Mutagenesis of beta -Glu-195 of the Rhodospirillum rubrum F1-ATPase and Its Role in Divalent Cation-dependent Catalysis*

Lubov NathansonDagger and Zippora Gromet-Elhanan§

From the Department of Biochemistry, The Weizmann Institute of Science, Rehovot 76100, Israel

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

We introduced mutations at the fully conserved residue Glu-195 in subunit beta  of Rhodospirillum rubrum F1-ATPase. The activities of the expressed wild type (WT) and mutant beta  subunits were assayed by following their capacity to assemble into the earlier prepared beta -depleted, membrane-bound R. rubrum enzyme (Philosoph, S., Binder, A., and Gromet-Elhanan, Z. (1977) J. Biol. Chem. 252, 8742-8747) and to restore ATP synthesis and/or hydrolysis activity. All three mutations, beta -E195K, beta -E195Q, and beta -E195G, were found to bind as the WTbeta into the beta -depleted enzyme. They restored between 30 and 60% of the WT restored photophosphorylation activity and 16, 45, and 105%, respectively of the CaATPase activity. The mutants required, however, much higher concentrations of divalent cations and could not restore any significant MgATPase or MnATPase activities. Only beta -E195G could restore some of these activities when assayed in the presence of 100 mM sulfite and high MgCl2 or MnCl2 concentrations. These results suggest that the observed difference in restoration of ATP synthesis and CaATPase, as compared with MgATPase and MnATPase, can be due to the tight regulation of the last two activities, resulting in their inhibition at cation/ATP ratios above 0.5. The R. rubrum F1beta -E195 is equivalent to the mitochondrial F1beta -E199, which points into the tunnel leading to the F1 catalytic nucleotide binding sites (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). Our findings indicate that this residue, although not an integral part of the F1 catalytic sites, affects divalent cation binding and release of inhibitory MgADP, suggesting its participation in the interconversion of the F1 catalytic sites between different conformational states.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

All respiratory and photosynthetic cells contain a membrane-embedded F0F1 ATP synthase that generates ATP at the expense of the electrochemical proton gradient formed during electron transport. The catalytic F1 component of the enzyme has been solubilized as a functional ATPase from many bacterial, mitochondrial, and chloroplast sources. It is a very conserved multimeric assembly with a stoichiometry of alpha 3beta 3gamma delta epsilon and has up to six nucleotide binding sites. Three of them are catalytic sites, residing mainly on the beta  subunits, and three are noncatalytic, located mainly on the alpha  subunits (1-6).

Two recently published x-ray crystallographic structures of rat liver mitochondrial MF1 at 3.6 Å (7) and bovine heart MF1 at 2.8 Å (8) have confirmed the alternate arrangement of the six large alpha  and beta  subunits in a closed hexamer. The structure at 2.8 Å resolution provides direct proof for the earlier suggested location of all six nucleotide binding sites at alpha /beta interfaces (see Ref. 9). It has also resolved two long C- and N-terminal helical domains of the gamma  subunit, which are embedded within the internal cavity of the alpha 3beta 3 hexamer, and impose on it an asymmetric structure. This asymmetry is also reflected in different conformational states displayed by the three catalytic sites on the beta  subunits (8). The binding change mechanism (10) suggested that ATP synthesis and hydrolysis involves transitions between such different conformational states via rotation of the gamma -subunit relative to an alpha 3beta 3 subassembly. So this structural information initiated a drive to demonstrate that such rotation is possible (11-13).

These important observations do not explain how rotation drives catalytic site transitions, which must involve intricate protein-protein interactions within the alpha /beta catalytic interfaces as well as between them and various domains on the gamma  subunit. Full elucidation of the unique mechanism of action of the ATP synthases will therefore require clear definition of the role of amino acid residues that are involved in catalysis, as well as identification and detailed characterization of all interacting subdomains on the alpha , beta , and gamma  subunits, and their relation to the catalytic sites.

Genetic/biochemical assay systems that are crucial for such studies have been developed in respiratory bacteria and yeast mitochondria (see Refs. 1, 2, and 6), but the application of this approach to photosynthetic systems lagged behind the respiratory ones. Although the conserved structure of all isolated F1-ATPases suggests a common catalytic mechanism, there are clear differences in various properties between the respiratory and photosynthetic F0F1 and F1-complexes, for instance in regulation of activity and sensitivity to some inhibitors (3-5). It is, therefore, important to develop recombinant molecular techniques and direct in vivo and/or in vitro assays of activity for photosynthetic complexes containing mutated F1-subunits. Two such systems have recently been reported for Chlamydomonas reinhardtii and spinach chloroplast F1-beta subunits (14-17).

The F1-beta subunit of the photosynthetic bacterium Rhodospirillum rubrum (RrF1beta )1 provides an especially suitable system for mutational analysis, based on the available MF1 crystal structure. Thus, the published sequence of the RrF1 operon (18) revealed that RrF1beta is most closely related to MF1beta . Their amino acid sequences show >76% identity, as compared with only 72% for EcF1beta , 69% for tobacco CF1beta (18), and 68% for TF1beta (19). The very high similarity between RrF1beta and MF1beta extends also to the R. rubrum and mitochondrial F0F1 and F1 which, unlike the chloroplast and Escherichia coli enzymes, exhibit an identical sensitivity to inhibition by oligomycin and efrapeptin (20-22). Furthermore, a large number of in vitro assays of activity have been developed for the RrF1beta subunit that was isolated in functional form from the chromatophore membrane-bound RrF0F1. They include the ability of the isolated RrF1beta to bind ATP, ADP, and Pi (23, 24) as well as to rebind to the beta -depleted enzyme and restore its lost ATP synthesis and hydrolysis activities (25, 26).

Baltscheffsky et al. (27) have cloned the RrF1beta gene and expressed it in E. coli as a fusion protein with glutathione S-transferase but did not carry out any assays of activity. We have recently cloned this gene and have expressed the RrF1beta subunit in E. coli lacking the whole unc operon as a soluble protein that could restore ATP synthesis to beta -depleted chromatophores (28). Here we describe the expression and full purification of RrF1beta mutated in Glu-195. A detailed comparison of the activities of these mutants with those of the expressed WT beta  subunit revealed that RrF1beta -E195 plays an important role in divalent cation-dependent ATP synthesis and hydrolysis.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Bacterial Strains-- R. rubrum cells were grown as described previously (25). Three unc operon-deleted E. coli strains, LM2800, LM3115 (29), and DK8 (30), were used as hosts for recombinant plasmids (28) carrying the WT and Glu-195 mutated RrF1beta genes. The transformed cells were grown in LB medium supplemented with thymine (50 µg/ml), thiamine (2 µg/ml), 0.2 mM isoleucine, 0.2 mM valine, 0.2% glucose, and 5% glycerol.

Cloning and Site-directed Mutagenesis-- The RrF1beta gene was amplified from R. rubrum genomic DNA by PCR and cloned as described in (28). Since the PCR is known to cause mutations, two PCR products, one obtained with Vent and the second with Taq DNA polymerase, were cloned and fully sequenced by the Taq dye deoxy chain termination method, using a 373A DNA Sequenase. Both clones were found to contain one mutated nucleotide. In the first clone, nucleotide 222 was changed, altering the published ACC codon of threonine 74 (18) into an ACA one that also codes for threonine. So this clone encodes the WT RrF1beta polypeptide. But the second clone had a mutation in nucleotide 584, which changed the GAG codon of glutamic acid 195 into the GGG codon of glycine. This clone thus encodes an RrF1beta -E195G mutant polypeptide.

Additional mutations at RrF1beta -E195 were introduced by site-directed mutagenesis with the U.S.E. mutagenesis kit (Pharmacia Biotech Inc.) according to the instructions of the supplier, using the EcoRI-BamHI PCR-prepared fragment containing the complete WT RrF1beta gene (28) cloned into pUC18. The mutagenic primers, which must anneal to the same strand of the heat-denatured plasmid DNA as the U.S.E. selection primer, were therefore designed with the 5'-end to right. The oligonucleotides used for the E195K and E195Q substitutions were, respectively: 3'-A-GAA-ATA-GTG-TTC-TAC-TAG-CTA-CGG-CCC-TAA-T-5' and 3'-A-GAA-ATA-GTG-GTC-TAC-TAG-CTA-CGG-CCC-TAA-T-5'. They contained a new, underlined site for ClaI, introduced by a single base change, indicated by a bold letter as the bases changed to give the Lys or Gln codons.

The U.S.E. selection primer eliminates the ScaI site in the pUC18 amp gene, but the repair defective E. coli NM 522 mutS strain, transformed with the ScaI-resistant mutated plasmid DNA according to the instructions of the supplier, failed to grow in liquid medium. The transformed cells did, however, form ampicillin-resistant colonies when plated on solid agar. Introduction of the mutations into the RrF1beta gene was confirmed by hybridization of these colonies with a 32P-labeled mutagenic primer. The mutations were further confirmed by ClaI restriction analysis, using the ClaI site introduced into the mutagenic primers, and by full DNA sequencing.

The beta -E195Q gene had only the stated mutation. But beta -E195K had two additional changes: 1) in nucleotide 159, altering the codon from GTG to GTT, both coding for valine 53 (18); and 2) in nucleotide 1069, altering the codon from CTG to TTG, both coding for leucine 357 (18). So this gene also encodes only the stated mutation.

Identification of Expressed RrF1beta -- Cultures of the various transformed unc-depleted E. coli strains were centrifuged and resuspended in buffer containing 50 mM Tricine-NaOH, pH 8.0; 4 mM MgATP, and 10% glycerol, which is optimal for isolating active native RrF1beta (25). The protease inhibitors benzamidine, phenylmethanesulfonyl fluoride, and Nalpha -p-tosyl-L-lysine chloromethyl ketone were added at 1.5 mM, 1 mM, and 30 µM, respectively, and the cells were disrupted under argon in a Yeda press (26). The soluble cytoplasmic and insoluble membrane fractions were analyzed for the presence of expressed RrF1beta by SDS-PAGE (31, 32) and Western immunoblotting (33).

Preparation of beta -less R. rubrum Chromatophores and Their Reconstitution with RrF1beta -- beta -less chromatophores were obtained as described in (26). This technique releases all their RrF1beta (Refs. 25 and 34; see Fig. 4, lane 6) together with trace amounts of RrF1alpha (28, 35), leading to loss of their ATP synthesis and hydrolysis activity. Reconstitution was carried out by incubating beta -less chromatophores, at 5 µg of Bchl, for 1 h at 35 °C in 0.2 ml of a reaction mixture containing 50 mM Tricine-NaOH (pH 8.0), 25 mM MgCl2, 4 mM ATP, 1 mM dithiothreitol, and unless otherwise stated, 1 µg of RrF1alpha and saturating amounts of RrF1beta at various stages of purification.

Assays of Restored ATP Synthesis and Hydrolysis-- ATP synthesis was usually assayed by a 5-fold dilution of the 0.2-ml mixture of reconstituted chromatophores into an assay mixture which contained in 1 ml a final concentration of 50 mM Tricine-NaOH (pH 8.0), 5 mM MgCl2, 4 mM sodium phosphate (containing 0.4-0.8 × 106 cpm of 32Pi), 2 mM ADP, 15 mM glucose, 24 units of hexokinase, and 66 µM N-methylphenazonium methosulfate. When assaying the Mg2+ requirement of the restored ATP synthesis (and hydrolysis), about 3 ml of the reconstituted chromatophores were subjected to two rounds of centrifugation and resuspension in 50 mM Tricine-NaOH (pH 8.0) and 10% glycerol to remove the 25 mM MgCl2, which are essential for the reconstitution (25, 26). These washed chromatophores, at 3-5 µg of Bchl, were added to the synthesis assay mixture, together with the indicated concentrations of MgCl2, and preequilibrated for 5 min at 35 °C in the dark. ATP synthesis was started by illumination and stopped after 3 min by turning off the lights and adding 0.1 ml of 2 M trichloroacetic acid. The synthesized [gamma -32P]ATP was determined as described by Avron (36).

For assaying restored ATP hydrolysis, the washed chromatophores, at 3-5 µg of Bchl, were preincubated for 5 min at 35 °C in 0.66 ml of 50 mM Tricine-NaOH (pH 8.0) with the divalent cation concentrations indicated in the text. Hydrolysis was started by addition of 40 µl of ATP to a final concentration of 4 mM and stopped by 0.1 ml of 2 M trichloroacetic acid. The released Pi was measured according to (37).

Other Procedures-- For measurement of ATP binding, purified RrF1beta preparations were depleted of bound MgATP by elution-centrifugation through Sephadex G-50 columns (38) preequilibrated with TGN buffer containing 50 mM Tricine-NaOH (pH 8.0), 20% glycerol, and 50 mM NaCl. Binding was assayed by incubating 10 µM depleted RrF1beta for 90 min at 23 °C in TG buffer with the indicated concentrations of MgCl2 and ATP, containing 1-2 × 106 cpm of [2,8-3H]ATP. Incubation was started by addition of RrF1beta and stopped by subjecting 50-µl samples to elution-centrifugation, and the effluent was assayed for protein and radioactivity as described by Gromet-Elhanan and Kananshvili (23).

Protein concentrations were measured either by the BCA method (39) or according to Lowry et al. (40). The Bchl content of chromatophores was determined at 880 nm using the absorption coefficient in vivo given by Clayton (41).

Materials-- E. coli DK8 was a gift of Dr. M. Futai, Institute of Scientific and Industrial Research, Osaka University. E. coli LM2800 and LM3115 were a gift of Dr. P. R. Jensen, The Netherlands Cancer Institute, Amsterdam.

Oligonucleotides were synthesized by Dr. Ora Goldberg, Biological Services, The Weizmann Institute of Science. Restriction enzymes, Vent DNA polymerase, and T4 ligase were from New England Biolabs. Taq DNA polymerase, and dNTP were purchased from Boehringer Mannheim. Plasmids pBSKS+ and pBTacI were from Stratagene and Boehringer Mannheim, respectively. The U.S.E. Mutagenesis Kit was obtained from Pharmacia Biotech Inc.. [2,8-3H]ATP was purchased from NEN Life Science Products. All other reagents were of the highest purity available.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Construction of RrF1beta -E195 Mutants-- The beta -E195G mutation was obtained as a mistake, which changed the GAG codon of glutamic acid to the GGG codon of glycine, during amplification of the RrF1beta gene from genomic R. rubrum DNA by PCR. Since the equivalent MF1beta -E199 is described in the crystal structure as pointing into the conical tunnel leading to the catalytic nucleotide binding site (8), it was interesting to check whether the E195G mutation affects activity. Using the system described previously (28), the expressed, partially purified mutated beta  subunit was found to restore a much lower rate of ATP synthesis in beta -less chromatophores than the native or expressed WT RrF1beta . We have therefore prepared two additional RrF1 beta -E195 mutants by oligonucleotide-directed mutagenesis: from glutamic acid to lysine (beta -E195K), carrying a positive charge, and to glutamine (beta -E195Q), carrying no charge.

Expression and Purification of WT and Mutant RrF1beta Subunits-- Cloned WT and mutant beta  genes were ligated into the expression vector pBTacI, and the recombinant plasmid was transformed into E. coli LM3115 (28). This strain was found to express larger amounts of RrF1beta than two other unc-deleted strains, LM2800 (29) and DK8 (30), under all tested growth conditions (not shown). Optimal conditions for expression of large amounts of RrF1beta as a soluble protein include growth of E. coli LM3115 at 22 °C in the presence of 5% glycerol to about A0.65. Growth at higher temperatures or to a higher optical density increased the fraction of expressed beta  subunit appearing in inclusion bodies (42).

The soluble cytoplasmic fraction of E. coli LM3115 cells containing the expressed WT and mutated RrF1beta was subjected to ammonium sulfate fractionation as described for native RrF1beta (34). The 30-60% ammonium sulfate precipitate was dissolved in TGN buffer to a concentration of about 10 mg of protein/ml and thoroughly dialyzed against the same buffer. The WT and beta -E195G mutant were further purified by dye-ligand chromatography on a Red A column. As is illustrated in Fig. 1, the dialyzed WT beta  subunit was loaded on a column preequilibrated in TGN buffer at 4 °C. After 1 h, the column was washed with TGN buffer, to elute all the unbound protein (Fig. 1, A, peak I, and B, lane 3). The RrF1beta was then eluted with TGN buffer containing 1 mM MgATP (Fig. 1, A, peak II, and B, lane 4). Further washing with TGN buffer containing 1 mM MgATP and 1.5 M NaCl did not elute any additional RrF1beta subunit (Fig. 1, A, peak III, and B, lane 5).


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 1.   Purification of the expressed WT RrF1beta on a dye ligand Red A column. A, elution profile of the RrF1beta from a Red A column. B, SDS-PAGE profile of pooled peaks I, II and III. Lane 1, 2 µg of purified native RrF1beta ; lane 2, 10 µg of the loaded protein; lanes 3-5, 10 µg of pooled peaks I, II, and III.

This column yielded a highly purified expressed WT RrF1beta (Fig. 1B, lane 4), which contained, of course, no trace of RrF1alpha . On the other hand, the earlier prepared native RrF1beta , which was removed by LiCl extraction of R. rubrum chromatophores (25) and purified by chromatography through two ion-exchange columns (26), was recently found to contain 5-10% of RrF1alpha (28, 35). Most of this alpha  subunit was removed by dye-ligand chromatography, leaving a pure native RrF1beta containing less than 1% of RrF1alpha .

Activities of the Highly Purified Native, Expressed WT and E195G RrF1beta -- All three types of RrF1beta subunits were found to bind, in the presence of Mg2+, up to 2 mol of ATP/mol of beta . But, whereas the binding of ATP to the expressed WT as to pure native RrF1beta (Fig. 2), saturated at 200 µM ATP and showed a pronounced cooperativity, with a Hill coefficient (nH) of 2.2, the RrF1beta -E195G mutant required a higher concentration of ATP for saturation and showed a much lower cooperativity with a Hill coefficient of only 1.5. The native RrF1beta , which contained at least 5% of RrF1alpha (28, 35), was earlier shown to bind up to 2 mol of ATP/mol (23) but with somewhat different properties than those reported here for the highly purified native beta  subunit.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 2.   Binding of ATP to the native (square ) and expressed mutant RrF1beta -E195G (black-triangle). Binding was carried out as described under "Experimental Procedures," using a ratio of Mg2+/ATP of 0.5. The points are experimental, and the lines are the best-fit curves using the calculated Hill coefficients of 2.2 for the WT and 1.5 for the mutant RrF1beta , obtained from the Hill plots in the inset.

The difference, between the native RrF1beta containing 5% of RrF1alpha and the highly purified one containing ~1% RrF1alpha , was much more pronounced in assays of assembly into beta -less chromatophores and restoration of their lost activities. RrF1beta with at least 5% of RrF1alpha could restore these activities (35), whereas pure native and expressed WT RrF1beta , containing either 1% or no alpha , respectively, could not restore any ATP synthesis by beta -less chromatophores (Fig. 3). They could, however, restore high rates of ATP synthesis when a fixed trace amount of RrF1alpha , which by itself was completely inactive, was added to the reconstitution mixture together with increasing amounts of the beta  subunits. Under these conditions, the degree of restoration of ATP synthesis (Fig. 3) and hydrolysis (not shown) was dependent on the amount of RrF1beta present during the reconstitution of a fixed amount of beta -less chromatophores. An identical saturation curve, which leveled off around a ratio of 1 µg of added RrF1beta /1 µg of Bchl, was obtained for both pure native and expressed WT RrF1beta (Fig. 3).


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 3.   Restoration of ATP synthesis in beta -less chromatophores reconstituted with increasing amounts of pure native or various expressed RrF1beta is activated by a fixed trace amount of RrF1alpha . beta -less chromatophores, at 5 µg of Bchl, were reconstituted in the absence (closed symbols) or presence (open symbols) of 1 µg of RrF1alpha and the indicated amounts of pure native (bullet ---bullet open circle ---open circle ) or expressed WT (black-square---black-squaresquare ---square ) and E195G RrF1beta (triangle ---triangle ). Photophosphorylation was assayed as described under "Experimental Procedures." Unreconstituted beta -less chromatophores had a residual activity of 27 µmol of ATP formed/h/mg of Bchl.

The RrF1beta -E195G mutant exhibited the same pattern of concentration-dependent saturation curve, in the presence of a fixed trace amount of RrF1alpha , but the restored rate was much lower, reaching only 30% of the rate restored by pure native or expressed WT RrF1beta (Fig. 3). This lower rate was not due to a lower capacity of the E195G mutant to bind into beta -less chromatophores. Fig. 4 illustrates that reconstitution of these chromatophores with the same, saturating amounts of WT or the E195G mutant, resulted in binding of similar amounts of RrF1beta (Fig. 4, lanes 2 and 3). Moreover, the other two RrF1beta -E195Q and -E195K mutants were also found to bind in similar amounts into the beta -less chromatophores (Fig. 4, lanes 4 and 5). The presence of alpha  in these beta -less chromatophores (Fig. 4, lane 6) indicates that the additional trace of soluble RrF1alpha , required for an effective reconstitution of all types of tested RrF1beta (Fig. 3), might indeed exert a chaperonin-like activity, keeping the pure RrF1beta correctly folded during reconstitution, as has earlier been suggested for CF1alpha (43).


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 4.   All WT and Glu-195 mutant RrF1beta bind to the beta -less chromatophores. Reconstitution was carried out as described under "Experimental Procedures." The reconstituted chromatophores were centrifuged to remove all unbound RrF1beta subunits and resuspended in buffer containing 50 mM Tricine-NaOH, pH 8.0, and 10% glycerol. Chromatophores corresponding to 3 µg of Bchl were incubated with 1% SDS at 100 °C for 5 min and applied to SDS-PAGE. After electrophoresis, the gel was transferred to nitrocellulose (33) and probed with antisera produced against native RrF1alpha and beta  subunits. Lane 1, control chromatophores; lanes 2-5, chromatophores reconstituted with WT, E195G, E195Q, and E195K RrF1beta , respectively; lane 6, unreconstituted beta -less chromatophores.

Divalent Cation-dependent Restoration of ATP Hydrolysis and Synthesis by WT and Several RrF1beta -E195 Mutants-- The similar binding of WT and the E195G mutant RrF1beta to beta -less chromatophores, but the much lower ability of the mutant to restore ATP synthesis, suggested that beta -E195 plays an important role in the catalytic activity of the ATP synthase. To test this possibility, we compared the capacity of WT and all three beta -E195 mutants to restore ATP hydrolysis (Fig. 5). ATP hydrolysis, unlike its synthesis, was shown to occur in control R. rubrum chromatophores in the presence of Ca2+ as well as Mg2+ and Mn2+ but was coupled to proton translocation only in the presence of the last two divalent cations (44). Reconstitution of beta -less chromatophores with WT RrF1beta restored their capacity to hydrolyze ATP with all three divalent cations (Fig. 5, A-C). As in control chromatophores (44), hydrolysis in the presence of Mg2+ and Mn2+ showed a narrow range of dependence on the cation concentration, resulting in maximal activity at cation/ATP ratios around 0.5 followed by a drastic inhibition at a cation/ATP ratio of 2 (Fig. 5, A and B). The CaATPase activity in control (44), as well as WT RrF1beta reconstituted, chromatophores (Fig. 5C) was much less sensitive to inhibition by an excess of free Ca2+-ions, remaining optimal at cation/ATP ratios between 0.5 and 2.0. Even with 32 mM CaCl2, at a cation/ATP ratio of 8, this restored ATPase activity was inhibited by only 35%.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 5.   Divalent cation requirement of ATP hydrolysis restored in beta -less chromatophores reconstituted with expressed WT and mutant RrF1beta at position 195. beta -less chromatophores, reconstituted with the following RrF1beta subunits: WT (square ---square ), E195G (black-triangle---black-triangle), E195Q (open circle ---open circle ), and E195K (diamond ---diamond ) were washed and assayed for restored ATP hydrolysis as described under "Experimental Procedures," using 4 mM ATP and the indicated concentrations of MgCl2 (A), MnCl2 (B), and CaCl2 (C).

Chromatophores reconstituted with the beta -E195 mutants showed very little MgATPase or MnATPase activities at the whole range of tested cation concentrations (Fig. 5, A and B). They did, however, exhibit a CaATPase activity that required higher CaCl2 concentrations than the activity restored with WT RrF1beta . At 32 mM CaCl2, the activities restored by RrF1beta -E195G, -E195Q, or -E195K mutants reached 105, 45, and 16%, respectively, of the activity restored with WT RrF1beta already at 4 mM CaCl2 (Fig. 5C). These results suggest that the Glu-195 mutants also might require higher concentrations of MgCl2 and MnCl2 for restoration of ATP hydrolysis. But, since high concentrations of these cations are already inhibitory (Fig. 5, A and B), the mutants could not restore MgATPase or MnATPase activity.

ATP synthesis in control chromatophores was found to be much less sensitive than the MgATPase activity to inhibition by high MgCl2 concentrations, remaining optimal at MgCl2/ADP ratios between 0.4 and 4.0 (44). Furthermore, beta -less chromatophores reconstituted with the beta -E195G mutant could clearly restore some ATP synthesis (Fig. 3). We have therefore investigated the Mg2+ requirement for restoration of ATP synthesis in chromatophores reconstituted with WT and all three RrF1beta -E195 mutants. Fig. 6 does indeed demonstrate that all tested RrF1beta -E195 mutants can restore at least some Mg-dependent ATP synthesis but that they require higher concentrations of MgCl2 than the activity restored with WT RrF1beta . In this reaction, unlike in CaATPase (Fig. 5C), both beta -E195G and beta -E195Q could restore at 5 mM MgCl2 up to 60%, whereas beta -E195K restored less than 25%, of the WT restored ATP synthesis (Fig. 6). However, higher concentrations of 10 mM MgCl2 already started to inhibit the WT restored rate, and 40 mM MgCl2 inhibited by about 50% the ATP synthesis restored by the WT and all three RrF1beta -E195 mutants (Fig. 6).


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 6.   Mg2+ requirement of photophosphorylation restored in beta -less chromatophores reconstituted with WT and mutant RrF1beta at position 195. Washed chromatophores reconstituted with WT (square ---square ), E195G (black-triangle---black-triangle), E195Q (open circle ---open circle ), and E195K (diamond ---diamond ) were assayed for restored ATP synthesis using the indicated MgCl2 concentrations.

The results summarized in Figs. 5 and 6 have indicated that the inability of the RrF1beta -E195 mutants to restore MgATPase and MnATPase activities while restoring ATP synthesis might be due to their requirement of high concentrations of Mg2+ and Mn2+ ions, which inhibit ATP hydrolysis much more than its synthesis. Since anions, such as sulfite were found to stimulate the MgATPase activity of RrF1 by relieving the inhibition caused by excess free Mg2+ ions (45), we have compared the effect of sulfite on the MgATPase and MnATPase activities in chromatophores reconstituted with WT and the E195 mutant RrF1beta (Fig. 7, A and B). Addition of 20 mM sulfite stimulated by 3-4-fold both restored ATPase activities in chromatophores reconstituted with WT RrF1beta , reducing the inhibition by high concentrations of MgCl2 and MnCl2 and shifting the cation/ATP ratio for maximal activity to 1 (compare Fig. 5, A and B, and Fig. 7, A and B). This effect of sulfite was already saturated at 20 mM since 100 mM caused no further stimulation of the WT RrF1beta restored activities. Addition of sulfite to chromatophores reconstituted by beta -E195G did indeed uncover both MgATPase and MnATPase activities; but in this case, 100 mM sulfite was much more effective than 20 mM, and maximal activity was obtained at a cation/ATP ratio of 3. With beta -E195Q and beta -E195K, even 100 mM sulfite did not uncover any MgATPase or MnATPase activities (Fig. 7, A and B), and higher sulfite concentrations did already inhibit the activities restored by WT RrF1beta .


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of sulfite on the Mg2+ and Mn2+ requirement of ATP hydrolysis restored in beta -less chromatophores reconstituted with WT and mutant RrF1beta at position 195. Washed reconstituted chromatophores were assayed for restored ATP hydrolysis using the indicated concentrations of MgCl2 (A) and MnCl2 (B). Sulfite was added to the assay mixtures to a final concentration of 20 mM (closed symbols, solid lines) or 100 mM (open symbols, dashed lines). WT (black-square---black-squaresquare ---square ); E195G (black-triangle---black-triangletriangle ---triangle ); E195Q (open circle ---open circle ), and E195K (diamond ---diamond ).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Glutamic acid 195 of RrF1beta is equivalent to MF1beta -Glu-199, which in the bovine heart crystal structure (8) is described as pointing into the conical tunnel leading to the nucleotide binding sites. This fully conserved glutamic acid residue was shown to bind the inhibitor DCCD in MF1beta (46) and EcF1beta (47). But a mutation in the equivalent EcF1beta -E192 to Gln was found to grow on succinate and showed normal assembly of F0F1, with only 6-8-fold inhibition of its membrane ATPase activity and small perturbations in rate constants and equilibrium constants of unisite catalysis (48). It was, therefore, suggested that the full inhibition of F0F1 by DCCD could be due to the presence of the two bulky cyclohexyl moieties in the conical tunnel (8).

The system developed in R. rubrum chromatophores enables reconstitution of beta -less chromatophores with RrF1beta and detailed characterization of ATP hydrolysis as well as synthesis restored to the same preparation of reconstituted chromatophores (25, 26). This system was used here for a direct comparison of the activities restored to beta -less chromatophores reconstituted with WT and three Glu-195 mutants of RrF1beta and provided rather unexpected results. ATP synthesis was restored to about 60% by beta -E195G and beta -E195Q and to 30% by beta -E195K (Fig. 6), whereas the Mg- and Mn-dependent ATP hydrolysis was not restored by E195Q and E195K mutants, even when assayed in the presence of saturating concentrations of sulfite (Fig. 7, A and B). The E195G mutant could restore some MgATPase and MnATPase activities in the presence of 100 mM sulfite, but they showed different divalent cation dependences and reached a much lower maximal rate than the activities restored by WT RrF1beta .

Similar properties were recently reported for the MgATPase activity of S-carboxymethyl-beta 185 EcF1 (49). This residue is equivalent to MF1beta -E192, which is located at the catalytic nucleotide binding sites of F1, whereas the RrF1beta -E195 residue is pointing into the tunnel leading to these sites (8). Our results indicate that residues within the tunnel might also affect divalent cation and nucleotide binding, suggesting their participation in the interconversion of the catalytic sites between different conformational states. Interestingly, two earlier reports describing mutations in EcF1beta -E192 to V (50) and TF1beta -E201 to Q (51), which are equivalent to RrF1beta -E195, have observed some type of interaction between this glutamic acid and EcF1-G149 or TF1beta -E190, which are located in the catalytic site (8). It would be worthwhile to check whether other amino acid residues that are located in the tunnel show similar effects.

The strange results obtained with the RrF1beta -E195 mutants, which enable restoration of ATP synthesis but not hydrolysis, could be explained by differences in affinity of the catalytic sites to MgADP as compared with MgATP. These differences are especially important during ATP hydrolysis in photosynthetic organisms, which is highly regulated to limit wasteful hydrolysis of low concentrations of ATP in the dark (3-5). One aspect of this regulation involves the onset of inhibition of ATPase activity, and of exchange of ADP tightly bound at a catalytic site with medium nucleotides, by added free Mg2+ (52). Sulfite has been shown to overcome the Mg2+-induced inhibition of both ATPase activity and release of the tightly bound MgADP (53, 54). Addition of a saturating sulfite concentration did indeed enable low MgATPase and MnATPase activities in the beta -E195G mutant, but not in beta -E195Q or beta -E195K (Fig. 7, A and B). These mutations might impede more severely the release of the inhibitory MgADP as compared with the release of the more loosely bound MgATP.

The overall higher activities restored by beta -E195G (Figs. 5C, 6, and 7) can be explained by the capacity of glycine to adopt a much wider range of conformations than the other residues and thus allow unusual main chain conformations in proteins (55). In RrF1beta -E195G, it could enable conformational changes that compensate the change of charge and size, and lead to the higher activity of this mutant.

An additional interesting effect observed with the RrF1beta -E195G and -E195Q, is their capacity to restore CaATPase, but not MgATPase and MnATPase activities (compare Fig. 5, A, B, and C). CaATPase, unlike the MgATPase and MnATPase activity, is not coupled to proton translocation and not subject to the tight regulation observed with MgATP and MnATP hydrolysis (44). Moreover CaCl2 does not enable ATP synthesis. So the binding of Ca2+ to the catalytic sites on RrF1beta might convert it to a different conformational state that is less impeded by the Glu-195 mutations. Work with RrF1beta -T159 mutants has provided direct experimental support for this conclusion.2 It would be most interesting to crystallize F1 in the presence of CaCl2 since any differences between the CaCl2- and MgCl2-containing crystals might illuminate the domains, and/or specific amino acid residues, that are involved in the coupling of proton translocation to ATP synthesis and hydrolysis. This information is essential for the understanding of the mechanism of ATP synthesis.

    FOOTNOTES

* This work was supported by Grants from the Basic Research Foundation administered by the Israel Academy of Sciences and Humanities and by the Avron-Wilstätter Minerva Center for Research in Photosynthesis, Rehovot, Israel.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Dept. of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, FL 33101-6129.

§ To whom correspondence should be addressed. Tel.: 972 8 9342729; Fax: 972 8 9344118.

1 The abbreviations used are: RrF1beta , RrF1alpha , CF1beta , CF1alpha , EcF1beta , MF1beta , and TF1beta , beta  and alpha  subunits of the F1-ATPase of R. rubrum, chloroplasts, E. coli, mitochondria, and thermophilic Bacillus PS3, respectively; Bchl, bacteriochlorophyll; PCR, polymerase chain reaction; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; WT, wild type; DCCD, dicyclohexylcarbodiimide.

2 L. Nathanson and Z. Gromet-Elhanan, manuscript in preparation.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Futai, M., Noumi, T., and Madea, M. (1989) Annu. Rev. Biochem. 58, 111-136[CrossRef][Medline] [Order article via Infotrieve]
  2. Penefsky, H. S., and Cross, R. L. (1991) Adv. Enzymol. Relat. Areas Mol. Biol. 64, 173-214[Medline] [Order article via Infotrieve]
  3. Gromet-Elhanan, Z. (1995) in Anoxygenic Photosynthetic Bacteria (Blankenship, R. E., Madigan, M. T., and Bauer, C. E., eds), pp. 807-839, Kluwer Academic Publishers, Dordrecht, The Netherlands
  4. McCarty, R. E. (1996) in Oxygenic Photosynthesis: The Light Reaction (Ort, D. R., and Yocum, C. F., eds), pp. 439-451, Kluwer Academic Publishers, Dordrecht, The Netherlands
  5. Richter, M. L., and Mills, D. A. (1996) in Oxygenic Photosynthesis: The Light Reaction (Ort, D. R., and Yocum, C. F., eds), pp. 453-468, Kluwer Academic Publishers, Dordrecht, The Netherlands
  6. Weber, J., and Senior, A. E. (1997) Biochim. Biophys. Acta 1319, 19-58[Medline] [Order article via Infotrieve]
  7. Bianchet, M., Ysern, X., Hullihen, J., Pedersen, P. L., and Amzel, L. M. (1991) J. Biol. Chem. 266, 21197-21201[Abstract/Free Full Text]
  8. Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628[CrossRef][Medline] [Order article via Infotrieve]
  9. Gromet-Elhanan, Z. (1992) J. Bioenerg. Biomembr. 24, 447-452[Medline] [Order article via Infotrieve]
  10. Boyer, P. D. (1993) Biochim. Biophys. Acta 1140, 215-250[Medline] [Order article via Infotrieve]
  11. Duncan, T. M., Bulygin, V. V., Zhou, Y., Hutcheon, M. L., and Cross, R. L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10964-10968[Abstract]
  12. Sabbert, D., Engelbrecht, S., and Junge, W. (1996) Nature 381, 623-625[CrossRef][Medline] [Order article via Infotrieve]
  13. Noji, H., Yasuda, R., Yoshida, M., and Kinosita, K. (1997) Nature 386, 299-302[CrossRef][Medline] [Order article via Infotrieve]
  14. Avni, A., Anderson, J. D., Holland, N., Rochaix, J. D., Gromet-Elhanan, Z., and Edelman, M. (1992) Science 257, 1245-1247[Medline] [Order article via Infotrieve]
  15. Hu, D., Fiedler, H. R., Golan, T., Edelman, M., Strotmann, H., Shavit, N., and Leu, S. (1997) J. Biol. Chem. 272, 5457-5463[Abstract/Free Full Text]
  16. Burkovski, A., Lill, H., and Engelbrecht, S. (1994) Biochim. Biophys. Acta 1186, 243-246[CrossRef][Medline] [Order article via Infotrieve]
  17. Chen, Z., Spies, A., Hein, R., Zhou, X., Thomas, B-C., Richter, M. L., and Gegenheimer, P. (1995) J. Biol. Chem. 270, 17124-17132[Abstract/Free Full Text]
  18. Falk, G., Hampe, A., and Walker, J. E. (1985) Biochem. J. 228, 391-407[Medline] [Order article via Infotrieve]
  19. Ohta, S., Yohda, M., Ishizuka, M., Hirata, H., Hamamoto, T., Otawara-Hamamoto, Y., Matsuda, K., and Kagawa, Y. (1988) Biochim. Biophys. Acta 933, 141-155[Medline] [Order article via Infotrieve]
  20. Johansson, B. C., Baltscheffsky, M., and Baltscheffsky, H. (1971) in Proceedings of the IInd International Congress on Photosynthetic Research (Forti, G., Avron, M., and Melandri, A., eds), Vol. 2, pp. 1203-1209, Dr. W. Junk N. V., The Hague, The Netherlands
  21. Bengis-Garber, C., and Gromet-Elhanan, Z. (1979) Biochemistry 18, 3577-3581[Medline] [Order article via Infotrieve]
  22. Weiss, S., McCarty, R. E., and Gromet-Elhanan, Z. (1994) J. Bioenerg. Biomembr. 26, 573-581[Medline] [Order article via Infotrieve]
  23. Gromet-Elhanan, Z., and Kananshvili, D. (1984) Biochemistry 23, 1022-1028
  24. Khananshvili, D., and Gromet-Elhanan, Z. (1985) Biochemistry 24, 2482-2487
  25. Philosoph, S., Binder, A., and Gromet-Elhanan, Z. (1977) J. Biol. Chem. 252, 8747-8752[Medline] [Order article via Infotrieve]
  26. Gromet-Elhanan, Z., and Khananshvili, D. (1986) Methods Enzymol. 126, 528-538
  27. Baltscheffsky, M., Nadanciva, S., and Harris, D. A. (1992) in Research in Photosynthesis (Mutata, N., ed), Vol. III, pp. 385-388, Kluwer Academic Publishers, Dordrecht, The Netherlands
  28. Nathanson, L., and Gromet-Elhanan, Z. (1995) in Photosynthesis: From Light to Biosphere (Mathis, P., ed), Vol. III, pp. 51-54, Kluwer Academic Publishers, Dordrecht, The Netherlands
  29. Jensen, P. R., and Michelsen, O. (1992) J. Bacteriol. 174, 7635-7641[Abstract]
  30. Klionsky, D. J., Brusilow, W. S. A., and Simoni, R. D. (1984) J. Bacteriol. 160, 1055-1060[Medline] [Order article via Infotrieve]
  31. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
  32. Flings, S. P., and Gregerson, D. S. (1986) Anal. Biochem. 155, 83-88[Medline] [Order article via Infotrieve]
  33. Gershoni, J. M. (1988) Methods Biochem. Anal. 33, 1-58[Medline] [Order article via Infotrieve]
  34. Philosoph, S., and Gromet-Elhanan, Z. (1981) Eur. J. Biochem. 119, 107-113[Abstract]
  35. Gromet-Elhanan, Z., and Sokolov, M. (1995) Photosynth. Res. 46, 79-86
  36. Avron, M. (1960) Biochim. Biophys. Acta 40, 257-272[CrossRef]
  37. Takeyama, M., Ihara, K., Moriyama, Y., Ida, K., Tomioka, N., Itai, A., Maeda, M., and Futai, M. (1953) J. Biol. Chem. 265, 21279-21284[Abstract/Free Full Text]
  38. Penefsky, H. S. (1977) J. Biol. Chem. 252, 2891-2899[Abstract]
  39. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Anal. Biochem. 150, 76-85[Medline] [Order article via Infotrieve]
  40. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275[Free Full Text]
  41. Clayton, R. K. (1963) in Bacterial Photosynthesis (Gest, H., San Pietro, A., and Vernon, L. P., eds), pp. 495-500, Antioch Press, Yellow Springs, OH
  42. Nathanson, L. (1997) Cloning, Expression and Activity of Wild Type and Mutant B Subunits of the Rhodospirillum rubrum F0F1 ATP Synthase, Ph.D. thesis, The Weizmann Institute of Science
  43. Avni, A., Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7317-7320[Abstract/Free Full Text]
  44. Gromet-Elhanan, Z., and Weiss, S. (1989) Biochemistry 28, 3645-3650
  45. Webster, G. D., Edwards, P. A., and Jackson, J. B. (1977) FEBS Lett. 76, 29-35[CrossRef][Medline] [Order article via Infotrieve]
  46. Esch, F. S., Bohlen, P., Otsuka, A. S., Yoshida, M., and Allison, W. S. (1981) J. Biol. Chem. 256, 9084-9089[Abstract/Free Full Text]
  47. Yoshida, M., Allison, W. S., Esch, F. S., and Futai, M. (1982) J. Biol. Chem. 257, 10033-10037[Free Full Text]
  48. Senior, A. E., and al-Shawi, M. K. (1992) J. Biol. Chem. 267, 21471-21478[Abstract/Free Full Text]
  49. Omote, H., Le, N. P., Park, M.-Y., Maeda, M., and Futai, M. (1995) J. Biol. Chem. 270, 25656-25660[Abstract/Free Full Text]
  50. Iwamoto, A., Park, M. Y., Maeda, M., and Futai, M. (1993) J. Biol. Chem. 268, 3156-3160[Abstract/Free Full Text]
  51. Amano, T., Tozawa, K., Yoshida, M., and Murakami, H. (1994) FEBS Lett. 348, 93-98[CrossRef][Medline] [Order article via Infotrieve]
  52. Feldman, R. I., and Boyer, P. D. (1985) J. Biol. Chem. 260, 13088-13094[Abstract/Free Full Text]
  53. Larson, E. M., Umbach, A., and Jagendorf, A. T. (1989) Biochim. Biophys. Acta 973, 75-85
  54. Du, Z., and Boyer, P. D. (1990) Biochemistry 29, 402-407[Medline] [Order article via Infotrieve]
  55. Richardson, J. S. (1981) Adv. Protein Chem. 34, 168-339


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.