Reconstitution of the H+-ATPase complex of Rhodospirillum rubrum by the beta subunit of the chloroplast coupling factor 1

A method is described for isolating the beta subunit from spinach chloroplast F1 (CF1). The isolated beta subunit reconstituted an active F1 hybrid with the F1 of Rhodospirillum rubrum chromatophores from which the beta subunit had been removed. The CF1 beta subunit was similar to the isolated beta subunit of Escherichia coli F1 (Gromet-Elhanan, Z., Khananshivili, D., Weiss, S., Kanazawa, H., and Futai, M. (1985) J. Biol. Chem. 260, 12635-12640) in that it restored a substantial rate of ATP hydrolysis and low, but significant light-dependent ATP synthesis to the beta-less chromatophores. The low rate of photophosphorylation observed with the hybrid enzyme probably resulted from a looser coupling of the CF1 beta subunit to proton translocation in the R. rubrum Fo-F1 complex. The hybrid enzyme exhibited a high specificity for Mg2+-ATP as substrate for ATP hydrolysis and both ATP synthesis and hydrolysis were strongly inhibited by the antibiotic tentoxin. In contrast, chromatophores reconstituted with the native R. rubrum beta subunit actively hydrolyzed both Mg2+-ATP and Ca2+-ATP and were insensitive to tentoxin. These results indicate a close functional homology between the beta subunits of the prokaryotic and eukaryotic H+-ATPases and suggest a role for the beta subunit in conferring the different metal ion specificities and inhibitor sensitivities upon the enzymes. They also demonstrate the feasibility of isolating the beta subunit from CF1 in a reconstitutively active form.     

Formation and properties of hybrid photosynthetic F1-ATPases. Demonstration of different structural requirements for stimulation and inhibition by tentoxin.

A hybrid ATPase composed of cloned chloroplast ATP synthase beta and gamma subunits (betaC and gammaC) and the cloned alpha subunit from the Rhodospirillum rubrum ATP synthase (alphaR) was assembled using solubilized inclusion bodies and a simple single-step folding procedure. The catalytic properties of the assembled alpha3Rbeta3CgammaC were compared to those of the core alpha3Cbeta3CgammaC complex of the native chloroplast coupling factor 1 (CF1) and to another recently described hybrid enzyme containing R. rubrum alpha and beta subunits and the CF1 gamma subunit (alpha3Rbeta3RgammaC). All three enzymes were similarly stimulated by dithiothreitol and inhibited by copper chloride in response to reduction and oxidation, respectively, of the disulfide bond in the chloroplast gamma subunit. In addition, all three enzymes exhibited the same concentration dependence for inhibition by the CF1 epsilon subunit. Thus the CF1 gamma subunit conferred full redox regulation and normal epsilon binding to the two hybrid enzymes. Only the native CF1 alpha3Cbeta3CgammaC complex was inhibited by tentoxin, confirming the requirement for both CF1 alpha and beta subunits for tentoxin inhibition. However, the alpha3Rbeta3CgammaC complex, like the alpha3Cbeta3CgammaC complex, was stimulated by tentoxin at concentrations in excess of 10 microm. In addition, replacement of the aspartate at position 83 in betaC with leucine resulted in the loss of stimulation in the alpha3Rbeta3CgammaC hybrid. The results indicate that both inhibition and stimulation by tentoxin require a similar structural contribution from the beta subunit, but differ in their requirements for alpha subunit structure.     

Role of the ATP synthase alpha-subunit in conferring sensitivity to tentoxin.

Tentoxin, produced by phytopathogenic fungi, selectively affects the function of the ATP synthase enzymes of certain sensitive plant species. Binding of tentoxin to a high affinity (K(i) approximately 10 nM) site on the chloroplast F(1) (CF(1)) strongly inhibits catalytic function, whereas binding to a second, lower affinity site (K(d) > 10 microM) leads to restoration and even stimulation of catalytic activity. Sensitivity to tentoxin has been shown to be due, in part, to the nature of the amino acid residue at position 83 on the catalytic beta subunit of CF(1). An aspartate in this position is required, but is not sufficient, for tentoxin inhibition. By comparison with the solved structure of mitochondrial F(1) [Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628], Asp83 is probably located at an interface between alpha and beta subunits on CF(1) where residues on the alpha subunit could also participate in tentoxin binding. A hybrid core F(1) enzyme assembled with beta and gamma subunits of the tentoxin-sensitive spinach CF(1), and an alpha subunit of the tentoxin-insensitive photosynthetic bacterium Rhodospirillum rubrum F(1) (RrF(1)), was stimulated but not inhibited by tentoxin [Tucker, W. C., Du, Z., Gromet-Elhanan, Z. and Richter, M. L. (2001) Eur. J. Biochem. 268, 2179-2186]. In this study, chimeric alpha subunits were prepared by introducing short segments of the spinach CF(1) alpha subunit from a poorly conserved region which is immediately adjacent to beta-Asp83 in the crystal structure, into equivalent positions in the RrF(1) alpha subunit using oligonucleotide-directed mutagenesis. Hybrid enzymes containing these chimeric alpha subunits had both the high affinity inhibitory tentoxin binding site and the lower affinity stimulatory site. Changing beta-Asp83 to leucine resulted in loss of both inhibition and stimulation by tentoxin in the chimeras. The results indicate that tentoxin inhibition requires additional alpha residues that are not present on the RrF(1) alpha subunit. A structural model of a putative inhibitory tentoxin binding pocket is presented.     

Isolation and purification of an active gamma-subunit of the F0.F1-ATP synthase from chromatophore membranes of Rhodospirillum rubrum. The role of gamma in ATP synthesis and hydrolysis as compared to proton translocation

We have earlier shown that extraction of Rhodospirillum rubrum chromatophores with LiCl removed completely the beta-subunit of their coupling factor ATPase complex leaving the other four subunits attached to the membrane (Philosoph, S., Binder, A., and Gromet-Elhanan, Z. (1977) J. Biol. Chem. 252, 8747-8752). Further treatment of these beta-less chromatophores with LiBr, under the described optimal conditions, resulted in specific removal of one additional subunit, the gamma-subunit, and both subunits were purified to homogeneity. The beta, gamma-less chromatophores as well as the beta-less ones lost their ATP-linked activities, but retained their light-induced proton uptake, resulting in the formation of an electrochemical gradient of protons composed of both a pH gradient and a membrane potential. These results indicate that the removed beta and gamma subunits cannot be an integral part of an H+ gate in the R. rubrum chromatophore membrane. Each of the removed subunits could bind to the beta, gamma-less chromatophores, but such separate reconstitution of either beta or gamma alone did not lead to restoration of any ATP-linked activity. ATP synthesis and hydrolysis could be restored to the same extent to these chromatophores by their reconstitution with both beta and gamma. It is thus concluded that the presence of both subunits is required for ATP synthesis as well as hydrolysis by the R. rubrum F0.F1 complex. The identical degree of elimination and restoration of ATP synthesis and hydrolysis upon removal and reconstitution of beta and gamma indicates that in R. rubrum at least, there seems to be no reason for suggesting the operation of different catalytic sites for the two activities.     

Extraction and purification of the beta subunit and an active alpha beta-core complex from the spinach chloroplast CFoF1-ATP synthase.

Incubation of Rhodospirillum rubrum chromatophores with 2 M LiCl in the presence of MgATP has been shown to remove their F1 beta subunit leaving inactive but fully reconstitutable beta-less chromatophores (Gromet-Elhanan, Z., and Khanashvili, D., (1986) Methods Enzymol, 126, 528-538). A similar treatment of thoroughly washed spinach thylakoids has now been shown to release the CF1 beta subunit (CF1 beta) together with a complex containing equal amounts of CF1 alpha and CF1 beta (CF1 (alpha beta]. The purified CF1 (alpha beta) complex can reconstitute an active membrane-bound hybrid F0F1-ATPase with beta-less R. rubrum chromatophores and also catalyzes a low but very reproducible soluble MgATPase. Purified CF1 beta shows none of these activities although it can bind as efficiently as CF1 (alpha beta) to the beta-less chromatophores. By subjecting the crude spinach 2 m LiCl extract to dissociating conditions an enriched CF1 beta preparation is released. It contains traces of CF1 alpha and CF1 delta, is able to reconstitute an active hybrid F0F1-ATPase but, as the pure CF1 beta shows no soluble ATPase activity. These results indicate that trace amounts of CF1 alpha are enough for endowing CF1 beta with a reconstitutive capacity, but for exhibition of a significant soluble ATPase activity equivalent amounts of CF1 alpha and beta are required. The CF 1 (alpha beta) complex isolated and purified in this report thus represents the minimal catalytic core of the CF1-ATPase.     

Isolation and characterisation of a functional alpha beta heterodimer from the ATP synthase of Rhodospirillum rubrum.

An alpha beta heterodimer of the F1-ATPase of Rhodospirillum rubrum was isolated by extraction of chromatophores with LiCl. Each alpha beta heterodimer contains one tightly bound ADP, which is released upon removal of medium Mg2+. The dimer can be reversibly dissociated by removal of Mg(2+)-ions. The alpha beta heterodimer restores both ATP-synthetic and -hydrolytic activities to LiCl-treated chromatophores, saturation being achieved at approximately 2 mmol alpha beta.mol BChl-1. The heterodimer itself hydrolyses Mg-ATP with an activity distinct from RF1, being unaffected by azide or sulphite ions. The Vmax and Km (ATP) for this Mg(2+)-dependent activity were 110 +/- 10 nmol.min-1.mg protein-1 and 100 +/- 30 microM, respectively. The Km did not differ significantly from that of RF1.     

Assembled F1-(alpha beta ) and Hybrid F1-alpha 3beta 3gamma -ATPases from Rhodospirillum rubrum alpha, wild type or mutant beta, and chloroplast gamma subunits. Demonstration of Mg2+versus Ca2+-induced differences in catalytic site structure and function

Refolding together the expressed alpha and beta subunits of the Rhodospirillum rubrum F(1)(RF(1))-ATPase led to assembly of only alpha(1)beta(1) dimers, showing a stable low MgATPase activity. When incubated in the presence of AlCl(3), NaF and either MgAD(T)P or CaAD(T)P, all dimers associated into closed alpha(3)beta(3) hexamers, which also gained a low CaATPase activity. Both hexamer ATPase activities exhibited identical rates and properties to the open dimer MgATPase. These results indicate that: a) the hexamer, as the dimer, has no catalytic cooperativity; b) aluminium fluoride does not inhibit their MgATPase activity; and c) it does enable the assembly of RrF(1)-alpha(3)beta(3) hexamers by stabilizing their noncatalytic alpha/beta interfaces. Refolding of the RrF(1)-alpha and beta subunits together with the spinach chloroplast F(1) (CF(1))-gamma enabled a simple one-step assembly of two different hybrid RrF(1)-alpha(3)beta(3)/CF(1)gamma complexes, containing either wild type RrF(1)-beta or the catalytic site mutant RrF(1)beta-T159S. They exhibited over 100-fold higher CaATPase and MgATPase activities than the stabilized hexamers and showed very different catalytic properties. The hybrid wild type MgATPase activity was, as that of RrF(1) and CF(1) and unlike its higher CaATPase activity, regulated by excess free Mg(2+) ions, stimulated by sulfite, and inhibited by azide. The hybrid mutant had on the other hand a low CaATPase but an exceptionally high MgATPase activity, which was much less sensitive to the specific MgATPase effectors. All these very different ATPase activities were regulated by thiol modulation of the hybrid unique CF(1)-gamma disulfide bond. These hybrid complexes can provide information on the as yet unknown factors that couple ATP binding and hydrolysis to both thiol modulation and rotational motion of their CF(1)-gamma subunit.     

ATP synthesis and hydrolysis by a hybrid system reconstituted from the beta-subunit of Escherichia coli F1-ATPase and beta-less chromatophores of Rhodospirillum rubrum

Photophosphorylation and ATPase activities were restored to beta-less Rhodospirillum rubrum chromatophores by their reconstitution with purified beta-subunits of either R. rubrum F1-ATPase (Rr beta) or Escherichia coli F1-ATPase (Ec beta). In the homologous reconstituted system both activities were restored to the same extent, whereas in the hybrid system ATP synthesis was restored to about 10% when the hydrolysis was restored to 200%. This difference in rates of synthesis and hydrolysis was not due to any general uncoupling effect of Ec beta leading to an increased membrane permeability to protons, because with both hybrid and homologous systems an identical light-induced quenching of quinacrine fluorescence was observed. They differed, however, in ATP-driven quenching of quinacrine fluorescence, which was much lower in the hybrid system. These results suggest that the hybrid has a decreased capacity for proton-translocation through the membrane-bound Fo channel during ATP hydrolysis, and probably also during ATP synthesis. The very high ATPase activity of the hybrid system indicates that it might enable the released protons to leak to the outside medium rather than to move inside through the Fo channel. The activities restored by Rr beta and Ec beta exhibit a similar sensitivity to dicyclohexylcarbodiimide, but different sensitivities to oligomycin and to an anti-E. coli F1 (EcF1) antibody. Oligomycin inhibited only the homologous R. rubrum system whereas anti-EcF1 was a much more effective inhibitor of the hybrid system. It is therefore concluded that Rr beta plays a role, that the Ec beta cannot fulfill, in conferring oligomycin sensitivity to the RrFo X F1-ATP synthase-ATPase complex.     

Reactivation of the chloroplast CF1-ATPase beta subunit by trace amounts of the CF1 alpha subunit suggests a chaperonin-like activity for CF1 alpha.

Incubation of tobacco and lettuce thylakoids with 2 M LiCl in the presence of MgATP removes the beta subunit from their CF1-ATPase (CF1 beta) together with varying amounts of the CF1 alpha subunit (CF1 alpha). These 2 M LiCl extracts, as with the one obtained from spinach thylakoids (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072), could form active hybrid ATPases when reconstituted into inactive beta-less Rhodospirillum rubrum chromatophores. Pure CF1 beta fractions that have been isolated from these extracts could not form such active hybrids by themselves, but could do so when supplemented with trace amounts (less than 5%) of CF1 alpha. A mitochondrial F1-ATPase alpha subunit was recently reported to be a heat-shock protein, having two amino acid sequences that show a highly conserved identity with sequences found in molecular chaperones (Luis, A. M., Alconada, A., and Cuezva, J. M. (1990) J. Biol. Chem. 265, 7713-7716). These sequences are also conserved in CF1 alpha isolated from various plants, but not in F1 beta subunits. The above described reactivation of CF1 beta by trace amounts of CF1 alpha could thus be due to a chaperonin-like function of CF1 alpha, which involves the correct, active folding of isolated pure CF1 beta.     

Nucleotide sequences of the genes for the alpha,beta and epsilon subunits of wheat chloroplast ATP synthase

Summary The nucleotide sequences of the chloroplast genes for the alpha, beta and epsilon subunits of wheat chloroplast ATP synthase have been determined. Open reading frames of 1512 bp, 1494 bp and 411 bp are deduced to code for polypeptides of molecular weights 55201, 53796 and 15200, identified as the alpha, beta and epsilon subunits respectively by homology with the subunits from other sources and by amino acid sequencing of the epsilon subunit. The genes for the beta and epsilon subunits overlap by 4 bp. The gene for methionine tRNA is located 118 bp downstream from the epsilon subunit gene. Comparisons of the deduced amino acid sequences of the alpha and beta subunits with those from other species suggest regions of the proteins involved in adenine nucleotide binding.     

Covalent binding of 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (BzATP) to the isolated alpha and beta subunits and the alpha 3 beta 3 core complex of TF1. Covalent binding of BzATP prevents association of alpha and beta subunits and induces dissociation of the alpha 3 beta 3 core complex.

Binding of the photoreactive ATP analog, 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (BzATP), to the isolated alpha and beta subunits of TF1 and to the alpha 3 beta 3 "core" complex of the holoenzyme is described. About 1 mol of BzATP/mol of subunit was incorporated to isolated alpha and beta subunits. The incorporation of BzATP was prevented by ATP. Covalent binding of BzATP to the alpha subunit was in general somewhat lower than that observed with the beta subunit. No complex was formed upon mixing of either of the modified subunits with the complementary nontreated subunits. Covalent binding of 3 mol of BzATP/alpha 3 beta 3 complex completely inhibited ATPase activity and resulted in the dissociation of the complex. The labeled nucleotide analog was specifically incorporated into the beta subunit of the complex. The holoenzyme TF1, in contrast to the core complex, did not dissociate to the individual subunits upon covalent binding of BzATP. These results are discussed in relation to the location of the catalytic nucleotide binding site(s) and the conformation stability of the alpha 3 beta 3 core complex of TF1.     

The alpha 1 beta 1 heterodimer of ATP synthase

The alpha 3 beta 3 hexamer was reconstituted from the alpha and beta subunits of TF1 portion of ATP synthase of thermophilic bacterium (Kagawa et al. (1989) FEBS Lett. 249, 67). The alpha 1 beta 1 heterodimer of ATP synthase was isolated by high performance liquid chromatography (HPLC) of the alpha 3 beta 3 hexamer in the presence of AT(D)P-Mg. On polyacrylamide gel electrophoresis, both bands corresponding to the dimer and hexamer showed ATPase activity. The alpha 1 beta 1 dimer was dissociated into the equal amounts of the alpha and beta monomers by sodium dodecyl sulfate. The alpha and beta monomers were practically inactive. The alpha 2 and beta 2 homodimers were not detected by electrophoresis and HPLC.     

Stepping rotation of F(1)-ATPase with one, two, or three altered catalytic sites that bind ATP only slowly

F(1)-ATPase is an ATP hydrolysis-driven motor in which the gamma subunit rotates in the stator cylinder alpha(3)beta(3). To know the coordination of three catalytic beta subunits during catalysis, hybrid F(1)-ATPases, each containing one, two, or three "slow" mutant beta subunits that bind ATP very slowly, were prepared, and the rotations were observed with a single molecule level. Each hybrid made one, two, or three steps per 360 degrees revolution, respectively, at 5 microm ATP where the wild-type enzyme rotated continuously without step under the same observing conditions. The observed dwell times of the steps are explained by the slow binding rate of ATP. Except for the steps, properties of rotation, such as the torque forces exerted during rotary movement, were not significantly changed from those of the wild-type enzyme. Thus, it appears that the presence of the slow beta subunit(s) does not seriously affect other normal beta subunit(s) in the same F(1)-ATPase molecule and that the order of sequential catalytic events is faithfully maintained even when ATP binding to one or two of the catalytic sites is retarded.     

Nucleotide binding drives conformational changes in the isolated alpha and beta subunits of the F(1)-ATPase from Escherichia coli

The modeling of the rotatory mechanism performed by the F(1)-ATPase complex during ATP synthesis shows that the beta, but not the alpha subunit, undergoes large conformational changes that depend on the occupancy of the catalytic site. Here we determined by fluorescence spectroscopy the changes in tertiary structure and hydrophobic exposed area of the isolated alpha and beta subunits of the F(1)-ATPase complex from Escherichia coli upon adenine nucleotide binding. The results show that in the absence of intersubunit contacts, the two subunits exhibit markedly similar conformational movements.     

Molecular mechanisms of rotational catalysis in the F(0)F(1) ATP synthase

Rotation of the F(0)F(1) ATP synthase gamma subunit drives each of the three catalytic sites through their reaction pathways. The enzyme completes three cycles and synthesizes or hydrolyzes three ATP for each 360 degrees rotation of the gamma subunit. Mutagenesis studies have yielded considerable information on the roles of interactions between the rotor gamma subunit and the catalytic beta subunits. Amino acid substitutions, such as replacement of the conserved gammaMet-23 by Lys, cause altered interactions between gamma and beta subunits that have dramatic effects on the transition state of the steady state ATP synthesis and hydrolysis reactions. The mutations also perturb transmission of specific conformational information between subunits which is important for efficient conversion of energy between rotation and catalysis, and render the coupling between catalysis and transport inefficient. Amino acid replacements in the transport domain also affect the steady state catalytic transition state indicating that rotation is involved in coupling to transport.     

Reactions of a fluorescent ATP analog, 2'-(5-dimethyl-aminonaphthalene-1-sulfonyl) amino-2'-deoxyATP, with E. coli F1-ATPase and its subunits: the roles of the high affinity binding site in the alpha subunit and the low affinity binding site in the beta subunit

We performed kinetic studies on the reactions of a fluorescent ATP analog, 2'-(5-dimethyl-aminonaphthalene-1-sulfonyl) amino-2'-deoxyATP (DNS-ATP), with E. coli F1-ATPase (EF1) and its subunits, to clarify the role of each subunit in the ATPase reaction. The following results were obtained. 1. One mol of EF1, which contains nonexchangeable 2 mol ATP and 0.5 mol ADP, binds 3 mol of DNS-ATP. The apparent dissociation constant, in the presence of Mg2+, was 0.23 microM. Upon binding, the fluorescence intensity of DNS-ATP at 520 nm increased exponentially with t1/2 of 35 s, and reached 3.5 times the original fluorescence level. Following the fluorescence increase, DNS-ATP was hydrolyzed, and the fluorescence intensity maintained its enhanced level. 2. The addition of an excess of ATP over the EF1-DNS-nucleotide complex, in the presence of Mg2+, decreased the fluorescence intensity rapidly, indicating the acceleration of DNS-nucleotide release from EF1. ADP and GTP also decreased the fluorescence intensity. 3. DCCD markedly inhibited the accelerating effect of ATP on DNS-nucleotide release from EF1 and the EF1-DNS-ATPase or -ATPase activity in a steady state. On the other hand, DCCD only slightly inhibited the fluorescence increase of DNS-ATP, due to its binding to EF1, and the rate of single cleavage of 1 mol of DNS-ATP per mol of alpha subunit of EF1. 4. In the presence of Mg2+, 0.65-0.82 mol of DNS-ATP binds to 1 mol of the isolated alpha subunit of EF1 with an apparent dissociation constant of 0.06-0.07 microM. Upon binding, the fluorescence intensity of DNS-ATP at 520 nm increased 1.55 fold very rapidly (t1/2 less than 1 s). No hydrolysis of DNS-ATP was observed upon the addition of the isolated alpha subunit. The fluorescence intensity of DNS-ATP was unaffected by the addition of the isolated beta subunit. DNS-ATP was also unhydrolyzed by the isolated beta subunit. 5. EF1-ATPase was reconstituted from alpha, beta, and gamma subunits in the presence of Mg2+ and ATP. The kinetic properties of the fluorescence change of DNS-ATP in the reaction with the reconstituted EF1-ATPase were quite similar to those of native EF1. Most of our findings are consistent with a simple mechanism that the high affinity catalytic site and low affinity regulatory site exist in the alpha subunit and beta subunit, respectively. However, the findings mentioned in (4) suggest that the binding of the alpha and beta subunit, which is mediated by the gamma subunit, induces conformational change(s) in the ATP binding site located probably in the alpha subunit, and that the conformational change(s) is essential to exert the full hydrolyzing activity.     

Cytoplasmic inheritance of chloroplast coupling factor 1 subunits

An analysis of interspecific hybrids of Nicotiana spp. in which one of the parents was sensitive to tentoxin showed that this sensitivity was transmitted only through the female parent. Since tentoxin acts by selectively binding to the alpha,beta subunit complex of chloroplast coupling factor 1, the gene(s) specifying either one or both of these subunits is located in the cytoplasm.     

Mutations within the C-terminus of the gamma subunit of the photosynthetic F1-ATPase activate MgATP hydrolysis and attenuate the stimulatory oxyanion effect

Two highly conserved amino acid residues near the C-terminus within the gamma subunit of the mitochondrial ATP synthase form a "catch" with an anionic loop on one of the three beta subunits within the catalytic alphabeta hexamer of the F1 segment [Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628]. Forming the catch is considered to be an essential step in cooperative nucleotide binding leading to gamma subunit rotation. The analogous residues, Arg304 and Gln305, in the chloroplast F1 gamma subunit were changed to leucine and alanine, respectively. Each mutant gamma was assembled together with alpha and beta subunits from Rhodospirillum rubrum F1 into a hybrid photosynthetic F1 that carries out both MgATPase and CaATPase activities and ATP-dependent gamma rotation [Tucker, W. C., Schwarcz, A., Levine, T., Du, Z., Gromet-Elhanan, Z., Richter, M. L. and Haran, G. (2004) J. Biol. Chem. 279, 47415-47418]. Surprisingly, changing Arg304 to leucine resulted in a more than 2-fold increase in the kcat for MgATP hydrolysis. In contrast, changing Gln305 to alanine had little effect on the kcat but completely abolished the well-known stimulatory effect of the oxyanion sulfite on MgATP hydrolysis. The MgATPase activities of combined mutants with both residues substituted were strongly inhibited, whereas the CaATPase activities were inhibited, but to a lesser extent. The results indicate that the C-terminus of the photosynthetic F1 gamma subunit, like its mitochondrial counterpart, forms a catch with the alpha and beta subunits that modulates the nucleotide binding properties of the catalytic site(s). The catch is likely to be part of an activation mechanism, overcoming inhibition by free mg2+ ions, but is not essential for cooperative nucleotide exchange.     

C-Terminal mutations in the chloroplast ATP synthase gamma subunit impair ATP synthesis and stimulate ATP hydrolysis

Two highly conserved amino acid residues, an arginine and a glutamine, located near the C-terminal end of the gamma subunit, form a "catch" by hydrogen bonding with residues in an anionic loop on one of the three catalytic beta subunits of the bovine mitochondrial F1-ATPase [Abrahams, J. P., Leslie, A. G., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628]. The catch is considered to play a critical role in the binding change mechanism whereby binding of ATP to one catalytic site releases the catch and induces a partial rotation of the gamma subunit. This role is supported by the observation that mutation of the equivalent arginine and glutamine residues in the Escherichia coli F1 gamma subunit drastically reduced all ATP-dependent catalytic activities of the enzyme [Greene, M. D., and Frasch, W. D. (2003) J. Biol. Chem. 278, 5194-5198]. In this study, we show that simultaneous substitution of the equivalent residues in the chloroplast F1 gamma subunit, arginine 304 and glutamine 305, with alanine decreased the level of proton-coupled ATP synthesis by more than 80%. Both the Mg2+-dependent and Ca2+-dependent ATP hydrolysis activities increased by more than 3-fold as a result of these mutations; however, the sulfite-stimulated activity decreased by more than 60%. The Mg2+-dependent, but not the Ca2+-dependent, ATPase activity of the double mutant was insensitive to inhibition by the phytotoxic inhibitor tentoxin, indicating selective loss of catalytic cooperativity in the presence of Mg2+ ions. The results indicate that the catch residues are required for efficient proton coupling and for activation of multisite catalysis when MgATP is the substrate. The catch is not, however, required for CaATP-driven multisite catalysis or, therefore, for rotation of the gamma subunit.     

A homologous sequence between H+-ATPase (F0F1) and cation-transporting ATPases. Thr-285----Asp replacement in the beta subunit of Escherichia coli F1 changes its catalytic properties

A sequence of 10 amino acids (I-C-S-D-K-T-G-T-L-T) of ion motive ATPases such as Na+/K+-ATPase is similar to the sequence of the beta subunit of H+-ATPases, including that of Escherichia coli (I-T-S-T-K-T-G-S-I-T) (residues 282-291). The Asp (D) residue phosphorylated in ion motive ATPase corresponds to Thr (T) of the beta subunit. This substitution may be reasonable because there is no phosphoenzyme intermediate in the catalytic cycle of F1-ATPase. We replaced Thr-285 of the beta subunit by an Asp residue by in vitro mutagenesis and reconstituted the alpha beta gamma complex from the mutant (or wild-type) beta and wild-type alpha and gamma subunits. The uni- and multisite ATPase activities of the alpha beta gamma complex with mutant beta subunits were about 20 and 30% of those with the wild-type subunit. The rate of ATP binding (k1) of the mutant complex under uni-site conditions was about 10-fold less than that of the wild-type complex. These results suggest that Thr-285, or the region in its vicinity, is essential for normal catalysis of the H+-ATPase. The mutant complex could not form a phosphoenzyme under the conditions where the H+/K+-ATPase is phosphorylated, suggesting that another residue(s) may also be involved in formation of the intermediate in ion motive ATPase. The wild-type alpha beta gamma complex had slightly different kinetic properties from the wild-type F1, possibly because it did not contain the epsilon subunit.