Inhibition of ATP hydrolysis by thermoalkaliphilic F1Fo-ATP synthase is controlled by the C terminus of the epsilon subunit

The F(1)F(o)-ATP synthases of alkaliphilic bacteria exhibit latent ATPase activity, and for the thermoalkaliphile Bacillus sp. strain TA2.A1, this activity is intrinsic to the F(1) moiety. To study the mechanism of ATPase inhibition, we developed a heterologous expression system in Escherichia coli to produce TA2F(1) complexes from this thermoalkaliphile. Like the native F(1)F(o)-ATP synthase, the recombinant TA2F(1) was blocked in ATP hydrolysis activity, and this activity was stimulated by the detergent lauryldimethylamine oxide. To determine if the C-terminal domain of the epsilon subunit acts as an inhibitor of ATPase activity and if an electrostatic interaction plays a role, a TA2F(1) mutant with either a truncated epsilon subunit [i.e., TA2F(1)(epsilon(DeltaC))] or substitution of basic residues in the second alpha-helix of epsilon with nonpolar alanines [i.e., TA2F(1)(epsilon(6A))] was constructed. Both mutants showed ATP hydrolysis activity at low and high concentrations of ATP. Treatment of the purified F(1)F(o)-ATP synthase and TA2F(1)(epsilon(WT)) complex with proteases revealed that the epsilon subunit was resistant to proteolytic digestion. In contrast, the epsilon subunit of TA2F(1)(epsilon(6A)) was completely degraded by trypsin, indicating that the C-terminal arm was in a conformation where it was no longer protected from proteolytic digestion. In addition, ATPase activity was not further activated by protease treatment when compared to the untreated control, supporting the observation that epsilon was responsible for inhibition of ATPase activity. To study the effect of the alanine substitutions in the epsilon subunit in the entire holoenzyme, we reconstituted recombinant TA2F(1) complexes with F(1)-stripped native membranes of strain TA2.A1. The reconstituted TA2F(o)F(1)(epsilon(WT)) was blocked in ATP hydrolysis and exhibited low levels of ATP-driven proton pumping consistent with the F(1)F(o)-ATP synthase in native membranes. Reconstituted TA2F(o)F(1)(epsilon(6A)) exhibited ATPase activity that correlated with increased ATP-driven proton pumping, confirming that the epsilon subunit also inhibits ATPase activity of TA2F(o)F(1).     

Genetic fusions of globular proteins to the epsilon subunit of the Escherichia coli ATP synthase: Implications for in vivo rotational catalysis and epsilon subunit function

The rotational mechanism of ATP synthase was investigated by fusing three proteins from Escherichia coli, the 12-kDa soluble cytochrome b(562), the 20-kDa flavodoxin, and the 28-kDa flavodoxin reductase, to the C terminus of the epsilon subunit of the enzyme. According to the concept of rotational catalysis, because epsilon is part of the rotor a large domain added at this site should sterically clash with the second stalk, blocking rotation and fully inhibiting the enzyme. E. coli cells expressing the cytochrome b(562) fusion in place of wild-type epsilon grew using acetate as the energy source, indicating their capacity for oxidative phosphorylation. Cells expressing the larger flavodoxin or flavodoxin reductase fusions failed to grow on acetate. Immunoblot analysis showed that the fusion proteins were stable in the cells and that they had no effect on enzyme assembly. These results provide initial evidence supporting rotational catalysis in vivo. In membrane vesicles, the cytochrome b(562) fusion caused an increase in the apparent ATPase activity but a minor decrease in proton pumping. Vesicles bearing ATP synthase containing the larger fusion proteins showed reduced but significant levels of ATPase activity that was sensitive to inhibition by dicyclohexylcarbodiimide (DCCD) but no proton pumping. Thus, all fusions to epsilon generated an uncoupled component of ATPase activity. These results imply that a function of the C terminus of epsilon in F(1)F(0) is to increase the efficiency of the enzyme by specifically preventing the uncoupled hydrolysis of ATP. Given the sensitivity to DCCD, this uncoupled ATP hydrolysis may arise from rotational steps of gammaepsilon in the inappropriate direction after ATP is bound at the catalytic site. It is proposed that the C-terminal domain of epsilon functions to ensure that rotation occurs only in the direction of ATP synthesis when ADP is bound and only in the direction of hydrolysis when ATP is bound.     

The role of subunit epsilon in the catalysis and regulation of FOF1-ATP synthase

The regulation of ATP synthase activity is complex and involves several distinct mechanisms. In bacteria and chloroplasts, subunit epsilon plays an important role in this regulation, (i) affecting the efficiency of coupling, (ii) influencing the catalytic pathway, and (iii) selectively inhibiting ATP hydrolysis activity. Several experimental studies indicate that the regulation is achieved through large conformational transitions of the alpha-helical C-terminal domain of subunit epsilon that occur in response to membrane energization, change in ATP/ADP ratio or addition of inhibitors. This review summarizes the experimental data obtained on different organisms that clarify some basic features as well as some molecular details of this regulatory mechanism. Multiple functions of subunit epsilon, its role in the difference between the catalytic pathways of ATP synthesis and hydrolysis and its influence on the inhibition of ATP hydrolysis by ADP are also discussed.     

Real-time monitoring of conformational dynamics of the epsilon subunit in F1-ATPase

It has been proposed that C-terminal two alpha-helices of the epsilon subunit of F1-ATPase can undergo conformational transition between retracted folded-hairpin form and extended form. Here, using F(1) from thermophilic Bacillus PS3, we monitored this transition in real time by fluorescence resonance energy transfer (FRET) between a donor dye and an acceptor dye attached to N terminus of the gamma subunit and C terminus of the epsilon subunit, respectively. High FRET (extended form) of F1 turned to low FRET (retracted form) by ATP, which then reverted as ATP was hydrolyzed to ADP. 5'-Adenyl-beta,gamma-imidodiphosphate, ADP + AlF4-, ADP + NaN3, and GTP also caused the retracted form, indicating that ATP binding to the catalytic beta subunits induces the transition. The ATP-induced transition from high FRET to low FRET occurred in a similar time scale to the ATP-induced activation of ATPase from inhibition by the epsilon subunit, although detailed kinetics were not the same. The transition became faster as temperature increased, but the extrapolated rate at 65 degrees C (physiological temperature of Bacillus PS3) was still too slow to assign the transition as an obligate step in the catalytic turnover. Furthermore, binding affinity of ATP to the isolated epsilon subunit was weakened as temperature increased, and the dissociation constant extrapolated to 65 degrees C reached to 0.67 mm, a consistent value to assume that the epsilon subunit acts as a sensor of ATP concentration in the cell.     

Met23Lys mutation in subunit gamma of F(O)F(1)-ATP synthase from Rhodobacter capsulatus impairs the activation of ATP hydrolysis by protonmotive force

H(+)-F(O)F(1)-ATP synthase couples proton flow through its membrane portion, F(O), to the synthesis of ATP in its headpiece, F(1). Upon reversal of the reaction the enzyme functions as a proton pumping ATPase. Even in the simplest bacterial enzyme the ATPase activity is regulated by several mechanisms, involving inhibition by MgADP, conformational transitions of the epsilon subunit, and activation by protonmotive force. Here we report that the Met23Lys mutation in the gamma subunit of the Rhodobacter capsulatus ATP synthase significantly impaired the activation of ATP hydrolysis by protonmotive force. The impairment in the mutant was due to faster enzyme deactivation that was particularly evident at low ATP/ADP ratio. We suggest that the electrostatic interaction of the introduced gammaLys23 with the DELSEED region of subunit beta stabilized the ADP-inhibited state of the enzyme by hindering the rotation of subunit gamma rotation which is necessary for the activation.     

Epsilon subunit of Escherichia coli F1-ATPase: effects on affinity for aurovertin and inhibition of product release in unisite ATP hydrolysis

The epsilon subunit of Escherichia coli F1-ATPase is a tightly bound but dissociable partial inhibitor of ATPase activity. The effects of epsilon on the enzyme were investigated by comparing the ATPase activity and aurovertin binding properties of the epsilon-depleted F1-ATPase and the epsilon-replete complex. Kinetic data of multisite ATP hydrolysis were analyzed to give the best fit for one, two, or three kinetic components. Each form of F1-ATPase contained a high-affinity component, with a Km near 20 microM and a velocity of approximately 1 unit/mg. Each also exhibited a component with a Km in the range of 0.2 mM. The velocity of this component was 25 units/mg for epsilon-depleted ATPase but only 4 units/mg for epsilon-replete enzyme. The epsilon-depleted enzyme also contained a very low affinity component not present in the epsilon-replete enzyme. In unisite hydrolysis studies, epsilon had no effect on the equilibrium between substrate ATP and product ADP.P1 at the active site but reduced the rate of product release 15-fold. These results suggest that epsilon subunit slows a conformational change that is required to reduce the affinity at the active site, allowing dissociation of product. It is suggested that inhibition of multisite hydrolysis by epsilon is also due to a reduced rate of product release. epsilon-depleted F1-ATPase showed little of no modulation of aurovertin fluorescence by added ADP and ATP. Aurovertin fluorescence titrations in buffer containing ethylenediaminetetraacetic acid (EDTA) revealed that epsilon-depleted enzyme had high affinity for aurovertin (Kd less than 0.1 microM) regardless of the presence of nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)     

The epsilon subunit of the F(1)F(0) complex of Escherichia coli. cross-linking studies show the same structure in situ as when isolated.

Four double mutants in the epsilon subunit were generated, each containing two cysteines, which, based on the NMR structure of this subunit, should form internal disulfide bonds. Two of these were designed to generate interdomain cross-links that lock the C-terminal alpha-helical domain against the beta-sandwich (epsilonM49C/A126C and epsilonF61C/V130C). The second set should give cross-linking between the two C-terminal alpha-helices (epsilonA94C/L128C and epsilonA101C/L121C). All four mutants cross-linked with 90-100% efficiency upon CuCl(2) treatment in isolated Escherichia coli ATP synthase. This shows that the structure obtained for isolated epsilon is essentially the same as in the assembled complex. Functional studies revealed increased ATP hydrolysis after cross-linking between the two domains of the subunit but not after cross-linking between the C-terminal alpha-helices. None of the cross-links had any effect on proton pumping-coupled ATP hydrolysis, on DCCD sensitivity of this activity, or on ATP synthesis rates. Therefore, big conformational changes within epsilon can be ruled out as a part of the enzyme function. Protease digestion studies, however, showed that subtle changes do occur, since the epsilon subunit could be locked in an ADP or 5'-adenylyl-beta,gamma-imidodiphosphate conformation by the cross-linking with resulting differences in cleavage rates.     

The regulator of the F1 motor: inhibition of rotation of cyanobacterial F1-ATPase by the epsilon subunit

The chloroplast-type F(1) ATPase is the key enzyme of energy conversion in chloroplasts, and is regulated by the endogenous inhibitor epsilon, tightly bound ADP, the membrane potential and the redox state of the gamma subunit. In order to understand the molecular mechanism of epsilon inhibition, we constructed an expression system for the alpha(3)beta(3)gamma subcomplex in thermophilic cyanobacteria allowing thorough investigation of epsilon inhibition. epsilon Inhibition was found to be ATP-independent, and different to that observed for bacterial F(1)-ATPase. The role of the additional region on the gamma subunit of chloroplast-type F(1)-ATPase in epsilon inhibition was also determined. By single molecule rotation analysis, we succeeded in assigning the pausing angular position of gamma in epsilon inhibition, which was found to be identical to that observed for ATP hydrolysis, product release and ADP inhibition, but distinctly different from the waiting position for ATP binding. These results suggest that the epsilon subunit of chloroplast-type ATP synthase plays an important regulator for the rotary motor enzyme, thus preventing wasteful ATP hydrolysis.     

Epsilon subunit, an endogenous inhibitor of bacterial F(1)-ATPase, also inhibits F(0)F(1)-ATPase

Since the report by Sternweis and Smith (Sternweis, P. C., and Smith, J. B. (1980) Biochemistry 19, 526-531), the epsilon subunit, an endogenous inhibitor of bacterial F(1)-ATPase, has long been thought not to inhibit activity of the holo-enzyme, F(0)F(1)-ATPase. However, we report here that the epsilon subunit is exerting inhibition in F(0)F(1)-ATPase. We prepared a C-terminal half-truncated epsilon subunit (epsilon(DeltaC)) of the thermophilic Bacillus PS3 F(0)F(1)-ATPase and reconstituted F(1)- and F(0)F(1)-ATPase containing epsilon(DeltaC). Compared with F(1)- and F(0)F(1)-ATPase containing intact epsilon, those containing epsilon(DeltaC) showed uninhibited activity; severalfold higher rate of ATP hydrolysis at low ATP concentration and the start of ATP hydrolysis without an initial lag at high ATP concentration. The F(0)F(1)-ATPase containing epsilon(DeltaC) was capable of ATP-driven H(+) pumping. The time-course of pumping at low ATP concentration was faster than that by the F(0)F(1)-ATPase containing intact epsilon. Thus, the comparison with noninhibitory epsilon(DeltaC) mutant shed light on the inhibitory role of the intact epsilon subunit in F(0)F(1)-ATPase.     

The C-terminal domain of the epsilon subunit of the chloroplast ATP synthase is not required for ATP synthesis

The epsilon subunit of the ATP synthases from chloroplasts and Escherichia coli regulates the activity of the enzyme and is required for ATP synthesis. The epsilon subunit is not required for the binding of the catalytic portion of the chloroplast ATP synthase (CF1) to the membrane-embedded part (CFo). Thylakoid membranes reconstituted with CF1 lacking its epsilon subunit (CF1-epsilon) have high ATPase activity and no ATP synthesis activity, at least in part because the membranes are very leaky to protons. Either native or recombinant epsilon subunit inhibits ATPase activity and restores low proton permeability and ATP synthesis. In this paper we show that recombinant epsilon subunit from which 45 amino acids were deleted from the C-terminus is as active as full-length epsilon subunit in restoring ATP synthesis to membranes containing CF1-epsilon. However, the truncated form of the epsilon subunit was significantly less effective as an inhibitor of the ATPase activity of CF1-epsilon, both in solution and bound to thylakoid membranes. Thus, the C-terminus of the epsilon subunit is more involved in regulation of activity, by inhibiting ATP hydrolysis, than in ATP synthesis.     

Role of the epsilon subunit of thermophilic F1-ATPase as a sensor for ATP

The epsilon subunit of F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) has been shown to bind ATP. The precise nature of the regulatory role of ATP binding to the epsilon subunit remains to be determined. To address this question, 11 mutants of the epsilon subunit were prepared, in which one of the basic or acidic residues was substituted with alanine. ATP binding to these mutants was tested by gel-filtration chromatography. Among them, four mutants that showed no ATP binding were selected and reconstituted with the alpha(3)beta(3)gamma complex of TF(1). The ATPase activity of the resulting alpha(3)beta(3)gammaepsilon complexes was measured, and the extent of inhibition by the mutant epsilon subunits was compared in each case. With one exception, weaker binding of ATP correlated with greater inhibition of ATPase activity. These results clearly indicate that ATP binding to the epsilon subunit plays a regulatory role and that ATP binding may stabilize the ATPase-active form of TF(1) by fixing the epsilon subunit into the folded conformation.     

F0F1-ATPase/synthase is geared to the synthesis mode by conformational rearrangement of epsilon subunit in response to proton motive force and ADP/ATP balance

The epsilon subunit in F0F1-ATPase/synthase undergoes drastic conformational rearrangement, which involves the transition of two C-terminal helices between a hairpin "down"-state and an extended "up"-state, and the enzyme with the up-fixed epsilon cannot catalyze ATP hydrolysis but can catalyze ATP synthesis (Tsunoda, S. P., Rodgers, A. J. W., Aggeler, R., Wilce, M. C. J., Yoshida, M., and Capaldi, R. A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 6560-6564). Here, using cross-linking between introduced cysteine residues as a probe, we have investigated the causes of the transition. Our findings are as follows. (i) In the up-state, the two helices of epsilon are fully extended to insert the C terminus into a deeper position in the central cavity of F1 than was thought previously. (ii) Without a nucleotide, epsilon is in the up-state. ATP induces the transition to the down-state, and ADP counteracts the action of ATP. (iii) Conversely, the enzyme with the down-state epsilon can bind an ATP analogue, 2',3'-O-(2,4,6-trinitrophenyl)-ATP, much faster than the enzyme with the up-state epsilon. (iv) Proton motive force stabilizes the up-state. Thus, responding to the increase of proton motive force and ADP, F0F1-ATPase/synthase would transform the epsilon subunit into the up-state conformation and change gear to the mode for ATP synthesis.     

The gammaepsilon-c subunit interface in the ATP synthase of Escherichia coli. cross-linking of the epsilon subunit to the c subunit ring does not impair enzyme function, that of gamma to c subunits leads to uncoupling

Mutants with a cysteine residue in the gamma subunit at position 207 and the epsilon subunit at position 31 were expressed in combination with a c-dimer construct, which contains a single cysteine at position 42 of the second c subunit. These mutants are called gammaY207C/cc'Q42C and epsilonE31C/cc'Q42C, respectively. Cross-linking of epsilon to the c subunit ring was obtained almost to completion without significant effect on any enzyme function, i.e. ATP hydrolysis, ATP synthesis, and ATP hydrolysis-driven proton translocation were all close to that of wild type. The gamma subunit could also be linked to the c subunit ring in more than 90% yield, but this affected coupling. Thus, ATP hydrolysis was increased 2. 5-fold, ATP synthesis was dramatically decreased, and ATP hydrolysis-driven proton translocation was abolished, as measured by the 9-amino-6-chloro-2-methoxyacridinequenching method. These results for epsilonE31C/cc'Q42C indicate that the c subunit ring rotates with the central stalk element. That the gamma-epsilon cross-linked enzyme retains ATPase activity also argues for a gammaepsilon-c subunit rotor. However, the uncoupling induced by cross-linking of gamma to the c subunit ring points to important conformational changes taking place in the gammaepsilon-c subunit interface during this. Blocking these structural changes by cross-linking leads to a proton leak within the F(0).     

Regulation of the F0F1-ATP synthase: the conformation of subunit epsilon might be determined by directionality of subunit gamma rotation

F(0)F(1)-ATP synthase couples ATP synthesis/hydrolysis with transmembrane proton transport. The catalytic mechanism involves rotation of the gamma epsilon c(approximately 10)-subunits complex relative to the rest of the enzyme. In the absence of protonmotive force the enzyme is inactivated by the tight binding of MgADP. Subunit epsilon also modulates the activity: its conformation can change from a contracted to extended form with C-terminus stretched towards F(1). The latter form inhibits ATP hydrolysis (but not synthesis). We propose that the directionality of the coiled-coil subunit gamma rotation determines whether subunit epsilon is in contracted or extended form. Block of rotation by MgADP presumably induces the extended conformation of subunit epsilon. This conformation might serve as a safety lock, stabilizing the ADP-inhibited state upon de-energization and preventing spontaneous re-activation and wasteful ATP hydrolysis. The hypothesis merges the known regulatory effects of ADP, protonmotive force and conformational changes of subunit epsilon into a consistent picture.     

The role of the epsilon subunit in the Escherichia coli ATP synthase. The C-terminal domain is required for efficient energy coupling

The role of the C-domain of the epsilon subunit of ATP synthase was investigated by fusing either the 20-kDa flavodoxin (Fd) or the 5-kDa chitin binding domain (CBD) to the N termini of both full-length epsilon and a truncation mutant epsilon(88-stop). All mutant epsilon proteins were stable in cells and supported F1F0 assembly. Cells expressing the Fd-epsilon or Fd-epsilon(88-stop) mutants were unable to grow on acetate minimal medium, indicating their inability to carry out oxidative phosphorylation because of steric blockage of rotation. The other forms of epsilon supported growth on acetate. Membrane vesicles containing Fd-epsilon showed 23% of the wild type ATPase activity but no proton pumping, suggesting that the ATP synthase is intrinsically partially uncoupled. Vesicles containing CBD-epsilon were indistinguishable from the wild type in ATPase activity and proton pumping, indicating that the N-terminal fusions alone do not promote uncoupling. Fd-epsilon(88-stop) caused higher rates of uncoupled ATP hydrolysis than Fd-epsilon, and epsilon(88-stop) showed an increased rate of membrane-bound ATP hydrolysis but decreased proton pumping relative to the wild type. Both results demonstrate the role of the C-domain in coupling. Analysis of the wild type and epsilon(88-stop) mutant membrane ATPase activities at concentrations of ATP from 50 mum to 8 mm showed no significant dependence of the ratio of bound/released ATPase activity on ATP concentration. These results support the hypothesis that the main function of the C-domain in the Escherichia coli epsilon subunit is to reduce uncoupled ATPase activity, rather than to regulate coupled activity.     

ATP Synthase of Chloroplast Thylakoid Membranes: A More in Depth Characterization of Its ATPase Activity

In contrast to everted mitochondrial inner membrane vesicles and eubacterial plasma membrane vesicles, the ATPase activity of chloroplast ATP synthase in thylakoid membranes is extremely low. Several treatments of thylakoids that unmask ATPase activity are known. Illumination of thylakoids that contain reduced ATP synthase (reduced thylakoids) promotes the hydrolysis of ATP in the dark. Incubation of thylakoids with trypsin can also elicit higher rates of ATPase activity. In this paper the properties of the ATPase activity of the ATP synthase in thylakoids treated with trypsin are compared with those of the ATPase activity in reduced thylakoids. The trypsin-treated membranes have significant ATPase activity in the presence of Ca²⁺, whereas the Ca²⁺-ATPase activity of reduced thylakoids is very low. The Mg²⁺-ATPase activity of the trypsinized thylakoids was only partially inhibited by the uncouplers, at concentrations that fully inhibit the ATPase activity of reduced membranes. Incubation of reduced thylakoids with ADP in Tris buffer prior to assay abolishes Mg²⁺-ATPase activity. The Mg²⁺-ATPase activity of trypsin-treated thylakoids was unaffected by incubation with ADP. Trypsin-treated membranes can make ATP at rates that are 75–80% of those of untreated thylakoids. The Mg²⁺-ATPase activity of trypsin-treated thylakoids is coupled to inward proton translocation and 10 mM sulfite stimulates both proton uptake and ATP hydrolysis. It is concluded that cleavage of the γ subunit of the ATP synthase by trypsin prevents inhibition of ATPase activity by the ε subunit, but only partially overcomes inhibition by Mg²⁺ and ADP during assay.     

Met23Lys mutation in subunit gamma of FOF1-ATP synthase from Rhodobacter capsulatus impairs the activation of ATP hydrolysis by protonmotive force

H⁺-F_OF₁-ATP synthase couples proton flow through its membrane portion, F_O, to the synthesis of ATP in its headpiece, F₁. Upon reversal of the reaction the enzyme functions as a proton pumping ATPase. Even in the simplest bacterial enzyme the ATPase activity is regulated by several mechanisms, involving inhibition by MgADP, conformational transitions of the ε subunit, and activation by protonmotive force. Here we report that the Met23Lys mutation in the γ subunit of the Rhodobacter capsulatus ATP synthase significantly impaired the activation of ATP hydrolysis by protonmotive force. The impairment in the mutant was due to faster enzyme deactivation that was particularly evident at low ATP/ADP ratio. We suggest that the electrostatic interaction of the introduced γLys23 with the DELSEED region of subunit β stabilized the ADP-inhibited state of the enzyme by hindering the rotation of subunit γ rotation which is necessary for the activation.     

Effects of sequential deletions of residues from the N- or C-terminus on the functions of epsilon subunit of the chloroplast ATP synthase

Ten truncated mutants of chloroplast ATP synthase epsilon subunit from spinach (Spinacia oleracea), which had sequentially lost 1-5 amino acid residues from the N-terminus and 6-10 residues from the C-terminus, were generated by PCR. These mutants were overexpressed in Escherichia coli, reconstituted with soluble and membrane-bound CF(1), and the ATPase activity and proton conductance of thylakoid membrane were examined. Deletions of as few as 3 amino acid residues from the N-terminus or 6 residues from the C-terminus of epsilon subunit significantly affected their ATPase-inhibitory activity in solution. Deletion of 5 residues from the N-terminus abolished its abilities to inhibit ATPase activity and to restore proton impermeability. Considering the consequence of interaction of epsilon and gamma subunit in the enzyme functions, the special interactions between the epsilon variants and the gamma subunit were detected in the yeast two-hybrid system and in vitro binding assay. In addition, the structures of these mutants were modeled through the SWISS-MODEL Protein Modeling Server. These results suggested that in chloroplast ATP synthase, both the N-terminus and C-terminus of the epsilon subunit show importance in regulation of the ATPase activity. Furthermore, the N-terminus of the epsilon subunit is more important for its interaction with gamma and some CF(o) subunits, and crucial for the blocking of proton leakage. Compared with the epsilon subunit from E. coli [Jounouchi, M., Takeyama, M., Noumi, T., Moriyama, Y., Maeda, M., and Futai, M. (1992) Arch. Biochem. Biophys. 292, 87-94; Kuki, M., Noumi, T., Maeda, M., Amemura, A., and Futai, M. (1988) J. Biol. Chem. 263, 4335-4340], the chloroplast epsilon subunit is more sensitive to N-terminal or C-terminal truncations.     

Probing conformations of the beta subunit of F0F1-ATP synthase in catalysis

A subcomplex of F0F1-ATP synthase (F0F1), alpha3beta3gamma, was shown to undergo the conformation(s) during ATP hydrolysis in which two of the three beta subunits have the "Closed" conformation simultaneously (CC conformation) [S.P. Tsunoda, E. Muneyuki, T. Amano, M. Yoshida, H. Noji, Cross-linking of two beta subunits in the closed conformation in F1-ATPase, J. Biol. Chem. 274 (1999) 5701-5706]. This was examined by the inter-subunit disulfide cross-linking between two mutant beta(I386C)s that was formed readily only when the enzyme was in the CC conformation. Here, we adopted the same method for the holoenzyme F0F1 from Bacillus PS3 and found that the CC conformation was generated during ATP hydrolysis but barely during ATP synthesis. The experiments using F0F1 with the epsilon subunit lacking C-terminal helices further suggest that this difference is related to dynamic nature of the epsilon subunit and that ATP synthesis is accelerated when it takes the pathway involving the CC conformation.