Metal Ligation by Walker Homology B Aspartate βD262 at Site 3 of the Latent but Not Activated Form of the Chloroplast F1-ATPase from Chlamydomonas reinhardtii *

Site-directed mutations D262C, D262H, D262N, and D262T were made to the β subunit Walker Homology B aspartate of chloroplast F1-ATPase in Chlamydomonas. Photoautotrophic growth and photophosphorylation rates were 3–14% of wild type as were ATPase activities of purified chloroplast F1 indicating that βD262 is an essential residue for catalysis. The EPR spectrum of vanadyl bound to Site 3 of chloroplast F1 as VO2+-ATP gave rise to two EPR species designated B and C in wild type and mutants. 51V-hyperfine parameters of species C, present exclusively in the activated enzyme state, did not change significantly by the mutations examined indicating that it is not an equatorial ligand to VO2+, nor is it hydrogen-bonded to a coordinated water at an equatorial position. Every mutation changed the ratio of EPR species C/B and/or the51V-hyperfine parameters of species B, the predominant conformation of VO2+-nucleotide bound to Site 3 in the latent (down-regulated) state. The results indicate that the Walker Homology B aspartate coordinates the metal of the predominant metal-nucleotide conformation at Site 3 in the latent state but not in the conformation present exclusively upon activation and elucidates one of the specific changes in metal ligation involved with activation.

F 0 F 1 -ATP synthases are found in the plasma membrane of bacteria, the inner membrane of mitochondria, and the thylakoid membrane of chloroplasts where they catalyze ATP synthesis driven by an electrochemical gradient (1). The F 0 portion contains membrane-spanning subunits and is responsible for translocating protons across the membrane. The F 1 1 portion is an extrinsic membrane complex, composed of five different subunits ␣, ␤, ␥, ␦, and ⑀, and retains the ability to hydrolyze ATP after purification from F 0 . In the crystal structure of F 1 from bovine heart mitochondria (2), the catalytic sites are located on each of the three ␤ subunits with some contribution from the proximal ␣ subunit. The enzyme crystallized with one catalytic site that contained Mg 2ϩ -AMPPNP, one that contained Mg 2ϩ -ADP, and one that was empty. Three noncatalytic sites, each located primarily on an ␣ subunit, contained bound Mg 2ϩ -AMPPNP in this structure.
Chloroplast F 1 has about four metal-nucleotides tightly bound upon purification from F 0 (6). 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 CF 1 by precipitation in ammonium sulfate and EDTA (7), and depletion of Sites 1 and 4 require the removal of the ⑀ subunit (8). Recent evidence indicates that Site 3 is catalytic (9). Fluorescence resonance energy transfer (FRET) measurements using TNP nucleotides enabled the mapping of the positions of Sites 1-3 relative to each other and to locations of fluorescent groups covalently modified to unique locations on CF 1 (10). This FRET map shows a close correspondence to the locations of the metal-nucleotides in the crystal structure of F 1 from bovine mitochondria. From this correspondence, Site 2 is a noncatalytic site and Site 1, like Site 3, is catalytic (7). The observation of unique identifiable locations for Sites 1-3 and the correspondence between the FRET map and the crystal structure indicate that each of the metal-nucleotide binding Sites 1-3 can be selectively filled with metalnucleotide complex (7).
The F 1 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 (3). The proton-motive force drives two sequential conformational changes of the catalytic site that decreases the affinity of the enzyme for ATP relative to ADP that facilitates the selective dissociation of ATP. This generates a chemical gradient in which the cellular concentration of ATP is much higher relative to ADP and phosphate than it would be at equilibrium. 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 as supported by the structure of F 1 (2).
Nucleotides bind the catalytic sites as a complex with Mg 2ϩ (4), which serves as a cofactor for the reaction. The decrease in affinity for ATP that results from the sequential conformational changes is directly dependent on the presence of Mg 2ϩ (5). These differences in affinity, which can be as much as 5 orders of magnitude for the F 1 from Escherichia coli, strongly suggest that the selective release of ATP results from changes in the metal ligands that are a consequence of the different conformations of the catalytic site.
Thylakoids maintain high rates of photophosphorylation by diverting some of the reducing equivalents derived from the light-driven electron transfer reactions to thioredoxin that in turn keeps a disulfide bond on the ␥ subunit of CF 1 reduced. Formation of this disulfide in darkness converts the enzyme from the activated to the latent state that has very low ATPase activity. Addition of ADP accelerates the dark decay of ATPase activity, suggesting that tightly bound ADP in a catalytic site serves a regulatory function. This is required as part of the mechanism to maintain the enzyme in its latent state in the dark (14,15). Conversion of this ADP from loosely to tightly bound correlates with formation of latent CF 1 (14). This regulatory interconversion only occurs upon subsequent addition of Mg 2ϩ (15,16). Thus, the metal that serves as a cofactor by binding as a complex with the nucleotide can also serve in a regulatory role by forming a nonfunctional conformation.
Vanadyl (V IV ϭO) 2ϩ has been used as a direct probe to identify the types of groups that serve as metal ligands at Sites 2 and 3 of CF 1 (11,13,27). The A and g tensors of 51 V hyperfine couplings from the EPR spectrum of the bound VO 2ϩ are a direct measure of the nature of the equatorial metal binding ligands (28). In a mixed ligand environment, each type of ligand contributes independently to the observed 51 V-hyperfine coupling (28,29). As a result, the 51 V-hyperfine parameters can provide information concerning the type of groups coordinated to the enzyme-bound metal.
When Site 2 of latent CF 1 is filled with VO 2ϩ -nucleotide, the bound VO 2ϩ -ATP gives rise to EPR species A (11). At Site 3, the majority of VO 2ϩ -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 (11,12). Upon activation of the enzyme, all of the signal intensity of species B converts to species C, suggesting that the latter results from the metal ligands when the enzyme is catalytically active. Titration of VO 2ϩ -nucleotide to CF 1 that had been depleted of metal-nucleotide only from Site 3 showed that the VO 2ϩ binds selectively to a single site (11,13).
It is possible for the Mg 2ϩ bound at each catalytic site to have up to six ligands. However, in the two catalytic sites in the crystal structure of F 1 from bovine mitochondria that contain Mg 2ϩ -nucleotide, only the oxygens of the phosphates and the hydroxyl of Thr-156 were within the 2.5 Å distance that would suggest that they were ligands. This threonine is a residue in a motif composed of GXXXXGKT known as Walker Homology A or phosphate-binding loop (P-loop) conserved among several enzymes that catalyze ATP hydrolysis.
The Walker Homology B (WHB) motif is also conserved among several Mg 2ϩ -nucleotide binding proteins including adenylate kinase, phosphofructokinase, human mdrI protein, ATP/ADP translocase, elongation factor Tu, as well as the ␣ and ␤ subunits of the F 1 -ATPases (17)(18)(19)(20)(21)(22). This motif, with the consensus sequence of four hydrophobic residues followed by an aspartate, terminates a ␤-strand with the carboxyl group facing the binding pocket for metal-nucleotide (2,20). The aspartate carboxyl of WHB has been suggested to hydrogen bond to a water that is coordinated to the metal, or to coordinate to a metal directly in several proteins including the ␤ subunit of the F 1 -ATPase (8,(23)(24)(25)(26). The crystal structure of the bovine mitochondrial F 1 -ATPase shows the closest carboxyl-oxygen of this residue to be 3.9 Å to 4.3 Å from the metal at catalytic sites that contain bound Mg 2ϩ -AMPPNP and Mg 2ϩ -ADP, respectively (2).
Recently, site-directed mutations of the P-loop threonine of the ␤ subunit in CF 1 from Chlamydomonas (␤T168) were compared to determine whether changes in the EPR spectra of VO 2ϩ bound to catalytic Site 3 could be detected (9). The mutations were found to cause changes in both the signal intensity and 51 V hyperfine parameters of the bound VO 2ϩ that gave rise to EPR species C in a manner that indicated that this residue was a metal ligand in the activated conformation. The lack of changes in EPR species B in the mutant CF 1 indicated that the P-loop threonine is not a ligand in the form that predominates in the latent state of the enzyme.
We now report an analysis of site-directed mutants of the WHB-aspartate (␤D262) of Chlamydomonas CF 1 by EPR spectroscopy of VO 2ϩ bound to catalytic Site 3. The results presented here indicate that ␤D262 participates in metal binding at Site 3 in the metal-nucleotide complex that predominates in the latent form, but not in the complex that occurs in the activated form of the enzyme.

Construction of Plasmids and Cell Strains Containing Mutations-
Chlamydomonas reinhardtii strains (CC-125 and CC-373) were obtained from the C. reinhardtii Culture Collection at Duke University. The plasmid pWT-373 (8) was used as a template for double-stranded, oligonucleotide-mediated, site-directed mutagenesis following the protocol described in Stratagene Chameleon double-stranded site-directed mutagenesis manual. Each mutagenesis reaction requires a selection primer and a mutagenic primer. The sequence of the selection primer is the same for every mutagenesis reaction, 5Ј-CGC CCC GAA GAA CGG ATC CCA ATG ATG AGC AC-3Ј. The sequences of the mutagenic primers for different mutated plasmids are listed as follows. 1) pD262C, 5Ј-TTA TTC TTC ATT TGT AAC ATT TTC CGG TTC GTA CAA GCT G; 2) pD262H, 5Ј-TTA TTC TTC ATT CAT AAC ATT TTC CGT TTC; 3) pD262N, 5Ј-TTA TTC TTC ATT AAC AAC ATT TTC CGT TTC; 4) pD262T, 5Ј-TAT TCT TCA TTA CAA ACA TTT TCC GGT TCG TAC AAG CTG G.
Mutated plasmid DNA was transformed into the C. reinhardtii chloroplast genome using biolistic transformation following procedures as described previously (8,30,31). Southern blot analyses and doublestranded DNA sequencing (32) were used to verify the presence of homoplasmic cell lines with desired mutations.
Biochemical Characterization of C. reinhardtii Mutants-Cell cultures and photoautotrophic growth curves of each C. reinhardtii strain were maintained and measured as per Hu et al. (8). Thylakoid membranes in which electron transfer and ATP synthesis were tightly coupled were prepared, and photophosphorylation assays were measured as per Hu et al. (8). The ability of purified thylakoids to generate a light-driven proton gradient was monitored using 9-aminoacridine fluorescence quenching as per Chen et al. (9).
Isolation of soluble CF 1 -ATPase from C. reinhardtii and the selective filling of VO 2ϩ -ATP into Site 3 were carried out as per Chen et al. (9). The ATPase activity was determined using the coupled ATPase assay including lactic dehydrogenase, and pyruvate kinase as described by Harris and Bashford (33). To activate the CF 1 , final concentrations of 50 mM dithiothreitol and 20% ethanol were incubated with purified CF 1 for more than one h at room temperature. The reaction rates were determined from the initial slopes, typically in the first 20 -30 s after adding the protein into the reaction mixture.
EPR Analyses-EPR experiments were carried out at X-band (9 GHz) using a Bruker 580E spectrometer with a TE 102 standard cavity and a liquid nitrogen flow cryostat operating at 125 K. Simulations of the CW-EPR spectra employed the program QPOWA (12,34).

Effects of Mutations on Yield and Composition of CF 1 and
Photoautotrophic Growth-Growth curves of wild type and mutant Chlamydomonas cultures were obtained under photoautotrophic conditions at 25°C at a light intensity of 80 microEinstein⅐M Ϫ2 s Ϫ1 . As shown in Table I, all of the mutations of ␤D262 caused dramatic decreases in the ability to grow photoautotrophically compared with wild type. The D262N, C, and T mutants grew at 5-10% of the wild type rate. The D262H mutant was completely incapable of photoautotrophic growth.
The effects of these mutations on rates of phenazine methylsulfate-dependent photophosphorylation of isolated thylakoids, and ATPase activity of purified CF 1 preparations are summarized in Table I. The results are consistent with the relative ability of the mutants to grow photoautotrophically. In all cases, the activities of the D262N, C, and T mutants were about 10% of wild type, whereas those of the D262H mutant were negligible.
The subunit composition of CF 1 isolated from wild type and mutants are compared by SDS-polyacrylamide gel electrophoresis in Fig. 1. All mutants were found to contain ␣, ␤, ␥, and ⑀ subunits as does the wild type. The abundance of the ␦ subunit relative to the other subunits was variable among preparations. This subunit is known to be weakly associated with the Chlamydomonas CF 1 and is easily lost when the enzyme preparation is stored (8,35). The variability of the abundance of the ␦ subunit among preparations did not differ from that of wild-type CF 1 . Other bands visible in these preparations are polypeptides that have been reported previously to copurify with CF 1 from Chlamydomonas (36). It is also noteworthy that no significant differences in the yields of purified CF 1 were observed between preparations from 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 was about the same in each of the mutants 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 wild type, it is unlikely that any of the mutations has caused a large conformational change that interferes with folding and assembly of the CF 1 F 0 complex.
Effects of Mutations on the EPR spectrum of VO 2ϩ bound at Site 3 of CF 1 - Fig. 3 shows parallel transitions of EPR spectra of VO 2ϩ bound to Site 3 of CF 1 from wild type Chlamydomonas. Because of the anisotropy that results from the oxo group of VO 2ϩ , the single unpaired electron and the nuclear spin I ϭ 7/2 result in an EPR spectrum that consists of 8 transitions from that fraction of molecules where the molecular axis (defined by the VϭO bond) is aligned parallel with the magnetic field of the spectrometer, and 8 transitions from the perpendicular alignment. The center of each group of 8 transitions and the spacing between them is determined by the values of g and A, respectively. The magnitude of these values depends on the strength of the hyperfine coupling between the unpaired electron and the 51 V nucleus. The 51 V-hyperfine coupling of VO 2ϩ is sensitive to the types of group coordinated at the equatorial positions. Of these parameters, the coupling constant for the equatorial ligand donor group, A ʈ , shows the largest and most easily discerned changes as a function of the types of groups that a Measured as the rate of increase in optical density (cell scattering) of the liquid culture at 720 nm in log phase at 25°C with a light intensity of 80 microEinstein ⅐ M Ϫ2 s Ϫ1 .
b Based on the rate of 250 mol of ATP (mg of chlorophyll ⅐ h) Ϫ1 using Mg 2ϩ -ADP and phosphate concentrations of 2 and 3 mM, respectively, with thylakoids from wild type.
c Based on the rate of 9.6 mol of ATP hydrolyzed (mg of CF 1 ⅐ min) Ϫ1 using 10 mM Mg 2ϩ -ATP with CF 1 purified from wild-type Chlamydomonas.  2. Light-driven proton gradient formation in thylakoids  purified from: a, wild type; b, D262C; c, D262N, d, D262T; and e, D262H mutants of Chlamydomonas measured by 9-aminoacridine fluorescence quenching.
FIG. 3. The parallel regions of the VO 2ϩ -EPR spectrum when VO 2ϩ -ATP is bound at Site 3 of wild type Chlamydomonas CF 1 (a). 1 mol equivalent of VO 2ϩ was added as a complex with ATP to 57.5 mg of CF 1 that had been depleted of metal-nucleotide from Site 3. 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 mW, temperature, 100 K; microwave frequency, 9.66278 GHz; number of scans, 200. Simulated spectra for species B (b) and species C (c) were generated using the program QPOWA with the experimental conditions above and the 51 V-hyperfine parameters from Table II. serve as equatorial ligands. These changes will be evident as differences in the spacing between the Ϫ7/2, Ϫ5/2, ϩ3/2, ϩ5/2, and ϩ7/2 transitions shown in Fig. 3, which do not overlap with perpendicular transitions.
Addition of an equivalent of VO 2ϩ -ATP to CF 1 under conditions in which all other higher affinity binding sites for metalnucleotides were filled with Mg 2ϩ -nucleotide complexes resulted in two sets of parallel transitions for VO 2ϩ . The simulated spectrum for each set (Fig. 3, spectra b and c) and the values of A ʈ and g ʈ used to generate these spectra are given in Table II. Spectra b and c correspond to EPR species B and C that result from the two specific binding environments for VO 2ϩ as a complex with nucleotide in Site 3 each with its own set of equatorial ligands. In the latent form of the enzyme, species B predominates, but activation induces the conversion of species B into species C. The ratios of the amplitudes of the simulated spectra for EPR species B and C from each mutant that, when summed, reproduced the experimental spectra are also shown in Table II. These data provide the ratio of the amount of vanadyl bound in the form that gives rise to species B versus species C.
The parallel transitions of the EPR spectrum from VO 2ϩ -ATP bound to Site 3 of CF 1 with the D262H mutation is shown in Fig. 4 along with the simulations of EPR species B and C that best fit the experimental data. The value of A ʈ for EPR species B in this mutant decreased 4.4 MHz from that of the wild type enzyme to a value of 493.8 MHz, whereas the 51 Vhyperfine components of species C remained unchanged (Table  II). This mutation also caused an increase in the species C:B ratio by more than 3-fold of that observed with the wild type enzyme.
No significant differences in A ʈ could be discerned for species B in the spectrum of VO 2ϩ bound to the D262N mutant (Fig. 5) versus those of wild type enzyme. However, much higher signal-to-noise in the EPR spectrum is required to resolve the small difference in A ʈ that would be anticipated if the asparagine side chain were to become a ligand (8) such that this experiment serves as a negative control. This conservative change in the side chain did increase the signal intensity of species B relative to species C by about 20% such that the ratio of the C:B signal intensity decreased to 0.68.
The EPR spectrum that resulted from VO 2ϩ -ATP bound to Site 3 of CF 1 that contained the D262T mutation is shown in Fig. 6. A value of 502.2 MHz for A ʈ was derived from the simulation of EPR species B, which represents a 4 MHz increase in A ʈ of species B from that of wild type. This mutation decreased the ratio of species C:B by 1.7-fold.
In the D262C mutant, if there were a single substitution of the sulfhydryl for the carboxyl in the equatorial ligands of the bound VO 2ϩ , a decrease in A ʈ of 31.6 MHz from that of the wild type is expected. However, A ʈ of species B remained unchanged (Fig. 7). This mutation did have the largest effect on the C:B ratio of any of the mutants, causing a decrease of more than 5-fold from that of wild type.
None of the mutations was found to change the 51 V-hyperfine parameters of EPR species C. This indicates that the WHBaspartate is not an equatorial ligand to VO 2ϩ bound at Site 3 in the species C conformation. This also serves as a negative control that indicates that the changes in 51 V-hyperfine parameters observed in species B are specific for that binding environment. All of the mutations were found to change the ratio of EPR species C:B from that observed in wild type CF 1 . Because A ʈ and g ʈ are not affected by the mutations, these changes are indicative of changes in the affinity of the VO 2ϩ -ATP complex for the conformation of Site 3 that gives rise to species B.

DISCUSSION
The results presented here indicate that ␤D262 serves as an essential residue of the chloroplast F 1 F 0 -ATP synthase in both a catalytic and regulatory capacity. Catalytic function of the enzyme was significantly affected by every mutation examined. In addition, every mutation changed the ratio of EPR species C:B and/or the 51 V-hyperfine parameters of EPR species B, the predominant conformation of VO 2ϩ -nucleotide bound to Site 3 of latent CF 1 . None of the crystal structures of F 1 determined to date (2,38,39) has provided any information concerning the conformation of the metal-nucleotide bound to CF 1 in the latent state.
Weber et al. (5) measured the binding affinities of nucleotides to the catalytic sites of the ␤Y331W mutant of E. coli F 1 by the fluorescence quenching that results from a direct interaction between the adenine ring of the nucleotide and the tryptophan residue. These studies revealed that Mg 2ϩ was responsible for the large differences in affinities of nucleotide among the three catalytic sites. When the WHB-aspartate was mutated to create a double mutant ␤Y331W/␤D242N of E. coli F 1 , the nucleotide binding affinities of the three catalytic sites were not increased by Mg 2ϩ but became closely similar to the lower affinity observed when nucleotide binds alone (26). Based on these results Weber et al. (26) concluded that there must be a water molecule not visible in the crystal structure that is both hydrogen-bonded to the WHB carboxyl and coordinated to the FIG. 4. The parallel regions of the VO 2ϩ -EPR spectrum when VO 2ϩ -ATP is bound at Site 3 of the ␤D262C mutant of Chlamydomonas CF 1 (a). 1 mol equivalent of VO 2ϩ was added as a complex with ATP to 36 mg of CF 1 that had been depleted of metal-nucleotide from Site 3. 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 mW, temperature, 100 K; microwave frequency, 9.66417 GHz; number of scans, 200. Simulated spectra for species B (b) and species C (c) were generated using the program QPOWA with the experimental conditions above and the 51 V-hyperfine parameters from Table II. The results presented here for EPR species C show that, in this conformation, the WHB carboxyl is neither an equatorial ligand to VO 2ϩ nor is it hydrogen-bonded to a water molecule coordinated at an equatorial position at Site 3. The 51 V-hyperfine parameters of EPR species C, the species present exclusively in the activated enzyme, were not changed significantly by any of the mutations examined. These mutations did inhibit catalytic function as observed for the D242N mutant in E. coli F 1 (26). Therefore, it is possible that the WHB carboxyl has hydrogen bonded to the vanadyl-oxo or to a water molecule coordinated at the axial position of the VO 2ϩ in Site 3. The loss of activity in these mutants could also be explained if this residue were to serve as a direct ligand in one of the other catalytic sites. This will be resolved as more of the residues that serve as metal ligands at each catalytic site are identified.
The EPR data that result from VO 2ϩ bound to Site 3 of mutant CF 1 provide insight into the metal ligation responsible for the changes in EPR species B in each of these mutants. Based on the measured coupling constants of A ʈ from model studies (28,37), the hyperfine coupling for a given group of equatorial ligands can be calculated from Eq. 1 where i counts the different types of equatorial ligand donor groups, n i (ϭ 1-4) is the number of ligands of type i, and A ʈi is the measured coupling constant for equatorial ligand donor group of type i (28). Similar equations can be written for g ʈ and for A iso , though the changes in A ʈ are the largest and most easily discerned. Table III shows the values of A ʈ and g ʈ calculated from Eq. 1 that give the closest fit to the experimental data derived from simulation of the spectra (Table II) and summarizes the equatorial ligands used for these calculations. The best fit of the data for species B at Site 3 of wild type CF 1 includes a water, as well as carboxyl, hydroxyl, and phosphate groups as equatorial ligands to VO 2ϩ (9). One interpretation of the data presented here is that the putative carboxyl ligand is D262. In this case, we expect to observe a change in 51 V-hyperfine parameters consistent with displacement of the carboxyl by the type of group substituted in the D262 mutation. Alternatively, D262 may be hydrogen-bonded to the putative water ligand derived from the best fit to EPR species B. No changes 51 V-hyperfine parameters are typically expected except in the rare event that the mutation causes a ligand to be displaced. Instead, the differences in the ability of the mutated groups to hydrogen bond to water are anticipated to change the ratio of EPR species C:B.
If D262 acts as a direct metal ligand, the 4.4 MHz change in A ʈ of species B observed with D262H can be easily explained as FIG. 5. The parallel regions of the VO 2ϩ -EPR spectrum when VO 2ϩ -ATP is bound at Site 3 of the ␤D262H mutant of Chlamydomonas CF 1 (a). 1 mol equivalent of VO 2ϩ was added as a complex with ATP to 20 mg of CF 1 that had been depleted of metal-nucleotide from Site 3. 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 mW, temperature, 100 K; microwave frequency, 9.66437 GHz; number of scans, 180. Simulated spectra for species B (b) and species C (c) were generated using the program QPOWA with the experimental conditions above and the 51 V-hyperfine parameters from Table II. FIG. 6. The parallel regions of the VO 2ϩ -EPR spectrum when VO 2ϩ -ATP is bound at Site 3 of the ␤D262N mutant of Chlamydomonas CF 1 (a). 1 mol equivalent of VO 2ϩ was added as a complex with ATP to 36 mg of CF 1 that had been depleted of metal-nucleotide from Site 3. 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 mW, temperature, 100 K; microwave frequency, 9.66434 GHz; number of scans, 660. Simulated spectra for species B (b) and species C (c) were generated using the program QPOWA with the experimental conditions above and the 51 V-hyperfine parameters from Table II. FIG. 7. The parallel regions of the VO 2ϩ -EPR spectrum when VO 2ϩ -ATP is bound at Site 3 of the ␤D262T mutant of Chlamydomonas CF 1 (a). 1 mol equivalent of VO 2ϩ was added as a complex with ATP to 51 mg of CF 1 that had been depleted of metal-nucleotide from Site 3. 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 mW, temperature, 100 K; microwave frequency, 9.66398 GHz; number of scans, 220. Simulated spectra for species B (b) and species C (c) were generated using the program QPOWA with the experimental conditions above and the 51 V-hyperfine parameters from Table II. a simple substitution of an imidazole nitrogen for a carboxyl oxygen as an equatorial ligand to the bound VO 2ϩ . This change in A ʈ is more difficult to explain if D262 is only hydrogenbonded to the coordinated water because this requires a double substitution of imidazole nitrogen for water and water for carboxyl.
The EPR data shown for the D262N mutation does not provide any information to distinguish between the possible roles of the carboxyl as a direct ligand, or as an indirect ligand hydrogen bonded to a coordinated water. An asparagine side chain coordinated to VO 2ϩ shows changes in A ʈ that can be resolved only at much higher signal-to-noise than that reported here (8). The ability of this side chain to hydrogen bond to water also allows for the latter possibility. It is noteworthy that the D262N mutation causes a small change in the ratio of EPR species C:B signal intensities.
If the ␤D262T mutation were to result solely in the substitution of a carboxyl for a hydroxyl oxygen, a decrease in A ʈ of about 22 MHz is expected. However, an increase in A ʈ of 4 MHz was observed in this mutant to a value of 502.2 MHz. These data are best fit to a set of equatorial ligands where the carboxyl group and the hydroxyl group have been substituted for an amino group and a tyrosine hydroxyl. Although these are somewhat unexpected substitutions, both the P-loop lysine (␤K157) and the catch-loop tyrosine (␤Y311) are in close proximity to the Mg 2ϩ in the catalytic sites of F 1 from bovine mitochondria (2). Clearly, the D262T mutation caused profound changes in the equatorial ligands of VO 2ϩ bound at catalytic Site 3. Because a hydroxyl group is capable of hydrogen bonding, the changes observed here are inconsistent with D262 serving only to hydrogen bond to a coordinated water.
The D262C mutation did not change the 51 V-hyperfine parameters. Although this appears to favor hydrogen bonding over direct ligation, sulfhydryl groups are relatively poor in forming hydrogen bonds. Consequently, if the group at this position is needed to hydrogen bond to the coordinated water, this mutant should increase the ratio of EPR species C:B. However, a large decrease in the ratio of EPR species C:B was observed. Furthermore, a double substitution of equatorial ligands of sulfhydryl sulfur for carboxyl oxygen and water oxygen for hydroxyl oxygen will also result in 51 V-hyperfine parameters closely similar to those of wild type (Table III).
The results presented here strongly favor the conclusion that the WHB carboxyl serves as an equatorial ligand to the VO 2ϩ bound in Site 3 in the form that gives rise to EPR species B, the form of Site 3 that predominates in latent CF 1 . Upon activation of the enzyme with dithiothreitol, the signal intensity of species B converts into species C. The lack of effects of the mutations to the WHB carboxyl on EPR species C indicates that activation of the enzyme causes the loss of this carboxyl group as an equatorial ligand. Chen et al. (9) showed that the P-loop threonine (␤T168 in Chlamydomonas) serves as an equatorial ligand to VO 2ϩ at Site 3 under conditions that give rise to species C but not to that which results in species B. When combined with the results presented here, it appears that activation changes the conformation of the enzyme to cause the substitution of the WHB carboxyl for the P-loop threonine as a metal ligand at Site 3.