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*

Ziyun DuDagger , Ward C. TuckerDagger §, Mark L. Richter§, and Zippora Gromet-ElhananDagger ||

From the Dagger  Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel and the § Department of Molecular Biosciences, the University of Kansas, Lawrence, Kansas 66045

Received for publication, August 18, 2000, and in revised form, November 29, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Refolding together the expressed alpha  and beta  subunits of the Rhodospirillum rubrum F1(RF1)-ATPase led to assembly of only alpha 1beta 1 dimers, showing a stable low MgATPase activity. When incubated in the presence of AlCl3, NaF and either MgAD(T)P or CaAD(T)P, all dimers associated into closed alpha 3beta 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 RrF1-alpha 3beta 3 hexamers by stabilizing their noncatalytic alpha /beta interfaces. Refolding of the RrF1-alpha and beta  subunits together with the spinach chloroplast F1 (CF1)-gamma enabled a simple one-step assembly of two different hybrid RrF1-alpha 3beta 3/CF1gamma complexes, containing either wild type RrF1-beta or the catalytic site mutant RrF1beta -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 RrF1 and CF1 and unlike its higher CaATPase activity, regulated by excess free Mg2+ 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 CF1-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 CF1-gamma subunit.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The F0F1-ATP synthases catalyze the synthesis of ATP from ADP and Pi at the expense of a transmembrane electrochemical proton gradient generated by the respiratory or photosynthetic electron transport chains. Its membrane-bound F0 sector functions as the proton pathway and has, in bacterial and photosynthetic cells, a subunit composition of a1b2c9-12 and a1b1b'1c9-12, respectively. The catalytic F1 sector, which functions as a soluble ATPase, has in all cells a subunit composition of alpha 3beta 3gamma 1delta 1epsilon 1 and six nucleotide-binding sites located on the alpha  and beta  subunits. Its three catalytic beta  sites show high negative cooperativity in substrate binding and strong positive cooperativity in catalysis (1-5). The minimal F1 subcomplex, which resembles the whole F1-ATPase in its catalytic properties, is the F1-alpha 3beta 3gamma -ATPase. Such subcomplexes were reconstituted from either native (6, 7) or recombinant (8, 9) individual subunits of the respiratory TF1-1 and EcF1-ATPases. Similar highly active pure photosynthetic subcomplexes have been assembled up to now only by incubating an isolated native CF1(alpha beta ) complex with a native (10) or recombinant (11) gamma C subunit.

A high resolution x-ray structure of the bovine heart mitochondrial MF1 (12) demonstrated the alternating arrangement of the alpha  and beta  subunits in a closed hexamer, with the resolved N- and C-terminal alpha -helices of the gamma  subunit embedded in its central cavity and all six nucleotide-binding sites residing at alternating alpha /beta interfaces. The three catalytic sites, located mainly on the beta  subunits, appeared in three different conformational states that, in association with the unique resolved part of the gamma  subunit, imposed an asymmetric structure on the alpha 3beta 3 hexamer (12). This MF1-alpha 3beta 3gamma structure is compatible with the binding change mechanism (3, 13), which proposed that ATP synthesis involves transitions between different but interacting catalytic sites, via energy-dependent affinity changes in substrate binding and product release. Such transitions were first suggested to occur via movement or rotation of a cluster of the catalytic alpha 3beta 3 subunits around a core of the single copy gamma  or epsilon  subunits (14). The reversible proton-translocating ATP hydrolysis was later proposed to generate rotation of the F1-gamma subunit which, when transmitted to F0, could result in pumping of protons back across the membrane (15), possibly via coupled rotation of the F1-gamma subunit with the F0-c subunits (3, 16). MgATPase-induced rotation of gamma  within immobilized alpha 3beta 3 hexamers was observed in genetically engineered respiratory TF1- and EcF1-alpha 3beta 3gamma complexes (17, 18). But its further coupling with F0-c rotation, which has been tested in some recent reports, did not yield clear results (19-21). So there is at present no direct correlation between gamma  rotation and proton-coupled ATP synthesis and hydrolysis.

Full elucidation of the detailed mechanism of action of the F0F1-ATP synthase will depend on the identification of the specific domains that participate in its proton-coupled ATP synthesis and hydrolysis as well as in the regulation of these reversible activities and their possible correlation with the gamma  or gamma -c rotation. Tight regulation of ATP hydrolysis is especially important in photosynthetic cells, where it prevents the depletion of essential cellular ATP pools in the dark (1, 4, 5). Plant chloroplasts and bacterial chromatophores have a number of such regulatory pathways, which operate in their membrane-bound F0F1- as well as the solubilized F1-ATPases. Both chloroplasts (22) and chromatophores (23) show a high sensitivity to inhibition by excess free Mg2+ ions, which results in a drastic decrease of their MgATPase activities at Mg/ATP ratios above 0.5. A unique chloroplast regulatory system, termed thiol modulation, involves reduction-oxidation of a disulfide bond (24) formed between Cys199 and Cys205 in a region of its gamma C subunit that does not appear in any other F1-gamma subunits (25). But there are as yet no assembled CF1-alpha 3beta 3gamma or similar photosynthetic complexes that can be engineered for studies aimed at elucidating the molecular mechanism of their regulatory systems and the possible ATPase-induced rotation of their gamma  subunit.

In this investigation, we have used our earlier developed procedure for refolding and assembly of the alpha R and beta R subunits of the photosynthetic bacterium Rhodospirillum rubrum into alpha R1beta R1 dimers (26) to follow their further association into alpha R3beta R3 hexamers, and with the recombinant chloroplast gamma C (11), into hybrid alpha R3beta R3gamma C-ATPases. Two types of such highly active hybrids were assembled, containing WT beta R or the catalytic site mutant beta R-T159S (27), and both retained the specific gamma C redox regulation. These hybrid complexes provide the first photosynthetic F1 assemblies that can be genetically engineered for probing rotational catalysis. Both hybrids show, besides a very high MgATPase also a CaATPase activity, that in the hybrid WT alpha R3beta R3gamma C is much higher than the MgATPase activity but does not respond to any tested MgATPase effector. This hybrid WT complex thus supplies an additional interesting system for assaying rotation, which has been induced up to now (17-21) only by MgATP hydrolysis.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- The recombinant alpha R (26, 28), WT beta R (29), and the mutant beta R-T159S (27) subunits were expressed in insoluble inclusion bodies and solubilized by urea, as described by Du and Gromet-Elhanan (26). Recombinant gamma C was expressed and refolded as described by Sokolov et al. (11). RrF1 was prepared from R. rubrum chromatophores according to Weiss et al. (30). All other reagents were of the highest purity available.

Assembly and Isolation of alpha R1beta R1 Dimers-- The dimers were assembled by refolding the urea-solubilized alpha R and beta R together according to the procedure developed for their optimal refolding into functional monomers (26). The refolded mixture was concentrated to about 1 mg/ml by Centriprep-10 (Amicon), precipitated with 60% saturated (NH4)2SO4, and resuspended in TGN buffer containing 50 mM Tricine-NaOH, pH 8.0, 20% glycerol, and 50 mM NaCl. The remaining insoluble aggregates were removed by centrifugation, and the refolded mixture was loaded on the size-exclusion HPLC Superdex-200 column (Amersham Pharmacia Biotech) and eluted with 100 mM NaPi, pH 7.0, containing 10% glycerol at a flow rate of 0.5 ml/min. The pooled alpha 1beta 1 dimer peak was concentrated, transferred to TGN buffer by elution-centrifugation through Sephadex G-50 columns, and stored at -80 °C.

Assembly of alpha R3beta R3 Hexamers-- The isolated alpha R1beta R1 dimers could be fully converted into the alpha R3beta R3 hexamers only when incubated, at >1 mg of protein/ml for 1 h at 22 °C, in TGN buffer containing also 10 mM NaF, and 0.5 mM AlCl3, in the presence of either 1 mM of CaCl2 or MgCl2 and 1 mM ATP or ADP (see Fig. 1D). Their activities could therefore be assayed directly by diluting samples assembled in the presence of each cation and ADP into the same cation-ATPase assay mixtures.

Assembly and Isolation of the Hybrid WT and Mutant alpha R3beta R3gamma C Complexes-- The hybrid WT alpha R3beta R3gamma C was assembled by two procedures as follows: 1) incubation of the isolated alpha R1beta R1 dimers with refolded gamma C for 1 h at 22 °C in TGN buffer in the presence of 1 mM MgCl2 or CaCl2 and 1 mM ADP which in this mixture, as in the hexamer assembly mixture, is as effective as ATP. Each incubated sample could therefore be assayed directly for its ATPase activity as well as size-exclusion HPLC. 2) Refolding the urea-solubilized recombinant alpha R and beta R each at 50 µg/ml, together with urea-solubilized gamma C at 20 µg/ml, according to the procedure developed above for assembly of alpha R1beta R1 dimers. The assembled hybrid complex was isolated by size-exclusion HPLC as described for the dimers, except that the hybrids were eluted with buffer containing 50 mM Tricine-NaOH, pH 8.0, 50 mM NaCl, and 10% glycerol. The peak containing the hybrid complex was pooled, concentrated, exchanged into TGN buffer as described for the dimers, and stored at -80 °C. The hybrid mutant alpha R3(beta R-T159S)3gamma C complex was assembled and isolated as described for the hybrid WT.

Assays of ATPase Activities-- The activities of RrF1 and both hybrid alpha R3beta R3gamma C WT and mutant complexes were measured with 4-20 µg of protein for 5 min at 35 °C in 0.5 ml of an assay mixture containing 50 mM Tricine-NaOH, pH 8.0, 50 mM NaCl, 4 mM ATP, and 2 mM of either MgCl2 or MnCl2 or 4 mM CaCl2. The activities of the isolated alpha R1 beta R1 dimers were measured with 30 µg of protein for 30 min under the conditions described above. To compare the ATPase activities of the alpha R3beta R3 hexamers with those of RrF1, the hexamers were first assembled by incubating the dimers as described above, and RrF1 was incubated under identical conditions. The ATPase activities of this RrF1 and the freshly assembled alpha R3beta R3 hexamers were measured by diluting each incubation mixture into the relevant assay mixtures, which also contained 10 mM NaF and 0.5 mM AlCl3. All ATPase activity assays were started by adding the protein complexes and stopped by adding 50 µl of M trichloroacetic acid, and the released Pi was measured as described by Taussky and Shorr (31). The effect of reduction or oxidation on the ATPase activities of RrF1 and the hybrid complexes was tested by their preincubation for 1 h at 35 °C in TGN buffer containing either 10 mM DTT or 100 µM CuCl2, followed by dilution into the relevant assay mixtures.

Other Procedures-- SDS-PAGE was carried out on the Novex Pre-Cast 10-20% Tris glycine gradient gels. The protein bands were visualized by staining with Coomassie Brilliant Blue R-250. Protein concentrations were determined by the Bradford method (32) or according to Lowry et al. (33), using bovine serum albumin as a standard.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Stepwise Assembly of RrF1-alpha R1beta R1 Dimers and alpha R3beta R3 Hexamers-- Active dimers, but no hexamers, have been assembled by incubation of the isolated alpha R and beta R monomers for 5 min at 35 °C in the presence of MgATP (26). These dimers showed a maximal MgATPase rate of 0.14 units/mg of protein, which remained linear for at least 1 h at 35 °C. In search for conditions that might enable the assembly of alpha R3beta R3 hexamers, the urea-solubilized alpha R and beta R subunits (26) were refolded together as described under "Experimental Procedures." They did indeed assemble directly, but again only into alpha R1beta R1 dimers with no indication for the appearance of any larger complexes (Fig. 1A). This simple one-step refolding/assembly procedure enabled the isolation of large amounts of the pooled concentrated dimer peak, which remained very stable in TGN buffer at protein concentrations above 1 mg/ml, even when incubated for 1 h at 22 °C (Fig. 1B). However, when diluted to the 10-20-fold lower protein concentrations used for ATPase activity assays, they remained stable only in presence of either MgADP or MgATP or even CaAD(T)P (not shown).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1.   Refolding of the RrF1-alpha R and beta R into alpha R1beta R1 dimers and their further association into stable alpha R3beta R3 hexamers in presence of aluminum fluoride. A, the urea-solubilized alpha R and beta R were folded together, and 15 µg of the concentrated product were loaded on a Superdex-200 column and eluted as described under "Experimental Procedures"; B-D, 15 µg of the pooled alpha R1beta R1 dimer peak, concentrated to 1.5 mg of protein/ml as described under "Experimental Procedures," were incubated in TGN buffer for 1 h at 22 °C with the following additions: B, none; C, 10 mM NaF and 0.5 mM AlCl3; D, as C plus 1 mM MgAD(T)P or CaAD(T)P; E and F were incubated as in D, diluted by 10-fold into TGN buffer, and further incubated for 1 h at 22 °C without (E) or with (F) 10 mM NaF, 0.5 mM AlCl3, and 1 mM MgAD(T)P or CaAD(T)P. All incubated samples were loaded on the column and eluted as described for A.

The closed alpha 3beta 3 hexamer, resolved in the x-ray crystallographic structure of bovine mitochondrial MF1 (12), has all six F1 nucleotide-binding sites arranged at alternating catalytic and noncatalytic alpha /beta interfaces. The isolated alpha R1beta R1 dimers could therefore have either one of these interfaces. But the fact that their MgATPase activity (26) is similar to that of CF1-alpha 3beta 3 (10, 34) indicates that these dimers contain the catalytic nucleotide-binding site at their alpha /beta interface. Their inability to associate into an alpha R3beta R3 hexamer therefore seems to reflect a very specific lower stability of the noncatalytic RrF1 alpha /beta interfaces. Indeed from the R. rubrum chromatophore-bound RrF0F1, only native dimers have been isolated (35). From chloroplast, on the other hand, only unstable alpha C3beta C3 hexamers could be obtained, and they readily dissociated into mixtures of their respective alpha  and beta  monomers (10, 36).

A search for compounds or conditions that can stabilize the noncatalytic RrF1 alpha /beta interfaces and enable the association of the alpha R1beta R1 dimers into hexamers has yielded the results demonstrated in Fig. 1, C and D. Incubation of the concentrated stable dimers (Fig. 1B) with NaF and AlCl3 that form aluminum fluoride (AlFx),2 a transition state analog of F1 nucleotide-binding sites (37-40), resulted in their partial conversion into the closed alpha R3beta R3 hexamers (Fig. 1C). Full association of these dimers into hexamers was obtained by their incubation with both NaF and AlCl3 in the presence of either Mg2+ or Ca2+ and either ADP or ATP at a cation/nucleotide ratio of 1 (Fig. 1D). AlFx was reported earlier to inhibit various F1-MgATPases but only after their very specific stepwise preincubation first with low ADP and high MgCl2 concentrations, then with NaF, and finally with AlCl3. The structure of bovine MF1, fully inhibited by this procedure, has recently been resolved (41). It shows that both aluminum trifluoride and Mg2+-ADP were bound, in a quasi-irreversible manner, to the catalytic nucleotide-binding site of the MF1-beta DP subunit. However, under our very different one-step incubation conditions, which associated practically all the alpha R1beta R1 dimers into alpha R3beta R3 hexamers (compare Fig. 1, B and D), the RrF1-MgATPase activity was hardly affected by AlFx (see Fig. 5). Furthermore, these alpha R3beta R3 hexamers remained fully stable only in the presence of AlFx and Mg- or CaAD(T)P at cation/AD(T)P ratios of 1 (Fig. 1, E and F). So under our association conditions AlFx does not bind irreversibly to the catalytic alpha /beta interface of the dimers and does not inhibit their MgATPase activity. It rather seems to bind in a reversible manner to the open noncatalytic nucleotide-binding site on the dimer alpha R subunit and facilitate its association with the beta R of another dimer, leading to their assembly into the closed hexameric structure.

Assembly of Hybrid WT and Mutant F1-alpha R3beta R3gamma C Complexes-- Incubation of the alpha R1beta R1 dimers with an F1-gamma subunit instead of AlFx resulted in their assembly into a stable, highly active alpha 3beta 3gamma complex (Fig. 2). A recombinant CF1-gamma C subunit was used for these studies since there is as yet no available native or recombinant gamma R. This gamma C was found to assemble with the native unstable CF1-alpha C3beta C3 into a stable highly active alpha C3beta C3gamma C complex (11), and its incubation with the isolated alpha R1beta R1 dimers resulted in their assembly into a hybrid alpha R3beta R3gamma C (Fig. 2, A and B). This assembly could also be followed by the dramatic 100-fold increase (Fig. 3) of the low 0.1-0.15 units/mg MgATPase activity of the alpha R1beta R1 dimers (26). But unlike the fast assembly of the alpha R and beta R monomers into dimers, which reached their maximal MgATPase activity after a 5-min incubation at 22 °C (26), this assembly was slow, requiring about 60 min for completion. A very similar time dependence was reported for the assembly of a CF1-(alpha beta gamma )-complex from isolated native CF1-(alpha beta ) and a CF1-gamma C (10). The increase in activity during the alpha R3beta R3gamma C assembly was also fully dependent on the amount of gamma C, saturating at a molar ratio of 2 gamma C/alpha 1beta 1 (Fig. 3, inset). The relatively high amount of gamma C required for obtaining this saturated activity was due to the tendency of both native and refolded gamma C to aggregate, since they remained soluble only when stored in a buffer containing 0.3 M LiCl at pH 9.5 (10, 11). So when gamma C was diluted into the incubation buffer with the alpha R1beta R1, it partially precipitated out during their slow assembly into the hybrid alpha R3beta R3gamma C complex. The time- and gamma C-dependent increase in the MgATPase activity during the assembly is therefore presented in units/mg alpha beta (Fig. 3).



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 2.   Size-exclusion HPLC demonstrates the assembly of a hybrid alpha R3beta R3gamma C complex by two different procedures. In the first procedure (A and B), the isolated alpha R1beta R1 (A) was incubated at 9.2 µg of protein with 5 µg of refolded gamma C for 1 h at 22 °C in 100 µl of TGN buffer containing 1 mM MgADP (B). In the second procedure (C and D), the individually expressed urea-solubilized alpha R, beta R, and gamma C subunits were refolded together as described under "Experimental Procedures" (C). The pooled alpha R3beta R3gamma C peak of C was concentrated and re-run in D. Each sample was loaded on the column and eluted as described in Fig. 1. Inset, SDS-PAGE profile of the isolated RrF1 and the pooled alpha R3beta R3gamma C peak.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3.   Time-dependent assembly of a highly active hybrid alpha R3beta R3gamma C-ATPase complex. 4.6 µg of the isolated alpha R1beta R1 dimers and 2.5 µg of a refolded gamma C were incubated at 22 °C in 50 µl of TGN buffer with 1 mM MgADP. The MgATPase activity of separate samples was assayed at the indicated intervals with the first point representing a sample mixed directly into the ATPase assay mixture containing 4 mM ATP, 2 mM MgCl2, and 50 mM sulfite. The ATPase activity was measured for 5 min at 35 °C as described under "Experimental Procedures." Inset, dependence of the increase in the hybrid ATPase activity on the amount of gamma C. Samples containing 9.2 µg of the isolated dimers were incubated for 1 h at 22 °C with increasing amounts of gamma C and assayed for their activity as described above.

The hybrid alpha R3beta R3gamma C was also assembled by refolding together all three urea-solubilized subunits under the conditions developed for refolding the alpha R1beta R1 dimers (see Fig. 1A). This much simpler one-step refolding procedure, which resulted in direct assembly of the alpha R3beta R3gamma C complex (Fig. 2C), enabled the isolation of large amounts of a pure, fully stable hybrid WT complex (Fig. 2D and inset). It was also used for the assembly of a hybrid mutant complex containing the beta R-T159S catalytic site mutant (27). This mutant beta R subunit was shown to bind in the presence of small amounts of monomeric alpha R into a beta -less chromatophore membrane-bound RrF0F1, which also lacked about 20% of its alpha  subunit and lost all ATP synthesis and hydrolysis activities. The reconstituted chromatophores regained all their Mg2+-dependent but none of the Ca2+-dependent activities (27).

Modulation of the Hybrid WT and Mutant F1-alpha R3beta R3gamma C-ATPase Activities by Their gamma  C Oxidation/Reduction-- A unique feature of the chloroplast CF1-ATPase activity is its high regulation by the reduction/oxidation of the disulfide bond formed between Cys199 and Cys205 in its gamma C subunit (24). The region containing these cysteine residues is completely missing from respiratory F1-gamma subunits, as well as from the gamma  subunit of cyanobacteria and purple photosynthetic bacteria, including the RrF1-gamma (25). All ATPase activities of RrF1 showed indeed no response to either reduction by DTT or oxidation by CuCl2 (Fig. 4A). However, both gamma C-containing hybrid WT and mutant F1-alpha 3Rbeta 3R3gamma C complexes showed clear redox regulation of their Mg2+-, Mn2+-, as well as Ca2+-dependent ATPase activities, which were about 2-fold higher in the reduced as compared with the oxidized states (Fig. 4, B and C). The MgATPase activity of a hybrid alpha 3beta 3gamma complex containing TF1-alpha and beta  subunits and the CF1-gamma C was also shown to be regulated by the gamma C redox state (42). These results indicate that the specific thiol modulation (24) of gamma C can be transferred to various hybrid alpha 3beta 3gamma complexes.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 4.   Modulation of the ATPase activities of RrF1 (A), and the assembled hybrid WT alpha R3beta R3gamma C (B), and mutant alpha R3(beta R-T159S)3gamma C (C) complexes by reduction and oxidation. All complexes were incubated for 1 h at 35 °C in TGN buffer without () or with either 10 mM DTT () or 100 µM CuCl2 (black-square), and their ATPase activities were measured with 4 mM ATP, 2 mM MgCl2 or MnCl2, or 4 mM CaCl2. The much lower RrF1 ATPase activities are presented in a 4-fold lower scale.

The Catalytic Properties of the Isolated RrF1 Dimers, Hexamers, and Both Hybrid WT and Mutant alpha R3beta R3gamma C Complexes-- An additional and more general tight regulation of the MgATPase activity of both chloroplasts and chromatophores is their sensitivity to inhibition by excess free Mg2+ ions (22, 23). Both isolated hybrid complexes showed the same optimal dependence on MgCl2 concentrations as the native RrF1, reaching maximal levels at a Mg/ATP ratio of 0.5 (Fig. 5 and inset). However, their much higher MgATPase activities showed a lower sensitivity than RrF1 to inhibition by excess free Mg2+ ions, being only 50% inhibited as compared with the RrF1 80% at an Mg/ATP ratio of 2.5. On the other hand, the similar, very low MgATPase activities of the dimers and the AlFx-stabilized hexamers were not subject to any regulation by excess free Mg2+ ions. They showed only a simple saturation curve, with no inhibition even at a Mg/ATP ratio of 2.5 (Fig. 5, inset). The MgATPase activity of RrF1 (+AlFx), which was incubated and assayed under the conditions used for assembly of the alpha R3beta R3 hexamers in presence of AlFx, was only slightly lower than that of native RrF1 and retained its full pattern of inhibition by excess free Mg2+ ions (Fig. 5, inset). These results indicate that the completely different response of both dimers and hexamers, as compared with RrF1, to increasing MgCl2 concentrations is due to absence of the gamma  subunit and not to the presence of AlFx.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of increasing MgCl2 concentrations on the ATPase activities of RrF1, its assembled alpha R1beta R1 dimers, alpha R3beta R3hexamers, and hybrid WT alpha R3beta R3gamma C and mutant alpha R3(beta R-T159S)3gamma C complexes. The MgATPase activities of RrF1 (), and the dimers (down-triangle), hybrid WT (open circle ), and mutant (diamond ) complexes were measured as described under "Experimental Procedures" using 4 mM ATP and the indicated concentrations of MgCl2. For comparing the activities of RrF1 (black-square) and the hexamers (black-triangle), the dimers were first assembled into the hexamers by incubation in the presence of 10 mM NaF, 0.5 mM AlCl3, and 1 mM MgADP as described under "Experimental Procedures," and RrF1 underwent the same treatment. Both complexes were diluted into the assay mixtures described above, except that all of them contained also 10 mM NaF and 0.5 mM AlCl3. The highly active hybrid WT (open circle ) and mutant (diamond ) complexes are presented in the main figure. Inset presents the much lower activities of RrF1 (, black-square), the dimers (down-triangle), and hexamers (black-triangle).

The properties of the CaATPase activities of the RrF1 and both hybrid WT and mutant complexes were very different from their MgATPase activities (compare Figs. 5 and 6). They were dependent on the presence of CaCl2, reaching saturation at a Ca/ATP ratio of 1.0, but showed no inhibition even at a ratio of 5.0. They were thus rather similar to the MgATPase activities of the dimers and hexamers (see Fig. 5, inset).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of increasing CaCl2 concentrations on the ATPase activity of RrF1 () and the hybrid WT alpha R3beta R3gamma C (open circle ) and mutant alpha R3(beta R-T159S)3gamma C (diamond ) complex. The CaATPase activities were measured as described under "Experimental Procedures" with 4 mM ATP and the indicated concentrations of CaCl2.

A detailed comparison of the Ca2+- and Mg2+-dependent ATPase activities of RrF1 and all assembled complexes (Table I) demonstrated a number of additional most interesting differences as follows:


                              
View this table:
[in this window]
[in a new window]
 
Table I
Ca2+- and Mg2+-dependent ATPase activities of RrF1 and the assembled RrF1-(alpha beta ) dimers, hexamers, and hybrid (alpha beta gamma ) complexes containing the chloroplast CF1gamma
Assembly and/or isolation of all complexes is described under "Experimental Procedures." The alpha R3beta R3 hexamers were assembled and assayed in the presence AlCl3 and NaF, which form AlFx, and the RrF1(+AlFx) was incubated and assayed under identical conditions. The ATPase activities were measured as described under "Experimental Procedures" with 4 mM ATP and 2 mM MgCl2 or 4 mM CaCl2, either with no additions (None) or with 50 mM sulfite or 2 mM azide.

1) Between the dimers and hexamers. The dimers have practically no CaATPase activity, although their MgATPase is similar in its rates and properties to that of the hexamers (Fig. 5 and Table I). These results illuminate clear differences in the structure of catalytic nucleotide-binding sites occupied by Ca2+ versus Mg2+, since CaATP can bind to these sites and enable the appearance of CaATPase activity only in the closed hexamer. On the other hand, the identical and very low dimer and hexamer MgATPase activities, which in the dimers cannot have any catalytic cooperativity, suggest its absence also in the hexamers. Indeed, both dimer and hexamer MgATPases do not respond to the usual MgATPase effectors of CF1 and RrF1. They are not stimulated by sulfite nor inhibited by azide (Table I).

2) Between both dimers and hexamers and the RrF1 (±AlFx) as well as the hybrid WT complex. The gamma -containing complexes show much higher Ca- and MgATPase activities as well as clear differences between the functional properties of these two ATPase activities. Their MgATPases are tightly regulated by excess free Mg2+ ions (Fig. 5), highly stimulated by sulfite and methanol (not shown) and inhibited by azide (Table I). But their 3-10-fold higher CaATPase activities are not regulated (Fig. 6) and, as both dimer and hexamer MgATPase activities, do not respond to any tested MgATPase effectors (Table I).

3) Between the RrF1 (+AlFx) Ca2+- and Mg2+-dependent ATPase activities. Surprisingly, although AlFx does not affect the RrF1-MgATPase (Fig. 5 and Table I), it inhibits by 70% the 10-fold higher CaATPase (Table I). In light of these results the similar Mg- and CaATPase activities in the alpha 3Rbeta R3 hexamers might be misleading, because their low CaATPase is the AlFx-inhibited activity. There is no possible way to confirm this suggestion in the RrF1 hexamers since they can assemble only in presence of AlFx (Fig. 1). But in the TF1-alpha 3beta 3 hexamers, which were assembled without AlFx, the CaATPase activity is 5-fold higher than their MgATPase, although the whole native TF1 has a 10-fold lower Ca- than MgATPase activity (43). The specific inhibition of the RrF1-CaATPase by AlFx, which has not been tested on any other F1-CaATPase activity, provides an additional clear difference of functional properties of the RrF1-CaATPase and MgATPase activities.

4) Between the hybrid WT and mutant complexes. Both RrF1 and the hybrid WT showed a 3-10-fold higher CaATPase than MgATPase activities. On the other hand, the hybrid mutant showed a much higher MgATPase activity than either RrF1 or the hybrid WT complex but a lower CaATPase (Figs. 5 and 6). Its CaATPase, as all other CaATPase activities, was not regulated and did not respond to any tested MgATPase effector. But the very high MgATPase activity of this mutant was also much less responsive to these effectors (Table I).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study large amounts of highly active hybrid WT and mutant photosynthetic F1-alpha 3beta 3gamma complexes were assembled by refolding the recombinant R. rubrum RrF1-alpha R and WT beta R (26) or mutant beta R-T159S subunits (27) together with the spinach CF1-gamma C (11). All ATPase activities of both isolated hybrid complexes showed, unlike those of RrF1, the specific thiol modulation (24) of their unique gamma C disulfide bond. Also all ATPase activities of the hybrid WT alpha R3beta R3gamma C, which were between 9- and 30-fold higher than those of the epsilon -containing RrF1 (Table I), retained the catalytic properties of both RrF1 and CF1-ATPases. This includes the specific regulation of the photosynthetic F1-ATPases by excess free Mg2+ ions (Refs. 22 and 23 and Fig. 5), which is released by sulfite resulting in a 2-3-fold stimulation of activity. (This MgATPase activity is also fully inhibited by azide (Table I). This also includes a much higher Ca2+- than Mg2+-dependent ATPase activity that is not inhibited by increasing Ca2+ concentrations nor by azide and is not stimulated by sulfite.

This hybrid WT alpha R3beta R3gamma C complex provides a most suitable candidate for studies aimed at elucidating the molecular mechanism involved in the gamma C thiol modulation of its high, ~40 units/mg, Mg2+- and Ca2+-dependent ATPase activities. Two other hybrid alpha 3beta 3gamma subcomplexes, exhibiting the regulatory thiol modulation of gamma C, were constructed with the TF1-alpha and beta  subunits. In the first report (42) the TF1 subunits were mixed with a recombinant gamma C, but the isolated hybrid showed at least a 10-fold lower MgATPase activity than the hybrid WT alpha R3beta R3gamma C complex. In a more recent report (44) a mutant TF1-alpha 3beta 3gamma complex was constructed by replacing 111 amino acid residues from the central region of the TF1-gamma with 148 residues of the homologous region from spinach gamma C, including the regulatory stretch with Cys199 and Cys205. This mutant complex was expressed and purified in large amounts and responded to the gamma C thiol modulation, but even its DTT-reduced MgATPase activity reached at the most 5 units/mg. Furthermore, no CaATPase activity has been reported in this mutant TF1-alpha 3beta 3gamma complex, probably because in the native TF1, unlike in RrF1 (Table I) and CF1 (30), the CaATPase activity is 10-fold lower than its MgATPase activity (43).

The hybrid WT alpha 3Rbeta 3R3gamma C complex provides also a promising system for following the possible CaATPase as well as MgATPase-induced gamma C rotation, which has not been measured as yet in any photosynthetic F1 complex. The CaATPase activity has not been tested as an inducer of any F1-gamma rotation. It is, however, a most important candidate for such assays because, although it reaches in both RrF1 and the hybrid WT complexes even higher rates than those of the MgATPase, it has very different catalytic properties. CaATPase, unlike the MgATPase, appears only in the AlFx-stabilized, closed alpha R3beta R3 hexameric structure (Table I). These results suggest that the Ca2+ binding affinity to the RrF1 catalytic nucleotide-binding sites is lower than that of Mg2+, which induces a similar MgATPase activity in the open dimers as in the closed hexamers. A lower binding affinity of Ca2+ could lead to its lower catalytic cooperativity. Indeed, the RrF1-CaATPase, unlike its MgATPase, is not inhibited at all by azide (Table I). Furthermore, the similar low MgATPase activity of the RrF1-alpha R1beta R1 dimers and alpha R3beta R3 hexamers, which in the open dimers has certainly no catalytic cooperativity, is also fully resistant to inhibition by azide (Table I). Azide, which was used as the inhibitor of gamma  rotation inside the alpha 3beta 3 hexamer cavity (17, 18), has recently been suggested to block the signal transmission between catalytic sites, which leads to positive catalytic cooperativity in all F1-MgATPases (45).

An additional, most important difference between Ca2+- and Mg2+- or Mn2+-dependent ATPase activities was recorded in the membrane-bound RrF0F1 where the CaATPase was not coupled to proton translocation and Ca2+ did not enable any ATP synthesis (23). So in the hybrid WT alpha R3beta R3gamma C the catalytic nucleotide-binding site in presence of Ca2+ has no clear catalytic cooperativity and is decoupled from any proton translocation. In a recent study on gamma  rotation in a genetically engineered EcF1-alpha 3beta 3gamma containing an uncoupled mutation of gamma Met23 to Lys (18), the mutant gamma  was found to rotate rather similarly to the WT gamma . This unexpected capacity of the uncoupled gamma  subunit to rotate was explained by suggesting that its defective coupling might be after gamma  rotation, possibly in the interactions between the F1 and F0 sectors (46). This explanation cannot hold for a catalytic site bound CaATP that cannot induce any proton translocation. The highly active CaATPase of the hybrid WT alpha R3beta R3gamma C is therefore a very interesting target for comparing the capacity of its Ca2+ versus Mg2+-occupied catalytic nucleotide-binding sites to induce catalytic cooperativity and/or rotational catalysis.

Another interesting target for such studies is the hybrid mutant alpha R3(beta R-T159S)3gamma C. The beta R-Thr159 is equivalent to the MF1 beta -Thr163, which was identified as a ligand to Mg2+ in the catalytic nucleotide-binding sites of the bovine heart crystal structure (12). This fully conserved residue has been mutated to serine in several respiratory F1-ATPases (47-49) as well as in Chlamydomonas reinhardtii CF1 (50). All of them showed, as our hybrid mutant alpha R3(beta R-T159S)gamma C (Table I), a very high MgATPase activity, with a much lower sensitivity to stimulation by sulfite or methanol and to inhibition by azide (Table I). But our hybrid mutant also has a 3-4-fold lower CaATPase activity that has not been followed in the other mutants. Since the hydroxyl group of serine is less nucleophilic than that of threonine, it would lower the bond energy between serine and both divalent cations and decrease their binding affinity. In the hybrid mutant, the Mg2+-occupied binding site would thus become rather similar to the lower affinity Ca2+-occupied catalytic site of our hybrid WT complex, which shows very high rates but lower or no catalytic cooperativity. But the mutant Ca2+-occupied catalytic sites would reach an even lower affinity that does already drastically reduce its overall activity (Table I).

In the membrane-bound RrF1 this beta R-T159S mutant was, however, as effective as the WT beta R in restoring the proton-translocating Mg2+-dependent ATP synthesis and hydrolysis. But it could not restore any membrane-bound CaATPase activity (27). These results demonstrate that there must be two sets of clear differences in the geometry of either the WT or mutant beta R catalytic sites when occupied by Ca2+ as compared with Mg2+. One is operating in the soluble state and a different one in the membrane-bound state, whose beta R-T159S-containing RrF0F1 shows the maximal difference between the fully operative proton-coupled Mg2+-occupied sites and the complete absence of any active Ca2+-occupied catalytic sites. The similar catalytic properties of the highly active hybrid mutant MgATPase and the hybrid WT CaATPase make them very promising tools for obtaining information on the as yet unknown factors that couple ATP binding and hydrolysis to rotational motion of the gamma  subunit of the catalytic F1-ATPase. Since azide does not inhibit the CaATPase activity, another inhibitor will be required for such comparative studies. A very suitable candidate is the specific CF1 effector tentoxin, which at low concentrations inhibits but at high concentrations stimulates both CF1 Ca- and MgATPase activities (30). RrF1 is, however, completely resistant to tentoxin. We have therefore assembled another set of hybrids composed of WT and mutated alpha R together with beta C and gamma C.3 They provide the possibility of assaying both gamma C thiol modulation and rotational catalysis in the presence of inhibitory as well as stimulating tentoxin concentrations.


    FOOTNOTES

* This work was supported in part by grants from the United States-Israel Binational Science Foundation, Jerusalem, and from 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.

Recipient of National Institutes of Health Predoctoral Training Grant GM08545.

|| To whom correspondence should be addressed. Tel.: 972-8-9342729; Fax: 972-8-9344118; E-mail: z.gromet-elhanan@weizmann.ac.il.

Published, JBC Papers in Press, January 12, 2001, DOI 10.1074/jbc.M007568200

2 The exact species of aluminum fluoride in the incubated mixture of AlCl3 and NaF that exerts the stabilizing effect on the noncatalytic RrF1 alpha /beta interfaces is as yet unknown. We therefore refer to it here as AlFx.

3 W. C. Tucker, Z. Du, Z. Gromet-Elhanan, and M. L. Richter, submitted for publication.


    ABBREVIATIONS

The abbreviations used are: CF1, EcF1, MF1, RrF1, and TF1, F1-ATPases from chloroplasts, E. coli, mitochondria, R. rubrum, and thermophilic Bacillus PS3, respectively; alpha R and beta R, the alpha  and beta  subunits of the RrF1-ATPase; gamma C, the gamma  subunit of spinach chloroplast CF1-ATPase; DTT, dithiothreitol; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; WT, wild type.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Gromet-Elhanan, Z. (1995) in Anoxygenic Photosynthetic Bacteria (Blankenship, R. E. , Madigan, M. T. , and Bauer, C. E., eds) , pp. 807-830, Kluwer Academic Publishers Group, Dordrecht, The Netherlands
2. Weber, J., and Senior, A. E. (1997) Biochim. Biophys. Acta 1319, 19-58[Medline] [Order article via Infotrieve]
3. Boyer, P. D. (1997) Annu. Rev. Biochem. 66, 717-749[CrossRef][Medline] [Order article via Infotrieve]
4. Richter, M. L., Hein, R., and Huchzermeyer, B. (2000) Biochim. Biophys. Acta 1458, 326-342[Medline] [Order article via Infotrieve]
5. McCarty, R. E., Evron, Y., and Johnson, E. A. (2000) Annu. Rev. Plant Physiol. Plant Mol. Biol. 51, 83-109[CrossRef]
6. Yoshida, M., Sone, N., Hirata, H., and Kagawa, Y. (1977) J. Biol. Chem. 252, 3480-3485[Abstract]
7. Futai, M. (1977) Biochem. Biophys. Res. Commun. 79, 1231-1237[Medline] [Order article via Infotrieve]
8. Noumi, T., Azuma, M., Shimomura, S., Maeda, M., and Futai, M. (1987) J. Biol. Chem. 262, 14978-14982[Abstract/Free Full Text]
9. Yohda, M., Ohta, S., Hisabory, T., and Kagawa, Y. (1988) Biochim. Biophys. Acta 933, 156-164[Medline] [Order article via Infotrieve]
10. Gao, F., Lipscomb, B., Wu, I., and Richter, M. (1995) J. Biol. Chem. 270, 9763-9769[Abstract/Free Full Text]
11. Sokolov, M., Lu, L., Tucker, W., Gao, F., Gegenheimer, P. A., and Richter, M. L. (1999) J. Biol. Chem. 274, 13824-13829[Abstract/Free Full Text]
12. Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628[CrossRef][Medline] [Order article via Infotrieve]
13. Boyer, P. D. (1993) Biochim. Biophys. Acta 1140, 215-250[Medline] [Order article via Infotrieve]
14. Boyer, P. D., and Kohlbrenner, W. E. (1981) in Energy Coupling in Photosynthesis (Selman, B. R. , and Selman-Reisner, S., eds) , pp. 231-240, Elsevier/North Holland
15. Fillingame, R. H. (1990) The Bacteria , Vol. XII , pp. 345-391, Academic Press
16. Vik, S., and Antonio, B. (1994) J. Biol. Chem. 269, 30364-30369[Abstract/Free Full Text]
17. Noji, H., Yasuda, R., Yoshida, M., and Kinosita, K. J. (1997) Nature 386, 299-302[CrossRef][Medline] [Order article via Infotrieve]
18. Omote, H., Sambonmatsu, N., Saito, K., Sambongi, Y., Iwamoto, K. A., Yanagida, T., Wada, Y., and Futai, M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 7780-7784[Abstract/Free Full Text]
19. Sambongi, Y., Iko, Y., Tanabe, M., Omote, H., Iwamoto-Kihara, A., Ueda, I., Yanagida, T., Wada, Y., and Futai, M. (1999) Science 286, 1722-1724[Abstract/Free Full Text]
20. Tsunoda, S. P., Aggeler, R., Noji, H., Kinosita, K. J., Yoshida, M., and Capaldi, R. A. (2000) FEBS Lett. 470, 244-248[CrossRef][Medline] [Order article via Infotrieve]
21. Panke, O., Gumbiowski, K., Junge, W., and Engelbrecht, S. (2000) FEBS Lett. 472, 34-38[CrossRef][Medline] [Order article via Infotrieve]
22. Hochman, Y., Lanir, A., and Carmeli, C. (1976) FEBS Lett. 61, 255-259[CrossRef][Medline] [Order article via Infotrieve]
23. Gromet-Elhanan, Z., and Weiss, S. (1989) Biochemistry 28, 3645-3650
24. Nalin, C. M., and McCarty, R. E. (1984) J. Biol. Chem. 259, 7275-7280[Abstract/Free Full Text]
25. Miki, J., Maeda, M., Mukohata, Y., and Futai, M. (1988) FEBS Lett. 232, 221-226[CrossRef][Medline] [Order article via Infotrieve]
26. Du, Z., and Gromet-Elhanan, Z. (1999) Eur. J. Biochem. 263, 430-437[Abstract/Free Full Text]
27. Nathanson, L., and Gromet-Elhanan, Z. (2000) J. Biol. Chem. 275, 901-905[Abstract/Free Full Text]
28. Du, Z., and Gromet-Elhanan, Z. (1995) in Photosynthesis: From Light to Biosphere (Mathis, P., ed), Vol. 3 , pp. 27-30, Kluwer Academic Publishers Group, Dordrecht, The Netherlands
29. Nathanson, L., and Gromet-Elhanan, Z. (1998) J. Biol. Chem. 273, 10933-10938[Abstract/Free Full Text]
30. Weiss, S., McCarty, R. E., and Gromet-Elhanan, Z. (1994) J. Bioenerg. Biomembr. 26, 573-581[Medline] [Order article via Infotrieve]
31. Taussky, H. H., and Shorr, E. (1953) J. Biol. Chem. 202, 675-685[Free Full Text]
32. Bradford, M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
33. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275[Free Full Text]
34. Gromet-Elhanan, Z., and Sokolov, M. (1995) Photosynth. Res. 46, 79-86
35. Andralojc, P. J., and Harris, D. A. (1992) FEBS Lett. 310, 187-192[CrossRef][Medline] [Order article via Infotrieve]
36. Sokolov, M., and Gromet-Elhanan, Z. (1996) Biochemistry 35, 1242-1248[CrossRef][Medline] [Order article via Infotrieve]
37. Lunardi, J., Dupuis, A., Garin, J., Issartel, J. P., Michel, L., Chabre, M., and Vignais, P. V. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8958-8962[Abstract]
38. Issartel, J. P., Dupuis, A., Lunardi, J., and Vignais, P. V. (1991) Biochemistry 30, 4726-4733[Medline] [Order article via Infotrieve]
39. Dou, C., Grodsky, N. B., Matsui, T., Yoshida, M., and Allison, W. S. (1997) Biochemistry 36, 3719-3727[CrossRef][Medline] [Order article via Infotrieve]
40. Nadanaciva, S., Weber, J., and Senior, A. E. (1999) J. Biol. Chem. 274, 7052-7058[Abstract/Free Full Text]
41. Braig, K., Menz, R. I., Montgomery, M. G., Leslie, A. G. W., and Walker, J. E. (2000) Structure 6, 567-573
42. Hisabori, T., Kato, Y., Motohashi, K., Kroth, P. P., Strotmann, H., and Amano, T. (1997) Eur. J. Bichem. 247, 1158-1165[Abstract]
43. Miwa, K., and Yoshida, M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6484-6487[Abstract]
44. Bald, D., Noji, H., Stumpp, M. T., Yoshida, M., and Hisabori, T. (2000) J. Biol. Chem. 275, 12757-12762[Abstract/Free Full Text]
45. Weber, J., and Senior, A. E. (1998) J. Biol. Chem. 273, 33210-33215[Abstract/Free Full Text]
46. Futai, M., Omote, H., Sambongi, Y., and Wada, Y. (2000) Biochim. Biophys. Acta 1458, 276-288[Medline] [Order article via Infotrieve]
47. Mueller, D. M. (1989) J. Biol. Chem. 264, 16552-16556[Abstract/Free Full Text]
48. Omote, H., Maeda, M., and Futai, M. (1992) J. Biol. Chem. 267, 20571-20576[Abstract/Free Full Text]
49. Jault, J. M., Dou, C., Grodsky, N. B., Matsui, T., Yoshida, M., and Allison, W. S. (1996) J. Biol. Chem. 271, 28818-28824[Abstract/Free Full Text]
50. Hu, D., Strotmann, H., Shavit, N., and Leu, S. (1998) FEBS Lett. 421, 65-68[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.