From the The Center for the Study of Early Events in Photosynthesis, Arizona State University, Tempe, Arizona 85287-1601
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ABSTRACT |
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Site-directed mutations were made to the
phosphate-binding loop threonine in the 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
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 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 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 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:
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
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).
Effects of the Mutations on Enzyme Assembly and ATP
Synthesis--
The yield of CF1 purified from each of the
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
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.
Coordination of Metal at Site 3 in
The values of A
In the
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).
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 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 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
A-subunit of the chloroplast
F1-ATPase in Chlamydomonas (
T168).
Rates of photophosphorylation and ATPase-driven proton translocation
measured in coupled thylakoids purified from
T168D,
T168C, and
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
-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).
-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
-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).
-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).
-subunit of CF1 from Chlamydomonas
reinhardtii (
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
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
T179D, 5'- TTCGGTGCCGGTGTAGGCAAAGACGTTTTAATTATG-3';
T179C,
5'-TTCGGTGCCGGTGTAGGCAAATGTGTTTTAATTATG-3'; and
T179L,
5'-GTGTAGGCAAATTAGTTTTAATTATGGAAC-3'. The selective primer
that changed restriction site Xmnl to BamHI was
Ps (5'-CGCCCCGAAGAACGGATCCCAATGATGAGCAC-3').
5/2 line of the EPR signal intensity per scan at a
gain of 105.
RESULTS
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
-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, T168C; lane 3,
T168D;
lane 4,
T168L mutants of Chlamydomonas.
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.
Functional comparison of wild-type and mutants
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Fig. 2.
Light-driven proton gradient formation in
thylakoids purified from (a) wild type, (b)
T168C, (c)
T168D, and (d)
T168L
mutants of Chlamydomonas measured by 9-aminoacridine
fluorescence quenching.
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
+5/2
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)
T168D, (c)
T168C, and (d)
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;
T168D, 9.5609 GHz, 84 scans, and 44.3 mg;
T168C, 9.5624 GHz, 128, scans, and 38.1 mg; and
T168L, 9.5623 GHz, 88 scans, and 28.2 mg, respectively.
and
g
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.
VO2+ coordination at Site N3 in CF1 from wild-type and
mutants
Mole ratios of vanadyl bound to CF1 upon addition of a single
equivalent of VO2+ as a complex with ATP
T168C mutant, the value of A
increased by
about 13 MHz. In addition, the EPR signal intensity of species C
relative to species B decreased by about 2-fold. The
T168D mutant
increased A
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.
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Fig. 4.
Initial rates of
Mg2+-ATPase-driven proton translocation catalyzed by
thylakoid membranes isolated from wild type ( ,
),
T168D (
,
),
T168C (
,
), and
T168L (
,
) 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
-subunit (
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.
T156D,
T156C, and
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.
from model studies (34, 36), the hyperfine
coupling for a given group of equatorial ligands can be calculated from
the equation
(Eq. 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 A
i
is the measured coupling constant for the equatorial ligand donor group
of type i (34). Similar equations can be written for
g
and for Aiso,
although the changes in A
are the largest and
most easily discerned.
Table II shows the values of A and
g
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
T168C mutant, values of
A
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
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 T168D mutation were to result solely in the substitution of a
carboxyl for a hydroxyl oxygen, an increase in
A
of about 22 MHz would be expected. Whereas
an increase in A
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 A
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
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 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 TS
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
3
3
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 TS 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.
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ACKNOWLEDGEMENT |
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We thank David Lowry for excellent technical support.
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FOOTNOTES |
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* 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.
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.
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ABBREVIATIONS |
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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.
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REFERENCES |
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