Investigation of the role and mechanism of IF1 and STF1 proteins,twin inhibitory peptides which interact with the yeast mitochondrial ATP synthase

Inhibition of the yeast F(0)F(1)-ATP synthase by the regulatory peptides IF1 and STF1 was studied using intact mitochondria and submitochondrial particles from wild-type cells or from mutants lacking one or both peptides. In intact mitochondria, endogenous IF1 only inhibited uncoupled ATP hydrolysis and endogenous STF1 had no effect. Addition of alamethicin to mitochondria readily made the mitochondrial membranes permeable to nucleotides, and bypassed the kinetic control exerted on ATP hydrolysis by the substrate carriers. In addition, alamethicin made the regulatory peptides able to cross mitochondrial membranes. At pH 7.3, F(0)F(1)-ATPase, initially inactivated by either endogenous IF1 or endogenous STF1, was completely reactivated hours or minutes after alamethicin addition, respectively. Previous application of a membrane potential favored the release of endogenous IF1 and STF1. These observations showed that IF1 and STF1 can fully inhibit ATP hydrolysis at physiological concentrations and are sensitive to the same effectors. However, ATP synthase has a much lower affinity for STF1 than for IF1, as demonstrated by kinetic studies of ATPase inhibition in submitochondrial particles by externally added IF1 and STF1 at pHs ranging from 5.5 to 8.0. Our data do not support previously proposed effects of STF1, like the stabilization of the IF1-F(0)F(1) complex or the replacement of IF1 on its binding site in the presence of the proton-motive force or at high pH, and raise the question of the conditions under which STF1 could regulate ATPase activity in vivo.     

Solution structure of a C-terminal coiled-coil domain from bovine IF(1): the inhibitor protein of F(1) ATPase

Bovine IF(1) is a basic, 84 amino acid residue protein that inhibits the hydrolytic action of the F(1)F(0) ATP synthase in mitochondria under anaerobic conditions. Its oligomerization state is dependent on pH. At a pH value below 6.5 it forms an active dimer. At higher pH values, two dimers associate to form an inactive tetramer. Here, we present the solution structure of a C-terminal fragment of IF(1) (44-84) containing all five of the histidine residues present in the sequence. Most unusually, the molecule forms an anti-parallel coiled-coil in which three of the five histidine residues occupy key positions at the dimer interface.     

The structure of bovine IF(1), the regulatory subunit of mitochondrial F-ATPase.

In mitochondria, the hydrolytic activity of ATP synthase is regulated by an inhibitor protein, IF(1). Its binding to ATP synthase depends on pH, and below neutrality, IF(1) is dimeric and forms a stable complex with the enzyme. At higher pH values, IF(1) forms tetramers and is inactive. In the 2.2 A structure of the bovine IF(1) described here, the four monomers in the asymmetric unit are arranged as a dimer of dimers. Monomers form dimers via an antiparallel alpha-helical coiled coil in the C-terminal region. Dimers are associated into oligomers and form long fibres in the crystal lattice, via coiled-coil interactions in the N-terminal and inhibitory regions (residues 14-47). Therefore, tetramer formation masks the inhibitory region, preventing IF(1) binding to ATP synthase.     

The mitochondrial ATP synthase inhibitor protein binds near the C-terminus of the F1 beta-subunit

The specific, mitochondrial ATP synthase protein (IF1) was covalently cross-linked to its binding site on the catalytic sector of the enzyme (F1-ATPase). The cross-linked complex was selectively cleaved, leaving IF1 intact to facilitate the subsequent purification of the F1 fragment to which IF1 was cross-linked. This fragment was identified by sequence analysis as comprising residues 394-459 on the F1 beta-subunit, near the C-terminus. This finding is discussed in the light of secondary structure predictions for both IF1 and the F1 beta-subunit, and sequence homologies between mitochondrial and other ATP synthases.     

An Inhibitor of the F1 subunit of ATP synthase (IF1) modulates the activity of angiostatin on the endothelial cell surface

Angiostatin binds to endothelial cell (EC) surface F(1)-F(0) ATP synthase, leading to inhibition of EC migration and proliferation during tumor angiogenesis. This has led to a search for angiostatin mimetics specific for this enzyme. A naturally occurring protein that binds to the F1 subunit of ATP synthase and blocks ATP hydrolysis in mitochondria is inhibitor of F1 (IF1). The present study explores the effect of IF1 on cell surface ATP synthase. IF1 protein bound to purified F(1) ATP synthase and inhibited F(1)-dependent ATP hydrolysis consistent with its reported activity in studies of mitochondria. Although exogenous IF1 did not inhibit ATP production on the surface of EC, it did conserve ATP on the cell surface, particularly at low extracellular pH. IF1 inhibited ATP hydrolysis but not ATP synthesis, in contrast to angiostatin, which inhibited both. In cell-based assays used to model angiogenesis in vitro, IF1 did not inhibit EC differentiation to form tubes and only slightly inhibited cell proliferation compared with angiostatin. From these data, we conclude that inhibition of ATP synthesis is necessary for an anti-angiogenic outcome in cell-based assays. We propose that IF1 is not an angiostatin mimetic, but it can serve a protective role for EC in the tumor microenvironment. This protection may be overridden in a concentration-dependent manner by angiostatin. In support of this hypothesis, we demonstrate that angiostatin blocks IF1 binding to ATP synthase and abolishes its ability to conserve ATP. These data suggest that there is a relationship between the binding sites of IF1 and angiostatin on ATP synthase and that IF1 could be employed to modulate angiogenesis.     

The inhibitor protein (IF1) promotes dimerization of the mitochondrial F1F0-ATP synthase

The effect of increased expression or reconstitution of the mitochondrial inhibitor protein (IF1) on the dimer/monomer ratio (D/M) of the rat liver and bovine heart F1F0-ATP synthase was studied. The 2-fold increased expression of IF1 in AS-30D hepatoma mitochondria correlated with a 1.4-fold increase in the D/M ratio of the ATP synthase extracted with digitonin as determined by blue native electrophoresis and averaged densitometry analyses. Removal of IF1 from rat liver or bovine heart submitochondrial particles increased the F1F0-ATPase activity and decreased the D/M ratio of the ATP synthase. Reconstitution of recombinant IF1 into submitochondrial particles devoid of IF1 inhibited the F1F0-ATPase activity by 90% and restored partially the D/M ratio of the whole F1F0 complex as revealed by blue native electrophoresis and subsequent SDS-PAGE or glycerol density gradient centrifugation. Thus, the inhibitor protein promotes or stabilizes the dimeric form of the intact F1F0-ATP synthase. A possible location of the IF1 protein in the dimeric structure of the rat liver F1F0 complex is proposed. According to crystallographic and electron microscopy analyses, dimeric IF1 could bridge the F1-F1 part of the dimeric F1F0-ATP synthase in the inner mitochondrial membrane.     

Homologous and heterologous inhibitory effects of ATPase inhibitor proteins on F-ATPases

In Saccharomyces cerevisiae, at least three proteins (IF(1), STF(1), and STF(2)) appear to be involved in the regulation of ATP synthase. Both IF(1) and STF(1) inhibit F(1), whereas the proposed function for STF(2) is to facilitate the binding of IF(1) and STF(1) to F(1). The oligomerization properties of yeast IF(1) and STF(1) have been investigated by sedimentation equilibrium analytical ultracentrifugation and by covalent cross-linking. Both techniques confirm that IF(1) and STF(1) oligomerize in opposite directions in relation to pH, suggesting that both proteins might regulate yeast F(1)F(0)-ATPase under different conditions. Their effects on bovine F-ATPases are also described. Whereas bovine IF(1) inhibits yeast F(1)-ATPase even better than yeast IF(1) or STF(1), the capability of yeast IF(1) to inhibit the bovine enzyme is very low and decreases with time. Such an effect is also observed in the study of the homologous inhibition of yeast F(1)-ATPase. Yeast inhibitors are not as effective as their bovine counterpart, and the complex seems to dissociate gradually.     

Modulation of the oligomerization state of the bovine F1-ATPase inhibitor protein, IF1, by pH

Bovine IF(1), a basic protein of 84 amino acids, is involved in the regulation of the catalytic activity of the F(1) domain of ATP synthase. At pH 6.5, but not at basic pH values, it inhibits the ATP hydrolase activity of the enzyme. The oligomeric state of bovine IF(1) has been investigated at various pH values by sedimentation equilibrium analytical ultracentrifugation and by covalent cross-linking. Both techniques confirm that the protein forms a tetramer at pH 8, and below pH 6.5, the protein is predominantly dimeric. By covalent cross-linking, it has been found that at pH 8.0 the fragment of IF(1) consisting of residues 44-84 forms a dimer, whereas the fragment from residues 32-84 is tetrameric. Therefore, some or all of the residues between positions 32 and 43 are necessary for tetramer formation and are involved in the pH-sensitive interconversion between dimer and tetramer. One important residue in the interconversion is histidine 49. Mutation of this residue to lysine abolishes the pH-dependent activation-inactivation, and the mutant protein is active and dimeric at all pH values investigated. It is likely from NMR studies that the inhibitor protein dimerizes by forming an antiparallel alpha-helical coiled-coil over its C-terminal region and that at high pH values, where the protein is tetrameric, the inhibitory regions are masked. The mutation of histidine 49 to lysine is predicted to abolish coiled-coil formation over residues 32-43 preventing interaction between two dimers, forcing the equilibrium toward the dimeric state, thereby freeing the N-terminal inhibitory regions and allowing them to interact with F(1).     

Supercomplexes and subcomplexes of mitochondrial oxidative phosphorylation

Dimerization or oligomerization of ATP synthase has been proposed to play an important role for mitochondrial cristae formation and to be involved in regulating ATP synthase activity. We found comparable oligomycin-sensitive ATPase activity for monomeric and oligomeric ATP synthase suggesting that oligomerization/monomerization dynamics are not directly involved in regulating ATP synthase activity. Binding of the natural IF1 inhibitor protein has been shown to induce dimerization of F1-subcomplexes. This suggested that binding of IF1 might also dimerize holo ATP synthase, and possibly link dimerization and inhibition. Analyzing mitochondria of human rho zero cells that contain mitochondria but lack mitochondrial DNA, we identified three subcomplexes of ATP synthase: (i) F1 catalytic domain, (ii) F1-domain with bound IF1, and (iii) F1-c subcomplex with bound IF1 and a ring of subunits c. Since both IF1 containing subcomplexes were present in monomeric state and exhibited considerably reduced ATPase activity as compared to the third subcomplex lacking IF1, we postulate that inhibition and induction of dimerization of F1-subcomplexes by IF1 are independent events. F1-subcomplexes were also found in mitochondria of patients with specific mitochondrial disorders, and turned out to be useful for the clinical differentiation between various types of mitochondrial biosynthesis disorders. Supramolecular associations of respiratory complexes, the "respirasomes", seem not to be the largest assemblies in the structural organization of the respiratory chain, as suggested by differential solubilization of mitochondria and electron microscopic analyses of whole mitochondria. We present a model for a higher supramolecular association of respirasomes into a "respiratory string".     

Functional transitions of F0F1-ATPase mediated by the inhibitory peptide IF1 in yeast coupled submitochondrial particles.

The mechanism of inhibition of yeast F(0)F(1)-ATPase by its naturally occurring protein inhibitor (IF1) was investigated in submitochondrial particles by studying the IF1-mediated ATPase inhibition in the presence and absence of a protonmotive force. In the presence of protonmotive force, IF1 added during net NTP hydrolysis almost completely inhibited NTPase activity. At moderate IF1 concentration, subsequent uncoupler addition unexpectedly caused a burst of NTP hydrolysis. We propose that the protonmotive force induces the conversion of IF1-inhibited F(0)F(1)-ATPase into a new form having a lower affinity for IF1. This form remains inactive for ATP hydrolysis after IF1 release. Uncoupling simultaneously releases ATP hydrolysis and converts the latent form of IF1-free F(0)F(1)-ATPase back to the active form. The relationship between the different steps of the catalytic cycle, the mechanism of inhibition by IF1 and the interconversion process is discussed.     

Insight into the bind-lock mechanism of the yeast mitochondrial ATP synthase inhibitory peptide

The mechanism of yeast mitochondrial F1-ATPase inhibition by its regulatory peptide IF1 was investigated with the noncatalytic sites frozen by pyrophosphate pretreatment that mimics filling by ATP. This allowed for confirmation of the mismatch between catalytic site occupancy and IF1 binding rate without the kinetic restriction due to slow ATP binding to the noncatalytic sites. These data strengthen the previously proposed two-step mechanism, where IF1 loose binding is determined by the catalytic state and IF1 locking is turnover-dependent and competes with IF1 release (Corvest, V., Sigalat, C., Venard, R., Falson, P., Mueller, D. M., and Haraux, F. (2005) J. Biol. Chem. 280, 9927-9936). They also demonstrate that noncatalytic sites, which slightly modulate IF1 access to the enzyme, play a minor role in its binding. It is also shown that loose binding of IF1 to MgADP-loaded F1-ATPase is very slow and that IF1 binding to ATP-hydrolyzing F1-ATPase decreases nucleotide binding severely in the micromolar range and moderately in the submillimolar range. Taken together, these observations suggest an outline of the total inhibition process. During the first catalytic cycle, IF1 loosely binds to a catalytic site with newly bound ATP and is locked when ATP is hydrolyzed at a second site. During the second cycle, blocking of ATP hydrolysis by IF1 inhibits ATP from becoming entrapped on the third site and, at high ATP concentrations, also inhibits ADP release from the second site. This model also provides a clue for understanding why IF1 does not bind ATP synthase during ATP synthesis.     

Downmodulation of mitochondrial F0F1 ATP synthase by diazoxide in cardiac myoblasts: a dual effect of the drug

Similar to ischemic preconditioning, diazoxide was documented to elicit beneficial bioenergetic consequences linked to cardioprotection. Inhibition of ATPase activity of mitochondrial F(0)F(1) ATP synthase may have a role in such effect and may involve the natural inhibitor protein IF(1). We recently documented, using purified enzyme and isolated mitochondrial membranes from beef heart, that diazoxide interacts with the F(1) sector of F(0)F(1) ATP synthase by promoting IF(1) binding and reversibly inhibiting ATP hydrolysis. Here we investigated the effects of diazoxide on the enzyme in cultured myoblasts. Specifically, embryonic heart-derived H9c2 cells were exposed to diazoxide and mitochondrial ATPase was assayed in conditions maintaining steady-state IF(1) binding (basal ATPase activity) or detaching bound IF(1) at alkaline pH. Mitochondrial transmembrane potential and uncoupling were also investigated, as well as ATP synthesis flux and ATP content. Diazoxide at a cardioprotective concentration (40 muM cell-associated concentration) transiently downmodulated basal ATPase activity, concomitant with mild mitochondria uncoupling and depolarization, without affecting ATP synthesis and ATP content. Alkaline stripping of IF(1) from F(0)F(1) ATP synthase was less in diazoxide-treated than in untreated cells. Pretreatment with glibenclamide prevented, together with mitochondria depolarization, inhibition of ATPase activity under basal but not under IF(1)-stripping conditions, indicating that diazoxide alters alkaline IF(1) release. Diazoxide inhibition of ATPase activity in IF(1)-stripping conditions was observed even when mitochondrial transmembrane potential was reduced by FCCP. The results suggest that diazoxide in a model of normoxic intact cells directly promotes binding of inhibitor protein IF(1) to F(0)F(1) ATP synthase and enhances IF(1) binding indirectly by mildly uncoupling and depolarizing mitochondria.     

Binding properties of 9K protein to F1-ATPase: a counterpart ligand to the ATPase inhibitor

A regulatory subunit of yeast mitochondrial ATP synthase, 9K protein, formed an equimolar complex with F1-ATPase in the presence of ATP and Mg2+, indicating that the binding of the protein to the enzyme took place in a similar manner to that of ATPase inhibitor. The ATP-hydrolyzing activity of F1-ATPase decreased 40% on binding of the 9K protein, and the remaining activity was resistant to external ATPase inhibitor. The apparent dissociation constant of the F1-ATPase-9K complex was determined by gel permeation chromatography to be 3.7 X 10(-6) M, which was in the same order of magnitude as that of enzyme-ATPase inhibitor complex (4.2 x 10(-6) M). When added simultaneously the binding of the inhibitor and 9K protein to F1-ATPase were competitive and the sum of their bindings did not exceed 1 mol per mol of enzyme. However, the binding of each protein ligand to F1-ATPase took more than 1 min for completion, and when one of these two proteins was added 10 min after the other, it did not replace the other. These observations strongly suggest that membrane-bound F1-ATPase always binds to either the 9K protein or ATPase inhibitor in intact mitochondria and that the complexes with the two ligands are active and inactive counterparts, respectively.     

Formation of the yeast F1F0-ATP synthase dimeric complex does not require the ATPase inhibitor protein,Inh1

The yeast F1F0-ATP synthase forms dimeric complexes in the mitochondrial inner membrane and in a manner that is supported by the F0-sector subunits, Su e and Su g. Furthermore, it has recently been demonstrated that the binding of the F1F0-ATPase natural inhibitor protein to purified bovine F1-sectors can promote their dimerization in solution (Cabezon, E., Arechaga, I., Jonathan P., Butler, G., and Walker J. E. (2000) J. Biol. Chem. 275, 28353-28355). It was unclear until now whether the binding of the inhibitor protein to the F1 domains contributes to the process of F1F0-ATP synthase dimerization in intact mitochondria. Here we have directly addressed the involvement of the yeast inhibitor protein, Inh1, and its known accessory proteins, Stf1 and Stf2, in the formation of the yeast F1F0-ATP synthase dimer. Using mitochondria isolated from null mutants deficient in Inh1, Stf1, and Stf2, we demonstrate that formation of the F(1)F(0)-ATP synthase dimers is not adversely affected by the absence of these proteins. Furthermore, we demonstrate that the F1F0-ATPase monomers present in su e null mutant mitochondria can be as effectively inhibited by Inh1, as its dimeric counterpart in wild-type mitochondria. We conclude that dimerization of the F1F0-ATP synthase complexes involves a physical interaction of the membrane-embedded F0 sectors from two monomeric complexes and in a manner that is independent of inhibitory activity of the Inh1 and accessory proteins.     

Mitochondrial adenosinetriphosphatase inhibitor protein: reversible interaction with complex V (ATP synthetase complex)

Mitochondrial ATPase inhibitor protein (IF1) reacts reversibly with complex V and inhibits up to 90% of its ATPase activity. Both the rate and extent of inhibition are pH and temperature dependent and increase as the pH is lowered from pH 8 tp 6.7 (the lowest pH examined) or as the temperature is increased from 4 to 36 degrees C. Nucleotide triphosphates plus Mg2+ ions are required for inhibition of complex V ATPase activity by IF1. In the presence of Mg2+ ions, the effectiveness order of nucleotides is ATP greater than ITP greater than GTP greater than UTP. Highly purified complex V, which requires added phospholipids for expressing ATPase and ATP-Pi exchange activities, cannot be inhibited by IF1 plust ATP-Mg2+ unless phospholipids are also added. This indicates that the active state of the enzyme is necessary for the IF1 effect to be manifested, because F1-ATPase, which does not contain nor require phospholipids for catalyzing ATP hydrolysis, can be inhibited by IF1 plus ATP-Mg2+ in the absence of added phospholipids. The IF1-inhibited complex V, but not IF1-inhibited F1-ATPase, can be reactivated by incubation at pH greater than 7.0 in the absence of ATP-Mg2+. The reactivation rate is pH dependent and is influenced by temperature and enzyme concentration. Complex V preparations contain small and variable amounts of IF1. This endogenous IF1 behaves the same as added IF1 with respect to conditions described above for inhibition and reactivation and can result in 25-50% inhibition in different complex V preparations. However, complex V lacking endogenous IF1 can be reconstituted from F0, F1, oligomycin sensitivity conferring protein, and phospholipids. Inhibition of this reconstituted preparation in the presence of ATP-Mg2+ depends entirely on addition of IF1. In general, the ATP-Pi exchange activity of complex V is more sensitive to the chemical inhibitors of F1-AtPase tha its ATPase activity. This is not so, however, for IF1. Under conditions that IF1 caused approximately 75% inhibition of ATPase activity of complex V, no more than 10% of the ATP-Pi exchange activity was inhibited.     

Mutations in the dimerization domain of the b subunit from the Escherichia coli ATP synthase. Deletions disrupt function but not enzyme assembly

The b subunit dimer of Escherichia coli ATP synthase serves essential roles as an assembly factor for the enzyme and as a stator during rotational catalysis. To investigate the functional importance of its coiled coil dimerization domain, a series of internal deletions including each individual residue between Lys-100 and Ala-105 (b(deltaK100)-b(deltaA105)), b(deltaK100-A103), and b(deltaK100-Q106) as well as a control b(K100A) missense mutation were prepared. All of the mutants supported assembly of ATP synthase, but all single-residue deletions failed to support growth on acetate, indicating a severe defect in oxidative phosphorylation, and b(deltaK100-Q106) displayed moderately reduced growth. The membrane-bound ATPase activities of these strains showed a related reduction in sensitivity to dicyclohexylcarbodiimide, indicative of uncoupling. Analysis of dimerization of the soluble constructs of b(deltaK100) and the multiple-residue deletions by sedimentation equilibrium revealed reduced dimerization compared with wild type for all deletions, with b(deltaK100-Q106) most severely affected. In cross-linking studies it was found that F1-ATPase can mediate the dimerization of some soluble b constructs but did not mediate dimerization of b(deltaK100) and b(deltaK100-Q106); these two forms also were defective in F1 binding analyses. We conclude that defective dimerization of soluble b constructs severely affects F1 binding in vitro, yet allows assembly of ATP synthase in vivo. The highly uncoupled nature of enzymes with single-residue deletions in b indicates that the b subunit serves an active function in energy coupling rather than just holding on to the F1 sector. This function is proposed to depend on proper, specific interactions between the b subunits and F1.     

The binding mechanism of the yeast F1-ATPase inhibitory peptide: role of catalytic intermediates and enzyme turnover

The mechanism of inhibition of yeast mitochondrial F(1)-ATPase by its natural regulatory peptide, IF1, was investigated by correlating the rate of inhibition by IF1 with the nucleotide occupancy of the catalytic sites. Nucleotide occupancy of the catalytic sites was probed by fluorescence quenching of a tryptophan, which was engineered in the catalytic site (beta-Y345W). Fluorescence quenching of a beta-Trp(345) indicates that the binding of MgADP to F(1) can be described as 3 binding sites with dissociation constants of K(d)(1) = 10 +/- 2 nm, K(d2) = 0.22 +/- 0.03 microm, and K(d3) = 16.3 +/- 0.2 microm. In addition, the ATPase activity of the beta-Trp(345) enzyme followed simple Michaelis-Menten kinetics with a corresponding K(m) of 55 microm. Values for the K(d) for MgATP were estimated and indicate that the K(m) (55 microm) for ATP hydrolysis corresponds to filling the third catalytic site on F(1). IF1 binds very slowly to F(1)-ATPase depleted of nucleotides and under unisite conditions. The rate of inhibition by IF1 increased with increasing concentration of MgATP to about 50 mum, but decreased thereafter. The rate of inhibition was half-maximal at 5 microm MgATP, which is 10-fold lower than the K(m) for ATPase. The variations of the rate of IF1 binding are related to changes in the conformation of the IF1 binding site during the catalytic reaction cycle of ATP hydrolysis. A model is proposed that suggests that IF1 binds rapidly, but loosely to F(1) with two or three catalytic sites filled, and is then locked in the enzyme during catalytic hydrolysis of ATP.     

Inhibitory and Anchoring Domains in the ATPase Inhibitor Protein IF1 of Bovine Heart Mitochondrial ATP Synthase

The inhibitor protein IF1 is a basic protein of 84 residues which inhibits the ATPase activity of the mitochondrial FoF1-ATP synthase complex without having any effect on ATP synthesis. Results of cross-linking and limited proteolysis experiments are presented showing that in the intact FoF1 complex "in situ," in the inner membrane of bovine heart mitochondria, the central segment of IF1 (residues 42-58) binds to the alpha and beta subunits of F1 in a pH dependent process, and inhibits the ATPase activity. The C-terminal region of IF1 binds, simultaneously, to the OSCP subunit of Fo in a pH-independent process. This binding keeps IF1 anchored to the complex, both under inhibitory conditions, at acidic pH, and noninhibitory conditions at alkaline pH.     

The structure of bovine F1-ATPase in complex with its regulatory protein IF1

In mitochondria, the hydrolytic activity of ATP synthase is prevented by an inhibitor protein, IF1. The active bovine protein (84 amino acids) is an alpha-helical dimer with monomers associated via an antiparallel alpha-helical coiled coil composed of residues 49-81. The N-terminal inhibitory sequences in the active dimer bind to two F1-ATPases in the presence of ATP. In the crystal structure of the F1-IF1 complex at 2.8 A resolution, residues 1-37 of IF1 bind in the alpha(DP)-beta(DP) interface of F1-ATPase, and also contact the central gamma subunit. The inhibitor opens the catalytic interface between the alpha(DP) and beta(DP) subunits relative to previous structures. The presence of ATP in the catalytic site of the beta(DP) subunit implies that the inhibited state represents a pre-hydrolysis step on the catalytic pathway of the enzyme.     

Isolated epsilon subunit of thermophilic F1-ATPase binds ATP

F1-ATPase, a soluble part of the F0F1-ATP synthase, has subunit structure alpha3beta3gammadeltaepsilon in which nucleotide-binding sites are located in the alpha and beta subunits and, as believed, in none of the other subunits. However, we report here that the isolated epsilon subunit of F1-ATPase from thermophilic Bacillus strain PS3 can bind ATP. The binding was directly demonstrated by isolating the epsilon subunit-ATP complex with gel filtration chromatography. The binding was not dependent on Mg2+ but was highly specific for ATP; however, ADP, GTP, UTP, and CTP failed to bind. The epsilon subunit lacking the C-terminal helical hairpin was unable to bind ATP. Although ATP binding to the isolated epsilon subunits from other organisms has not been detected under the same conditions, a possibility emerges that the epsilon subunit acts as a built in cellular ATP level sensor of F0F1-ATP synthase.