The preferred stoichiometry of c subunits in the rotary motor sector of Escherichia coli ATP synthase is 10

The stoichiometry of c subunits in the H(+)-transporting F(o) rotary motor of ATP synthase is uncertain, the most recent suggestions varying from 10 to 14. The stoichiometry will determine the number of H(+) transported per ATP synthesized and will directly relate to the P/O ratio of oxidative phosphorylation. The experiments described here show that the number of c subunits in functional complexes of F(o)F(1) ATP synthase from Escherichia coli can be manipulated, but that the preferred number is 10. Mixtures of genetically fused cysteine-substituted trimers (c(3)) and tetramers (c(4)) of subunit c were coexpressed and the c subunits crosslinked in the plasma membrane. Prominent products corresponding to oligomers of c(7) and c(10) were observed in the membrane and purified F(o)F(1) complex, indicating that the c(10) oligomer formed naturally. Oligomers larger than c(10) were also observed in the membrane fraction of cells expressing c(3) or c(4) individually, or in cells coexpressing c(3) and c(4) together, but these larger oligomers did not copurify with the functional F(o)F(1) complex and were concluded to be aberrant products of assembly in the membrane.     

The two rotor components of yeast mitochondrial ATP synthase are mechanically coupled by subunit delta

The mitochondrial ATP synthase is made of a membrane-integrated F0 component that forms a proton-permeable pore through the inner membrane and a globular peripheral F1 domain where ATP is synthesized. The catalytic mechanism is thought to involve the rotation of a 10-12 c subunit ring in the F0 together with the gamma subunit of F1. An important and not yet resolved question is to define precisely how the gamma subunit is connected with the c-ring. In this study, using a doxycycline-regulatable expression system, we provide direct evidence that the rest of the enzyme can assemble without the delta subunit of F1, and we show that delta-less mitochondria are uncoupled because of an F0-mediated proton leak. Based on these observations, and taking into account high-resolution structural models, we propose that subunit delta plays a key role in the mechanical coupling of the c-ring to subunit gamma.     

Engineering rotor ring stoichiometries in the ATP synthase

ATP synthase membrane rotors consist of a ring of c-subunits whose stoichiometry is constant for a given species but variable across different ones. We investigated the importance of c/c-subunit contacts by site-directed mutagenesis of a conserved stretch of glycines (GxGxGxGxG) in a bacterial c(11) ring. Structural and biochemical studies show a direct, specific influence on the c-subunit stoichiometry, revealing c(<11), c(12), c(13), c(14), and c(>14) rings. Molecular dynamics simulations rationalize this effect in terms of the energetics and geometry of the c-subunit interfaces. Quantitative data from a spectroscopic interaction study demonstrate that the complex assembly is independent of the c-ring size. Real-time ATP synthesis experiments in proteoliposomes show the mutant enzyme, harboring the larger c(12) instead of c(11), is functional at lower ion motive force. The high degree of compliance in the architecture of the ATP synthase rotor offers a rationale for the natural diversity of c-ring stoichiometries, which likely reflect adaptations to specific bioenergetic demands. These results provide the basis for bioengineering ATP synthases with customized ion-to-ATP ratios, by sequence modifications.     

Subunit rotation of ATP synthase embedded in membranes: a or beta subunit rotation relative to the c subunit ring

ATP synthase F(o)F(1) (alpha(3)beta(3)gammadelta epsilon ab(2)c(10-14)) couples an electrochemical proton gradient and a chemical reaction through the rotation of its subunit assembly. In this study, we engineered F(o)F(1) to examine the rotation of the catalytic F(1) beta or membrane sector F(o) a subunit when the F(o) c subunit ring was immobilized; a biotin-tag was introduced onto the beta or a subunit, and a His-tag onto the c subunit ring. Membrane fragments were obtained from Escherichia coli cells carrying the recombinant plasmid for the engineered F(o)F(1) and were immobilized on a glass surface. An actin filament connected to the beta or a subunit rotated counterclockwise on the addition of ATP, and generated essentially the same torque as one connected to the c ring of F(o)F(1) immobilized through a His-tag linked to the alpha or beta subunit. These results established that the gamma epsilon c(10-14) and alpha(3)beta(3)deltaab(2) complexes are mechanical units of the membrane-embedded enzyme involved in rotational catalysis.     

Energy-driven subunit rotation at the interface between subunit a and the c oligomer in the FO sector of Escherichia coli ATP synthase

Subunit rotation within the F(1) catalytic sector of the ATP synthase has been well documented, identifying the synthase as the smallest known rotary motor. In the membrane-embedded F(O) sector, it is thought that proton transport occurs at a rotor/stator interface between the oligomeric ring of c subunits (rotor) and the single-copy a subunit (stator). Here we report evidence for an energy-dependent rotation at this interface. F(O)F(1) was expressed with a pair of substituted cysteines positioned to allow an intersubunit disulfide crosslink between subunit a and a c subunit [aN214C/cM65C; Jiang, W. & Fillingame, R. H. (1998) Proc. Natl. Acad. Sci. USA 95, 6607--6612]. Membranes were treated with N,N'-dicyclohexyl-[(14)C]carbodiimide to radiolabel the D61 residue on less than 20% of the c subunits. After oxidation to form an a--c crosslink, the c subunit properly aligned to crosslink to subunit a was found to contain very little (14)C label relative to other members of the c ring. However, exposure to MgATP before oxidation significantly increased the radiolabel in the a-c crosslink, indicating that a different c subunit was now aligned with subunit a. This increase was not induced by exposure to MgADP/P(i). Furthermore, preincubation with MgADP and azide to inhibit F(1) or with high concentrations of N,N'-dicyclohexylcarbodiimide to label most c subunits prevented the ATP effect. These results provide evidence for an energy-dependent rotation of the c ring relative to subunit a.     

Activation of pausing F1 motor by external force

A rotary motor F(1), a catalytic part of ATP synthase, makes a 120 degrees step rotation driven by hydrolysis of one ATP, which consists of 80 degrees and 40 degrees substeps initiated by ATP binding and probably by ADP and/or P(i) dissociation, respectively. During active rotations, F(1) spontaneously fails in ADP release and pauses after a 80 degrees substep, which is called the ADP-inhibited form. In the present work, we found that, when pushed >+40 degrees with magnetic tweezers, the pausing F(1) resumes its active rotation after releasing inhibitory ADP. The rate constant of the mechanical activation exponentially increased with the pushed angle, implying that F(1) weakens the affinity of its catalytic site for ADP as the angle goes forward. This finding explains not only its unidirectional nature of rotation, but also its physiological function in ATP synthesis; it would readily bind ADP from solution when rotated backward by an F(o) motor in the ATP synthase. Furthermore, the mechanical work for the forced rotation was efficiently converted into work for expelling ADP from the catalytic site, supporting the tight coupling between the rotation and catalytic event.     

Rotation scheme of V1-motor is different from that of F1-motor

V(1), a water-soluble portion of vacuole-type ATPase (V-ATPase), is an ATP-driven rotary motor, similar to F(1)-ATPase. Hydrolysis of ATP is coupled to unidirectional rotation of the central rotor D and F subunits relative to the A(3)B(3) cylinder. In this study, we analyzed the rotation kinetics of V(1) in detail. At low ATP concentrations, the D subunit rotated stepwise, pausing every 120 degrees . The dwell time between steps revealed that V(1) consumes one ATP per 120 degrees step. V(1) generated torque of approximately 35 pN nm, slightly lower than the approximately 46 pN nm measured for F(1). Noticeably, the angles for both ATP cleavage and binding were apparently the same in V(1), in sharp contrast to F(1), which cleaves ATP at 80 degrees posterior to the binding of ATP. Thus, the mechanochemical cycle of V(1) has marked differences to that of F(1).     

Comparison of the H+/ATP ratios of the H+-ATP synthases from yeast and from chloroplast

F(0)F(1)-ATP synthases use the free energy derived from a transmembrane proton transport to synthesize ATP from ADP and inorganic phosphate. The number of protons translocated per ATP (H(+)/ATP ratio) is an important parameter for the mechanism of the enzyme and for energy transduction in cells. Current models of rotational catalysis predict that the H(+)/ATP ratio is identical to the stoichiometric ratio of c-subunits to β-subunits. We measured in parallel the H(+)/ATP ratios at equilibrium of purified F(0)F(1)s from yeast mitochondria (c/β = 3.3) and from spinach chloroplasts (c/β = 4.7). The isolated enzymes were reconstituted into liposomes and, after energization of the proteoliposomes with acid-base transitions, the initial rates of ATP synthesis and hydrolysis were measured as a function of ΔpH. The equilibrium ΔpH was obtained by interpolation, and from its dependency on the stoichiometric ratio, [ATP]/([ADP]·[P(i)]), finally the thermodynamic H(+)/ATP ratios were obtained: 2.9 ± 0.2 for the mitochondrial enzyme and 3.9 ± 0.3 for the chloroplast enzyme. The data show that the thermodynamic H(+)/ATP ratio depends on the stoichiometry of the c-subunit, although it is not identical to the c/β ratio.     

Stoichiometry of vectorial H+ movements coupled to electron transport and to ATP synthesis in mitochondria

In order to verify more directly our earlier measurements showing that, on the average, close to four vectorial H(+) are rejected per pair of electrons passing each of the three energy-conserving sites of the mitochondrial electron transport chain, direct tests of the H(+)/2e(-) ratio for sites 2 and 3 were carried out in the presence of permeant charge-compensating cations. Site 2 was examined by utilizing succinate as electron donor and ferricyanide as electron acceptor from mitochondrial cytochrome c; the directly measured H(+)/2e(-) ratio was close to 4. Energy-conserving site 3 was isolated for study with ferrocyanide or ascorbate plus tetramethylphenylenediamine as electron donors to cytochrome c and with oxygen as electron acceptor. The directly measured H(+)/2e(-) ratio for site 3 was close to 4. The H(+)/ATP ratio (number of vectorial H(+) ejected per ATP hydrolyzed) was determined with a new method in which the steady-state rates of both H(+) ejection and ATP hydrolysis were measured in the presence of K(+) + valinomycin. The H(+)/ATP ratio was found to approach 3.0. A proton cycle for oxidative phosphorylation is proposed, in which four electrochemical H(+) equivalents are ejected per pair of electrons passing each energy-conserving site; three of the H(+) equivalents pass inward to derive ATP synthesis from ADP and phosphate and the fourth H(+) is used to bring about the energy-requiring electrogenic expulsion of ATP(4-) in exchange for extramitochondrial ADP(3-), via the H(+)/H(2)PO(4) (-) symporter.     

ATP-driven stepwise rotation of FoF1-ATP synthase

FoF1-ATP synthase (FoF1) is a motor enzyme that couples ATP synthesis/hydrolysis with a transmembrane proton translocation. F1, a water-soluble ATPase portion of FoF1, rotates by repeating ATP-waiting dwell, 80 degrees substep rotation, catalytic dwell, and 40 degrees -substep rotation. Compared with F1, rotation of FoF1 has yet been poorly understood, and, here, we analyzed ATP-driven rotations of FoF1. Rotation was probed with an 80-nm bead attached to the ring of c subunits in the immobilized FoF1 and recorded with a submillisecond fast camera. The rotation rates at various ATP concentrations obeyed the curve defined by a Km of approximately 30 microM and a Vmax of approximately 350 revolutions per second (at 37 degrees C). At low ATP, ATP-waiting dwell was seen and the kon-ATP was estimated to be 3.6 x 10(7) M(-1) x s(-1). At high ATP, fast, poorly defined stepwise motions were observed that probably reflect the catalytic dwells. When a slowly hydrolyzable substrate, adenosine 5'-[gamma-thio]triphosphate, was used, the catalytic dwells consisting of two events were seen more clearly at the angular position of approximately 80 degrees . The rotational behavior of FoF1 resembles that of F1. This finding indicates that "friction" in Fo motor is negligible during the ATP-driven rotation. Tributyltin chloride, a specific inhibitor of proton translocation, slowed the rotation rate by 96%. However, dwells at clearly defined angular positions were not observed under these conditions, indicating that inhibition by tributyltin chloride is complex.     

Catalysis and rotation of F1 motor: cleavage of ATP at the catalytic site occurs in 1 ms before 40 degree substep rotation

F1, a water-soluble portion of FoF1-ATP synthase, is an ATP hydrolysis-driven rotary motor. The central gamma-subunit rotates in the alpha 3 beta 3 cylinder by repeating the following four stages of rotation: ATP-binding dwell, rapid 80 degrees substep rotation, interim dwell, and rapid 40 degrees substep rotation. At least two 1-ms catalytic events occur in the interim dwell, but it is still unclear which steps in the ATPase cycle, except for ATP binding, correspond to these events. To discover which steps, we analyzed rotations of F1 subcomplex (alpha 3 beta 3 gamma) from thermophilic Bacillus PS3 under conditions where cleavage of ATP at the catalytic site is decelerated: hydrolysis of ATP by the catalytic-site mutant F1 and hydrolysis of a slowly hydrolyzable substrate ATP gamma S (adenosine 5'-[gamma-thio]triphosphate) by wild-type F1. In both cases, interim dwells were extended as expected from bulk phase kinetics, confirming that cleavage of ATP takes place during the interim dwell. Furthermore, the results of ATP gamma S hydrolysis by the mutant F1 ensure that cleavage of ATP most likely corresponds to one of the two 1-ms events and not some other faster undetected event. Thus, cleavage of ATP on F1 occurs in 1 ms during the interim dwell, and we call this interim dwell catalytic dwell.     

Critical role of a transmembrane lysine in aminophospholipid transport by mammalian photoreceptor P4-ATPase ATP8A2

ATP8A2 is a P(4)-ATPase ("flippase") located in membranes of retinal photoreceptors, brain cells, and testis, where it mediates transport of aminophospholipids toward the cytoplasmic leaflet. It has long been an enigma whether the mechanism of P(4)-ATPases resembles that of the well-characterized cation-transporting P-type ATPases, and it is unknown whether the flippases interact directly with the lipid and with counterions. Our results demonstrate that ATP8A2 forms a phosphoenzyme intermediate at the conserved aspartate (Asp(416)) in the P-type ATPase signature sequence and exists in E(1)P and E(2)P forms similar to the archetypical P-type ATPases. Using the properties of the phosphoenzyme, the partial reaction steps of the transport cycle were examined, and the roles of conserved residues Asp(196), Glu(198), Lys(873), and Asn(874) in the transport mechanism were elucidated. The former two residues in the A-domain T/D-G-E-S/T motif are involved in catalysis of E(2)P dephosphorylation, the glutamate being essential. Transported aminophospholipids activate the dephosphorylation similar to K(+) activation of dephosphorylation in Na(+),K(+)-ATPase. Lys(873) mutants (particularly K873A and K873E) display a markedly reduced sensitivity to aminophospholipids. Hence, Lys(873), located in transmembrane segment M5 at a "hot spot" for cation binding in Ca(2+)-ATPase and Na(+),K(+)-ATPase, appears to participate directly in aminophospholipid binding or to mediate a crucial interaction within the ATP8A2-CDC50 complex. By contrast, Lys(865) is unimportant for aminophospholipid sensitivity. Binding of Na(+), H(+), K(+), Cl(-), or Ca(2+) to the E(1) form as a counterion is not required for activation of phosphorylation from ATP. Therefore, phospholipids could be the only substrate transported by ATP8A2.     

H+/ATP ratio during ATP hydrolysis by mitochondria: modification of the chemiosmotic theory.

The stoichiometry of H+ ejection by mitochondria during hydrolysis of a small pulse of ATP (the H+/ATP ratio) has been reexamined in the light of our recent observation that the stoichiometry of H+ ejection during mitochondrial electron transport (the H+/site ratio) was previously underestimated. We show that earlier estimates of the H+/ATP ratio in intact mitochondria were based upon an invalid correction for scaler H+ production and describe a modified method for determination of this ratio which utilizes mersalyl or N-ethylmaleimide to prevent complicating transmembrane movements of phosphate and H+. This method gives a value for the H+/ATP ratio of 2.0 without the need for questionable corrections, compared with a value of 3.0 for the H+/site ratio also obtained by pulse methods. A modified version of the chemiosmotic theory is presented, in which 3 H+ are ejected per pair of electrons traversing each energy-conserving site of the respiratory chain. Of these, 2 H+ return to the matrix through the ATPase to form ATP from ADP and phosphate, and 1 H+ returns through the combined action of the phosphate and adenine nucleotide exchange carriers of the inner membrane to allow the energy-requiring influx of Pi and ADP3- and efflux of ATP4-. Thus, up to one-third of the energy input into synthesis of extramitochondrial ATP may be required for transport work. Since other methods suggest that the H+/site significantly exceeds 3.0, an alternative possibility is that 4 h+ are ejected per site, followed by return of 3 H+ through the ATPase and 1 H+ through the operation of the proton-coupled membrane transport systems.     

Prevalence of ATP7B Gene Mutations in Iranian Patients With Wilson Disease

Background

Wilson disease (WD) is an autosomal recessive disorder. The WD gene, ATP7B, encodes a copper-transporting ATPase involved in the transport of copper into the plasma protein ceruloplasmin and in excretion of copper from the liver. ATP7B mutations cause copper to accumulate in the liver and brain.

Objectives

We examined the ATP7B mutation spectrum in Wilson disease patients in Iran.

Patients and Methods

Genomic DNA was extracted from patients with Wilson disease. The entire coding region of the ATP7B gene was amplified using PCR and analyzed using direct sequencing.

Results

We identified five novel mutations in 5 Iranian patients with Wilson disease. The first was a transversion, c.2363C > T, which led to an amino acid change from threonine to isoleucine. The second mutation was a deletion, c.2532delA (Val845Ser), which occurred in exon 10. The third mutation was a transition mutation, c.2311C > G (Leu770Leu), which occurred in the TM4 domain of the ATP7B protein. The fourth mutation was a transversion, (c.3061G > A) (Lys1020Lys), in exon 14. Lastly, we identified a transversion, c.3206C > A (His1069Asn) in exon 14 which led to a change in function of the ATP loop domain of the ATP7B protein. The H1069Q mutation was identified as the most common mutation in our study population.

Conclusions

Based on our findings, the H1069Q may be a biomarker that can be used in a rapid detection assay for diagnosing WD patients     

Large conformational changes of the epsilon subunit in the bacterial F1F0 ATP synthase provide a ratchet action to regulate this rotary motor enzyme

The F(1)F(0) ATP synthase is the smallest motor enzyme known. Previous studies had established that the central stalk, made of the gamma and epsilon subunits in the F(1) part and c subunit ring in the F(0) part, rotates relative to a stator composed of alpha(3)beta(3)deltaab(2) during ATP hydrolysis and synthesis. How this rotation is regulated has been less clear. Here, we show that the epsilon subunit plays a key role by acting as a switch of this motor. Two different arrangements of the epsilon subunit have been visualized recently. The first has been observed in beef heart mitochondrial F(1)-ATPase where the C-terminal portion is arranged as a two-alpha-helix hairpin structure that extends away from the alpha(3)beta(3) region, and toward the position of the c subunit ring in the intact F(1)F(0). The second arrangement was observed in a structure determination of a complex of the gamma and epsilon subunits of the Escherichia coli F(1)-ATPase. In this, the two C-terminal helices are apart and extend along the gamma to interact with the alpha and beta subunits in the intact complex. We have been able to trap these two arrangements by cross-linking after introducing appropriate Cys residues in E. coli F(1)F(0), confirming that both conformations of the epsilon subunit exist in the enzyme complex. With the C-terminal domain of epsilon toward the F(0), ATP hydrolysis is activated, but the enzyme is fully coupled in both ATP hydrolysis and synthesis. With the C-terminal domain toward the F(1) part, ATP hydrolysis is inhibited and yet the enzyme is fully functional in ATP synthesis; i.e., it works in one direction only. These results help explain the inhibitory action of the epsilon subunit in the F(1)F(0) complex and argue for a ratchet function of this subunit.     

Evidence that catecholamine transport into chromaffin vesicles is coupled to vesicle membrane potential

The effects of ATP, Mg(2+), and various agents on pH gradient, membrane potential, and catecholamine transport across membranes of intact bovine chromaffin vesicles were investigated. Methylamine and thiocyanate (SCN(-)) distributions across the vesicle membrane were used to estimate the H(+) concentration gradient and membrane potential, respectively. The H(+) concentration ratio (intravesiculanmedium) equals 16 when the medium pH is 6.9 and is unaltered by ATP and Mg(2+). In the absence of ATP and Mg(2+), the steady-state intravesicular S(14)CN(-) concentration is lower than the medium concentration. ATP and Mg(2+) cause an increased influx and a decreased efflux of SCN(-) that results in SCN(-) being concentrated in the vesicles 6- to 8-fold over the medium. The findings are consistent with an ATP,Mg(2+)-induced potential of approximately 50 mV (intravesicular side positive). Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), a H(+) translocater, and N-ethylmaleimide (NEM), a sulfhydryl reagent, decrease the SCN(-) ratio and, thus, the membrane potential in the presence of ATP and Mg(2+). They have no effect on the H(+) concentration gradient. The rate of catecholamine uptake into vesicles is increased 4- to 6-fold by ATP and Mg(2+). The ATP,Mg(2+)-stimulated uptake is inhibited by FCCP and NEM over the same concentration ranges that reduce the SCN(-) distribution (membrane potential). FCCP increases and NEM decreases vesicular membrane ATPase activity. Thus, catecholamine uptake is correlated to an inside-positive membrane potential, and not to ATPase activity. If catecholamine uptake is coupled to membrane potential, then a charged species must be involved in the transport mechanism. Reserpine and rotenone inhibit catecholamine influx but have no effect on the H(+) electrochemical gradient; they probably act at a step before coupling to the membrane potential (or the H(+) electrochemical gradient). Atractyloside, an inhibitor of nucleotide transport, has no effects on catecholamine transport or the H(+) electrochemical gradient.     

Glucose triggers protein kinase A-dependent insulin secretion in mouse pancreatic islets through activation of the K+ATP channel-dependent pathway

OBJECTIVE: To assess the significance of protein kinase A (PKA) in glucose triggering of ATP-sensitive K(+) (K(+)(ATP)) channel-dependent insulin secretion and in glucose amplification of K(+)(ATP) channel-independent insulin secretion. METHODS: Insulin release from cultured perifused mouse pancreatic islets was determined by radioimmunoassay. RESULTS: In islets cultured at 5.5 mmol/l glucose, and then perifused in physiological Krebs-Ringer medium, the PKA inhibitors, H89 (10 micromol/l) and PKI 6-22 amide (30 micromol/l) did not inhibit glucose (16.7 mmol/l)-induced insulin secretion, but inhibited stimulation by the adenylyl cyclase activator, forskolin (10 micromol/l). In the presence of 60 mmol/l K(+) and 250 micromol/l diazoxide, which stimulates maximum Ca(2+) influx independently of K(+)(ATP) channels, H89 (10 micromol/l) inhibited Ca(2+)-evoked insulin secretion, but failed to prevent glucose amplification of K(+)(ATP) channel-independent insulin secretion. In the presence of 1 mmol/l ouabain and 250 micromol/l diazoxide, which cause modest Ca(2+) influx, glucose amplification of K(+)(ATP) channel-independent insulin secretion was observed without concomitant Ca(2+) stimulation of PKA activity. In islets cultured at 16.7 mmol/l glucose, glucose (16.7 mmol/l)-induced insulin secretion in physiological Krebs-Ringer medium was augmented and now inhibited by H89 (10 micromol/l), implicating that culture at 16.7 mmol/l glucose may increase Ca(2+)-sensitive adenylyl cyclase activity and hence PKA activity. In accordance, Ca(2+)-evoked insulin secretion at 60 mmol/l K(+) and 250 micromol/l diazoxide was improved, whereas glucose amplification of K(+)(ATP) channel-independent insulin secretion was unaffected. CONCLUSIONS: Glucose may activate PKA through triggering of the K(+)(ATP) channel-dependent pathway. Glucose amplification of K(+)(ATP) channel-independent insulin secretion, on the other hand, occurs by PKA-independent mechanisms.     

The essential carboxyl group in subunit c of the F1F0 ATP synthase can be moved and H(+)-translocating function retained.

The proteolipid subunit c of F1F0-type H(+)-transporting ATP synthases [ATP phosphohydrolase (H(+)-transporting), EC 3.6.1.34] contains a conserved Asp/Glu residue that is thought to function in H+ translocation. To test the importance of the position of this residue in the Escherichia coli enzyme, we used oligonucleotide-directed mutagenesis to move the carboxyl side chain from position 61 to position 58, 60, or 62. Mutant cells with these changes were incapable of growth via oxidative phosphorylation on succinate. An Asp-61----Glu mutant grew on succinate but at 50% the efficiency of wild type. Hence, even minor changes in the position of the carboxyl group can significantly reduce function. In a second approach, slow-growing revertants to an Asp-61----Gly mutant were isolated. In one such revertant, Ala-24 was changed to Asp, while the original Asp-61----Gly mutation remained unchanged. The Asp-24-Gly-61 double mutant grew on succinate at 60% the efficiency of wild type. Hence the essential carboxyl group of subunit c can function when anchored at either position 24 or position 61, and this supports the idea that these residues may neighbor each other when subunit c is folded in the membrane. The rate of ATP-driven H+ translocation by mutant membrane vesicles was estimated by the quenching of 9-amino-6-chloro-2-methoxyacridine fluorescence and corresponded to actual H+ pumping rates less than 25% that of wild type.