EPR Spectroscopy of VO2+-ATP Bound to Catalytic Site 3 of Chloroplast F1-ATPase from Chlamydomonas Reveals Changes in Metal Ligation Resulting from Mutations to the Phosphate-binding Loop Threonine (beta T168)*

Wei Chen, Russell LoBrutto, and Wayne D. FraschDagger

From the The Center for the Study of Early Events in Photosynthesis, Arizona State University, Tempe, Arizona 85287-1601

    ABSTRACT
Top
Abstract
Introduction
References

Site-directed mutations were made to the phosphate-binding loop threonine in the beta -subunit of the chloroplast F1-ATPase in Chlamydomonas (beta T168). Rates of photophosphorylation and ATPase-driven proton translocation measured in coupled thylakoids purified from beta T168D, beta T168C, and beta T168L mutants had <10% of the wild type rates, as did rates of Mg2+-ATPase activity of purified chloroplast F1-ATPase (CF1). The EPR spectra of VO2+-ATP bound to Site 3 of CF1 from wild type and mutants showed that EPR species C, formed exclusively upon activation, was altered in CF1 from each mutant in both signal intensity and in 51V hyperfine parameters that depend on the equatorial VO2+ ligands. These data provide the first direct evidence that Site 3 is a catalytic site. No significant differences between wild type and mutants were observed in EPR species B, the predominant form of the latent enzyme. Thus, the phosphate-binding loop threonine is an equatorial metal ligand in the activated conformation but not in the latent conformation of Site 3. The metal-nucleotide conformation that gives rise to species B is consistent with the Mg2+-ADP complex that becomes entrapped in a catalytic site in a manner that regulates enzymatic activity. The lack of catalytic function of CF1 with entrapped Mg2+-ADP may be explained in part by the absence of the phosphate-binding loop threonine as a metal ligand.

    INTRODUCTION
Top
Abstract
Introduction
References

The F1F0-ATP synthase uses a nonequilibrium transmembrane proton gradient to synthesize ATP from ADP and phosphate to form a nonequilibrium chemical gradient of products versus substrates (1). The F11 portion binds substrate with high affinity in a manner that allows rapid interconversion of ADP and phosphate with bound ATP in the absence of the proton-motive force (2). The proton-motive force drives two sequential conformational changes of the catalytic site that decrease the affinity of the enzyme for ATP relative to ADP, thereby facilitating the selective dissociation of ATP to generate the chemical gradient. The conformation of each of three catalytic sites on the enzyme is staggered such that the enzyme contains a catalytic site in each of the three sequential conformations at any instant.

The nucleotides bind the catalytic site as a complex with Mg2+ (3), which serves as a cofactor for the reaction. Several metals will substitute in a functional manner for Mg2+ including Mn2+, Ca2+, and VO2+ (4). In the absence of Mg2+, the three catalytic sites of F1 from Escherichia coli bind ATP with the same affinity (5). However, the affinity of each site for the Mg2+-ATP complex changes such that there is a difference of more than 5 orders of magnitude between the sites with highest and lowest affinity. This strongly suggests that the changes in coordination of the metal at the catalytic site play a critical role in the protein conformational changes that promote the selective release of ATP from the enzyme.

The catalytic activity of chloroplast F1 purified from F0 and the thylakoid membrane is latent, although ATPase activity can be activated via reduction of a disulfide bond on the gamma -subunit (6). The oxidation state of this disulfide provides one of multiple levels of interrelated mechanisms to regulate the enzyme to minimize ATPase activity once the proton gradient dissipates in the absence of light-driven proton translocation. The dark decay of ATPase activity in thylakoids is accelerated by the addition of ADP that becomes tightly bound in the latent state (7-9). Formation of this tightly bound ADP and inhibition of ATPase activity only occur upon the addition of Mg2+ (10). Under these conditions, Mg2+ apparently induces the formation of a Mg2+-ADP complex at the catalytic site that is bound to the enzyme in a manner that prevents further catalysis. This binding of Mg2+-ADP has been found to serve a regulatory function in the F1-ATPases from other organisms as well (11, 12).

Of the six possible metal ligands at each catalytic site of the bovine mitochondrial F1-ATPase, only the oxygens from the nucleotide phosphates and from T156 on the beta -subunit were within 2.5 Å of the metal in the crystal structure of the enzyme from bovine mitochondria (13). This residue is part of the phosphate-binding loop (P-loop) motif GXXXXGKT that is common to many enzymes that catalyze the hydrolysis of ATP (14). Based on available crystal structures, the hydroxyl group of the threonine typically coordinates to the Mg2+ cofactor of the enzyme-bound Mg2+-nucleotide complex (15-20). Mutation of this residue in the beta -subunit of EF1 decreases the Mg2+-dependent binding affinity of nucleotide (21). Although several other residues have been suggested to serve as metal ligands at the catalytic site of F1 based on site-directed mutagenesis studies, these side chains are all at least 5 Å away from the metal in the crystal structure (13).

Recently, vanadyl has been used as a direct probe to identify the types of groups that serve as metal ligands at one catalytic site (Site 3) of the latent and activated forms of the chloroplast F1-ATPase (4, 22, 23). Vanadyl (VIV=O)2+ is a cation composed of vanadium (IV) double-bonded to oxygen to give a net charge of 2+, such that it has four equatorial ligands and one axial ligand that is trans to the vanadium-oxygen bond. Vanadyl has a single unpaired electron and a nuclear spin (I = 7/2) that gives rise to a 16-line EPR spectrum due to the anisotropy resulting from the V=O bond. The 51V hyperfine parameters of VO2+ are sensitive to the type of groups that serve as equatorial ligands. Each of these four ligands contributes independently to the 51V hyperfine parameters such that the overall coupling is the average of couplings observed for a uniform environment, weighted by the number of each type of ligand.

Purified CF1 contains four tightly bound metal-nucleotides (24). The metal-nucleotide bound to the site designated Site 3 can be removed by gel filtration chromatography, whereas depletion of Site 2 requires partial unfolding of CF1 by precipitation in ammonium sulfate and EDTA (25), and depletion of Sites 1 and 4 requires the removal of the epsilon -subunit (26). Sites 1 and 3 have been postulated to be catalytic, whereas Site 2 has been suggested to be noncatalytic (25). Fluorescence resonance energy transfer measurements using 2í(3í)-trinitrophenyl-nucleotides enabled the positions of Sites 1-3 relative to each other and to locations of fluorescent groups covalently modified to unique locations on CF1 to be mapped (27). The observation of unique, identifiable locations for Sites 1-3 and the closeness with which the fluorescence resonance energy transfer map corresponds to the locations of the metal-nucleotides in the crystal structure of F1 from bovine mitochondria indicate that each of the metal-nucleotide binding Sites 1-3 can be selectively filled with metal-nucleotide complex (25).

When Site 2 of latent CF1 is filled with VO2+-nucleotide, the bound VO2+-ATP gives rise to EPR species A (22). At Site 3, the majority of VO2+-nucleotide binds in a ligand environment that gives rise to EPR species B, whereas a smaller fraction binds to Site 3 in a form that gives rise to EPR species C (22, 28). Upon activation of the enzyme, all of species B converts to species C, indicating that the latter results from the metal ligands responsible for catalytic competence. The titration of VO2+-nucleotide to CF1 that had been depleted of metal-nucleotide only from Site 3 showed that the VO2+ had bound selectively to a single site (4, 22). The best fits of the 51V hyperfine coupling parameters to EPR species at Site 3 strongly suggest that this site contains hydroxyl groups from serine or threonine as metal ligands in both the latent and activated states.

We have now characterized site-directed mutants of the P-loop threonine in the beta -subunit of CF1 from Chlamydomonas reinhardtii (beta T168). The effects of these mutations on the EPR spectra of VO2+ bound to Site 3 of CF1 were examined to determine whether changes could be observed in a manner that would indicate that this residue is a metal ligand. The results indicate that beta T168 contributes a hydroxyl group as an equatorial metal ligand when VO2+ binds to Site 3 in the manner that gives rise to EPR species C, the activated enzyme conformation. This is the first direct evidence to indicate that Site 3 is a catalytic site. However, this group is not an equatorial ligand to VO2+ under conditions that give rise to EPR species B, the predominant form in the latent state.

    EXPERIMENTAL PROCEDURES

Construction of CF1 Mutants in C. reinhardtii-- The recombinant plasmid carrying wild type gene atpB of C. reinhardtii was constructed as described by Hu et al. (28). The 5.3-kb EcoRI-BamHI fragment of the chloroplast genome containing atpB was cloned into pUC18, and then a 1.4-kb atpA-aadA cassette was inserted into the plasmid at a KpnI site of the 5.3-kb fragment. The final recombinant plasmid was named pBam 10.3-spec and used as the template to make mutants. The atpA-aadA cassette included the promoter of the atpA gene and aadA that encodes a protein against the antibiotic spectinomycin.

Stratagene kit 200508 was used to generate atpB mutations on pBam 10.3-spec following the provided protocol that required two primers for each mutant. The mutagenesis primers were as follows: beta T179D, 5'- TTCGGTGCCGGTGTAGGCAAAGACGTTTTAATTATG-3'; beta T179C, 5'-TTCGGTGCCGGTGTAGGCAAATGTGTTTTAATTATG-3'; and beta T179L, 5'-GTGTAGGCAAATTAGTTTTAATTATGGAAC-3'. The selective primer that changed restriction site Xmnl to BamHI was Ps (5'-CGCCCCGAAGAACGGATCCCAATGATGAGCAC-3').

Sequences of DNA were obtained using ABI PRISM automatic sequencing to confirm the mutations in the plasmids. The sequence primer was 5'-CCGTACAGCTCCTGCTTTCG-3'. The mutated plasmids were purified by ultracentrifugation with cesium chloride and diluted to 1 mg/ml.

The CC-373 and CC-125 strains of C. reinhardtii were obtained from the Chlamydomonas culture collection center at Duke University. The CC-373 strain has a 2.5-kb deletion that starts close to the 5'-end of atpB. The transformation of CC-373 with mutated pBam 10.3-spec was performed using the PDS-1000 particle delivery system (DuPont New England Nuclear) as described by Hu et al. (28). Southern blots were done as described by Sambrook et al. (29) using 32P-labeled pBam 10.3-spec as the probe to determine that the mutants were homoplasmic. The presence of the desired mutation was confirmed by sequencing the PCR product from the chloroplast genome of each mutant.

Biochemical Characterization of C. reinhardtii Mutants-- Methods to measure photoautotrophic growth curves, to prepare coupled thylakoid membranes, and to purify CF1 from strains of Chlamydomonas were carried out as described by Hu et al. (28). Assays of photosystem I-dependent electron transfer activity, photophosphorylation activity with coupled thylakoids, and ATPase activity with purified CF1 were also performed as described by Hu et al. (28).

The ability of purified thylakoids to generate a light-driven proton gradient was monitored using 9-aminoacridine fluorescence quenching. The reaction buffer contained 50 mM Tricine (pH 8.3), 10 mM NaCl, 0.08 mM phenazine methylsulfate, 0.5 µM 9-aminoacridine, and 30 µg of chlorophyll in a total volume of 2 ml. Fluorescence quenching was measured at room temperature with a Spex FluoroMax Model DM3000 with excitation at 400 nm and emission at 450 nm. Actinic light was supplied by an Oriel-66181 lamp using a 600 nm cut-off filter via a light pipe.

To measure ATPase-driven proton pumping, thylakoid membranes were suspended in 50 mM Tricine, pH 8.3, and 50 mM KCl. The membranes were incubated at 4 °C for 15 min after the addition of 9-aminoacridine to a final concentration of 5 µM. The membranes were then pelleted by centrifugation and resuspended in 50 mM Tricine, pH 8.3, and 50 mM KCl. The thylakoid membranes were then diluted to a final chlorophyll concentration of 5 mg chlorophyll/ml. Fluorescence quenching was measured as described above. The initial rate of 9-aminoacridine fluorescence quenching was measured with 2 ml of reaction buffer that contained 50 mM Tricine, pH 8.3, 50 mM KCl, 10 mM dithiothreitol, 0.5 µg/ml valinomycin, 20 µg of chlorophyll, and the indicated concentration of Mg2+-ATP.

EPR Measurements and Sample Preparation-- To purify CF1 from Chlamydomonas thylakoids, thylakoids were incubated with 0.75 mM EDTA (pH 8.0) for 15 min at room temperature. The suspension was centrifuged at 30,000 × g for 20 min to remove the thylakoids. This EDTA extraction was repeated three times. DEAE-Sephadex A-50 that had been pre-equilibrated in 20 mM Tris-NaOH (pH 8.0), 10 mM ammonium sulfate, and 1 mM EDTA was added to the combined supernatants containing CF1. The suspension was stirred slowly for 30 min at room temperature and then loaded into a chromatography column. The protein was eluted from this column with 300 ml of buffer that contained 20 mM Tris-NaOH (pH 8.0), 1.0 mM ATP, 2 mM EDTA, and 500 mM NaCl. The fractions that contained CF1 were concentrated by pressure dialysis using an Amicon YM-100 membrane to a volume of about 3 ml. It has been shown previously (24) that CF1 purified in this manner contains about four tightly bound Mg2+-nucleotides.

The purified CF1 was depleted of the Mg2+-nucleotide from Site 3 by chromatography on a 3 × 30-cm Sephadex G-50 column equilibrated in 50 mM HEPES (pH 8.0), 1 mM ATP, and 500 mM NaCl. The eluent was concentrated again via pressure dialysis using a YM-100 membrane to a volume of about 1 ml. The sample was then diluted in buffer containing 50 mM HEPES (pH 8.0) to a final NaCl concentration of 175 mM. This treatment has been shown to deplete CF1 of Site 3 without removing significant amounts of metal-nucleotide from the other tight binding sites (25). The protein was concentrated to 0.3 ml, and the extinction coefficient of 0.483 mg CF1/ml (25) was used to determine the concentration. A 1:1 mole ratio solution of VO2+-ATP, prepared as described by Houseman et al. (4), was added to the Site 3-depleted CF1 samples to a final mole ratio of VO2+:CF1 of 1:1. The CF1 concentration of the EPR samples prepared in this manner was typically about 0.3 mM. The millimolar concentration of vanadyl bound to CF1 was determined as described by Houseman et al. (4) as 3.247 times the integrated area of the -5/2   line of the EPR signal intensity per scan at a gain of 105.

EPR experiments were carried out at X-band (9 GHz) using a Bruker 300E spectrometer and a liquid nitrogen flow cryostat operating at 100 K. Simulations of these spectra were made as described by Houseman et al. (4) using the QPOWA program (30, 31).

    RESULTS

Effects of the Mutations on Enzyme Assembly and ATP Synthesis-- The yield of CF1 purified from each of the beta T168 mutants of Chlamydomonas was approximately the same as that isolated from the wild type. The subunit composition of the CF1 from wild type and mutants was compared by SDS-polyacrylamide gel electrophoresis as shown in Fig. 1. Preparations of the enzyme from the mutant and wild type contained all five subunits including the delta -subunit, which is known to dissociate easily from Chlamydomonas CF1 (28). These results suggest that the mutations do not affect the synthesis and assembly of the enzyme significantly.


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Fig. 1.   Polypeptide composition of CF1 isolated from mutant and wild type strains of C. reinhardtii as determined by Coomassie Blue-stained SDS-polyacrylamide gel electrophoresis using (A) 15% acrylamide and (B) 10% acrylamide. Lane 1, wild type; lane 2, beta T168C; lane 3, beta T168D; lane 4, beta T168L mutants of Chlamydomonas.

The relative ability of the mutant and wild type strains of Chlamydomonas to grow under photoautotrophic conditions is shown in Table I. The site-directed mutants of beta T168 were essentially incapable of photoautotrophic growth. The rates of phenazine methosulfate-dependent photophosphorylation of thylakoids from the wild type and mutant preparations are also summarized in Table I. Although the rates remained linear in all cases for up to 5 min, the ATP synthase activity of the mutants was <10% that of the wild type. The results are consistent with the inability of the mutants to grow photoautotrophically.

                              
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Table I
Functional comparison of wild-type and mutants

The low rates of photophosphorylation were not the result of the inability of the thylakoids to form a light-driven proton gradient. Fig. 2 shows the relative fluorescence quenching of 9-aminoacridine in the wild type and mutant thylakoids upon illumination. The rate and extent of fluorescence quenching in each of the mutants were about the same as in the wild type. Thus, none of the mutations caused the membranes to become uncoupled. Combined with the observations that the yield and subunit composition of the mutant proteins are the same as those of the wild type, it is unlikely that any of the mutations caused a large conformational change that interferes with the folding and assembly of the CF1F0 complex.


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Fig. 2.   Light-driven proton gradient formation in thylakoids purified from (a) wild type, (b) beta T168C, (c) beta T168D, and (d) beta T168L mutants of Chlamydomonas measured by 9-aminoacridine fluorescence quenching.

Coordination of Metal at Site 3 in beta T179 Mutants of CF1-- A single eq of VO2+ was added to CF1 as a complex with ATP under conditions in which all other higher affinity binding sites for metal-nucleotides were filled with Mg2+-nucleotide complexes as described under "Experimental Procedures." Fig. 3 shows the parallel features of the EPR spectra of VO2+ bound in this manner to the CF1 from wild type and mutant Chlamydomonas. The differences in EPR species B and C between wild type and mutants are clearly resolved with the +5/2parallel line. None of the mutations was found to change the 51V hyperfine parameters of EPR species B. However, EPR species C was altered from the wild type in each of the mutants examined.


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Fig. 3.   The parallel regions of VO2+ EPR spectra of the VO2+-ATP complex bound to Site 3 of CF1 from (a) wild type, (b) beta T168D, (c) beta T168C, and (d) beta T168L mutants of Chlamydomonas. EPR conditions were as follows: field modulation frequency, 100 kHz; modulation amplitude, 0.5 mT; sweep rate, 0.95 mT/s; time constant, 82 ms; microwave power, 1.0 milliwatt, temperature, 100 K. 1 mol eq of VO2+ was added as a complex with ATP to CF1 that had been depleted of metal-nucleotide from Site 3. The microwave frequency, the number of scans, and the amount of CF1 in each sample was as follows: wild type, 9.5530 GHz, 118 scans, and 57.5 mg; beta T168D, 9.5609 GHz, 84 scans, and 44.3 mg; beta T168C, 9.5624 GHz, 128, scans, and 38.1 mg; and beta T168L, 9.5623 GHz, 88 scans, and 28.2 mg, respectively.

The values of Aparallel and gparallel derived from each spectrum in Fig. 3 by simulation of the entire spectrum are summarized in Table II. The total amount of bound VO2+ can be measured from the EPR signal intensity under nonsaturating conditions based on the proportion of the signal intensity to the amount of CF1-bound vanadium as determined by atomic absorption spectroscopy (4). Table III shows the amount of VO2+ per CF1 bound upon the addition of 1 eq of VO2+ as a complex with ATP. The addition of 1 eq of VO2+ to wild type CF1 under the conditions described here resulted in about 30% occupancy of Site 3 (Table III). The ratio of the amplitudes of the simulated spectra for species B and C from each mutant that, when summed, reproduced the experimental spectra is shown in Table III. Based on these data, the mole ratios of bound VO2+:CF1 as species B or species C were calculated.

                              
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Table II
VO2+ coordination at Site N3 in CF1 from wild-type and mutants

                              
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Table III
Mole ratios of vanadyl bound to CF1 upon addition of a single equivalent of VO2+ as a complex with ATP

In the beta T168C mutant, the value of Aparallel increased by about 13 MHz. In addition, the EPR signal intensity of species C relative to species B decreased by about 2-fold. The beta T168D mutant increased Aparallel of species C by about 4 MHz but had little effect on the ratio of the species C to species B signal intensities. When leucine was substituted for the P-loop threonine, the amount of VO2+ bound to the latent protein in the conformation that gives rise to species C was insignificant.

Substitution of hydrophobic leucine for the P-loop threonine did not affect the total amount of VO2+ bound to Site 3, but all of the bound VO2+ was in the form that gives rise to species B (Table III). Mutations that introduced either the sulfhydryl or carboxyl groups were found to bind the form of VO2+ that gives rise to species C to the same extent as the wild type enzyme. However, these mutations increased the amount of VO2+ bound in the species B form such that there was a net increase in the occupancy of Site 3 upon the addition of a single equivalent of the metal.

Effects of Mutations on ATPase-related Function-- The effects of the mutations on the ability of CF1F0 to catalyze ATPase-driven proton translocation was determined by fluorescence quenching of 9-aminoacridine using thylakoids purified from wild type and mutant strains of Chlamydomonas. Shown as a function of the Mg2+-ATP concentration (Fig. 4), the initial rate of proton translocation was severely affected in thylakoids purified from the mutants such that there was little difference in fluorescence quenching between thylakoids from the mutants and the azide-inhibited wild type. These results were similar to the rates of Mg2+-ATPase activity observed with CF1 purified from each mutant (data not shown).


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Fig. 4.   Initial rates of Mg2+-ATPase-driven proton translocation catalyzed by thylakoid membranes isolated from wild type (open circle , ), beta T168D (triangle , black-triangle), beta T168C (diamond , black-diamond ), and beta T168L (, black-square) strains of Chlamydomonas in the absence (open symbols) and presence (closed symbols) of 5 mM sodium azide. The initial rate of fluorescence quenching of 9-aminoacridine was determined as described under "Experimental Procedures." Data are expressed as the percentage of the rate of the wild type observed at 10 mM Mg2+-ATP.


    DISCUSSION

Vanadyl has been used to estimate the types of groups that serve as metal-ligands in F1-ATPase (4, 22, 23) and other proteins (32) because the g and A tensors of the 51V hyperfine couplings are approximately a linear combination of tensors from each type of group that contributes an equatorial ligand (33, 34). The results presented here show for the first time that site-directed mutations of a metal ligand on F1-ATPase will cause measurable changes in the EPR spectrum of enzyme-bound VO2+ and thereby provide direct evidence of a specific residue as a metal ligand.

The presence of hydroxyl groups from serine or threonine were suggested as equatorial ligands to VO2+ bound at Site 3 of CF1 in the forms that predominate in both the latent and activated states (23, 28). We have now shown that one of the putative hydroxyl ligands in the activated enzyme form is the P-loop threonine of the beta -subunit (beta T168). This concurs with the role of the P-loop threonine inferred from the crystal structure of F1-ATPase from bovine mitochondria (13). Site 3 was previously suggested to be a catalytic site (25). The results presented here show conclusively that this assignment is correct.

The crystal structure of mitochondrial F1 is not able to provide information concerning the conformation of the metal-nucleotide bound to CF1 in the latent state. The results presented here indicate that the P-loop threonine is not an equatorial ligand to VO2+ bound as the VO2+-ATP complex at Site 3 in the ligand environment that predominates in the latent enzyme conformation (EPR species B).

In addition to the changes observed to the 51V hyperfine coupling of VO2+ bound at Site 3, the mutations of the P-loop threonine of CF1 to a carboxyl, sulfhydryl, or leucine side chain significantly affected the function of the enzyme. The E. coli F1 P-loop mutations beta T156D, beta T156C, and beta T156A also decreased the ability of EF1F0 to catalyze ATP synthesis and ATPase-dependent proton translocation, as well as the ability of purified EF1 to catalyze ATP hydrolysis (35). Although the magnitude of the decrease in ATP-dependent H+-gradient formation was comparable between the mutants of EF1F0 and Chlamydomonas CF1F0, the mutations of the former enzyme were 100-fold more effective in eliminating the other activities.

The EPR data in Fig. 4 and Table II that result from VO2+ bound to Site 3 of mutant CF1 also provide insight into the metal ligation responsible for the functional changes in each of these mutants. Based on the measured coupling constants of Aparallel from model studies (34, 36), the hyperfine coupling for a given group of equatorial ligands can be calculated from the equation
A<SUB>∥calc</SUB>=&Sgr;n<SUB>i</SUB><UP>A<SUB>∥</SUB></UP><SUB>i</SUB><UP>/4</UP> (Eq. 1)
i=1
where i counts the different types of equatorial ligand donor groups, ni (ni = 1-4) is the number of ligands of type i, and Aparallel i is the measured coupling constant for the equatorial ligand donor group of type i (34). Similar equations can be written for gparallel and for Aiso, although the changes in Aparallel are the largest and most easily discerned.

Table II shows the values of Aparallel and gparallel calculated from Equation 1 that give the closest fit to the experimental data derived from simulation of the spectra and summarizes the equatorial ligands used for these calculations. When VO2+ was bound to Site 3 of CF1 from wild type and the beta T168C mutant, values of Aparallel were 457.5 and 469.5 MHz, respectively. As published previously (23, 28), species C is best fit to an equatorial ligand set that contains two hydroxyl groups from serine or threonine. The ligand set that gives the best fit to the data from the beta T168C mutant contains a sulfhydryl and a phosphate or carboxyl oxygen as ligands in lieu of the hydroxyl oxygens. Additional information (e.g. superhyperfine coupling from 31P) is required to distinguish phosphate and carboxyl oxygens as ligands to VO2+.

If the beta T168D mutation were to result solely in the substitution of a carboxyl for a hydroxyl oxygen, an increase in Aparallel of about 22 MHz would be expected. Whereas an increase in Aparallel was observed in this mutant, it only increased by 4-461.5 MHz. The closest fit of the data is derived from an equatorial ligand set of three imidazole-nitrogens (histidines) and one sulfhydryl (cysteine). This makes no sense when compared with the side chains available at the catalytic site in the crystal structure of mitochondrial F1 from bovine mitochondria, nor can it be explained via unconserved residues unique to CF1 from Chlamydomonas. The closest set of equatorial ligands that makes any sense with regard to the known groups in the site is when the oxygens from a hydroxyl, and the carboxyl/phosphate of the wild type are substituted for two amine nitrogens. These substitutions result in Aparallel of 462.6 MHz, which is within 1.1 MHz of that observed experimentally. With the information currently available, we can not assign a set of equatorial ligands for this one mutant. Clearly, the beta T168D mutation has caused profound changes in the equatorial ligands of VO2+ bound at catalytic Site 3.

The introduction of a hydrophobic leucine side chain at position beta 168 reduced the amount of VO2+ bound to Site 3 in the EPR species C conformation by more than 95% relative to that of the wild type enzyme. Consequently, the signal intensity of the residual species C was insufficient to suggest a possible equatorial ligand set. In EF1, these mutants decreased the affinity of the catalytic sites for ATP under unisite conditions and multisite conditions (21, 35). The EPR results presented here were obtained under conditions in which the amount of VO2+-ATP added was stoichiometric to metal-nucleotide-depleted, catalytic Site 3 when the enzyme contained Mg2+-nucleotide bound to at least one other catalytic site.2 Under these conditions, the T168L mutant bound the same total amount of vanadyl, but all of it was in the form that gave rise to EPR species B. This supports previous results that species B and C are derived from VO2+ bound in two conformations at the same location.

Whereas substitution of the P-loop threonine with a hydrophobic side chain did not affect the affinity of Site 3 for VO2+, the presence of a charged or polar group did. In the T168D or T168C mutants, the amount of VO2+ bound in the species B form increased. The Mg2+-ATPase activity of the P-loop Tright-arrowS mutation in F1 or F1-subcomplexes has also been characterized in several organisms. In yeast (37, 38), E. coli (35), thermophilic bacteria (39), and Chlamydomonas (40), this mutation, which maintained a polar group in this position, increased the Vmax of purified F1 Mg2+-ATPase activity severalfold above that of wild type. Evidence suggests that the capacity to entrap inhibitory Mg2+-ADP in a catalytic site is significantly impaired in the alpha 3beta 3gamma subcomplex of thermophilic bacterial F1 with this substitution. The mutations to the P-loop threonine reported here cause a loss of catalytic activity concurrent with an increase in the proportion of VO2+ that binds to Site 3 in the species B form relative to species C. This implies that the bound VO2+-nucleotide complex that gives rise to species B may be the form that is entrapped as part of the regulatory mechanism.

Hu et al. (40) reported that the P-loop Tright-arrowS substitution in Chlamydomonas CF1 had a much higher Mg2+-ATPase activity in the absence of dithiothreitol than did the wild type. Although data suggested that this mutation in CF1 also interferes with the entrapment of tightly bound Mg2+-ADP by a catalytic site that regulates ATPase activity, at least one ADP remains tightly bound to the purified enzyme. This strongly implies that catalytic Site 1 (25) and perhaps Site 4 are not the site(s) of entrapment. Rather, this suggests that the catalytic Site 3, and thus the VO2+-ADP conformation that gives rise to EPR species B, is the regulatory site. More experiments need to be done to test this hypothesis.

    ACKNOWLEDGEMENT

We thank David Lowry for excellent technical support.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM50202.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 To whom correspondence should be addressed. Tel.: 602-965-8663; Fax: 602-965-6899; E-mail: frasch{at}asu.edu.

2 Under the experimental conditions, Sites 1, 2, and 4 contain Mg2+-nucleotide. Because the data presented here confirm that Site 3 is catalytic, a comparison of the fluorescence resonance energy transfer map of CF1 with the crystal structure of F1 from bovine mitochondria indicates that Sites 1 and 2 are catalytic and noncatalytic, respectively. Because Site 4 does not bind 2í(3í)-trinitrophenyl-nucleotides, its location relative to Sites 1-3 has not been determined. Data available at this time suggest that Site 4 is also a catalytic site.

    ABBREVIATIONS

The abbreviations used are: F1, the extrinsic membrane portion of the F1F0-ATP synthase; CF1, chloroplast F1; EF1, Escherichia coli F1; P-loop, phosphate-binding loop; kb, kilobase.

    REFERENCES
Top
Abstract
Introduction
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