©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
In Vitro Assembly of the Core Catalytic Complex of the Chloroplast ATP Synthase (*)

Fei Gao , Brian Lipscomb , Inmin Wu , Mark L. Richter (§)

From the (1) Department of Biochemistry, The University of Kansas, Lawrence, Kansas 66045

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The regulatory subunit and an complex were isolated from the catalytic Fportion of the chloroplast ATP synthase. The isolated subunit was devoid of catalytic activity, whereas the complex exhibited a very low ATPase activity (200 nmol/min/mg of protein). The complex migrated as a hexameric complex during ultracentrifugation and gel filtration but reversibly dissociated into and monomers after freezing and thawing in the presence of ethylenediamine tetraacetic acid and in the absence of nucleotides. Conditions are described in which the and preparations were combined to rapidly and efficiently reconstitute a fully functional catalytic core enzyme complex. The reconstituted enzyme exhibited normal tight binding and sensitivity to the inhibitory subunit and to the allosteric inhibitor tentoxin. However, neither the complex nor the isolated subunit alone could bind the subunit or tentoxin with high affinity. Similarly, high affinity binding sites for ATP and ADP, which are characteristic of the core enzyme, were absent from the complex. The results indicate that when the subunit binds to the complex, it induces a three-dimensional conformation in the enzyme, which is necessary for tight binding of the inhibitors and for high-affinity, asymmetric nucleotide binding.


INTRODUCTION

The chloroplast coupling factor 1 (CF)() utilizes the energy of a transmembrane proton gradient to catalyze ATP synthesis. CFis composed of five different subunits designated to in order of decreasing molecular weight and with a subunit stoichiometry of 3, 3, 1, 1, and 1 (1) . CFis a latent ATPase, requiring activation either by removing the subunit and/or reducing the only disulfide bond of the enzyme located on the subunit (2) . The subunit is readily removed from the enzyme by treatment with ethyl alcohol, while CFis bound to an anion-exchange resin. The resulting -deficient enzyme is recovered from the resin as a fully active ATPase (3) . The subunit can also be removed by similar treatment (2, 4) with no apparent effect on the catalytic activity of isolated CF. Thus the maximum number of subunits required for ATPase activity is .

There are six nucleotide binding sites on the enzyme (5, 6, 7) probably located at interfaces between the and subunits (8, 9) . Freshly isolated CFhas up to two molecules of ADP bound with sufficient strength to resist removal during successive passage through several gel filtration columns (10) . Addition of MgATP results in occupancy of four sites, two with ADP tightly bound and two with ATP tightly bound. The remaining two sites do not retain nucleotides during gel filtration and are thus assumed to be of lower affinity. Tightly bound ADP can be released from the enzyme upon binding of MgATP at another site, probably one of the lower affinity sites (10, 11, 12) . This intersite cooperativity is considered to be involved in an alternating sites mechanism in which two (12) or three (13) catalytic sites switch properties with each other with respect to their affinity for nucleotides.

The apparent asymmetry among the nucleotide binding sites on CFis considered to result from asymmetric interactions between the three and three subunits and the smaller single copy subunits, , , and. Since the asymmetry remains intact following removal of the two smallest subunits, and , the subunit is sufficient to provide the necessary asymmetric interaction(s). However, some recent studies of the reconstituted and subunits of the Fof the thermophylic bacterium PS3 (14, 15, 16) indicated that an complex retained asymmetric features resembling those of the normal enzyme but in the complete absence of small subunits.

The Fcomplexes of several bacteria have been successfully disassembled in vitro and reassembled from their individual subunits (17, 18, 19, 20) . In vitro assembly of an Fenzyme from a eukaryotic source, however, has not yet been reported in spite of intensive efforts in many laboratories. This goal remains a very important one for effectively studying subunit interactions for these enzymes, especially since the eukaryotic enzymes exhibit several properties, including subunit composition and regulatory mechanisms, that are different from their bacterial counterparts (for reviews, see Refs. 1, 21, and 22). Development of an in vitro assembly system is also essential for our studies of CFthat involve assembling genetically engineered subunits into an active enzyme complex for probe-labeling studies aimed at examining protein dynamics (23, 24) .

In addition to the selective removal of the and subunits from CF(3, 4) , conditions have been described (25) for isolating the CF subunit and reconstituting it with -deficient membranes of Rhodospirrillum rubrum to form an active hybrid Fcomplex. In this paper, we report the isolation of the subunit and an complex from CF. The two protein preparations were readily reconstituted with each other to form a fully functional core enzyme complex. Using this reconstitution system, we have been able to demonstrate that an complex is likely to be the minimum structure required for asymmetric nucleotide binding and, therefore, for cooperative catalysis by CF. Formation of the core complex is also prerequisite for proper binding of the inhibitory subunit and for binding of the allosteric CF-specific inhibitor tentoxin.


EXPERIMENTAL PROCEDURES

Materials

CFand CFlacking the and subunits, CF(), were prepared from fresh market spinach as described previously (2, 26) and stored as ammonium sulfate precipitates. Prior to use, the proteins were desalted on Sephadex G-50 centrifuge columns (27) . The isolated subunit was stored in the isolation buffer at 4 °C (2, 3) .

Nucleotides and tentoxin were purchased from Sigma. Stock solutions of tentoxin were prepared by dissolving the inhibitor in ethanol to a final concentration of 5 mM, and stored at 70 °C. Urea and guanidine hydrochloride were purchased from Fluka. Hydroxylapatite was purchased from Bio-Rad.

Preparation of an Complex

1-10 mg of CF() were desalted on Sephadex G-50 centrifuge columns equilibrated with 30 mM potassium phosphate (pH 7.0). ATP and MgClwere added to final concentrations of 5.5 mM and 5 mM, respectively. LiCl was added slowly from a 10 M stock solution to a final concentration of 2 M. The final protein concentration was kept below 1 mg/ml. The solution was incubated on ice for 1.5 h and then applied to a 1.5 10-cm hydroxylapatite column equilibrated with a solution containing 30 mM potassium phosphate (pH 7.0), 1 mM MgATP, and 0.3 M LiCl at 4 °C. The column was washed with the same buffer solution. A mixture of and subunits, and usually a trace amount of contaminating subunit, was washed straight through the column. This fraction was collected and dialyzed at 4 °C against at least 5 volumes of a solution containing 10% (v/v) glycerol, 20 mM Hepes-NaOH (pH 7.0), 1 mM MgATP, and 2 mM dithiothreitol. The complex was further purified by application to a diethylaminoethyl-cellulose column equilibrated with a solution containing 20 mM Hepes-NaOH (pH 6.5), 1 mM ATP, and 1 mM EDTA. A small amount of free subunit was washed from the column with the equilibration buffer containing 0.12 M NaCl. The mixture eluted when the column was washed with the same buffer containing 0.17 M NaCl. Any residual undissociated remained tightly bound to the column until the NaCl concentration was raised to 0.4 M. The mixture was dialyzed at 4 °C against at least 5 volumes of a solution containing 20% glycerol, 20 mM Hepes-NaOH (pH 7.0), and 1 mM MgATP and stored at 70 °C for further use. More than 50% of the and subunits of the CF() were routinely recovered in the complex. Isolation of the Subunit from CF ()-CF() was desalted on a centrifuge column of Sephadex G-50 equilibrated with a solution containing 15 mM sodium phosphate (pH 7.0). ATP, MgCl, and urea were added to final concentrations of 5.5 mM, 5 mM, and 4 M, respectively. The protein concentration was kept at or slightly below 1 mg/ml. The solution was stirred gently on ice for 1 h and then applied to a 1.5 7-cm hydroxylapatite column equilibrated with a solution containing 15 mM sodium phosphate (pH 7.0), 1 mM ATP, and 0.5 mM MgClat 4 °C. The protein () was washed through the column with 50 ml of buffer containing 4 M urea, 15 mM sodium phosphate (pH 7.0), 1 mM ATP, and 0.5 mM MgCl. The column was washed with 20 ml of 15 mM NaHPO(pH 7.0) followed by 30 ml of 200 mM NaHPO(pH 7.0). The subunit was eluted as a sharp peak by washing the column with a solution containing 4 M urea and 300 mM sodium phosphate (pH 7.0). The subunit tended to aggregate when the protein concentration at this step was too high, requiring dilution with the elution buffer. The fraction was dialyzed at 4 °C against a buffer containing 20% glycerol, 50 mM NaHCO(pH 9.5), 5 mM dithiothreitol, and 0.3 M LiCl. The protein (1 mg/ml) was stored indefinitely at 70 °C without loss of reconstitutive activity.

Reconstitution of the and Fractions

The purified mixture was diluted to about 100 µg/ml with a solution containing 20% glycerol, 50 mM Hepes-NaOH (pH 7.0), 1 mM MgCl, 1 mM ATP, and 2 mM dithiothreitol and kept on ice. The subunit was added slowly to the and gently mixed. The mixture was left to sit at room temperature (22 °C) for at least 1 h before assaying ATPase activity. In some experiments, any unreconstituted subunits were separated from the reconstituted by DEAE chromatography as described in the preceding section.

Analysis of Bound Nucleotides

Release of bound nucleotides upon denaturation and precipitation of the and CF() complexes was achieved essentially as described elsewhere (10) . 50 µl of 1.2 M HClOwere added to each 100 µl of protein sample containing between 100 and 200 µg of protein. Precipitated material was removed by centrifugation in a Beckman Microfuge at full speed for 5 min. 25 µl of 1.3 M KCOwere added immediately to the supernatant to neutralize it. The centrifugation was repeated, and the supernatant was subjected to HPLC analysis on a Beckman System Gold gradient system equipped with a Shimadzu CR601 integrator. Nucleotides were separated isocratically on a Beckman 5-µm ODS column with an eluting solvent containing 50 mM potassium phosphate and 5 mM tetrabutylammonium hydroxide (pH 6.5) in 92.2% (v/v) distilled water, 7.8% (v/v) acetonitrile (10) . The nucleotide absorbance was monitored at 254 nm. Concentrations of nucleotides in extracts were determined by comparing peak areas with those of standard nucleotides of known concentration.

Other Procedures

ATPase activities were determined by measuring phosphate release (28) for 5-10 min at 37 °C. The assay mixture for calcium-dependent ATPase activity contained 50 mM Tris-HCl (pH 8), 5 mM ATP, and 5 mM CaCl. That for magnesium-dependent ATPase activity contained 40 mM Tricine-NaOH (pH 8), 4 mM ATP, 2 mM MgCl, and 50 mM NaSO(29) . Protein concentrations were determined by the method of Lowry et al. (30) or by the method of Bradford (31) .

Analytical ultracentrifugation was performed on a Beckman model E664 analytical ultracentrifuge equipped with schlieren optics. Electron microscopy was performed as described elsewhere (32) .


RESULTS

Isolation and Properties of the Subunit and Subunit Complex

Treatment of CF() with 2 M LiClfollowed by hydroxylapatite chromatography under the conditions described under ``Experimental Procedures'' led to isolation of two protein fractions, one containing the subunit with an almost equal concentration of contaminating subunit, and another containing a freely soluble and highly purified mixture of and subunits. Between 50 and 70% of the and subunits were recovered from the starting material. A highly purified subunit was obtained using a similar procedure in which the LiCl was replaced by 4 M urea. The fraction obtained using this alternative procedure was inactive in reconstitution. Therefore, the LiCl fraction and the urea fraction were used for reconstitution studies. Gel electrophoresis of these fractions is shown in Fig. 1. Preliminary studies (not shown) indicated that when the fraction shown in Fig. 1was subjected to analytical ultracentrifugation, it sedimented as a single species with an apparent molecular weight of approximately 350,000 (the calculated molecular weight of the complex is 328,000). The fraction was also shown to co-chromatograph with CF() on a 1 40-cm column of Sephadex G-200, a considerable distance in front of standard bovine serum albumin that was shown earlier (33) to co-elute with free subunit under the same conditions.


Figure 1: SDS gel electrophoresis of isolated and subunits. Proteins were separated by electrophoresis on 12% polyacrylamide gels and stained with Coomassie Brilliant Blue G. Lane 1, CF() before dissociation; lane 2, purified complex; lane 3, isolated subunit; lane 4, complex reconstituted from the and fractions shown in lanes 2 and 3 and purified by anion-exchange chromatography as described under ``Experimental Procedures.''



The high molecular weight of the fraction suggested that it may exist as a hexameric complex similar to that isolated from the thermophilic bacterial F(34) and to that isolated from CFfollowing treatment of thylakoid membranes with LiCl (35) . However, when the protein was examined by electron microscopy, we did not observe the usual hexameric structure, which is routinely observed in preparations of CFand CF() (32) . This was true despite a number of trials in which the protein concentration, the ionic strength, and the nucleotide concentration of the suspension medium were varied. A likely explanation for the lack of structure is that the complex is less stable than the complex and fell apart upon dehydration and/or binding to the carbon grid when preparing the protein for electron microscopy. That the mixture is less stable than CFor CF() was evident when the protein was analyzed by HPLC as shown in Fig. 2. In the experiment shown in Fig. 2B, the mixture was chromatographed in the presence of Mgand ATP, both of which are required for reconstitution with the subunit (see the following section). Under these conditions most of the protein migrated with an apparent molecular weight expected for a hexameric complex. A small fraction of the protein migrated as a species with a molecular weight of approximately 50,000, which was presumed to be monomeric and subunits. When the mixture was frozen and thawed in the absence of nucleotides and in the presence of EDTA, only the lower molecular weight monomeric form was present (Fig. 2 C). We did not observe significant amounts of material migrating between these two forms, which would have suggested the presence of stable heterodimers.


Figure 2: Gel filtration chromatography of the complex. Standard proteins and the complex were chromatographed on a Phenomenex Biosep-SEC-S-2000, 600 mm 7.8-mm column equilibrated with 50 mM sodium phosphate (pH 7) using a Spectra Physics SP8700 HPLC system. Proteins were separated isocratically in the column equilibration buffer and detected by their absorbance at 280 nm. A, a mixture of molecular mass markers containing bovine thioglobulin (679 kDa, peak 1), bovine globulin (158 kDa, peak 2), chicken ovalbumin (44 kDa, peak 3), equine myoglobin (17 kDa, peak 4), and hydroxycobalamin (1.4 kDa, peak 5). B, freshly isolated complex. C, the complex in B was desalted into 20 mM sodium phosphate buffer (pH 7) containing 1 mM EDTA to remove unbound metal and nucleotide, frozen at 20 °C, and thawed prior to HPLC.



The complex exhibited a low rate of magnesium- or calcium-dependent ATP hydrolysis (). The activity persisted even after the was recycled through the isolation procedure to remove any residual traces of the subunit and after further purification by anion-exchange chromatography as described under ``Experimental Procedures.'' In addition, the activity was totally insensitive to the inhibitor tentoxin and to added subunit (results not shown) under conditions where both strongly inhibit the catalytic activity of CF(). These observations precluded the possibility that the residual activity of the complex resulted from the presence of a small amount of contaminating CF().

We found that the subunit aggregated upon removal of the urea, which was used to elute it from the hydroxylapatite column during isolation. In order to keep soluble, it was necessary to slowly exchange the urea for a buffer containing a relatively low concentration of chaotropic ions (0.3 M LiCl) at high pH (9.5). Under these conditions, could be concentrated to as much as 5 mg/ml without apparent aggregation or loss of reconstitutive activity. The cleanest preparations of the subunit were completely devoid of any catalytic activity yet were fully functional in reconstitution.

Reconstitution of and Subunits

By mixing the and fractions, we were able to recover more than 80% of the catalytic activity of the starting material (). The reconstitution occurred equally well regardless of whether the subunits were in the hexameric or monomeric form. Purification of the reconstituted complex by anion-exchange chromatography resulted in an enzyme with essentially identical properties to CF() (). The reconstitution was complete within 2-3 h of mixing (Fig. 3) and was not sensitive to variations in pH between 6 and 8 (). However, if the pH of the reconstitution mixture was kept at pH 9 or above, such as the conditions used to store the subunit, almost no activity was recovered ().


Figure 3: Time-dependence for reconstitution of and subunits. The isolated complex and subunit were reconstituted and assayed under the conditions described under ``Experimental Procedures'' except that the time allowed for reconstitution was varied. Magnesium-dependent ATPase activities were determined at the times indicated.



The reconstituted core enzyme complex was fully sensitive to inhibition by tentoxin and was strongly inhibited by the subunit ( Fig. 4and ). It was observed that exposure of the subunit to urea-containing solutions for several hours or overnight resulted in a significant loss of sensitivity to inhibition by the subunit, without a significant loss of -dependent reconstitution of ATPase activity. This suggested that part of the binding site for is present on the subunit and that this site may have been chemically modified ( e.g. carbamylation of one or more amino groups) during prolonged incubation of in the presence of urea. This explained the higher residual activity observed for the reconstituted complex than for CF() following addition of normally saturating concentrations of the subunit (). To avoid such modification, the subunit isolation procedure was performed as quickly as possible, and more recently (results not shown) it was found that 2 M guanidine hydrochloride can effectively substitute for urea in the isolation procedure, eliminating the time-dependent loss of binding.


Figure 4: Sensitivity of the reconstituted complex to tentoxin. The and subunits were reconstituted as described under ``Experimental Procedures.'' 10-µg samples of reconstituted complex () or CF() () were preincubated for 5 min at 22 °C with the indicated amounts of tentoxin in 0.5 ml of a solution containing 50 mM Tricine-NaOH and 1 mM ATP. At the end of the preincubation, 0.5 ml of double strength calcium-ATPase assay mixture, prewarmed to 37 °C, was added to initiate the assay. The control rate was 17.5 µmol/min/mg of protein.



MgATP was present throughout the preparation and storage of the complex, so in order to test the effects of magnesium and ATP on reconstitution, the complex was first desalted by column centrifugation. Reconstitution was found to be almost completely dependent on the presence of both magnesium and ATP (). The small residual activity observed in the absence of added nucleotide probably resulted from a small amount of ATP or ADP remaining bound to the complex during the desalting procedure (see I). ADP could substitute quite well for ATP, whereas GTP was a poor substitute. AMP and CTP were ineffective.

The Complex Lacks Tight ADP Binding Sites

The protein complex was routinely isolated and stored in the presence of both magnesium and ATP. To examine the complex for the presence of tightly bound nucleotides, free and loosely bound nucleotides were first removed by passing the protein sequentially through two centrifuge columns. The columns were warmed to room temperature prior to addition of the protein to avoid dissociation of the into monomers. Nucleotides that remained bound to the protein following chromatography on two consecutive Sephadex G-50 centrifuge columns were identified using a HPLC procedure similar to that described by Shapiro et al. (10) . The results are shown in I. The complex retained approximately 1 mol of MgATP/mol of under these conditions. When EDTA was included in the buffer used to equilibrate the centrifuge columns, the residual MgATP was completely removed. This is in contrast to CF(), which contains nearly four tightly bound nucleotides/, two of which remain tightly bound even after continuous exposure to EDTA during isolation (I). The nucleotide binding properties of CF() were essentially identical to those described previously for CF(10, 26) .

High Affinity Binding of Tentoxin and Subunit Requires the Subunit

The isolated subunit alone was shown (26) not to bind tentoxin with sufficient strength to compete with CF() for binding of the toxin. This result suggested that CFsubunits other than, or in addition to, the subunit are required for tentoxin binding. We undertook similar competition experiments to test for a binding interaction between tentoxin and the subunit or the subunit complex. As shown in Fig. 5, neither the complex nor the subunit, when preincubated with tentoxin, could reduce the effective concentration of the toxin, even at molar concentrations more than 10-fold higher than the concentration of latent CF, which was needed to completely block tentoxin inhibition. As an additional control, some of the CF() was pretreated with DCCD (see the legend to Fig. 6) before it was reconstituted with the subunit. The resulting complex was devoid of catalytic activity, but it was still capable of binding tentoxin and thus effectively blocking tentoxin inhibition of the active CF() (results not shown).


Figure 5: Competition between CF(), the complex, and the isolated subunit for tentoxin binding. The effectiveness of the complex (), the free subunit (), or latent CF() to compete with CF() for tentoxin binding was determined by preincubating fixed concentrations of tentoxin with varying amounts of the competing protein. Each incubation mixture contained, in a total volume of 0.5 ml, 50 mM Tricine-NaOH, 1 mM ATP, 0.6 µM tentoxin (except for the control sample), 10 µg of CF() plus the indicated amounts of competing protein. After 5 min of incubation at 22 °C, 0.5 ml of double-strength CaATPase assay mixture, preincubated at 37 °C, was added to begin the ATPase reaction. Controls samples lacking the CF() were performed at each concentration of the added competing proteins (, , and latent CF) in order to adjust for the low catalytic activities (all <1 µmol/min/mg of protein) of these protein preparations.




Figure 6: Competition between CF(), the complex, and the free subunit for binding. The effectiveness of the complex (), the free subunit (), or DCCD-inhibited CF() () to compete with CF() for subunit binding, was determined by preincubating a fixed concentration of the subunit with varying amounts of the competing protein. Each incubation mixture contained, in a total volume of 0.5 ml, 50 mM Tricine-NaOH, 1 mM ATP, 10 µg of CF(), varying amounts of the competing protein, and 1.9 µg of purified subunit (added last to all but the control sample), which was sufficient to inhibit the activity of the CF() by 51%. After 5 min of incubation at 22 °C, 0.5 ml of double strength CaATPase assay mixture, preincubated at 37 °C, was added to begin the ATPase reaction. The DCCD-inhibited CF() sample was prepared by treating CF() with 200 µM DCCD for 1 h in buffer containing 50 mM Hepes-NaOH (pH. 7.0) (47). The CaATPase activity of the DCCD-treated protein was <0.1 µmol/min/mg of protein.



Competition experiments were also used to examine the structural requirement for binding. This was done by preincubating the subunit with the subunit, or with the complex, prior to addition of CF(). If the subunit binds to either of these fractions, then their presence should reduce the effective concentration of the subunit, thus reducing the extent of inhibition of the ATPase activity of CF(). The results (Fig. 6) indicate that at high concentrations the subunit did somewhat reduce the effectiveness of the subunit, suggesting a weak binding interaction between these two subunits. The complex, however, even at a very high molar excess, did not reduce the effective concentration of the subunit, suggesting the absence of a strong binding interaction between the subunit and the complex. In contrast, inactivated (DCCD-treated) CF() bound the subunit strongly, effectively competing for free subunit (Fig. 6).


DISCUSSION

The subunit has been isolated in reconstitutively active form from the Fenzymes of several different bacterial species including Escherichia coli (17) , the thermophilic bacterium PS3 (18) , and R. rubrum (19) . In each case, the subunit could be reconstituted with the other For F-Fsubunits to restore, to varying extents, the catalytic activity to the -less enzymes. Repeating these experiments with eukaryotic Fenzymes has proven much more difficult, due largely to the aggregative properties of the and subunits, especially under the conditions that were effective in reconstitution of the bacterial subunits. In this report, we have described conditions for isolating the CF subunit in a highly purified form, which can be stored indefinitely at a high concentration without aggregation and which retains its ability to reconstitute with the and subunits to form an active core enzyme complex. Large amounts of reconstituted, fully active complex could be obtained using a remarkably simple and quick protocol.

Conditions were also identified for isolating large amounts of a complex, which contained equal amounts of the and subunits and which migrated as an hexamer during analytical ultracentrifugation and gel filtration. The complex is very similar to that described by Avital and Gromet-Elhanan (30) , which was one of several CFsubcomplexes isolated following treatment of thylakoid membranes with 2 M LiCl. Their complex chromatographed as two species, one with a high molecular weight, consistent with an structure, and a lower molecular weight species, which migrated during gel filtration as an heterodimer. In our case, the higher molecular weight form predominates unless the protein is frozen and thawed in the absence of metal ions and nucleotides, in which case it reversibly disassembles into and monomers. Both the hexameric and monomeric forms could be reconstituted with the subunit to form an active core enzyme complex.

An complex has been assembled from the Fsubunits of the thermophilic bacterium PS3. The structure of the complex was confirmed by electron microscopy as an hexamer (34) , which dissociated into heterodimers in the presence of micromolar concentrations of nucleotides (14, 34) . Removal of the nucleotides resulted in reassembly of the hexameric species (36) . A stable complex has also been isolated from the photosynthetic bacterium R. rubrum following treatment of chromatophore membranes with 2 M LiCl (37) . The chromatographic properties of the complex suggested that it was an heterodimer. All of the preparations described have been reported to exhibit low levels of ATPase activity ranging between about 30 nmol/min/mg of protein for the heterodimers to about 300 nmol/min/mg of protein for the hexamers. With the exception of PS3 F(34) , these catalytic rates represent only a small percentage of the maximum catalytic rates obtained with the parent Fenzymes. For the CF complex, the activity reached a maximum of about 0.5% of the fully activated CFcomplex.

Nearly all Fenzymes have been shown to contain six nucleotide binding sites. The recently published crystal structure of the beef heart mitochondrial Fat 2.8 Å resolution revealed that the six sites are located at the six interfaces formed between the alternating and subunits of the hexameric Fcomplex (9) . The subunits contribute the bulk of the structure of three putative catalytic sites, the subunit contributing a small section of the adenine binding portion of the catalytic pocket. This situation is reversed for the three putative noncatalytic sites, which reside mainly on the subunits with a small contribution from each of the three subunits. Since the and subunits are well conserved among different organisms (38) it should be safe to assume that the arrangement of the six nucleotide binding sites is essentially identical in all Fenzymes. The fact that both subunits contribute to nucleotide binding at each site would explain why the and subunits isolated from various Fenzymes are unable to catalyze ATP hydrolysis on their own even though they are able to bind nucleotides (39, 40, 41, 42, 43) . It also explains the observation that complexes containing both and subunits are catalytically active, albeit at very low levels.

The results presented in this study show that the subunit is required, in addition to the and subunits, for the high rates of ATP hydrolysis that are normally associated with activated CF. Similar results have been obtained for several bacterial systems in which it has been possible to reconstitute the active ATPase complex from isolated subunits (17, 18, 19, 20) . High catalytic rates are usually associated with cooperative interactions among different nucleotide binding sites that involve either two (12) or three (for review, see Ref. 13) catalytic sites alternately switching their conformational states during the course of a single catalytic cycle. The occurrence of heterogeneous nucleotide binding sites requires structural asymmetry, which is generally assumed to result from binding of the smaller, single-copy subunits to the hexamer. Since the core complex of CFis fully active (26) , the necessary asymmetry must be provided by binding of the subunit to the hexamer. Indeed, the crystal structure of the beef heart Frevealed a direct interaction between the subunit and one of the three subunits, suggesting that the subunit forces that subunit into an alternative conformation, altering its nucleotide binding properties, and thereby introducing structural and functional heterogeneity among the three potential catalytic sites (9) .

Four of the six nucleotide binding sites on CFor CF() can, at any one time, be occupied by nucleotides (2 ADP and 2 ATP) that remain firmly bound following extensive desalting of the enzyme (12) . In contrast, the isolated (33, 40) and () subunits bind nucleotides with a lower affinity (dissociation constants in the range of 0.5-2 µM), such that the nucleotides dissociate completely from the proteins during gel filtration. Our results have shown that the hexamer lacks the very tight ADP binding sites that are characteristic of the core complex (I). Therefore, the complex lacks the asymmetry necessary for tight ADP binding and consequently must lack the intersite catalytic cooperativity in which tightly bound ADP is considered to be an essential intermediate (13, 11) . The fact that some MgATP (<1 mol/mol of ) remained bound to the complex after desalting in the absence of EDTA may reflect a slightly enhanced affinity of pairs for MgATP over that of the individual and subunits.

The low ATPase activity observed with the complex probably results from the presence of three structurally and functionally equivalent catalytic sites/hexamer that are capable of slow catalytic turnover. Each site presumably operates independently of the other two, since the heterodimers from CF(35) exhibit catalytic rates of the same order of magnitude, indicating that formation of the complex does not result in a significant cooperative rate enhancement. Thus, the observed activity represents single-site catalysis. This is in contrast to ``unisite'' catalysis (for review, see Refs. 13 and 21), which involves one site on the Fcomplex that exhibits single site catalytic turnover but binds nucleotides with a very high affinity ( K<10 M). We have shown that formation of such tight sites on CFrequires the presence of the subunit. These results are at odds with studies of the PS3 Fhexamer, which suggest that the activities of the catalytic sites on this complex are interdependent. Inhibition of a single site by chemical modification of a tyrosine residue on one of the three subunits, blocks the function of the entire complex (16) . Interestingly, the isolated PS3 Ffurther differs from other Fenzymes in that it is devoid of tightly bound endogenous nucleotides (44) .

The catalytic activity that is manifested by the chloroplast subunit complexes is atypical of CFin that it is insensitive to the potent inhibitors tentoxin and azide (35) and also insensitive to the subunit that is a naturally occurring ATPase inhibitor of CF(3) . Avital and Gromet-Elhanan (35) reported that, although the activity of the complex was not inhibited by tentoxin, tentoxin was still able to bind to the complex as it co-eluted with the complex during chromatography on a Sephadex centrifuge column. Our results have shown that the chloroplast complex is unable to effectively compete with the core complex for tentoxin binding and, therefore, that the complex has a significantly lower (at least 2 orders of magnitude) affinity for tentoxin than the core complex, which is known to bind tentoxin with an apparent dissociation constant of between 5 and 100 10 M(45, 26) . We had shown earlier (25) that reconstitution of spinach chloroplast subunit with -deficient chromatophores of R. rubrum conferred tentoxin sensitivity upon the normally tentoxin-insensitive R. rubrum F-Fcomplex. This result indicated that the subunit may contain all or part of the tentoxin binding domain. Studies by Avni et al. (46) confirmed this result and identified a region of the N terminus of the subunit that is important for tentoxin binding. More recently, however, we showed that the isolated subunit is unable to compete with CFfor tentoxin binding, suggesting that the subunit provides only part of the tentoxin binding site (26) . The fact that the complex binds tentoxin with a much lower affinity than the complex indicates that the same structure that is required for cooperative interactions among nucleotide binding sites must also be required for normal tight binding of the inhibitor.

The subunit binds to CFwith an apparent dissociation constant of <10 M(2) . Like tentoxin binding, normal high affinity binding of the subunit also required the minimum structure, and could not bind tightly to the subunit or the complex alone. Tentoxin (26) and the subunit (3) are both allosteric effectors that inhibit the cooperative interactions between nucleotide binding sites on CF. The fact that neither inhibitor binds with high affinity to the complex nor effects its low catalytic activity is in accord with the lack of structural asymmetry in the complex. Thus the normal high affinity binding of both inhibitors requires an asymmetric conformational state of CFthat results from an interaction between the subunit and the and subunits.

In conclusion, we have described and characterized procedures for isolating and reconstituting the subunit with an subunit mixture of the chloroplast ATP synthase. The reconstitution procedure is unique in that it is simple and rapid and therefore will be a valuable tool for studying interactions among the different CFsubunits. It is noteworthy that, while this manuscript was under review, Chen and Jagendorf (48) published a method for reconstituting cloned, over-expressed CFsubunits with the help of molecular chaperonins. Together, these techniques have opened the way for studies, which are currently underway in our laboratory, that involve incorporating genetically engineered , and subunits into an active core eukaryotic Fenzyme complex.

  
Table: Reconstitution of an active core enzyme complex from isolated and subunits of CF


  
Table: Conditions for reconstitution of the complex with the subunit


  
Table: Analysis of tightly bound nucleotides



FOOTNOTES

*
This work was supported by Grants from the National Science Foundation (OSR-9255223), from the United States Department of Agriculture (93-37306-9633), and from the University of Kansas (GRF 3063). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviations used are: CF, chloroplast coupling factor 1; CF(), CFlacking the and subunits; Tricine, N-[2-hydroxy-1-bis(hydroxymethyl)ethyl]glycine; HPLC, high performance liquid chromatography; DCCD, N, N`-dicyclohexylcarbodiimide.

I. Wu and M. Richter, unpublished results.


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