangement was also seen in the low-resolution structure of the E. coli F₁ (194). This behavior of the ε subunit does not appear to occur in the mitochondrial enzyme.

Fig. 5ATwo conformations of the ε subunit. The N-terminal domain of ε is yellow, and the C-terminal domain is cyan. The tenth beta-strand is red. The Protein Data Bank ID code is 1aqt (446).

Fig. 5BTwo conformations of the ε subunit. When bound to the γ subunit (dark blue), the C-terminal helices of ε can extend. The hinge for this motion is in the tenth beta-strand. The helices extend toward the catalytic sites. The Protein Data Bank ID code is 1fs0 (375).

The structure of an N-terminal fragment of the δ subunit, residues 1 to 134, was solved by NMR, revealing a six-helix bundle, along with a less stable seventh helix. The location of the δ subunit has not been seen in any crystal structure of F₁, but it has been visualized by microscopy. For example, both δ in E. coli (492), and its homolog in yeast (377) were shown to reside at the membrane distal part of F ₁. The δ subunit forms part of the slender, peripheral stalk that is composed primarily of the two b subunits in E. coli (48, 488). This structure had been overlooked in earlier studies by electron cryomicroscopy (162, 285).

Structures of F_o Components

The wider, central stalk is composed of ε and the globular domain of γ. These two subunits interact with the ring of c subunits, and together constitute the rotor. The relative placements of the γ-ε heterodimer and the decameric ring of c subunits was visualized in a low-resolution structure of the yeast F₁F_o (430). The structures of monomeric c subunits from E. coli, both wild type and mutants, had been solved by NMR in aqueous organic solvent, and revealed a helical hairpin, with two long alpha-helices of about 35 residues, each separated by a short turn (100, 102, 161, 370). Two structures are shown in Fig. 6A and B. The crystal structure of an undecameric ring of c subunits from a sodium ion translocating ATP synthase was solved at 2.4 Å resolution (305), shown in Fig. 7A, B, and C. This structure was similar to models that had been built (103, 370) based on monomeric structures, in that the N-terminal alpha-helices were packed in an inner ring, and the C-terminal helices were more widely spaced at the periphery. Electron spectroscopic imaging and immunoelectron microscopy have been used to detect subunit organization in F_o from E. coli (41).

Fig. 6ATwo conformations of the c subunit. At pH 5.0, the two helices are nearly straight. The N-terminal helix is magenta, and the C-terminal helix is cyan. Key residue Asp 61 is shown in ball-and-stick. The Protein Data Bank ID code is 1a91 (161).

Fig. 6BTwo conformations of the c subunit. At pH 8.0, the C-terminal helix adopts a different conformation. Asp 61 moves from one side to the other, as the helix has twisted. The Protein Data Bank ID code is 1c99 (370).

Fig. 7ACrystal structure of the sodium-translocating undecameric c subunits. A side view in space-filling, with each subunit colored differently, shows that the sodium ions, colored magenta, can be seen near the indentation. The Protein Data Bank ID code is 1yce (305).

Fig. 7BCrystal structure of the sodium-translocating undecameric c subunits. Two subunits are shown in ribbons, so that each sodium ion can be seen to be coordinated by two adjacent subunits. The Protein Data Bank ID code is 1yce (305).

Fig. 7CCrystal structure of the sodium-translocating undecameric c subunits. A view from the top shows that there is a central cavity known to be filled by phospholipids. The Protein Data Bank ID code is 1yce (305).

EVIDENCE FOR ROTATION OF SUBUNITS IN THE ATP SYNTHASE

Early Ideas and Evidence

Ideas about rotational catalysis in F₁ and F_o were seen in print in the 1980s (54, 80, 82, 169, 346, 347, 392). Experiments from the lab of Boyer, reviewed in reference 53, produced the binding-change mechanism, and motivated thinking about a rotational mechanism, in which the three catalytic sites were equivalent, but, at any particular instant, they were all distinct. Rotation nicely accommodates the interactions of a single, asymmetric subunit, with three equivalent catalytic subunits. A variety of other experiments indicated that the three β subunits are in different conformations, or that they interact differently with the minor subunits. Cryoelectron microscopy showed nucleotide-dependent movement of ε subunit relative to α and β (487). The nonequivalence of β subunits with respect to inhibitors or other subunits was demonstrated in the labs of Capaldi (174, 175, 192, 193, 281) and Bragg (58, 59, 424, 425). Asymmetry in the catalytic sites was shown by spin-labeled probes (248, 280). Cross and coworkers (66, 67, 107, 513) tested the movement of β subunits relative to γ or ε, using a scheme involving disassembly of the F₁ subunits after disulfide formation. They found that γ or ε subunits formed disulfide cross-links to different β subunits at βD380C after ATP hydrolysis or synthesis. Sabbert et al. (379) labeled γ with an eosin probe and detected rotation of γ of at least 280 degrees relative to a fixed α-β by measuring polarized absorption relaxation after photobleaching.

Video Rotation Assay

The most compelling evidence for rotation in the ATP synthase came from direct visualization studies of the Bacillus PS3 enzyme introduced by Noji et al. (335) in which α-β was immobilized and γ was cross-linked to a fluorescently labeled actin filament whose ATP-dependent rotation could be monitored in real time by a light microscope. Movies from the Yoshida lab are available at http://www.res.titech.ac.jp/~seibutu/projects/f1_e.html. The rotation of ε was similarly demonstrated (246). Subsequent improvements to the technique included replacing the actin filament with a single fluorophore (3), a bead (445), a gold particle (325), or a gold nanorod (420), to reduce viscous drag or improve time resolution. These improvements allowed detection of steps of 120 degrees of rotation (3, 503), and of substeps (504), thought to correspond to an ATP binding dwell and a product release dwell. Fluorescence resonance energy transfer (FRET) techniques have also been applied to the observation of rotation (47, 96, 502, 515).

Using the E. coli enzyme, others demonstrated rotation of γ in F₁ (334) and rotation involving subunit c in the F_o sector (331, 352, 382, 436). See Fig. 8 for a schematic view of this experiment. These results confirmed γ-ε-c oligomer as the rotor, but others (439) raised doubts that the a:c interface was intact during the assay. Subsequently, it was demonstrated the both a and β subunits rotated relative to an immobilized, and membrane-embedded c subunit, with full dicyclohexylcarbodiimide (DCCD) sensitivity, indicating an intact a:c interface (331).

Fig. 8Schematic of a video rotation assay. The α subunits are immobilized, and the fluorescently labeled actin filament is attached to a c subunit through streptavidin. Addition of ATP initiates the rotation. (Reproduced from Science [382] with permission from AAAS.)

Cross-Linking of Rotor and Stator Subunits

Further testing of the rotational model involved the construction of site-specific cross-links between subunits. In general, cross-links within the stator or within the rotor should not impair function, while those between the rotor and stator should block rotation. This concept was valid, but with a few exceptions. Conventional bifunctional cross-linkers are, in general, not specific enough for this purpose, since numerous different subunits might be cross-linked simultaneously. Nevertheless, native α-δ disulfide cross-links had been seen, which did not affect ATP hydrolysis (55, 438). Aggeler et al. (6) introduced a series of photoactivatable hetero-, bifunctional reagents, tetrafluorophenylazide maleimides (TFPAMs), that proved successful in linking two subunits with high specificity. One end of the reagent is targeted to a cysteine side chain, typically a mutation in a cysteine-free background. In this study they analyzed cross-linking from two cysteine mutations, S10C and S108C, introduced into ε, and from the naturally occurring cysteine residues in δ. The εS10C cross-linked to γ, and the εS108C cross-linked to α, both in a nucleotide-dependent fashion. Furthermore, the ε-γ cross-linking did not greatly affect the rate of ATP hydrolysis, while the ε-α cross-linking inhibited the rate in proportion to the yield of the cross-link. In contrast, the δ subunit could be cross-linked to both α and β subunits, but the yield did not depend on nucleotides, and the cross-linking did not inhibit the enzyme activity. Similar experiments (4) found that γ could be cross-linked to α, via γV286C, or to β, via γS8C, and both inhibited ATP hydrolysis, and that efficiency of the latter cross-link, but not the former, was nucleotide dependent. Following the publication of the crystal structure of the bovine F₁ (2), it became possible to design double-cysteine mutations that were capable of forming disulfide bonds, and this led to a multitude of new experiments. Other experiments using disulfide bond formation demonstrated that γ and ε were part of the rotor (7). An interesting exception to these experimental results was presented by Gumbioski et al. (179), who found that the disulfide between γA285C and αP280C did not impair ATP hydrolysis. They proposed that the high torque unfolded the alpha helix at the C terminus of γ and allowed rotation about a single bond in the polypeptide. This proposal was supported by molecular dynamics simulations.

From the crystal structure (2), it was seen that two β subunits were in close contact at residue 390 in the C-terminal, alpha-helical domain. When replaced by cysteine, using the Bacillus PS3 F₁, a disulfide formed, and it inhibited ATP hydrolysis (441). In this case, the cross-link was within the stator, but it was inhibitory since it restricted necessary conformational changes.

Further evidence that the δ subunit is part of the stator was provided by constructing the αQ2C mutation. Under oxidizing conditions, it formed disulfides with a naturally occurring cysteine of δ, primarily at residue 140. The cross-linking had limited effect on ATP hydrolysis or ATP-driven proton translocation. Similarly, cross-links between δM158C and bE155C or b158C (an extension of the normal b), or between bL156C and an intrinsic cysteine of δ, did not impair ATP hydrolysis. The latter cross-link did affect ATP-dependent proton translocation, indicating possible restricted motion of the cross-linked subunits.

Cross-links were also constructed with F_o subunits to distinguish between rotor and stator, but not all were guided by high-resolution structural models. For example, Zhang and Fillingame (510) showed that εE31C could form disulfides with cP43C, cQ42C and cA40C, but not cA39C. These mutants were constructed based on the identification of εE31G,V,K as second-site suppressors of cQ42C (511). These cross-links appeared to impair ATP-driven proton translocation. To circumvent the problem of disulfide formation between c subunit neighbors that reduced activity and lowered ε-c cross-linking, a genetically fused c-c dimer was developed. In this way only one Q42C need be introduced for every two c subunits. Using such a construct Tsunoda et al. (440), were able to form disulfides between γT217C and ε168C, and between εE31C and cQ42C. They showed that these disulfides had little or no effect on ATP hydrolysis, ATP-driven proton translocation, or ATP synthesis. These results confirmed that the rotor contained γ-ε and the c oligomer.

A cross-link was formed between aK74C and a b subunit by using a photoactivatable cross-linker, TFPAM (278). This linkage had little or no effect on ATP-driven proton translocation, confirming that both a and b are in the stator. Disulfides were formed between bN2C and cV78C (229), which confirmed their proximity. ATP-driven proton translocation was inhibited by this disulfide formation, as would be expected if b is part of the stator and c is part of the rotor. In contrast, ATP hydrolysis was not similarly inhibited, indicating that a decoupling between F₁ and F_o had occurred, probably at the γ-ε-c interface. Similar experiments were performed with the Bacillus PS3 enzyme (434), in which bL2C and cS2C were constructed. In this case the rate of ATP hydrolysis was significantly inhibited when the disulfide bond was formed. ATP-driven proton translocation was nearly normal when the sulfhydryl groups were reduced, and was substantially inhibited when the disulfide bond was formed. ATP-dependent motion between a and c subunits was shown by Hutcheon et al. (210), using the disulfide bond formed between aN214C and cM65C, previously identified by Jiang and Fillingame (225). They found that if DCCD was added after disulfide bond formation, the cross-linked c subunit had much lower levels of ¹⁴C-labeled DCCD than if the disulfide bond was reduced after DCCD addition, ATP hydrolysis was allowed to proceed, and then the disulfide bond was re-formed. These results confirmed that ATP hydrolysis moves c subunits away from the a-c interface.

FUNCTIONAL ASPECTS OF THE ATP SYNTHASE

New Approaches Support the Binding-Change Mechanism

ATP synthesis and hydrolysis occur in F₁, specifically requiring subunits α, β and γ. The three catalytic sites are formed primarily by β subunits and the three noncatalytic sites are formed primarily by α subunits. Boyer pointed out in 1993 that trimeric enzymes are uncommon (53), but in the intervening years there has been increase in the number of hexameric, or pseudohexameric enzymes that are known to utilize ATP hydrolysis for bacterial secretion systems (214), conjugation (163), transcription termination (374), nucleic acid helicases (357), AAA+ proteases (119, 517), or DNA clamp loaders (51). Common features seem to be cooperativity, movement of proteins or nucleic acids, and use of the central cavity.

Understanding of the mechanism of ATP hydrolysis by F₁ has been advanced in recent years by the results of three experimental approaches. First, starting in 1994, high-resolution crystal structures, primarily solved by the research group of Walker, have advanced our understanding of the nature of the catalytic sites, and how nucleotides, transition-state analogs, and other inhibitors interact with the enzyme. Second, the engineering of tryptophan fluorescence probes at the nucleotide binding sites, in the lab of Senior, has allowed true binding constants of nucleotides to be measured, instead of estimations of binding from K_m determinations. And third, the video rotation assay, first developed in the labs of Yoshida and Kinosita, allowed visualization of, and ultimately great insights into rotation.

Most studies of the ATP synthase have focused on its reaction in the direction of ATP hydrolysis. In E. coli, this enzyme can function in either direction, depending on the conditions. Evidence has indicated that both directions of the reaction proceed through the same transition state (16). The key difference between the two reactions is that ATP synthesis only occurs when a sufficient proton motive force is applied. It is unknown what effect this has on the structure of the enzyme, but it might account for some differences seen in the two reactions (432, 460). Possibilities include the creation of a phosphate binding site in the catalytic site, the opening of a docking site for the C-terminal helices of ε, and the control of access of protons to the c subunits.

The general concept under which the ATP synthase functions can be called the binding-change mechanism proposed by Boyer (for a comprehensive review, see reference 53). An example scheme is shown in Fig. 9. The key feature is that each of the three catalytic sites goes through a series of conformational changes as ATP is bound and hydrolyzed and products are released. At any moment each of the sites is at a different stage of the cycle. The enzyme is highly cooperative. With respect to binding, the cooperativity is negative. These issues were first examined in the mitochondrial enzyme for ATP hydrolysis (84, 177, 178) and for ATP synthesis (291). In the hydrolysis direction the first ATP binds with extremely high affinity with a K_d of <1 nM. The second and third ATPs bind with K_ds of ~1 μM and ~30 μM (481, 483). The cooperativity is positive with respect to enzyme activity. The rate of hydrolysis when only one ATP is bound, so-called uni-site catalysis, is <10⁻³ s⁻¹. This is accelerated by 5 orders of magnitude when higher concentrations of ATP allow the second and third sites to be filled (17, 19). Several lines of evidence support a model in which all three sites must be occupied with ATP before maximal rates are achieved, so-called tri-site catalysis, which is distinguished from bi-site catalysis in which only two sites must function for maximal rates. First, crystal structures of the bovine F₁ have shown that three sites can be occupied with nucleotides at once, and that two sites can have transition-state analogs (308) or AMP-PNP (49) bound. Second, simultaneous measurements of bound nucleotide using the fluorescence probes near the active sites have found that maximal rates are achieved only when all three sites are occupied (479). And third, by using a fluorescently tagged ATP, it was shown that ATP remains bound for a rotation of 240 degrees (332), consistent with tri-site catalysis, although a more recent report by the same group complicates this picture (381).

Another question is which steps in the catalytic mechanism, ATP binding, ATP hydrolysis, or product release, are coupled to torque generation in the γ subunit. In time-resolved video rotation experiments with the Bacillus PS3 enzyme (504), Yoshida and colleagues identified four phases of the ATP hydrolysis pathway. First, a pause in which the duration depended on ATP concentration was termed the ATP binding dwell. This was followed by a rapid 80-degree rotation. Then, another pause occurred during which ATP hydrolysis occurred (409) and eventually ADP was released. This was followed by a rapid rotation of 40 degrees. So, each 120-degree step of rotation is composed of two substeps. One is associated with the binding of ATP and the other with the release of ADP. This technique cannot determine whether the binding and release occur from the same sites or from different ones. These results are consistent with a cooperative mechanism in which binding of ATP leads to hydrolysis and the subsequent release of ADP at another site, accompanied by a rotation of γ by 120 degrees.

Description of the Catalytic Sites

Nucleotides are bound at the Rossmann fold domain of the α and β subunits. Mutagenesis of residues at the nucleotide binding sites showed that the β subunits contained the catalytic sites, while the α subunits contained noncatalytic sites (369, 403, 476). The adenine ring of the nucleotide packs along an alpha-helix, and the loop from the preceding beta-strand wraps around the terminal phosphates of the nucleotide. This so-called P-loop corresponds to the Walker sequence motif A (468), found in many ATP binding proteins. The Walker motif B forms a nearby beta-strand.

Each of the three catalytic sites binds a single Mg²⁺ ion. Magnesium binding appears to be essential for the asymmetry of the enzyme (39, 276), and it is essential for formation of the transition state (256). Results from mutagenesis (477) and the crystal structures (156, 308) indicate that the Mg²⁺ ions are coordinated by the hydroxyl O of βT156 (343) of the Walker A sequence, one water molecule that is H-bonded to βD242 (277) of the Walker B sequence, and two water molecules that are H-bonded to βE185. The octahedral coordination is completed by two O’s from the β- and γ-phosphates of ATP. Mutagenesis in the E. coli enzyme has confirmed the importance of these residues (477).

Several residues at the catalytic site have been shown to be important for binding and catalysis, as has been reviewed (408, 480). Aromatic residues βY331, βF404, and βF410 interact with the adenine ring of the nucleotide. Two positive residues are important in binding the γ-phosphate: βK155 (277, 319, 343) in the Walker A sequence and βR182 (321). βE181 appears to have a role in the transition state, in which it H-bonds, and perhaps activates the attacking water (319). In the transition state, the P-loop βA151 might also move closer to the γ-phosphate, as indicated by studies of the rat liver enzyme (74). The transition state, as in many phosphoryl transfer enzymes, is believed to be a pentavalent phosphorus, with a water attacking from one of the two equatorial positions (474). This geometry can be mimicked by the transition-state analogs ADP-AlF₃, ADP-AlF₄⁻, or ADP-vanadate. The α subunit contributes an important residue to the catalytic site, αR376 (2), which has been implicated in the transition state (322), and would be analogous to the arginine finger seen in the activation of G proteins. For an alternative view of the role of αR376, see reference 272.

A reevaluation of the binding of P_i by the E. coli F₁ revealed that by using an equilibrium-dialysis procedure, at least 2 mol/mol were bound, with a K_d of about 0.1 mM. Such weak binding canot be detected by the often-used centrifuge columns, because the off-rate is too high. An alternative procedure for estimating the binding of P_i by the E. coli enzyme is the ability of phosphate to protect the enzyme from reaction with 4-chloro-7-nitrobenzofurazan. Using this method, the molecular basis of phosphate binding was examined recently (9, 10, 11, 12). Mutagenesis of βR246 leads to a loss of phosphate binding, but it can be recovered if a nearby residue is changed to arginine, such as βN243 or αF291. All three mutants have lost catalytic activity. Mutagenesis of catalytic residues αR376, βK155, and βR182 leads to a loss of phosphate binding.

Numerous studies applying mutagenesis to α and β subunits have found mutations that are deleterious to cooperativity or assembly of F₁ (35, 170, 290, 337, 338, 342, 353, 494). Because cooperativity is a property of the system, it is typically difficult to dissect by mutagenesis. A special case is cooperativity exerted through the γ subunit, which interacts via rotation with each of the β and α subunits. The γM23K mutation, analyzed by Nakamoto and colleagues (324, 410), was inefficient at coupling ATP hydrolysis to proton translocation. Second-site suppressors were found in both the C terminus of γ and in the DELSEED region of the β subunit (249, 324). The lysine mutant was found to have a higher free energy of activation, consistent with the existence of an additional H bond in the ground state between γ and β. In ATP synthesis, the apparent K_m for phosphate was increased 7-fold. (16). Analysis of rotation (344) indicated that the γM23K mutant generates the normal amount of torque and, therefore, points to a defect in coupling the rotation to proton translocation, possibly via ε (15). Molecular dynamics simulations (288) have identified the importance of a track of arginine and lysine residues on the helical segments of γ that guide rotation within the α₃β₃ hexamer. Frasch and colleagues (44, 45, 166, 284) have investigated the effect of mutating residues in the helical segments of γ that appear to form H bonds with catch regions of β. The effects on ATP synthesis and hydrolysis have been found to be significant and indicate the importance of a network of polar interactions at this rotation interface. For example, elimination of the pair βD272 or γS12 drastically reduced ATP synthesis and ATP-driven proton translocation, but increased slightly the rate of ATP hydrolysis by isolated F₁.

Role of the γ Subunit

Numerous γ mutants have been studied, including many that had truncations at the N (235) or C terminus (209, 217). The mutant studied by Humbert and Altendorf (209) remains an interesting one, since, although it lacks about 40 amino acids at the C terminus, it allows some degree of assembly of the ATP synthase in a way that causes a proton leak through F_o. A series of C-terminal deletions have been analyzed in a video assay for torque generation, and even those lacking 12 amino acids were capable of normal torque generation (318). With larger deletions, the enzyme failed to assemble.

The γ subunit also has important interactions with ε and the ring of c subunits. Dunn (108) showed in 1982 that the isolated γ and ε subunits form a tight complex with a K_d of about 3 nM. The atomic interactions can now be seen in the crystal structures of the γ-ε complex from E. coli and the F₁ complex from bovine mitochondria. One face of the beta-sandwich packs against the N- and C-terminal alpha-helices of γ. The polypeptide chain preceding the long, C-terminal helix of γ provides a short beta-strand, more clearly seen in the E. coli γ-ε structure (375), residues 206 to 208, that adds to the interfacial five-stranded sheet in the ε subunit, as shown in Fig. 5B. This is the region of γ and ε that interacts with the ring of c subunits (471, 472, 473). When random mutagenesis was applied to this region of γ (250), one mutation stood out because of its temperature-dependent growth defect on succinate medium: γE208K. This mutation affected both the coupling of ATP hydrolysis to proton translocation and the relief from inhibition by ε. Partial suppressors were isolated in the c subunits, but not in ε. This region of γ was also studied by site-directed spin labeling (23), and the results indicated that residues 204, 208, and 210 of γ contacted F_o, probably subunit c.

Role of the ε Subunit

The ε subunit has long been known to be an inhibitor of ATP hydrolysis by F₁, and to be essential for binding F₁ to F_o (253, 267, 415, 416, 428, 429, 444). In E. coli, the role of ε is not entirely understood, but it is clear that its role is somewhat different from its mitochondrial homolog, called δ (156, 351). Although the isolated E. coli ε (446) is very similar in tertiary structure to the mitochondrial δ subunit as found in an F₁ complex (156), the E. coli protein can also be found with its C-terminal alpha-helices unpacked in an extended form, rather than a hairpin (194, 375). The precise docking site of the helices in the α₃β₃γ complex is unknown, but this can explain many of the various cross-links that had been seen between ε and α, and β and γ (5, 6, 87). Based on a study using two engineered cross-links between εA117C and cQ42C, and between εS118C and γL99C, Tsunoda et al. (442) proposed a ratchet model for ATP synthase function. The effects of polypeptide fusions at the N or C termini of ε have been studied. Fusions of 5 kDa (at the N terminus) or 12 kDa (at the C terminus) had no effect on function, but fusions of 20 or more kilodaltons at either terminus appeared to block rotation ( 76, 77). From these studies it was concluded that the role of the C-terminal helices is to reduce uncoupled ATPase activity, rather than to regulate coupled function. The kinetics of conformational changes in ε were investigated (67) using previously identified residues in the ε-c interaction (440), and εS108C-βE380C for a second cross-link. The results suggested that the conformational changes necessary for β-ε cross-linking might be fast enough to be catalytically competent. The cross-links indicted that the helices of ε are more extended than the crystal structure showed (375).

Related studies have been done with the Bacillus PS3 enzyme. Caution should be exercised when comparing the properties of ε from the two bacterial enzymes. In contrast to the E. coli enzyme, it has been shown that the Bacillus PS3 ε (247) and ε-γ complex (213) bind ATP, its inhibition can be relieved without dissociation from F₁ (244), and that it also inhibits F₁F_o (245), as well as F₁. Other differences were highlighted in a recent study of kinetics (127). It was shown that the C terminus of ε, when changed to cysteine, can form a disulfide bond with either γS3C or with εA85C, in the beta-barrel domain (432). Conditions that favor formation of the ε-γ cross-link are the lack of nucleotides, or proton motive force. Addition of ATP favors the ε-ε cross-link, i.e., the compact conformation,, and ADP counteracts ATP. The same group used FRET techniques to monitor conformational changes in real time (212). They found that ATP binding induced the transition from the helix-extended conformation to the compact conformation, but that the rate was too slow to be a catalytic step.

Early studies of mutations in ε were of random mutants (81), site-directed mutants of conserved residues (271, 413), and truncations of the N and C termini (232, 262, 414). Important results were that mutations of H38 could disrupt F₁-F_o interactions (271), and that the C terminus was not necessary for binding to F₁, but that it was important in inhibition (262). Later work identified residues that affected both binding to F₁ and coupling between F₁ and F_o (498, 499). In video rotation experiments, with a minimal viscous drag due to the use of fluorescent beads rather than filaments, ε was found to increase the duration of pauses (325).

Other lines of work focused on nucleotide-dependent conformational changes in ε (306, 307) that were shown to be related to trypsin sensitivity of the C-terminal helices. Movements of ε relative to a probe in the b subunit have been monitored by FRET (516) and were consistent with a transition in the enzyme between an active and an inactive state, which was associated with a conformational change in ε. Conformational changes in ε were probed by chemical modification of mono-cysteine mutants that were constructed throughout the protein (147). Results suggested that the most C-terminal helix has intrinsic flexibility and allows extensive labeling of the C-terminal domain.

Role of the δ Subunit

The peripheral, or stator, stalk in the E. coli ATP synthase, reviewed recently (475), is composed of the dimeric b subunits, which are anchored in the membrane with the a subunit, and at the top of F₁ by the δ subunit. The b subunits also contact α or β subunits. The δ subunit is a polypeptide of 177 amino acids, apparently in two domains. The structure of an N-terminal fragment, 1 to 135, was solved by NMR (491), and residues 1 to 105 were found to form a six-helix bundle. Previous work had shown that this region of δ could be cross-linked to the α subunit (340), and δ had been visualized at the top of F₁ by cryoelectron microscopy (492). Using the native fluorescence of δW28 or of other tryptophan mutants, the binding of δ to α₃β₃γε was measured, and the K_d was determined to be 1.4 nM (485). Interestingly, the δW28L mutant had a K_d of 4.6 nM and appeared to function normally, whereas the αG29D mutant had a K_d of 26 nM, and binding of δ to F₁ was severely impaired, indicating an apparent threshold value for strength of interaction. Mg²⁺ ions increased affinity, and high pH decreased affinity. A peptide consisting of the N-terminal 22 amino acids of α, called αN1-22, was synthesized, and shown to bind to δ with a K_d of 130 nM in a 1:1 stoichiometry (478). Residues of δ important for interaction with α were identified first by mutagenesis (484), and then by solving the NMR structure of the αN1-22 peptide complexed with δ_3-105 (486). The αN1-22 peptide forms an alpha-helix that interacts with helix I and helix V of δ, as shown in Fig. 10. Mutagenesis of the peptide, and of the entire polypeptide, showed that essentially the entire region of 22 amino acids is important (484). Other findings had implications for assembly. Isolated α subunit was found not to bind δ. Furthermore, its N terminus was not sensitive to proteolysis by trypsin, which readily occurs in F₁, and this proteolysis is known to prevent δ binding (113). Therefore, it appears that the N terminus of α is sequestered until α-β interactions occur and that δ organizes preassembled α₃β₃γε and ab₂c₁₀ units.

Fig. 10The interactions of δ and the N terminus of α. The δ subunit is magenta and the αN1-22 peptide is blue. The side chains are light blue and show the helical twist. The N terminus of α is pointing out. The Protein Data Bank ID code is 2a7u (486).

The C-terminal domain of δ appears to interact with the b subunits. Deletions of more than three residues from the C terminus of δ prevented the assembly of the ATP synthase on the membrane (231). Extensive mutagenesis of this subunit by three groups uncovered only a few missense mutations that severely compromised function. Loss of function by several criteria was found for δG150D (197), δG150N (196), and δD161P (421). These results bolstered the view that δ had a structural, rather than a catalytic role in the ATP synthase. Many studies of the interactions of δ and b have employed a soluble form of b that was introduced originally by Dunn (109). The b subunit is a polypeptide of 156 amino acids, with a hydrophobic, membrane-spanning domain of about 25 residues at the extreme N terminus. A truncated form, b_sol_25-156 was shown to be soluble and dimeric (109). It had a high content of alpha-helix by circular dichroism, and appeared elongated by analytical ultracentrifugation studies. It could bind to F₁, and it competed with F_o for F₁ binding. In another study (110), a b₂δ complex was formed from b_sol_34-156 and δ, which appeared very elongated by analytical ultracentrifugation, as if it were an end-to-end complex. Removal of amino acids from the C terminus of subunit b drastically reduced the ability of the protein to bind to δ or F₁ (302). A cross-linking agent, benzophenone-4-maleimide, when placed at the C terminus of b, generated covalent links to the extreme C terminus of δ. These findings support the earlier work in which proteolysis of b was shown to prevent binding to F₁ (202, 204, 361).

Role of the b Subunits

The dimeric nature of the b subunits was investigated by Dunn and colleagues (304, 372). They examined the tendency for cysteine mutations throughout the b_sol sequence to generate disulfide bonds. In general, dimerization was stronger toward the C-terminal end, and less so at residues N-terminal to about 53. Truncations at the C-terminal end of b at residues before 122 prevented dimerization. On the basis of these results, they described subunit b as having four domains: 1 to 24, transmembrane; 25 to 53, tether domain, 54 to 122, dimerization domain; and 123 to 156, δ binding (114), as is illustrated in Fig. 11.

Fig. 11Domain structure of the b subunit.

The tether domain was studied by Cain and colleagues (419), with the interesting finding that deletions and insertions of up to 11 residues can be made in this region without compromising function in the ATP synthase. Deletions were made in the 50- to 65-residue region, with little effect caused by up to 11 residues deleted. Insertions into the same region, which duplicated adjacent sequences, also had little effect up to 11 residues (417). The results indicate that the b subunit dimer is not taut in the ATP synthase, but they do not clarify what the b subunits might be doing during ATP synthesis or hydrolysis. Perhaps the normal length is a trade-off between two or more needs, such as efficiency of assembly and stator functioning, or between ATP synthesis and ATP hydrolysis (164). Using two different tags (165), this group demonstrated a functioning ATP synthase using one b subunit with a seven-residue deletion, and one b subunit with a seven-residue insertion. They then tested whether a deleterious mutation in b could be complemented by a second, but different deleterious mutation (164). They combined the bR36I mutation, previously shown to affect coupling of ATP hydrolysis to proton translocation (73), with the C-terminal truncation of four residues, described above (231). While both of the single mutants showed essentially zero ATP-driven proton tranlsocation, the heterozygous construct had intermediate levels of function. This indicates that the two b subunits do not have identical roles in the ATP synthase. This might be expected, to the extent that the b subunits make important contacts with single-copy subunits, such as δ and a.

The tether domain has also been studied using site-directed spin labeling (427). Distances between the same residue in both b subunits were measured, and the results were consistent with parallel helices separated by about 1.9 nm, or by closely packed helices that are out of register by several turns of the helix. The structure of a peptide consisting of residues 1 to 34 of the b subunit was determined by NMR spectroscopy in aqueous-organic solvent (98). Residues 4 to 22 formed an alpha-helix, followed by a sharp break, and another helix from 23 to 26. Cysteine residues introduced at positions 2, 6, and 10 showed the greatest tendency to form disulfides, consistent with a dimerization motif in the transmembrane region. As the helices exit the membrane, around residues 23 to 26, they would be well separated, consistent with the spin-labeling results above (427). Mutants bN2A, bT6A, and bQ10A had little effect individually, but together they were deficient in oxidative phosphorylation (182). Possibly the triple mutant is defective in F_o assembly, although it is found in membranes. A mutant studied much earlier (219, 366), bG9D, was partially suppressed by mutations in subunit a (264), aP240L or A, and in subunit c (263), cA62S.

The dimerization domain has also been probed by mutagenesis, crystallized, and studied by site-directed spin labeling. An early mutagenic study focused on a conserved region of the b subunit from residues 77 to 85 (300). Most mutations were not deleterious, but several to proline, including bA79P, were. Alignments of b subunits appeared to have heptad repeats in the vicinity of A79. In a follow-up study (301), the authors showed that higher expression of many of the mutant b subunits allowed them to function as well as the wild type. Dunn and colleagues (93) expressed the dimerization domain of b, b_62-122, and solved the crystal structure. Unfortunately, it crystallized as a monomeric alpha-helix, leaving the nature of its dimeric interactions to be determined by less direct methods. These authors favored a right-handed coiled coil, based on an hendecad repeat in the amino acid sequence, and an observed hydrophobic strip with a right-handed twist. They found that mutation of R83 to alanine stabilized the dimeric structure, consistent with their model. The results of further biophysical experiments were used to support this model (92, 371), and they proposed a scheme in which the right-handed coiled coil in dimeric b and the left-handed coiled coil in γ could be involved in elastic coupling. Other workers (22, 167) have emphasized the possible differences in structure between b_sol and full-length b incorporated into liposomes or detergents. Circular dichroism (CD) spectroscopy has shown a detergent dependence on helical content and evidence for significant nonhelical secondary structure.

Interactions between b subunits and F₁ subunits have been probed by site-directed cross-linking from bA92C (303). After reaction with benzophenone-4-maleimide, cross-linking to both α and β subunits was detected. They were confirmed by mass spectrometric analysis to be near residues 464 and 483 of the α subunit, which places b at an α/β interface near a noncatalytic site. Another mutant, bI109C, was similarly cross-linked to an unknown site in α. Results from site-direct spin-labeling studies (317) confirmed a large interaction surface between b subunits and α or β subunits. Spin labels were incorporated into b_sol constructs and added to F₁, with or without δ. The entire region from residue 80 to 114 appeared to be in proximity to F₁. The results indicated that dimeric b could bind to α/β in the absence of δ. The binding affinity of dimeric b_sol_34-156 was measured by Weber et al. (482), using native tryptophan fluorescence. They found that Mg²⁺ ions were a key factor in high-affinity binding. Without Mg²⁺ the K_d was 1.36 μM, but with Mg²⁺ the K_d decreased to 150 nM. This is consistent with the common practice of adding EDTA to remove F₁ from the plasma membrane of E. coli. Similar studies were carried out using fluorophores attached through cysteine mutations (95). Using T62C in the construct b_sol_34-156, they showed that the K_d for b dimerization was 1.8 μM, similar to previous measurements (371). The K_d for binding dimeric b to F₁ was determined to be between 0.2 and 14 nm, depending on the model used. Interactions between b and γ were examined in the Bacillus PS3 F₁ by construction of cysteine mutations (433). Cysteines were introcduced at the N terminus of a soluble b, in the tether domain, and at residue 109 of the γ subunit, which is most distal from the central shaft. Disulfide formation led to inhibition of ATP hydrolysis.

The binding of full-length b subunit (260), incorporated into liposomes, was measured to F₁. Dimeric b subunits and an isolated ab₂ complex (423), both gave K_ds of 9 to 10 nM, indicating that subunit a does not contribute to binding of F₁. When the entire F_o was used, ab₂c₁₀, the affinity increased to 2.7 nM, indicating that the c subunits do contribute to binding.

The b subunit has a clear role in the structure of the peripheral stalk, and in the binding of F₁ (91), but it is also important for the function of F_o. Early studies showed by genetics (143) and by reconstitution experiments (391, 393) that the b subunit was an essential component of F_o. Altendorf and colleagues investigated the role of the C-terminal domain of b using proteolysis (426). Using a synthetic peptide b_1-34, which is somewhat longer than the transmembrane domain, they were able to show that this is sufficient to reconstitute a proton-permeable F_o using a preformed ac complex (168). The b subunit is not thought to provide any residues that directly participate in proton movement. If so, it must provide some stability to the structure of subunit a.

Role of the a Subunit

The remaining subunits to be discussed, a and c, provide the pathway for proton translocation through an unusual interface. While a great deal is known about the structure of the c subunit from E. coli, and from other species, no high-resolution information exists about subunit a. A low-resolution picture of a membrane protein can be obtained simply by determining the location of transmembrane-spanning regions and the orientation of the protein in the membrane. Early studies used proteolysis (204) and antibody binding (88, 90, 160) to examine the orientation of a and c subunits. Later, methods with greater specificity were developed which allowed a more complete picture to emerge. The membrane topology of subunit a was investigated by six different groups, using three distinct approaches. Two groups (42, 273) applied the phoA fusion method to identify regions that are exposed on the periplasmic surface. Two groups raised antibodies against peptides within the a subunit sequence, and used them to probe right-side-out and inside-out membranes (218, 500). Two groups generated monocysteine mutants and used sulfhydryl reactivity to determine orientation (279, 449, 465). The results from the last two groups agreed that there are five membrane-spanning regions, with the N terminus in the periplasm and the C terminus in the cytoplasm. A transmembrane model is shown in Fig. 12. An advantage of the last method is that one can exhaustively change residues to cysteine to obtain a complete picture (278, 507, 508). Any single mutant, gene fusion, or antibody might give a false result.

Fig. 12Transmembrane model of subunit a. The dark blue residues are those that labeled strongly when mutated to cysteine. The light blue residues showed little or no label. The results are from references 278, 279, 465, 507, and 508.

Numerous groups have participated in mutagenic studies of subunit a. The key residue is a strictly conserved arginine, aR210, which is found near the middle of the fourth transmembrane span. Studies have shown that it cannot be replaced with lysine, isoleucine, valine, glutamic acid, or glutamine without loss of function (70, 123, 275). However, aR210A mutants are leaky to protons (190, 448), and the double mutant R210Q/Q252R retains some function (190). Other important residues are thought to be aH245 and aE219, which are found in many bacteria, and are reversed in other bacteria and in most mitochondria. Mutating 219 (69, 123, 274) or 245 (68, 69, 123, 186, 275, 458) generally leads to loss of activity, although aE219G and aE219K retain some function (189, 448, 454). In the Rhodobacter capsulatus enzyme it was shown (443) that the homologous mutant of aE219K had an altered dependence on the electrical portion of the proton motive force. The switched mutant, aH245E/E219H, has greater activity than do the single mutants, arguing for some kind of interaction (69). Other highly conserved residues in this C-terminal region can be replaced by a variety of amino acids without significant loss of function, such as aE196 (274, 455), aN214 (70, 456), aQ252 (123, 185, 454), and aY263 (123, 185, 457). A proposal was made by Vik and Antonio (454) that aR210 interacted with the key amino acid of the c subunit, cD61, as part of a rotary proton-translocation mechanism. Evidence for this interaction was provided by cysteine cross-linking experiments (225). A series of cysteine-cysteine double mutants were constructed, one cysteine was constructed in the vicinity of residue 210 in subunit a, and one in the vicinity of residue 61 in subunit c. The following yielded strong cross-linking: aS207C/cI55C, aN214C/cA62C, aN214C/cM65C, aI221C/cG69C, aI223C/cL72C, aL224C/cY73C, and aI225C/cY73C. The results are indicative of two interacting alpha-helices, centered at aR210 and cD61. Residues aE219 and aH245 would be placed in the bilayer, but toward the periplasm, according to the results above. A variety of other mutagenic analyses have supported the importance of this interaction, and of a proton pathway through subunit a to the cD61, including random and site-directed mutagenesis (150, 151, 360), chemical proteolysis (508), insertion scanning mutagenesis (94, 458, 470), and second-site suppression (140, 186, 266).

Details of possible proton pathways through subunit a have been provided by Angevine and Fillingame, who have analyzed the reactivities of a great number of cysteine mutants (24, 25, 26). They have used both direct reactivity with N-[¹⁴C]ethylmaleimide (NEM), and sensitivity of ATP-driven proton translocation to reaction with NEM and Ag¹⁺. The results indicated that three transmembrane helices, 2, 4, and 5, formed a proton pathway from the periplasm to the middle of the membrane, near aR210. The pathway to the cytoplasm seemed to involve only TM 4 and, therefore, might occur at the interface of TM 4 and the c subunit. Furthermore they have constructed a series of double-cysteine mutants within subunit a and have identified the following disulfide partners (398): L120C(TM 2) and S144C(TM 3), L120C(TM 2) and G218C(TM 4), L120C(TM 2) and H245C(TM 5), L120C(TM 2) and I246C(TM 5), N148C(TM 3) and E219C(TM 4), N148C(TM 3) and H245C(TM 5) G218C(TM 4) and I248C(TM 5), and D119C(TM 2) and G218C(TM 4). These results confirm a four-helix bundle, composed of TM 2, 3, 4, and 5, containing an access channel to the periplasm, and are summarized in Fig. 13. Preliminary NMR studies have been reported concerning the attempt to determine the structure of subunit a in a polar organic solvent (99, 101).

Fig. 13Aqueous access channels of subunit a. The access channels are indicated by residues that are sensitive to inhibition by Ag⁺, red (>90% inhibition), orange (71 to 90% inhibition), and brown (51 to 70% inhibition). (Reproduced from Angevine et al. [26] with permission from the American Society for Biochemistry and Molecular Biology.)

Mutations have been constructed in subunit a in an attempt to model the human mitochondrial disease, Leigh syndrome. The mitochondrial mutations T8993C and T8993G, which correspond in E. coli to aL207P and aL207R, respectively, have been analyzed by two groups (72, 187, 188, 341). By Western blotting, the levels of aL207R were very low or undetectable. Consistent with that finding, there was little or no ATP synthesis or ATP-driven proton translocation (72, 187, 188). The other allele, aL207P, is known to be less severe in humans (383), and the phenotype in E. coli was also more like the wild type (72, 188, 341). Low levels of ATP synthesis and ATP-driven proton translocation were seen, and the western blots showed greater amounts of a subunit in the membranes. Mitochondrial mutations T9176C and T9176G were also modeled in E. coli (72, 341). These mutations correspond to aL259P and aL259R, respectively. Both mutants showed the presence of subunit a on a western blot. The proline mutant was more like the wild type, but the arginine mutant had very low rates of ATP synthesis and ATP-driven proton translocation. Another mitochondrial mutation in a similar position, T9185C, corresponding to aL262P, also showed rather low rates of ATP synthesis and ATP-driven proton translocation (341).

Role of the c Subunits

Studies of the c subunit from E. coli began in the 1970s, as it was discovered that it could be purified following a chloroform-methanol extraction of whole cells (21, 130). This procedure led, much later (159, 314), to the use of NMR spectroscopy to determine its monomeric structure in a polar organic solvent. In a series of studies (157, 158, 161) it was shown that the protein forms an alpha-helical hairpin with two long helices, 1 to 40 and 45 to 79, connected by short turn consisting of conserved residues R41, Q42, P43, and D44, as shown. That placed the key residue, cD61, in the middle of the bilayer, with a pK_a estimated to be 7.1 (33), as shown in Fig. 6A. From this monomeric model, using molecular dynamics and distance constraints from disulfide mapping (228, 230), an oligomeric model of subunit c was constructed (103). The model supported several previous observations. Residues D61 of the C-terminal helix and A24 of the N-terminal helix from adjacent monomers were found in proximity. This explains the viability of the switched, double mutants, cA24D/D61G and cA24D/D61N (310, 509). Similarly, the proximity of A24 and T28 to D61 of an adjacent monomer is consistent with the effect on DCCD sensitivity of mutants cA24S and cT28I (131, 205) and cA25T (133).

Rastogi and Girvin (370) determined the monomeric structure of subunit c by NMR spectroscopy at pH 8.0 to examine the consequences of deprotonation of D61. The primary differences were that the C-terminal helix was rotated 140 degrees about its long axis, and that the four residues in the turn, 41 to 44, underwent a significant conformational change. The N-terminal helix was nearly identical, and locally, the C-terminal helix was little changed, as shown in Fig. 6B. These authors went on to model a four-helix bundle of subunit a, and an oligomeric c, with one deprotonated subunit at the interface with subunit a. The monomeric structures of two double mutants was also solved by NMR. The structure of cA20P/P64A showed how a proline residue could be accommodated in either the N- or C-terminal helices, as both locations are found in nature (102, 134). The structure of A24D/D61N, solved at low pH, had a conformation that resembled that of the wild-type protein solved at pH 8.0. This reinforced the idea that a c subunit, probably at the interface with subunit a, can readily convert between two conformations. Structure determination of the Bacillus PS3 monomeric subunit c by NMR showed a third conformation, suggesting that there might be a series of conformational states that interfacial subunit c must undergo (326).

The crystal structure of an undecameric ring of c subunits from a sodium-translocating ATP synthase (I. tartaricus) brought this oligomer into sharper focus (305). The structure is shown in Fig. 7A, B, and C. The conformation of the monomers most closely resembles that of the deprotonated c subunit from E. coli. Eleven sodium ions are bound, each of which is coordinated by amino acids from two adjacent subunits. The sodium ions are near the surface of an indentation that gives the oligomer an hourglass appearance. This structure also confirmed that the ring of c subunits has a hollow center. It was determined for the E. coli enzyme that this region contains phospholipids (339), as would be necessary to seal the membrane.

The interactions between ε and c subunits, which were first identified by intragenic suppressor analysis (511), were examined in disulfide cross-linking studies (199). The results indicated that a loop of ε consisting of a beta-hairpin from strands 3 and 4 packs in between two c subunits. These results were substantially confirmed by the low-resolution crystal structure of the yeast enzyme (430), which showed the homolog of ε positioned across the polar loops of the ring of c subunits. Labeling studies in the E. coli system showed that ε could protect 4 to 5 c subunits (471), also consistent with the low-resolution structure. In another study (40), the binding of monoclonal antibodies specific to the loop of subunit c were found to occur in the presence of F₁, indicating that not all c subunits were occluded by F₁. Furthermore, the binding of the antibodies did not prevent ATP hydrolysis and presumably rotation. Without direct visualization, one cannot know if they would be stripped off during the rotation driven by ATP hydrolysis, or if there is enough clearance for an antibody to remain bound. Other studies have shown that fusions of up to 12 kDa to the C terminus of ε can be tolerated (76).

Ealier mutagenesis studies (138, 139, 141, 309) of the polar loop of the c subunit indicated that it was important for coupling to ATP hydrolysis, including residues cR41, cQ42, and cP43. Others probed residues within the bilayer. For example, c subunit structure was probed by tryptophan-scanning mutagenesis (173, 396) in which tryptophan was used to replace all of the bilayer residues. The interpretation of these experiments was probably complicated by the fact that the c subunit has both a fixed architecture, and a dynamic interface with subunit a.

Inhibitors

The analysis of inhibitors of the ATP synthase has led to deeper understanding of its function and has provided useful tools for a variety of other investigations. Covalent modifiers of F₁, such as DCCD (385, 387) and 4-chloro-7-nitrobenzofurazan (56, 128, 287, 453), react with a single β subunit and therefore provide evidence of asymmetry. This was later confirmed by X-ray crystallography of the inhibited bovine enzyme with both DCCD (156) and with 4-chloro-7-nitrobenzofurazan (348).

Azide is an inhibitor of ATP hydrolysis (258), and has recently been shown to occupy the γ-phosphate position at the β_DP site filled with Mg-ADP (50). This explains why azide does not inhibit unisite catalysis (184, 336), and why it does not inhibit ATP synthesis, since this ADP is expected to be expelled by the enzyme during ATP synthesis (34, 435). It also explains why azide inhibition may develop slowly during an assay, since it requires the production of ADP. Thus, azide can be seen as a stabilizer of the Mg-ADP-inhibited form of the ATP synthase. Direct evidence for Mg-ADP inhibition of the E. coli enzyme has been difficult to obtain (211). One trademark of Mg-ADP inhibition is a lag phase in ATP hydrolysis, and this is generally not seen in the E. coli enzyme if care is taken to eliminate the inhibitory effects of ε (406). Recently, it was shown that the E. coli ATP synthase exhibits the behavior of a Mg-ADP-inhibited enzyme if assayed at low temperatures, but the lag phase disappears if a short incubation at 37°C precedes the assay (146).

Aurovertin is another inhibitor of the mitochondrial enzyme (270), and also of the E. coli enzyme (112, 386, 495). In crystallographic studies it was found to bind at two different β subunits, one of which is probably of higher affinity (452). In the third β subunit the equivalent site is occluded.

Fluoro-aluminate-based transition state analog inhibitors have been useful tools for study of the E. coli ATP synthase. Their inhibitory properties were first demonstrated in the bacterial ATP synthase by Vignais and colleagues (216, 286). Later it was shown that the Bacillus PS3 enzyme (104) and the E. coli enzyme (319, 320) can bind two such inhibitors at once. These compounds are transition-state analogs because they bind at the catalytic sites with ADP and take up the position of the γ-phosphate, with the Al-fluorides assuming a geometry similar to the P-oxygens in a transition state. Crystallographic studies have defined the binding of these agents at the catalytic sites (62, 308). Magnesium fluoride, recently shown to inhibit E. coli F₁, may act in a similar way (8).

The detergent lauryldimethylamine oxide (LDAO) has complex interactions with the ATP synthase. It was shown to activate ATP hydrolysis about fivefold by relieving the inhibitory function of ε (282). Later, it was shown that it provided some stimulation of activity even in the absence of ε (115). The concentration dependence is complex, and a mutant, γY205C, behaves differently from the wild type (363). In the Bacillus PS3 enzyme, it is also stimulatory, and it was shown to eliminate the lag phase of ATP hydrolysis due to Mg-ADP inhibition (220, 350). Assays of ATP hydrolysis by membrane preparations are often performed in the presence of LDAO to indicate the total amount of F₁ bound to membranes. The fold stimulation can be used to estimate the extent of inhibition by ε. However, the quantitative interpretation of this stimulation can be problematic, especially for subunit a mutants, which are known to cause partially assembled complexes. F₁ and F₁F_o are stimulated to different extents by LDAO (283, 363).

In the presence of Mg²⁺ ions, low concentrations of DCCD, up to 50 μM, will preferentially inhibit F_o rather than the β subunit. This results in covalent modification of subunit c at cD61, the proton carrier. Studies have shown that inhibition occurs with the reaction of approximately one of the c subunits (201), and there is probably a steric block of rotation. The extent of inhibition of ATP hydrolysis by DCCD is generally reported to be from 65 to 80%, and the reason for this is not clear. The uninhibited rate could represent nonrotary ATP hydrolysis, or it could reflect that DCCD-modified subunit c can still rotate at a slow rate. The rate of the DCCD reaction in detergent-solubilized enzyme was shown to decrease at high pH with a pK_a of 8, while no pH dependence was seen in the membrane-bound state, suggesting that the carboxyl group was not accessible to the aqueous medium (447). DCCD inhibition is often used as a diagnostic of the coupling of ATP hydrolysis to proton translocation, but the correlation is not exact. Several mutations of aE219 vary in the extent of ATP-driven proton translocation, but all have the same sensitivity to DCCD as the wild type (448). Mutations in ε (271) and a (458) that are found at the interface with c are known to affect the sensitivity of ATP hydrolysis to DCCD, and these do correlate with loss of proton-translocation rates.

The classical inhibitor of mitochondrial ATP synthase, oligomycin (368), acts on F_o and strongly inhibits ATP hydrolysis. Therefore, it is a useful indicator that an ATP synthase is tightly coupled with respect to ATP hydrolysis and proton translocation. This agent is not effective against the E. coli enzyme. It was shown that venturicidin is more effective, and that both inhibitors impede the reaction of DCCD with the c subunit (362).

MODELING THE MOLECULAR ROTOR

Rotary Models of F_o

Several different aspects of the rotary function of the ATP synthase have been modeled in recent years. Junge had conceived of the F_o as a Brownian rotor, with a biased rotation due to a pH gradient across the membrane, in the early 1990s, but did not publish any of his models until 1997 (233, 378, 380). Junge and colleagues had identified the mismatch of the stepping motors as an important issue, and hypothesized that accumulation of elastic energy in the stalks would solve this problem (75). Oster and colleagues (117) drew upon a Brownian rotor model proposed independently (454), and evaluated the energetics quantitatively with aR210 interacting electrostatically with cD61. It was determined that such a charge interaction was necessary to generate the observed torque. A detailed model was developed for rotation in F_o (14), using molecular dynamics simulations based on a modeled subunit c oligomer. It shows strong involvement of aSer206 and aN214 along with the essential aR210 at the interface with cD61 in the c subunit oligomer. The conformation of the c subunits in the crystal structure of the c ring from the sodium-translocating ATP synthase (305) are somewhat different from the conformations used in this study. It is not clear what effect that might have on the outcome, but perhaps little, because the model includes free rotation of the C-terminal helix of the c subunits at the a:c interface. The calculated model also requires two deprotonated c subunits at the interface with subunit a, a key feature of an early hypothesis of rotary proton translocation (454). A schematic model is shown in Fig. 14.

Fig. 14A schematic proton translocation mechanism. This diagram is based on material presented in the text. Protons enter subunit a through an access channel that ends near aR210, aN214, and cD61. When the cD61 is protonated, the ring of csubunits can move, since the charge is now neutralized, and the aR210 will engage with the adjacent cD61, which is ionized. Rotation will bring a new c subunit into the region near aR210 and aS206, where the proton will be lost to the cytoplasm.

Rotary Models of F₁

Using the crystal structure (2), Oster and colleagues produced a corresponding mechanical model for the operation of F₁ (469). For the ATP-hydrolysis reaction, they developed a "binding-zipper" mechanism (349), in which the motion of the shaft of γ is correlated with breaking or making interactions between ATP and the catalytic residues. This model was evaluated by using molecular dynamics with interpolated atomic structures derived from the three catalytic site conformations seen in the crystal structure (29). In a later model, they incorporated other properties of the enzyme (431), including features of γ and ε and of ADP inhibition ( 496), using a Markov-Fokker-Planck formalism, defined previously (497). Karplus and colleagues have developed the most detailed models of ATP synthesis and hydrolysis by using the high-resolution structures of F₁ (156, 308), and also other kinetic and thermodynamic data about the ATP synthase (148, 149, 288, 501).

CELL PHYSIOLOGY

Although the ATP synthase is a key enzyme in E. coli, experiments in the 1990s showed that it exerts little control over the cell's metabolism (221, 222, 223). For example, under growth on glucose minimal medium, the ATP synthase does not control the growth rate of the cells. It exerts a slightly positive control on growth yield, and it has a slightly negative influence on the respiration rate. This can be explained by an excess capacity of the ATP synthase. If the level of enzyme decreases, the available enzyme can work faster. Enzymatic analysis of membranes in vitro has also indicated that the enzyme operates below capacity (120).

Current technology allows one to monitor the levels of thousands of transcribed genes and proteins at once. A recent study (333) looked at cells that had no detectable amounts of F₁ ATPase due to an atpA mutation. They found increased rates of glucose consumption and of respiration, consistent with the earlier findings. They were also able to identify large numbers of other genes whose expression changed, such as the down-regulation of genes for flagellar biogenesis. This approach will eventually permit a better understanding of the interactions involved in the physiology of E. coli. One example would be the relationship between ATP synthesis and the transport of C4-dicarboxylates, such as succinate, and how they can sustain growth as a carbon source (46).

ACKNOWLEDGMENTS

Work in the author's laboratory was supported by National Institutes of Health Grant GM-40508 and Welch Foundation Award N-1378. Images of proteins shown in Fig. 3 to 7 and Fig. 10 were generated by Jmol (http://jmol.sourceforge.net/).

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