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.     

Identification of the subunits of F1F0-ATPase from bovine heart mitochondria.

An oligomycin-sensitive F1F0-ATPase isolated from bovine heart mitochondria has been reconstituted into phospholipid vesicles and pumps protons. this preparation of F1F0-ATPase contains 14 different polypeptides that are resolved by polyacrylamide gel electrophoresis under denaturing conditions, and so it is more complex than bacterial and chloroplast enzymes, which have eight or nine different subunits. The 14 bovine subunits have been characterized by protein sequence analysis. They have been fractionated on polyacrylamide gels and transferred to poly(vinylidene difluoride) membranes, and N-terminal sequences have been determined in nine of them. By comparison with known sequences, eight of these have been identified as subunits beta, gamma, delta, and epsilon, which together with the alpha subunit form the F1 domain, as the b and c (or DCCD-reactive) subunits, both components of the membrane sector of the enzyme, and as the oligomycin sensitivity conferral protein (OSCP) and factor 6 (F6), both of which are required for attachment of F1 to the membrane sector. The sequence of the ninth, named subunit e, has been determined and is not related to any reported protein sequence. The N-terminal sequence of a tenth subunit, the membrane component A6L, could be determined after a mild acid treatment to remove an alpha-N-formyl group. Similar experiments with another membrane component, the a or ATPase-6 subunit, caused the protein to degrade, but the protein has been isolated from the enzyme complex and its position on gels has been unambiguously assigned. No N-terminal sequence could be derived from three other proteins. The largest of these is the alpha subunit, which previously has been shown to have pyrrolidonecarboxylic acid at the N terminus of the majority of its chains. The other two have been isolated from the enzyme complex; one of them is the membrane-associated protein, subunit d, which has an alpha-N-acetyl group, and the second, surprisingly, is the ATPase inhibitor protein. When it is isolated directly from mitochondrial membranes, the inhibitor protein has a frayed N terminus, with chains starting at residues 1, 2, and 3, but when it is isolated from the purified enzyme complex, its chains are not frayed and the N terminus is modified. Previously, the sequences at the N terminals of the alpha, beta, and delta subunits isolated from F1-ATPase had been shown to be frayed also, but in the F1F0 complex they each have unique N-terminal sequences.(ABSTRACT TRUNCATED AT 400 WORDS)     

F1F0-ATPase from Escherichia coli with mutant F0 subunits. Partial purification and immunoprecipitation of F1F0 complexes

Previously identified mutations in subunits a and b of the F0 sector of the F1F0-ATPase from Escherichia coli are further characterized by isolating detergent-solubilized, partially purified F1F0 complexes from cells bearing these mutations. The composition of the various F1F0 complexes was judged by quantitating the amount of each subunit present in the detergent-solubilized preparations. The composition of the F0 sectors containing altered polypeptides was determined by quantitating the F0 subunits that were immunoprecipitated by antibodies directed against the F1 portion. In this way, the relative amounts of F0 subunits (a, b, c) which survived the isolation procedure bound to F1 were determined for each mutation. This analysis indicates that both missense mutations in subunit a (aser206----leu and ahis245----tyr) resulted in the isolation of F1F0 complexes with normal subunit composition. The nonsense mutation in subunit a (atyr235----end) resulted in isolation of a complex containing the b and c subunits. The bgly131----asp mutation in the b subunit results in an F0 complex which does not assemble or survive the isolation. The isolated F1F0 complex containing the mutation bgly9----asp in the b subunit was defective in two regards: first, a reduction in F1 content relative to F0 and second, the absence of the a subunit. Immunoprecipitations of this preparation demonstrated that F1 interacts with both c and mutant b subunits. A strain carrying the mutation, bgly9----asp, and the compensating suppressor mutation apro240----leu (previously shown to be partially unc+) yielded an F1F0 ++ complex that remained partially defective in F1 binding to F0 but normal in the subunit composition of the F0 sector. The assembly, structure, and function of the F1F0-ATPase is discussed.     

Identification of subunits required for the catalytic activity of the F1-ATPase

F₁() complexes containing equimolar ratios of the and subunits have been shown to function as active ATPases, whereas individually isolated and subunits show no real ATPase activity. These results indicate that the single-copy subunits are not required for F₁-ATPase activity. The minimal F₁()-core complexes exhibit, however, lower rates and some different properties from those of their parent whole F₁ or ₃₃ complexes. It is therefore concluded that for obtaining a full spectrum of the characteristic functional properties of an F₁-ATPase the presence of the F₁- subunit is also required. The implications of these findings on the subunit location of both catalytic and noncatalytic nucleotide binding sites is discussed.     

Modifiers of the oligomycin sensitivity of the mitochondrial F1F0-ATPase

The mitochondrial F₁F₀ complex is highly sensitive to macrolide antibiotics and especially targeted by oligomycins. These compounds bind to the membrane-embedded sector F₀ and block proton conductance through the inner membrane, thus inhibiting both ATP synthesis and hydrolysis. Oligomycin sensitivity is universally recognized as a clue of the functional integrity and matching between F₀ and F₁. Since oligomycin binding implies multiple interactions with amino acid residues of F₀, amino acid substitutions often affect the inhibition efficiency. Moreover, variegated factors spanning from membrane properties to xenobiotic incorporation and detachment of the oligomycin-insensitive F₁ sector can alter the oligomycin sensitivity of the enzyme complex. The overview on the multiple factors involved strengthens the link between altered oligomycin sensitivity and physiopathological conditions associated with defective ATPases. An improved understanding of the mechanisms involved may also favor drug design to counteract oxidative damage, which stems from most mitochondrial dysfunctions.     

Myocardial ischemic preconditioning and mitochondrial F1F0-ATPase activity

A short period of ischemia followed by reperfusion (ischemic preconditioning) is known to trigger mechanisms that contribute to the prevention of ATP depletion. In ischemic conditions, most of the ATP hydrolysis can be attributed to mitochondrial F₁F₀-ATPase (ATP synthase). The purpose of the present study was to examine the effect of myocardial ischemic preconditioning on the kinetics of ATP hydrolysis by F₁F₀-ATPase. Preconditioning was accomplished by three 3-min periods of global ischemia separated by 3 min of reperfusion. Steady state ATP hydrolysis rates in both control and preconditioned mitochondria were not significantly different. This suggests that a large influence of the enzyme on the preconditioning mechanism may be excluded. However, the time required by the reaction to reach the steady state rate was increased in the preconditioned group before sustained ischemia, and it was even more enhanced in the first 5 min of reperfusion (101 ± 3.0 sec in preconditioned vs. 83.4 ± 4.4 sec in controls, p 0.05). These results suggest that this transient increase in activation time may contribute to the cardioprotection by slowing the ATP depletion in the very critical early phase of post-ischemic reperfusion.     

Structural and functional characterization of subunits of the F0 sector of the mitochondrial F0F1-ATP synthase.

Proteolytic digestion of F1-depleted submitochondrial particles (USMP), reconstitution with isolated subunits and titration with inhibitors show that the nuclear-encoded PVP protein, previously identified as an intrinsic component of bovine heart F0 (F01) (Zanotti, F. et al. (1988) FEBS Lett. 237, 9-14), is critically involved in maintaining the proper H+ translocating configuration of this sector and its correct binding to the F1 catalytic moiety. Trypsin digestion of USMP, under conditions leading to cleavage of the carboxyl region of the PVP protein and partial inhibition of transmembrane H+ translocation, results in general loss of sensitivity of this process to F0 inhibitors. This is restored by addition of the isolated PVP protein. Trypsin digestion of USMP causes also loss of oligomycin sensitivity of the catalytic activity of membrane reconstituted soluble F1, which can be restored by the combined addition of PVP and OSCP, or PVP and F6. Amino acid sequence analysis shows that, in USMP, modification by [14C] N,N'-dicyclohexylcarbodiimide of subunit c of F0 induces the formation of a dimer of this protein, which retains the 14C-labelled group. Chemical modification of cysteine-64 of subunit c results in inhibition of H+ conduction by F0. The results indicate that proton conduction in mitochondrial F0 depends on interaction of subunit c with the PVP protein.     

Voltage-driven ATP synthesis by beef heart mitochondrial F0F1-ATPase

The F0F1-ATPase of the inner mitochondrial membrane catalyzes the conversion of a proton electrochemical energy into the chemical bond energy of ATP (Boyer, P.D., Chance, B., Ernster, L., Mitchell, P., Racker, E., and Slater, E.C. (1977) Annu. Rev. Biochem. 46, 955-1026). To assess the role of the membrane potential (delta psi) in this process and to study the effect of very short pulses on ATP synthesis, we employed a high voltage pulsation method (Kinosita, K., and Tsong, T.Y. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1923-1927) to induce a delta psi of controlled magnitude and duration in a suspension of submitochondrial particles and F0F1-ATPase vesicles. Cyanide-treated submitochondrial particles were exposed to electric pulses of 10-30 kV/cm of magnitude (generating a peak delta psi of 150-450 mV) and 1-100 microseconds duration. Net [32P]ATP synthesis from [32P]Pi and ADP was observed with maximal values of 410 pmol/mg X pulse for a 30 kV/cm-100-microseconds pulse. This corresponds to a yield of 10-12 mol of ATP per mol of F0F1 complex per pulse. As many as 4 nmol/mg were produced after pulsing the same sample 8 times. By varying the ionic strength of the suspending medium, and consequently the pulse width, it is clearly shown that the synthesis was electrically driven and did not correlate with Joule heating of the sample. Titrations using specific inhibitors and ionophores were performed. The voltage-induced ATP synthesis was 50% inhibited by 0.11 microgram/mg of oligomycin and 2.4 nmol/mg of N,N'-dicyclohexylcarbodiimide. Ionophores and uncouplers had varying degrees of inhibition. The dependence of ATP synthesis on pulse width was nonlinear, exhibiting a threshold at 10 microseconds and a biphasic behavior above this value. Isolated F0F1-ATPase reconstituted into asolectin vesicles also synthesized ATP when pulsed with electric fields. A 35 kV/cm pulse induced the synthesis of 115 pmol of ATP per mg of protein, which corresponds to approximately 0.34 mol of ATP per mol of F0F1-ATPase. This synthesis was also sensitive to oligomycin and dicyclohexylcarbodiimide. The possibility of turnover of the ATPase in microseconds is considered.     

Correspondence of minor subunits of plant mitochondrial F1ATPase to F1F0ATPase subunits of other organisms

In addition to two major alpha- and beta-subunits, the soluble oligomycin-insensitive F1ATPase purified from sweet potato root mitochondria contains four different minor subunits of gamma (Mr = 35,500), delta (Mr = 27,000), delta' (Mr = 23,000), and epsilon (Mr = 12,000) (Iwasaki, Y., and Asashi, T. (1983) Arch. Biochem. Biophys. 227, 164-173). Among these minor subunits, the delta-subunit specifically cross-reacted with an antibody against the delta-subunit of maize mitochondrial F1 which contains only three minor gamma-, delta- and epsilon-subunits like F1ATPases from other organisms, indicating that the delta'-subunit is an extra subunit of sweet potato F1 which is absent in the maize F1. All of the four minor subunits of sweet potato F1 were purified and their N-terminal amino acid sequences of 30-36 residues were determined. The N-terminal sequence of gamma-subunit was homologous to those of the gamma-subunits of bacterial F1 and mammalian mitochondrial F1. The N-terminal sequence of the delta-subunit was homologous to those of the delta-subunits of bacterial F1, chloroplast CF1, and oligomycin sensitivity conferring protein of bovine mitochondrial F1F0. A sequence homology was also observed between the sweet potato epsilon-subunit and the epsilon-subunit of bovine mitochondrial F1. The N-terminal sequence of the delta'-subunit did not show any significant sequence homology to known protein sequences. These subunit correspondences place plant mitochondrial F1 at an unique position in the evolution of F1ATPase.     

Effect of polyamines on mitochondrial F1-ATPase catalyzed reactions

The effect of polyamines on F1-ATPase catalyzed reactions has been studied through the use of submitochondrial particles and F1-ATPase. ATP degradation catalyzed by submitochondrial particles and F1-ATPase was inhibited by spermine and spermidine. Spermine's inhibition was much greater than spermidine's effect. In contrast, P1-ATP exchange and succinate dependent ATP synthesis catalyzed by submitochondrial particles were both stimulated by spermine. The inhibition of ATPase activity by polyamines probably occurs through polyamine's replacement of Mg2+ on ATP, for the following reasons. (a) The ATPase activity inhibited by spermine was partially recovered when Mg2+ was added. (b) Spermine bound to ATP and phospholipids but not to F1-ATPase; yet spermine inhibited the ATPase reaction catalyzed by F1-ATPase, a protein free of phospholipid. (c) The binding of spermine to ATP was inhibited by Mg2+. The ATP content in polyamine-deficient cells definitely was lower than that in normal cells. On the basis of these results, the possible role of spermine in keeping the ATP concentration at a high level is discussed.     

Reconstitution of mitochondrial F0.F1-ATPase with phosphatidylcholine using the nonionic detergent, octylglucoside

A reconstitution procedure has been developed for the incorporation of the mitochondrial F0.F1-ATPase into the bilayer of egg phosphatidylcholine vesicles. The nonionic detergent, octylglucoside, egg phosphatidylcholine, and the lipid-deficient, oligomycin-sensitive F0.F1-ATPase (Serrano, R., Kanner, B., and Racker, E. (1976) J. Biol. Chem. 251, 2453-2461) were combined in a 4770:320:1 detergent/phospholipid/protein molar ratio and then centrifuged on a discontinuous sucrose gradient to isolate the F0.F1-phosphatidylcholine complex. The specific activity of the reconstituted F0.F1-ATPase was as high as 14.5 mumol/min/mg protein, whereas with no added lipid the activity ranged between 1.4 and 2.2 mumol/min/mg protein. This reconstituted preparation exhibited greater than 90% oligomycin sensitivity which demonstrated the intactness of the multisubunit enzyme complex. The phosphatidylcholine/protein molar ratio of the reconstituted F0.F1 was 250:1 with less than 0.4% of the added octylglucoside remaining. Titrations with both phosphatidylcholine and octylglucoside demonstrated that the specific activity and oligomycin sensitivity were highly dependent on the concentrations of both phospholipid and detergent in the original reconstitution mixture. Analysis of the reconstituted ATPase by electron microscopy demonstrated that the catalytic portion of the enzyme complex projected from the phospholipid bilayer with an orientation similar to that observed with submitochondrial particles. The F0.F1-phosphatidylcholine complex was able to trap inulin, which suggests a vesicular structure impermeable to macromolecules. The electrophoretic mobility of the complex was identical to that for liposomes of egg phosphatidylcholine alone. The reconstitution conditions utilized give rise to an enzyme-phospholipid complex with very low ionic charge that demonstrates high oligomycin-sensitive ATPase activity.     

1H-NMR studies on nucleotide binding to the catalytic sites of bovine mitochondrial F1-ATPase

The conformation of adenine nucleotides bound to bovine mitochondrial F1-ATPase was investigated using transfer nuclear Overhauser enhancement measurements. It is shown that all nucleotides investigated adopt a predominantly anti conformation when bound to the catalytic sites. Furthermore, the experiment suggests that 8-azido-ADP and 8-azido-ATP, which are predominantly in the syn conformation in solution, are in the anti conformation when bound to F1 catalytic sites.     

A switch in enantiomer preference between mitochondrial F1F0-ATPase chemotypes

The preferred absolute configuration of two series of F(1)F(0)-ATP synthase inhibitors was determined. Although the configuration of the active enantiomer in each series is different, each series presents the same 'triaryl' pharmacophore to the enzyme binding site.     

Inhibition of yeast mitochondrial F1-ATPase, F0F1-ATPase and submitochondrial particles by rhodamines and ethidium bromide

ATP hydrolysis by F1-ATPase is strongly inhibited by cationic rhodamines; neutral rhodamines are very poor inhibitors. Rhodamine 6G is a noncompetitive inhibitor of purified F0F1-ATPase and submitochondrial particles, however, an uncompetitive inhibitor of F1-ATPase (KI approximately equal to 2.4 microM for all three enzyme forms). Ethidium bromide is a noncompetitive inhibitor of F0F1-ATPase, submitochondrial particles and also F1-ATPase (KI approximately equal to 270 microM). Neither of the inhibitors affects the negative cooperativity (nH approximately equal to 0.7). The non-identical binding sites for rhodamine 6G and ethidium bromide are located on the F1-moiety and are topologically distinct from the catalytic site. Binding of the inhibitors prevents the conformational changes essential for energy transduction. It is concluded that the inhibitor binding sites are involved in proton translocation. In F1-ATPase, binding of MgATP at a catalytic site causes conformational changes, which allosterically induce the correct structure of the rhodamine 6G binding site. In F0F1-ATPase, this conformation of the F1-moiety exists a priori, due to allosteric interactions with F0-subunits. The binding site for ethidium bromide on F1-ATPase does not require substrate binding at the catalytic site and is not affected by F0F1-subunit interactions.     

Insertion of mitochondrial DNA-encoded F1F0-ATPase subunit 8 across the mitochondrial inner membrane in vitro

Cytochrome oxidase subunits I, II, and III, the mitochondrial DNA-encoded proteins, are inserted across the inner membrane by the Oxa1p-containing translocator in a membrane potential-dependent manner. Oxa1p is also involved in the insertion of the cytoplasmically synthesized precursor of Oxa1p itself into the inner membrane from the matrix via the conservative sorting pathway. The mechanism of insertion of the other mitochondrially synthesized proteins, however, is unexplored. The insertion of the mitochondrial DNA-encoded subunit 8 of F(1)F(0)-ATPase (Su8) across the inner membrane was analyzed in vitro using the inverted inner membrane vesicles and the Escherichia coli lysate-synthesized substrate. This assay revealed that the N-terminal segment of Su8 inserted across the membrane to the intermembrane space and assumed the correct trans-cis topology depending on the mitochondrial matrix fraction. This translocation reaction was similar to those of Sec-independent, direct insertion pathways of E. coli and chloroplast thylakoid membranes. (i) It required neither nucleotide triphosphates nor membrane potential, and hydrophobic forces drove the process. (ii) It did not require protease-sensitive membrane components facing the matrix space. (iii) It could be inserted across liposomes in the correct topology in a matrix fraction-dependent manner. Thus, a novel mechanism conserved in bacteria and chloroplasts also functions in the insertion of Su8 across the mitochondrial inner membrane.     

Modulation of mitochondrial F0F1 catalysis by boundary and bulk phase phospholipids

The importance of boundary and bulk phase phospholipids was studied on a mitochondrial ATPase complex isolated by AH-Sepharose chromatography as described by Dreyfus et al (1984, Anal. Biochem. 142,215-220), this preparation was devoid of the adenine nucleotide carrier. The presence of isoelectric or acidic phospholipids during the purification in the column allows the exchange of tightly bound phospholipids up to 95%. ATP hydrolysis and oligomycin sensitivity were slightly affected by the nature of boundary and bulk phase phospholipids, while Pi-ATP exchange was highly inhibited.     

Inhibition of the mitochondrial F1F0-ATPase by ligands of the peripheral benzodiazepine receptor

Although PK11195 binds to the peripheral benzodiazepine receptor with nanomolar affinity, significant data exist which suggest that it has another cellular target distinct from the PBR. Here we demonstrate that PK11195 inhibits F(1)F(0)-ATPase activity in an OSCP-dependent manner, similar to the pro-apoptotic benzodiazepine Bz-423. Importantly, our data indicate that cellular responses observed with micromolar concentrations of PK11195, which are commonly attributed to modulation of the PBR, are likely a direct result of mitochondrial F(1)F(0)-ATPase inhibition.     

The delta'-subunit of higher plant six-subunit mitochondrial F1-ATPase is homologous to the delta-subunit of animal mitochondrial F1-ATPase.

Mitochondrial F1-ATPases purified from several dicotyledonous plants contain six different subunits of alpha, beta, gamma, delta, delta' and epsilon. Previous N-terminal amino acid sequence analyses indicated that the gamma-, delta-, and epsilon-subunits of the sweet potato mitochondrial F1 correspond to the gamma-subunit, the oligomycin sensitivity-conferring protein and the epsilon-subunit of animal mitochondrial F1F0 complex (Kimura, T., Nakamura, K., Kajiura, H., Hattori, H., Nelson, N., and Asahi, T. (1989) J. Biol. Chem. 264, 3183-3186). However, the N-terminal amino acid sequence of the delta'-subunit did not show any obvious homologies with known protein sequences. A cDNA clone for the delta'-subunit of the sweet potato mitochondrial F1 was identified by oligonucleotide-hybridization selection of a cDNA library. The 1.0-kilobase-long cDNA contained a 600-base pair open reading frame coding for a precursor for the delta'-subunit. The precursor for the delta'-subunit contained N-terminal presequence of 21-amino acid residues. The mature delta'-subunit is composed of 179 amino acids and its sequence showed similarities of about 31-36% amino acid positional identity with the delta-subunit of animal and fungal mitochondrial F1 and about 18-25% with the epsilon-subunit of bacterial F1 and chloroplast CF1. The sweet potato delta'-subunit contains N-terminal sequence of about 45-amino acid residues that is absent in other related subunits. It is concluded that the six-subunit plant mitochondrial F1 contains the subunit that is homologous to the oligomycin sensitivity-conferring protein as one of the component in addition to five subunits that are homologous to subunits of animal mitochondrial F1.     

Plant mitochondrial F0F1 ATP synthase. Identification of the individual subunits and properties of the purified spinach leaf mitochondrial ATP synthase.

Spinach leaf mitochondrial F0F1 ATPase has been purified and is shown to consist of twelve polypeptides. Five of the polypeptides constitute the F1 part of the enzyme. The remaining polypeptides, with molecular masses of 28 kDa, 23 kDa, 18.5 kDa, 15 kDa, 10.5 kDa, 9.5 kDa and 8.5 kDa, belong to the F0 part of the enzyme. This is the first report concerning identification of the subunits of the plant mitochondrial F0. The identification of the components is achieved on the basis of the N-terminal amino acid sequence analysis and Western blot technique using monospecific antibodies against proteins characterized in other sources. The 28-kDa protein crossreacts with antibodies against the subunit of bovine heart ATPase with N-terminal Pro-Val-Pro- which corresponds to subunit F0b of Escherichia coli F0F1. Sequence analysis of the N-terminal 32 amino acids of the 23-kDa protein reveals that this protein is similar to mammalian oligomycin-sensitivity-conferring protein and corresponds to the F1 delta subunit of the chloroplast and E. coli ATPases. The 18.5-kDa protein crossreacts with antibodies against subunit 6 of the beef heart F0 and its N-terminal sequence of 14 amino acids shows a high degree of sequence similarity to the conserved regions at N-terminus of the ATPase subunits 6 from different sources. ATPase subunit 6 corresponds to subunit F0a of the E. coli enzyme. The 15-kDa protein and the 10.5-kDa protein crossreact with antibodies against F6 and the endogenous ATPase inhibitor protein of beef heart F0F1-ATPase, respectively. The 9.5-kDa protein is an N,N'-dicyclohexylcarbodiimide-binding protein corresponding to subunit F0c of the E. coli enzyme. The 8.5-kDa protein is of unknown identity. The isolated spinach mitochondrial F0F1 ATPase catalyzes oligomycin-sensitive ATPase activity of 3.5 mumol.mg-1.min-1. The enzyme catalyzes also hydrolysis of GTP (7.5 mumol.mg-1.min-1) and ITP (4.4 mumol.mg-1.min-1). Hydrolysis of ATP was stimulated fivefold in the presence of amphiphilic detergents, however the hydrolysis of other nucleotides could not be stimulated by these agents. These results show that the plant mitochondrial F0F1 ATPase complex differs in composition from the other mitochondrial, chloroplast and bacterial ATPases. The enzyme is, however, more closely related to the yeast mitochondrial ATPase and to the animal mitochondrial ATPase than to the chloroplast enzyme. The plant mitochondrial enzyme, however, exhibits catalytic properties which are characteristic for the chloroplast enzyme.     

Effect of the epsilon-subunit on nucleotide binding to Escherichia coli F1-ATPase catalytic sites.

The influence of the epsilon-subunit on the nucleotide binding affinities of the three catalytic sites of Escherichia coli F1-ATPase was investigated, using a genetically engineered Trp probe in the adenine-binding subdomain (beta-Trp-331). The interaction between epsilon and F1 was not affected by the mutation. Kd for binding of epsilon to betaY331W mutant F1 was approximately 1 nM, and epsilon inhibited ATPase activity by 90%. The only nucleotide binding affinities that showed significant differences in the epsilon-depleted and epsilon-replete forms of the enzyme were those for MgATP and MgADP at the high-affinity catalytic site 1. Kd1(MgATP) and Kd1(MgADP) were an order of magnitude higher in the absence of epsilon than in its presence. In contrast, the binding affinities for MgATP and MgADP at sites 2 and 3 were similar in the epsilon-depleted and epsilon-replete enzymes, as were the affinities at all three sites for free ATP and ADP. Comparison of MgATP binding and hydrolysis parameters showed that in the presence as well as the absence of epsilon, Km equals Kd3. Thus, in both cases, all three catalytic binding sites have to be occupied to obtain rapid (Vmax) MgATP hydrolysis rates.