Properties of Bound Inorganic Phosphate on Bovine Mitochondrial F1F0-ATP Synthase

Beef-heart mitochondrial F₁F₀-ATP synthase contained six molecules of bound inorganic phosphate (P_i). This phosphate exchanged completely with exogenous ³²P_i when the enzyme was exposed to 30% (v/v) dimethyl sulfoxide (DMSO) and then returned to a DMSO-free buffer (Beharry and Bragg 2001). Only two molecules were replaced by ³²P_i when the enzyme was not pretreated with DMSO. These two molecules of ³²P_i were not displaced from the enzyme by the treatment with 1 mM ATP. Similarly, two molecules of bound ³²P_i remained on the DMSO-pretreated enzyme following addition of ATP, that is, four molecules of ³²P_i were displaced by ATP. The ATP-resistant ³²P_i was removed from the enzyme by pyrophosphate. It is proposed that these molecules of ³²P_i are bound at an unfilled adenine nucleotide-binding noncatalytic site on the enzyme. Brief exposure of the enzyme loaded with two molecules of ³²P_i to DMSO, followed by removal of the DMSO, resulted in the loss of the bound ³²P_i and in the formation of two molecules of bound ATP from exogenous ADP. A third catalytic site on the enzyme was occupied by ATP, which could undergo a P_i ATP exchange reaction with bound P_i The presence of two catalytic sites containing bound P_i is consistent with the X-ray crystallographic structure of F₁ (Bianchet, et al., 1998). Thus, five of the six molecules of bound P_i were accounted for. Three molecules of bound P_i were at catalytic sites and participated in ATP synthesis or P_i ATP exchange. Two other molecules of bound P_i were present at a noncatalytic adenine nucleotide-binding site. The location and role of the remaining molecule of bound P_i remains to be established. We were unable to demonstrate, using chemical modification of sulfhydryl groups by iodoacetic acid, any gross difference in the conformation of F₁F₀ in DMSO-containing compared with DMSO-free buffers.     

Structure of Dimeric F1F0-ATP Synthase

The structure of the dimeric ATP synthase from yeast mitochondria was analyzed by transmission electron microscopy and single particle image analysis. In addition to the previously reported side views of the dimer, top view and intermediate projections served to resolve the arrangement of the rotary c₁₀ ring and the other stator subunits at the F₀-F₀ dimeric interface. A three-dimensional reconstruction of the complex was calculated from a data set of 9960 molecular images at a resolution of 27 Å. The structural model of the dimeric ATP synthase shows the two monomers arranged at an angle of ∼45°, consistent with our earlier analysis of the ATP synthase from bovine heart mitochondria (Minauro-Sanmiguel, F., Wilkens, S., and Garcia, J. J. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 12356–12358). In the ATP synthase dimer, the two peripheral stalks are located near the F₁-F₁ interface but are turned away from each other so that they are not in contact. Based on the three-dimensional reconstruction, a model of how dimeric ATP synthase assembles to form the higher order oligomeric structures that are required for mitochondrial cristae biogenesis is discussed.     

Hypothyroidism Leads to a Decreased Expression of Mitochondrial F0F1-ATP Synthase in Rat Liver

In liver mitochondria isolated from hypothyroid rats, the rate of ATP synthesis is lower than in mitochondria from normal rats. Oligomycin-sensitive ATP hydrolase activity and passive proton permeability were significantly lower in submitochondrial particles from hypothyroid rats compared to those isolated from normal rats. In mitochondria from hypothyroid rats, the changes in catalytic activities of F₀F₁-ATP synthase are accompanied by a decrease in the amount of immunodetected -F₁, F₀1-PVP, and OSCP subunits of the complex. Northern blot hybridization shows a decrease in the relative cytosolic content of mRNA for -F₁ subunit in liver of hypothyroid rats. Administration of 3,5,3-triodo-L-thyronine to the hypothyroid rats tends to remedy the functional and structural defects of F₀F₁-ATP synthase observed in the hypothyroid rats. The results obtained indicate that hypothyroidism leads to a decreased expression of F₀F₁-ATP synthase complex in liver mitochondria and this contributes to the decrease of the efficiency of oxidative phosphorylation.     

Single-molecule Analysis of F0F1-ATP Synthase Inhibited by N,N-Dicyclohexylcarbodiimide

N,N-Dicyclohexylcarbodiimide (DCCD) is a classical inhibitor of the F₀F₁-ATP synthase (F₀F₁), which covalently binds to the highly conserved carboxylic acid of the proteolipid subunit (c subunit) in F₀. Although it is well known that DCCD modification of the c subunit blocks proton translocation in F₀ and the coupled ATP hydrolysis activity of F₁, how DCCD inhibits the rotary dynamics of F₀F₁ remains elusive. Here, we carried out single-molecule rotation assays to characterize the DCCD inhibition of Escherichia coli F₀F₁. Upon the injection of DCCD, rotations irreversibly terminated with first order reaction kinetics, suggesting that the incorporation of a single DCCD moiety is sufficient to block the rotary catalysis of the F₀F₁. Individual molecules terminated at different angles relative to the three catalytic angles of F₁, suggesting that DCCD randomly reacts with one of the 10 c subunits. DCCD-inhibited F₀F₁ sometimes showed transient activation; molecules abruptly rotated and stopped after one revolution at the original termination angle, suggesting that hindrance by the DCCD moiety is released due to thermal fluctuation. To explore the mechanical activation of DCCD-inhibited molecules, we perturbed inhibited molecules using magnetic tweezers. The probability of transient activation increased upon a forward forcible rotation. Interestingly, during the termination F₀F₁, showed multiple positional shifts, which implies that F₁ stochastically changes the angular position of its rotor upon a catalytic reaction. This effect could be caused by balancing the angular positions of the F₁ and the F₀ rotors, which are connected via elastic elements.     

The Structural and Functional Connection between the Catalytic and Proton Translocating Sectors of the Mitochondrial F 1 F 0 -ATP Synthase

The structural and functional connection between the peripheral catalytic F₁ sector and theproton-translocating membrane sector F₀ of the mitochondrial ATP synthase is reviewed. Theobservations examined show that the N-terminus of subunit , the carboxy-terminal and centralregion of F₀I-PVP(b), OSCP, and part of subunit d constitute a continuous structure, the lateralstalk, which connects the peripheries of F₁ to F₀ and surrounds the central element of thestalk, constituted by subunits and . The ATPase inhibitor protein (IF₁) binds at one sideof the F₁F₀ connection. The carboxy-terminal segment of IF₁ apparently binds to OSCP. The42L-58K segment of IF₁, which is per se the most active domain of the protein, binds at thesurface of one of the three / pairs of F₁, thus preventing the cyclic interconversion of thecatalytic sites required for ATP hydrolysis.     

The F0F1-ATP Synthase Complex Contains Novel Subunits and Is Essential for Procyclic Trypanosoma brucei

The mitochondrial F₀F₁ ATP synthase is an essential multi-subunit protein complex in the vast majority of eukaryotes but little is known about its composition and role in Trypanosoma brucei, an early diverged eukaryotic pathogen. We purified the F₀F₁ ATP synthase by a combination of affinity purification, immunoprecipitation and blue-native gel electrophoresis and characterized its composition and function. We identified 22 proteins of which five are related to F₁ subunits, three to F₀ subunits, and 14 which have no obvious homology to proteins outside the kinetoplastids. RNAi silencing of expression of the F₁ α subunit or either of the two novel proteins showed that they are each essential for the viability of procyclic (insect stage) cells and are important for the structural integrity of the F₀F₁-ATP synthase complex. We also observed a dramatic decrease in ATP production by oxidative phosphorylation after silencing expression of each of these proteins while substrate phosphorylation was not severely affected. Our procyclic T. brucei cells were sensitive to the ATP synthase inhibitor oligomycin even in the presence of glucose contrary to earlier reports. Hence, the two novel proteins appear essential for the structural organization of the functional complex and regulation of mitochondrial energy generation in these organisms is more complicated than previously thought.     

Regulation of the F1F0-ATP Synthase Rotary Nanomotor in its Monomeric-Bacterial and Dimeric-Mitochondrial Forms

The F₁F₀-adenosine triphosphate (ATP) synthase rotational motor synthesizes most of the ATP required for living from adenosine diphosphate, Pi, and a proton electrochemical gradient across energy-transducing membranes of bacteria, chloroplasts, and mitochondria. However, as a reversible nanomotor, it also hydrolyzes ATP during de-energized conditions in all energy-transducing systems. Thus, different subunits and mechanisms have emerged in nature to control the intrinsic rotation of the enzyme to favor the ATP synthase activity over its opposite and commonly wasteful ATPase turnover. Recent advances in the structural analysis of the bacterial and mitochondrial ATP synthases are summarized to review the distribution and mechanism of the subunits that are part of the central rotor and regulate its gyration. In eubacteria, the ε subunit works as a ratchet to favor the rotation of the central stalk in the ATP synthase direction by extending and contracting two α-helixes of its C-terminal side and also by binding ATP with low affinity in thermophilic bacteria. On the other hand, in bovine heart mitochondria, the so-called inhibitor protein (IF₁) interferes with the intrinsic rotational mechanism of the central γ subunit and with the opening and closing of the catalytic β-subunits to inhibit its ATPase activity. Besides its inhibitory role, the IF₁ protein also promotes the dimerization of the bovine and rat mitochondrial enzymes, albeit it is not essential for dimerization of the yeast F₁F₀ mitochondrial complex. High-resolution electron microscopy of the dimeric enzyme in its bovine and yeast forms shows a conical shape that is compatible with the role of the ATP synthase dimer in the formation of tubular the cristae membrane of mitochondria after further oligomerization. Dimerization of the mitochondrial ATP synthase diminishes the rotational drag of the central rotor that would decrease the coupling efficiency between rotation of the central stalk and ATP synthesis taking place at the F₁ portion. In addition, F₁F₀ dimerization and its further oligomerization also increase the stability of the enzyme to natural or experimentally induced destabilizing conditions.     

How the Nucleus and Mitochondria Communicate in Energy Production During Stress: Nuclear MtATP6, an Early-Stress Responsive Gene, Regulates the Mitochondrial F1F0-ATP Synthase Complex

A small number of stress-responsive genes, such as those of the mitochondrial F₁F₀-ATP synthase complex, are encoded by both the nucleus and mitochondria. The regulatory mechanism of these joint products is mysterious. The expression of 6-kDa subunit (MtATP6), a relatively uncharacterized nucleus-encoded subunit of F₀ part, was measured during salinity stress in salt-tolerant and salt-sensitive cultivated wheat genotypes, as well as in the wild wheat genotypes, Triticum and Aegilops using qRT-PCR. The MtATP6 expression was suddenly induced 3 h after NaCl treatment in all genotypes, indicating an early inducible stress-responsive behavior. Promoter analysis showed that the MtATP6 promoter includes cis-acting elements such as ABRE, MYC, MYB, GTLs, and W-boxes, suggesting a role for this gene in abscisic acid-mediated signaling, energy metabolism, and stress response. It seems that 6-kDa subunit, as an early response gene and nuclear regulatory factor, translocates to mitochondria and completes the F₁F₀-ATP synthase complex to enhance ATP production and maintain ion homeostasis under stress conditions. These communications between nucleus and mitochondria are required for inducing mitochondrial responses to stress pathways. Dual targeting of 6-kDa subunit may comprise as a mean of inter-organelle communication and save energy for the cell. Interestingly, MtATP6 showed higher and longer expression in the salt-tolerant wheat and the wild genotypes compared to the salt-sensitive genotype. Apparently, salt-sensitive genotypes have lower ATP production efficiency and weaker energy management than wild genotypes; a stress tolerance mechanism that has not been transferred to cultivated genotypes.     

mtDNA T8993G Mutation-Induced F1F0-ATP Synthase Defect Augments Mitochondrial Dysfunction Associated with hypoxia/reoxygenation: The Protective Role of Melatonin

Background

F1F0-ATP synthase (F1F0-ATPase) plays important roles in regulating mitochondrial function during hypoxia, but the effect of F1F0-ATPase defect on hypoxia/reoxygenation (H/RO) is unknown. The aim of this study was to investigate how mtDNA T8993G mutation (NARP)-induced inhibition of F1F0-ATPase modulates the H/RO–induced mitochondrial dysfunction. In addition, the potential for melatonin, a potent antioxidant with multiple mitochondrial protective properties, to protect NARP cells exposed to H/RO was assessed.

Methods And Findings

NARP cybrids harboring 98% of mtDNA T8993G genes were established as an in vitro model for cells with F1F0-ATPase defect; their parental osteosarcoma 143B cells were studied for comparison. Treating the cells with H/RO using a hypoxic chamber resembles ischemia/reperfusion in vivo. NARP significantly enhanced apoptotic death upon H/RO detected by MTT assay and the trypan blue exclusion test of cell viability. Based on fluorescence probe-coupled laser scanning imaging microscopy, NARP significantly enhanced mitochondrial reactive oxygen species (mROS) formation and mitochondrial Ca²⁺ (mCa²⁺) accumulation in response to H/RO, which augmented the depletion of cardiolipin, resulting in the retardation of mitochondrial movement. With stronger H/RO stress (either with longer reoxygenation duration, longer hypoxia duration, or administrating secondary oxidative stress following H/RO), NARP augmented H/RO-induced mROS formation to significantly depolarize mitochondrial membrane potential (ΔΨm), and enhance mCa²⁺ accumulation and nitric oxide formation. Also, NARP augmented H/RO-induced mROS oxidized and depleted cardiolipin, thereby promoting permanent mitochondrial permeability transition, retarded mitochondrial movement, and enhanced apoptosis. Melatonin markedly reduced NARP-augmented H/RO-induced mROS formation and therefore significantly reduced mROS-mediated depolarization of ΔΨm and accumulation of mCa²⁺, stabilized cardiolipin, and then improved mitochondrial movement and cell survival.

Conclusion

NARP-induced inhibition of F1F0-ATPase enhances mROS formation upon H/RO, which augments the depletion of cardiolipin and retardation of mitochondrial movement. Melatonin may have the potential to rescue patients with ischemia/reperfusion insults, even those associated with NARP symptoms.     

Mitochondrial membrane potential is dependent on the oligomeric state of F1F0-ATP synthase supracomplexes

The F1F0-ATP synthase in mitochondria, in addition to its function in energy transduction, has a structural role in determining cristae morphology. This depends on its ability to form dimeric and higher oligomeric supracomplexes. Here we show that mutants of the dimer-specific subunits e and g, which destabilize dimeric and oligomeric F1F0-ATP synthase supracomplexes, have a decreased mitochondrial membrane potential delta psi. The degree of destabilization correlated with the reduction of the membrane potential. The enzymatic activities of F1F0-ATP synthase and cytochrome c oxidase, maximal respiration rate, coupling of oxidative phosphorylation, and tubular mitochondrial morphology were not affected or only to a minor extent. In mutants lacking one or two coiled-coil domains of subunit e, the reduction of the mitochondrial membrane potential was not due to loss of mitochondrial DNA, a reduced capacity of oxidative phosphorylation, or to altered cristae morphology. We propose a role for the supracomplexes of the F1F0-ATP synthase in organizing microdomains within the inner membrane, ensuring optimal bioenergetic competence of mitochondria.     

Mitochondrial F0F1 H+-ATP synthase. Characterization of F0 components involved in H+ translocation

The membrane F0 sector of mitochondrial ATP synthase complex was rapidly isolated by direct extraction with CHAPS from F1-depleted submitochondrial particles. The preparation thus obtained is stable and can be reconstituted in artificial phospholipid membranes to result in oligomycin-sensitive proton conduction, or recombined with purified F1 to give the oligomycin-sensitive F0F1-ATPase complex. The F0 preparation and constituent polypeptides were characterized by SDS-polyacrylamide gel electrophoresis and immunoblot analysis. The functional role of F0 polypeptides was examined by means of trypsin digestion and reconstitution studies. It is shown that, in addition to the 8 kDa DCCD-binding protein, the nuclear encoded protein [(1987) J. Mol. Biol. 197, 89-100], characterized as an intrinsic component of F0 (F0I, PVP protein [(1988) FEBS Lett. 237,9-14]) [corrected] is involved in H+ translocation and the sensitivity of this process to the F0 inhibitors, DCCD and oligomycin.     

Modulation at a Distance of Proton Conductance through the Saccharomyces cerevisiae Mitochondrial F1F0-ATP Synthase by Variants of the Oligomycin Sensitivity-Conferring Protein Containing Substitutions near the C-Terminus

We have sought to elucidate how the oligomycin sensitivity-conferring protein (OSCP) of the mitochondrial F₁F₀-ATP synthase (mtATPase) can influence proton channel function. Variants of OSCP, from the yeast Saccharomyces cerevisiae, having amino acid substitutions at a strictly conserved residue (Gly166) were expressed in place of normal OSCP. Cells expressing the OSCP variants were able to grow on nonfermentable substrates, albeit with some increase in generation time. Moreover, these strains exhibited increased sensitivity to oligomycin, suggestive of modification in functional interactions between the F₁ and F₀ sectors mediated by OSCP. Bioenergetic analysis of mitochondria from cells expressing OSCP variants indicated an increased respiratory rate under conditions of no net ATP synthesis. Using specific inhibitors of mtATPase, in conjunction with measurement of changes in mitochondrial transmembrane potential, it was revealed that this increased respiratory rate was a result of increased proton flux through the F₀ sector. This proton conductance, which is not coupled to phosphorylation, is exquisitely sensitive to inhibition by oligomycin. Nevertheless, the oxidative phosphorylation capacity of these mitochondria from cells expressing OSCP variants was no different to that of the control. These results suggest that the incorporation of OSCP variants into functional ATP synthase complexes can display effects in the control of proton flux through the F₀ sector, most likely mediated through altered protein—protein contacts within the enzyme complex. This conclusion is supported by data indicating impaired stability of solubilized mtATPase complexes that is not, however, reflected in the assembly of functional enzyme complexes in vivo. Given a location for OSCP atop the F₁-₃₃ hexamer that is distant from the proton channel, then the modulation of proton flux by OSCP must occur at a distance. We consider how subtle conformational changes in OSCP may be transmitted to F₀.     

Modulation of F0F1-ATP synthase activity by cyclophilin D regulates matrix adenine nucleotide levels

Cyclophilin D was recently shown to bind to and decrease the activity of F(0)F(1)-ATP synthase in submitochondrial particles and permeabilized mitochondria [Giorgio V et al. (2009) J Biol Chem, 284, 33982-33988]. Cyclophilin D binding decreased both ATP synthesis and hydrolysis rates. In the present study, we reaffirm these findings by demonstrating that, in intact mouse liver mitochondria energized by ATP, the absence of cyclophilin D or the presence of cyclosporin A led to a decrease in the extent of uncoupler-induced depolarization. Accordingly, in substrate-energized mitochondria, an increase in F(0)F(1)-ATP synthase activity mediated by a relief of inhibition by cyclophilin D was evident in the form of slightly increased respiration rates during arsenolysis. However, the modulation of F(0)F(1)-ATP synthase by cyclophilin D did not increase the adenine nucleotide translocase (ANT)-mediated ATP efflux rate in energized mitochondria or the ATP influx rate in de-energized mitochondria. The lack of an effect of cyclophilin D on the ANT-mediated adenine nucleotide exchange rate was attributed to the ～ 2.2-fold lower flux control coefficient of the F(0)F(1)-ATP synthase than that of ANT, as deduced from measurements of adenine nucleotide flux rates in intact mitochondria. These findings were further supported by a recent kinetic model of the mitochondrial phosphorylation system, suggesting that an ～ 30% change in F(0)F(1)-ATP synthase activity in fully energized or fully de-energized mitochondria affects the ADP-ATP exchange rate mediated by the ANT in the range 1.38-1.7%. We conclude that, in mitochondria exhibiting intact inner membranes, the absence of cyclophilin D or the inhibition of its binding to F(0)F(1)-ATP synthase by cyclosporin A will affect only matrix adenine nucleotides levels.     

The molecular mechanism of ATP synthesis by F1F0-ATP synthase

ATP synthesis by oxidative phosphorylation and photophosphorylation, catalyzed by F1F0-ATP synthase, is the fundamental means of cell energy production. Earlier mutagenesis studies had gone some way to describing the mechanism. More recently, several X-ray structures at atomic resolution have pictured the catalytic sites, and real-time video recordings of subunit rotation have left no doubt of the nature of energy coupling between the transmembrane proton gradient and the catalytic sites in this extraordinary molecular motor. Nonetheless, the molecular events that are required to accomplish the chemical synthesis of ATP remain undefined. In this review we summarize current state of knowledge and present a hypothesis for the molecular mechanism of ATP synthesis.     

Sulfite inhibits the F1F0-ATP synthase and activates the F1F0-ATPase of Paracoccus denitrificans

The F₁F₀ complex of Paracoccus denitrificans (PdF₁F₀) is the fastest ATP synthase but the slowest ATPase. Sulfite exerts maximal activation of the PdF₁F₀-ATPase (Pacheco-Moisés, F., García, J. J., Rodríguez-Zavala, J. S., and Moreno-Sánchez, R. (2000). Eur. J. Biochem. 267, 993–1000) but its effect on the PdF₁F₀-ATP synthase activity remains unknown. Therefore, we studied the effect of sulfite on ATP synthesis and ³²Pi ATP exchange reactions of inside-out membrane vesicles of P. denitrificans. Sulfite inhibited both reactions under conditions of maximal pH and normal sensitivity to dicyclohexylcarbodiimide. Sulfite increased by 10- and 5-fold the K _0.5 for Mg²⁺-ADP and Pi during ATP synthesis, respectively, and by 4-fold the IC₅₀ of Mg²⁺-ADP for inhibition of the PdF₁F₀-ATPase activity. Thus, sulfite exerts opposite effects on the forward and reverse functioning of the PdF₁F₀ complex. These effects are not due to membrane or PdF₁F₀ uncoupling. Kinetic and structural modifications that could account for these results are discussed.     

ATP synthesis driven by proton transport in F1F0-ATP synthase

Topical questions in ATP synthase research are: (1) how do protons cause subunit rotation and how does rotation generate ATP synthesis from ADP+Pi? (2) How does hydrolysis of ATP generate subunit rotation and how does rotation bring about uphill transport of protons? The finding that ATP synthase is not just an enzyme but rather a unique nanomotor is attracting a diverse group of researchers keen to find answers. Here we review the most recent work on rapidly developing areas within the field and present proposals for enzymatic and mechanoenzymatic mechanisms.     

The b Subunits in the Peripheral Stalk of F1F0 ATP Synthase Preferentially Adopt an Offset Relationship

The peripheral stalk of F₁F₀ ATP synthase is essential for the binding of F₁ to F_O and for proper transfer of energy between the two sectors of the enzyme. The peripheral stalk of Escherichia coli is composed of a dimer of identical b subunits. In contrast, photosynthetic organisms express two b-like genes that form a heterodimeric peripheral stalk. Previously we generated chimeric peripheral stalks in which a portion of the tether and dimerization domains of the E. coli b subunits were replaced with homologous sequences from the b and b′ subunits of Thermosynechococcus elongatus (Claggett, S. B., Grabar, T. B., Dunn, S. D., and Cain, B. D. (2007) J. Bacteriol. 189, 5463–5471). The spatial arrangement of the chimeric b and b′ subunits, abbreviated Tb and Tb′, has been investigated by Cu²⁺-mediated disulfide cross-link formation. Disulfide formation was studied both in soluble model polypeptides and between full-length subunits within intact functional F₁F₀ ATP synthase complexes. In both cases, disulfides were preferentially formed between Tb_A83C and Tb′_A90C, indicating the existence of a staggered relationship between helices of the two chimeric subunits. Even under stringent conditions rapid formation of disulfides between these positions occurred. Importantly, formation of this cross-link had no detectable effect on ATP-driven proton pumping, indicating that the staggered conformation is compatible with normal enzymatic activity. Under less stringent reaction conditions, it was also possible to detect b subunits cross-linked through identical positions, suggesting that an in-register, nonstaggered parallel conformation may also exist.F₁F₀ ATP synthases are found in the inner mitochondrial membrane, the thylakoid membrane of chloroplasts, and the cytoplasmic membrane of bacteria (1–4). These enzymes are responsible for harnessing an electrochemical gradient of protons across the membranes for the synthesis of ATP. In Escherichia coli F₁F₀ ATP synthase, the membrane-embedded F₀ sector is composed of subunits ab₂c₁₀ and a soluble F₁ portion composed of subunits α₃β₃γδϵ. The F₀ sector houses a proton channel located principally in the a subunit, and the flow of protons through F₀ generates torque used to rotate the c₁₀ subunit ring relative to the ab₂ subunits. The F₁ γ and ϵ subunits are bound to the c₁₀ ring and form the central or rotor stalk. Catalytic sites are located at the interfaces of each αβ pair in F₁. The γ subunit extends into the center of the α₃β₃ hexamer, creating an asymmetry in the conformations of the αβ pairs (5). It is the rotation of the γ subunit and the resulting sequential conformational changes in each αβ pair that provides the driving force for the synthesis of ATP at the catalytic sites. The α₃β₃ hexamer is held stationary relative to the rotary stalk by the peripheral stalk consisting of the b₂δ subunits.The peripheral stalk is essential for binding F₁ to F₀ and for coupling proton translocation to catalytic activity (6–8). In the E. coli enzyme, the peripheral stalk is a dimer of identical b subunits. The stalk has been conceptually divided into functional domains called the membrane domain (b_M1-I33), the tether domain (b_E34-A61), the dimerization domain (b_T62-K122), and the F₁-binding domain (b_Q123-L156) (9). Although there is ample evidence of direct protein-protein interactions between b subunits within the membrane, dimerization, and F₁-binding domains, there is remarkably little evidence of tight packing between the b subunits in the tether domain. In fact, electron spin resonance studies suggested that the tether domains of the two b subunits may be separated by more than 20 Å in the F₁F₀ complex (10, 11). Much of what is known about the structure of the stalk has been inferred from analysis of the properties of polypeptides modeling segments of the b subunit. The structure of a peptide modeling the membrane domain, b_M1-E34, has been determined by NMR (12), and a peptide based on the dimerization domain, b_T62-K122, has been determined by x-ray diffraction (13). Both polypeptides assumed α-helical conformations, but neither structure directly revealed b subunit dimerization interactions. Recently, Priya et al. (14) reported a low resolution structure of a b_M22-L156 dimer, but the extended conformation appears to be slightly too long to accurately reflect the dimensions of the peripheral stalk within the F₁F₀ complex.Molecular modeling efforts supported by a variety of biochemical and biophysical experiments have yielded competing right-handed coiled coil (15, 16) and left-handed coiled coil (17, 18) models for the peripheral stalk. The parallel two-stranded left-handed coiled coli is a well known structure characterized by knobs-into-holes packing of the side chains of the two helices that are aligned in-register. An in-register conformation implies that any specific amino acid on the b subunit-subunit interface would occupy a position immediately adjacent to its counterpart in the other b subunit. In contrast, del Rizzo et al. (16) proposed a novel parallel right-handed coiled coil with the helices of the two b subunits offset by approximately one and a half turns of an α helix. This staggered model positions the two identical residues contributed by each of the b subunits in a homodimer into differing environments and at considerable distance from one another. Sequence analyses have been offered in support of both models (16, 18). In terms of experimental support, cross-linking studies of polypeptides have provided evidence that dimer packing could be in-register at many sites starting from residue Ala⁵⁹ and continuing to the carboxyl termini in model b_Y24–L156 dimers and in b_D53–K122 dimers (9, 16). Cross-linking at a few of the carboxyl-proximal positions has been confirmed within intact F₁F₀ ATP synthase complexes (19, 20). Recent electron spin resonance distance measurements on b_24–156 have also been interpreted as support for the in-register arrangement (17, 18). Conversely, work with polypeptides modeling the b subunit has generated evidence favoring a staggered conformation in this section of the dimer (15, 16, 21). In mixtures of dimerization domain polypeptides with cysteines incorporated at different sites, disulfides preferentially formed between positions that were 4–7 residues apart. For example, b_D53–L156 dimers were covalently locked into the offset conformation by the formation of disulfide bridges between cysteines introduced at positions b_A79 and b_R83 as well as b_R83 and b_A90 (15). These staggered dimers were more stable, melted with higher cooperativity, and bound soluble F₁ with higher affinity than b_D53–L156 dimers fixed in the in-register arrangement. Moreover, active and coupled F₁F₀ complexes were assembled with heterodimeric peripheral stalks using b subunits with tether domains varying in length by as many as 14 amino acids (22). These F₁F₀ complexes had peripheral stalks that were by definition out of register, at least within the tether domain.In contrast to the homodimer of identical b subunits observed in the peripheral stalk of E. coli, photosynthetic organisms express two b-like subunits, b and b′, that are thought to form heterodimeric peripheral stalks in F₁F₀ ATP synthase. Previously, we generated heterodimeric peripheral stalks within the E. coli F₁F₀ by constructing chimeric b subunits (23). Segments of the tether and dimerization domains of the E. coli b subunit were replaced with the homologous regions of the Thermosynechococcus elongatus b and b′ subunits. The chimeric subunits formed heterodimeric peripheral stalks that were incorporated into intact, functional F₁F₀ ATP synthase complexes. The most active chimeric enzymes had T. elongatus primary sequences replacing residues b_E39–I86 of the E. coli b subunit. For simplicity, these chimeric subunits will be referred to here as Tb and Tb′.The ability to generate F₁F₀ ATP synthases with Tb/Tb′ heterodimeric peripheral stalks provided a means to investigate the positions of the two subunits in the peripheral stalk. In the present work, we show that the Tb and Tb′ subunits assumed preferred positions relative to one another within the F₁F₀ complex. The staggered conformation appears to be a favored and functional conformation for the peripheral stalk. However, within a population of F₁F₀ complexes, some complexes with peripheral stalks in the in-register conformation are likely to exist.     

Subunit movement during catalysis by F1-F0-ATP synthases

The catalytic portion (F₁) of ATP synthases have the subunit composition ₃, ₃, , , . This composition imparts structural asymmetry to the entire complex that results in differences in nucleotide binding affinity among the six binding sites. Evidence that two or more sites participate in catalysis, alternating their properties, led to the notion that the interactions of individual pairs with the small subunits must change as binding site properties alternate. A rotation of the subunit within the ₃₃ hexamer has been proposed as a means of alternating the properties of catalytic sites. Evidence argues that the rotation of the complete subunit during ATP hydrolysis is not mandatory for activity. The subunit of chloroplast F₁ may be cleaved into three large fragments that remain bound to F₁. This cleavage enhances ATPase activity without loss of evidence of site-site interactions. Complexes of ₃₃ have been shown to have significant ATPase activity in the absence of . Mg²⁺ATP affects the interaction of with the different subunits, and induces other changes in F₁, but whether these changes are induced by catalysis, or are fast enough to be involved in the catalytic turnover of the enzyme has not been established. Likewise, changes in structure and in binding site properties induced in thylakoid membrane bound CF₁ by formation of an electrochemical proton gradient may activate the enzyme rather than be apart of catalysis. Mechanisms other than rotary catalysis should be considered.     

Identification of F0 subunits in the rat liver mitochondrial F0F1-ATP synthase.

In order to identify the subunits constituting the rat liver F0F1-ATP synthase, the complex prepared by selective extraction from the mitochondrial membranes with a detergent followed by purification on a sucrose gradient has been compared to that obtained by immunoprecipitation with an anti-F1 serum. The subunits present in both preparations that are assumed to be authentic components of the complex have been identified. The results show that the total rat liver F0F1-ATP synthase contains at least 13 different proteins, seven of which can be attributed to F0. The following F0 subunits have been identified: the subunit b (migrating as a 24 kDa band in SDS-PAGE), the oligomycin-sensitivity-conferring protein (20 kDa), and F6 (9 kDa) that have N-terminal sequences homologous to the beef-heart ones; the mtDNA encoded subunits 6 (20 kDa) and 8 (less than 7 kDa) that can be synthesized in isolated mitochondria; an additional 20 kDa protein that could be equivalent to the beef heart subunit d.