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.     

Reversible ischemic inhibition of F(1)F(0)-ATPase in rat and human myocardium

The physiological role of F(1)F(0)-ATPase inhibition in ischemia may be to retard ATP depletion although views of the significance of IF(1) are at variance. We corroborate here a method for measuring the ex vivo activity of F(1)F(0)-ATPase in perfused rat heart and show that observation of ischemic F(1)F(0)-ATPase inhibition in rat heart is critically dependent on the sample preparation and assay conditions, and that the methods can be applied to assay the ischemic and reperfused human heart during coronary by-pass surgery. A 5-min period of ischemia inhibited F(1)F(0)-ATPase by 20% in both rat and human myocardium. After a 15-min reperfusion a subsequent 5-min period of ischemia doubled the inhibition in the rat heart but this potentiation was lost after 120 min of reperfusion. Experiments with isolated rat heart mitochondria showed that ATP hydrolysis is required for effective inhibition by uncoupling. The concentration of oligomycin for 50% inhibition (I(50)) for oxygen consumption was five times higher than its I(50) for F(1)F(0)-ATPase. Because of the different control strengths of F(1)F(0)-ATPase in oxidative phosphorylation and ATP hydrolysis an inhibition of the F(1)F(0)-ATPase activity in ischemia with the resultant ATP-sparing has an advantage even in an ischemia/reperfusion situation.     

Suppression of mitochondrial ATPase inhibitor protein (IF1) in the liver of late septic rats

Sepsis and ensuing multiple organ failure continue to be the most leading cause of death in critically ill patients. Despite hepatocyte-related dysfunctions such as necrosis, apoptosis as well as mitochondrial damage are observed in the process of sepsis, the molecular mechanism of pathogenesis remains uncertain. We recently identified one of the differentially expressed genes, mitochondrial ATPase inhibitor protein (IF1) which is down-regulated in late septic liver. Hence, we further hypothesized that the variation of IF1 protein may be one of the causal events of the hepatic dysfunction during late sepsis. The results showed that the elevated mitochondrial F0F1-ATPase activity is concomitant with the decline of intramitochondrial ATP concentration in late septic liver. In addition, the key finding of this study showed that the mRNA and the mitochondrial content of IF1 were decreased in late sepsis while no detectable IF1 was found in cytoplasm. When analyzed by immunoprecipitation, it seems reasonable to imply that the association capability of IF1 with F1-ATPase beta-subunit is not affected. These results confirm the first evidence showing that the suppression of IF1 expression and subsequent elevated mitochondrial F0F1-ATPase activity might contribute to the bioenergetic failure in the liver during late sepsis.     

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.     

Farnesol-induced generation of reactive oxygen species dependent on mitochondrial transmembrane potential hyperpolarization mediated by F(0)F(1)-ATPase in yeast

An isoprenoid farnesol (FOH) inhibited cellular oxygen consumption and induced mitochondrial generation of reactive oxygen species (ROS) in cells of Saccharomyces cerevisiae in correlation with hyperpolarization of the mitochondrial transmembrane potential (mtDeltaPsi). The FOH-induced events were coordinately abolished with the F(1)-ATPase inhibitor sodium azide as well as the F(0)F(1)-ATPase inhibitor oligomycin, suggesting the dependence of ROS generation on mtDeltaPsi hyperpolarization mediated by the proton pumping function of F(0)F(1)-ATPase as a result of ATP hydrolysis. The role of F(1)-ATPase activity in mtDeltaPsi hyperpolarization was supported by the intracellular depletion of ATP in FOH-treated cells and its protection with sodium azide. An indirect mechanism was suggested to exist in the regulation of F(0)F(1)-ATPase by FOH to accelerate its ATP-hydrolyzing activity.     

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.     

Dimerization of bovine F1-ATPase by binding the inhibitor protein, IF1

In mitochondria, the hydrolytic activity of ATP synthase is regulated by a natural inhibitor protein, IF(1). The binding of IF(1) to ATP synthase depends on pH values, and below neutrality, IF(1) forms a stable complex with the enzyme. Bovine IF(1) has two oligomeric states, dimer and tetramer, depending on pH values. At pH 6.5, where it is active, IF(1) dimerizes by formation of an antiparallel alpha-helical coiled-coil in its C-terminal region. This arrangement places the inhibitory N-terminal regions in opposition, implying that active dimeric IF(1) can bind two F(1) domains simultaneously. Evidence of dimerization of F(1)-ATPase by binding to IF(1) is provided by gel filtration chromatography, analytical ultracentrifugation, and electron microscopy. At present, it is not known whether IF(1) can bring about the dimerization of the F(1)F(0)-ATPase complex.     

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.     

In vitro and in vivo studies of F(0)F(1)ATP synthase regulation by inhibitor protein IF(1) in goat heart

A method has been developed to allow the level of F(0)F(1)ATP synthase capacity and the quantity of IF(1) bound to this enzyme be measured in single biopsy samples of goat heart. ATP synthase capacity was determined from the maximal mitochondrial ATP hydrolysis rate and IF(1) content was determined by detergent extraction followed by blue native gel electrophoresis, two-dimensional SDS-PAGE and immunoblotting with anti-IF(1) antibodies. Anaesthetized open-chest goats were subjected to ischemic preconditioning and/or sudden increases of coronary blood flow (CBF) (reactive hyperemia). When hyperemia was induced before ischemic preconditioning, a steep increase in synthase capacity, followed by a deep decrease, was observed. In contrast, hyperemia did not affect synthase capacity when applied after ischemic preconditioning. Similar effects could be produced in vitro by treatment of heart biopsy samples with anoxia (down-regulation of the ATP synthase) or high-salt or high-pH buffers (up-regulation). We show that both in vitro and in vivo the same close inverse correlation exists between enzyme activity and IF(1) content, demonstrating that under all conditions tested the only significant modulator of the enzyme activity was IF(1). In addition, both in vivo and in vitro, 1.3-1.4 mol of IF(1) was predicted to fully inactivate 1 mol of synthase, thus excluding the existence of significant numbers of non-inhibitory binding sites for IF(1) in the F(0) sector.     

Mutant residues suppressing rho(0)-lethality in Kluyveromyces lactis occur at contact sites between subunits of F(1)-ATPase

Characterisation of 35 Kluyveromyces lactis strains lacking mitochondrial DNA has shown that mutations suppressing rho(0)-lethality are limited to the ATP1, 2 and 3 genes coding for the alpha-, beta- and gamma- subunits of mitochondrial F(1)-ATPase. All atp mutations reduce growth on glucose and three alleles, atp1-2, 1-3 and atp3-1, produce a respiratory deficient phenotype that indicates a drop in efficiency of the F(1)F(0)-ATP synthase complex. ATPase activity is needed for suppression as a double mutant containing an atp allele, together with a mutation abolishing catalytic activity, does not suppress rho(0)-lethality. Positioning of the seven amino acids subject to mutation on the bovine F(1)-ATPase structure shows that two residues are found in a membrane proximal region while five amino acids occur at a region suggested to be a molecular bearing. The intriguing juxtaposition of mutable amino acids to other residues subject to change suggests that mutations affect subunit interactions and alter the properties of F(1) in a manner yet to be determined. An explanation for suppressor activity of atp mutations is discussed in the context of a possible role for F(1)-ATPase in the maintenance of mitochondrial inner membrane potential.     

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.     

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.     

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.     

Mutations in the beta-subunit Thr(159) and Glu(184) of the Rhodospirillum rubrum F(0)F(1) ATP synthase reveal differences in ligands for the coupled Mg(2+)- and decoupled Ca(2+)-dependent F(0)F(1) activities

In the crystal structure of the mitochondrial F(1)-ATPase, the beta-Thr(163) residue was identified as a ligand to Mg(2+) and the beta-Glu(188) as directly involved in catalysis. We replaced the equivalent beta-Thr(159) of the chromatophore F(0)F(1) ATP synthase of Rhodospirillum rubrum with Ser, Ala, or Val and the Glu(184) with Gln or Lys. The mutant beta subunits were isolated and tested for their capacity to assemble into a beta-less chromatophore F(0)F(1) and restore its lost activities. All of them were found to bind into the beta-less enzyme with the same efficiency as the wild type beta subunit, but only the beta-Thr(159) --> Ser mutant restored the activity of the assembled enzyme. These results indicate that both Thr(159) and Glu(184) are not required for assembly and that Glu(184) is indeed essential for all the membrane-bound chromatophore F(0)F(1) activities. A detailed comparison between the wild type and the beta-Thr(159) --> Ser mutant revealed a rather surprising difference. Although this mutant restored the wild type levels and all specific properties of this F(0)F(1) proton-coupled ATP synthesis as well as Mg- and Mn-dependent ATP hydrolysis, it did not restore at all the proton-decoupled CaATPase activity. This clear difference between the ligands for Mg(2+) and Mn(2+), where threonine can be replaced by serine, and Ca(2+), where only threonine is active, suggests that the beta-subunit catalytic site has different conformational states when occupied by Ca(2+) as compared with Mg(2+). These different states might result in different interactions between the beta and gamma subunits, which are involved in linking F(1) catalysis with F(0) proton-translocation and can thus explain the complete absence of Ca-dependent proton-coupled F(0)F(1) catalytic activity.     

Suppression of mitochondrial ATPase inhibitor protein (IF1) in the liver of late septic rats

Sepsis and ensuing multiple organ failure continue to be the most leading cause of death in critically ill patients. Despite hepatocyte-related dysfunctions such as necrosis, apoptosis as well as mitochondrial damage are observed in the process of sepsis, the molecular mechanism of pathogenesis remains uncertain. We recently identified one of the differentially expressed genes, mitochondrial ATPase inhibitor protein (IF1) which is down-regulated in late septic liver. Hence, we further hypothesized that the variation of IF1 protein may be one of the causal events of the hepatic dysfunction during late sepsis. The results showed that the elevated mitochondrial F₀F₁-ATPase activity is concomitant with the decline of intramitochondrial ATP concentration in late septic liver. In addition, the key finding of this study showed that the mRNA and the mitochondrial content of IF1 were decreased in late sepsis while no detectable IF1 was found in cytoplasm. When analyzed by immunoprecipitation, it seems reasonable to imply that the association capability of IF1 with F₁-ATPase β-subunit is not affected. These results confirm the first evidence showing that the suppression of IF1 expression and subsequent elevated mitochondrial F₀F₁-ATPase activity might contribute to the bioenergetic failure in the liver during late sepsis.     

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.     

Energy-dependent transformation of F0.F1-ATPase in Paracoccus denitrificans plasma membranes

F(0).F(1)-ATP synthase in tightly coupled inside-out vesicles derived from Paracoccus denitrificans catalyzes rapid respiration-supported ATP synthesis, whereas their ATPase activity is very low. In the present study, the conditions required to reveal the Deltamu(H+)-generating ATP hydrolase activity of the bacterial enzyme have been elucidated. Energization of the membranes by respiration results in strong activation of the venturicidin-sensitive ATP hydrolysis, which is coupled with generation of Deltam?(H+). Partial uncoupling stimulates the proton-translocating ATP hydrolysis, whereas complete uncoupling results in inhibition of the ATPase activity. The presence of inorganic phosphate is indispensable for the steady-state turnover of the Deltam?(H+)-activated ATPase. The collapse of Deltam?(H+) brings about rapid deactivation of the enzyme, which has been subjected to pre-energization. The rate and extent of the deactivation depend on protein concentration, i.e. the more vesicles are present in the assay mixture, the higher the rate and extent of the deactivation is seen. Sulfite and the ADP-trapping system protect ATPase against the Deltam?(H+) collapse-induced deactivation, whereas phosphate delays the rate of deactivation. A low concentration of ADP (<1 microm) increases the rate of deactivation. Taken together, the results suggest that latent proton-translocating ATPase in P. denitrificans is kinetically equivalent to the previously characterized ADP(Mg2+)-inhibited, azide-trapped bovine heart mitochondrial F(0).F(1)-ATPase (Galkin, M. A., and Vinogradov, A. D. (1999) FEBS Lett. 448, 123-126). A Deltam?(H+)-sensitive mechanism operates in P. denitrificans that prevents physiologically wasteful consumption of ATP by F(0).F(1)-ATPase (synthase) complex when the latter is unable to maintain certain value of Deltam?(H+).     

The hydrogen ion-pumping adenosine triphosphatase of platelet dense granule membrane. Differences from F1F0- and phosphoenzyme-type ATPases

Using a coupled transport assay which detects only those ATPase molecules functionally inserted into the platelet dense granule membrane, we have characterized the inhibitor sensitivity, substrate specificity, and divalent cation requirements of the granule H+ pump. Under identical assay conditions, the granule ATPase was insensitive to concentrations of NaN3, oligomycin, and efrapeptin which almost completely inhibit ATP hydrolysis by mitochondrial membranes. The granule ATPase was inhibited by dicyclohexylcarbodiimide but only at concentrations much higher than those needed to maximally inhibit mitochondrial ATPase. Vanadate (VO3-) ion and ouabain also failed to inhibit granule ATPase activity at concentrations which maximally inhibited purified Na+,K+-ATPase. Two alkylating agents, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole and N-ethylmaleimide both completely inhibited H+ pumping by the granule ATPase under conditions where ATP hydrolysis by mitochondrial membranes or Na+,K+-ATPase was hardly affected. These results suggest that the H+-pumping ATPase of platelet granule membrane may belong to a class of ion-translocating ATPases distinct from both the phosphoenzyme-type ATPases present in plasma membrane and the F1F0-ATPases of energy-transducing membranes.     

Regulatory role of the ATPase inhibitor protein on proton conduction by mitochondrial H+-ATPase complex

This study shows that the natural inhibitor protein of mitochondrial H+-ATPase complex (IF1) inhibits, in addition to the catalytic activity, the proton conductivity of the complex. The inhibition of ATPase activity by IF1 is less effective in the purified F1 than in submitochondrial particles where F1 is bound to F0. No inhibition of H+ conductivity by F0 is observed in F1-depleted particles.     

Kinetic properties of F1-ATPase influence the ability of yeasts to grow in anoxia or absence of mtDNA

A mechanism for hypoxia survival by eukaryotic cells is suggested from studies on the petite mutation of yeasts. Previous work has shown that mutations in the alpha, beta and gamma subunit genes of F1-ATPase can suppress lethality due to loss of the mitochondrial genome from the petite-negative yeast Kluyveromyceslactis. Here it is reported that suppressor mutations appear to increase the affinity of F1-ATPase for ATP. Extension of this study to other yeasts shows that petite-positive species have a higher affinity for ATP in the hydrolysis reaction than petite-negative species. Possession of a F1-ATPase with a low K(m) for ATP is considered to be an adaptation for hypoxic growth, enabling maintenance of the mitochondrial inner membrane potential, deltapsi, by enhanced export of protons through F1F0-ATP synthase connected to increased ATP hydrolysis at low substrate concentration.