From the Department of Biochemistry, The Weizmann Institute of Science, Rehovot 76100, Israel
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ABSTRACT |
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We introduced mutations at the fully conserved
residue Glu-195 in subunit of Rhodospirillum
rubrum F1-ATPase. The activities of the
expressed wild type (WT) and mutant
subunits were assayed by
following their capacity to assemble into the earlier prepared
-depleted, membrane-bound R. rubrum enzyme
(Philosoph, S., Binder, A., and Gromet-Elhanan, Z. (1977)
J. Biol. Chem. 252, 8742-8747) and to restore ATP
synthesis and/or hydrolysis activity. All three mutations,
-E195K,
-E195Q, and
-E195G, were found to bind as the WT
into the
-depleted enzyme. They restored between 30 and 60% of the WT
restored photophosphorylation activity and 16, 45, and 105%,
respectively of the CaATPase activity. The mutants required, however, much higher concentrations of divalent cations and could not restore any significant MgATPase or MnATPase activities.
Only
-E195G could restore some of these activities when assayed in the presence of 100 mM sulfite and high MgCl2
or MnCl2 concentrations. These results suggest that
the observed difference in restoration of ATP synthesis and
CaATPase, as compared with MgATPase and MnATPase, can be due
to the tight regulation of the last two activities, resulting in their
inhibition at cation/ATP ratios above 0.5. The R. rubrum
F1
-E195 is equivalent to the mitochondrial
F1
-E199, which points into the tunnel leading to the
F1 catalytic nucleotide binding sites (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). Our findings indicate that
this residue, although not an integral part of the F1
catalytic sites, affects divalent cation binding and release of
inhibitory MgADP, suggesting its participation in the interconversion
of the F1 catalytic sites between different conformational
states.
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INTRODUCTION |
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All respiratory and photosynthetic cells contain a
membrane-embedded F0F1 ATP synthase that
generates ATP at the expense of the electrochemical proton gradient
formed during electron transport. The catalytic F1
component of the enzyme has been solubilized as a functional ATPase
from many bacterial, mitochondrial, and chloroplast sources. It is a
very conserved multimeric assembly with a stoichiometry of
3
3
and has up to six nucleotide
binding sites. Three of them are catalytic sites, residing mainly on
the
subunits, and three are noncatalytic, located mainly on the
subunits (1-6).
Two recently published x-ray crystallographic structures of rat liver
mitochondrial MF1 at 3.6 Å (7) and bovine heart
MF1 at 2.8 Å (8) have confirmed the alternate arrangement
of the six large and
subunits in a closed hexamer. The
structure at 2.8 Å resolution provides direct proof for the earlier
suggested location of all six nucleotide binding sites at
/
interfaces (see Ref. 9). It has also resolved two long C- and
N-terminal helical domains of the
subunit, which are embedded
within the internal cavity of the
3
3
hexamer, and impose on it an asymmetric structure. This asymmetry is
also reflected in different conformational states displayed by the
three catalytic sites on the
subunits (8). The binding change
mechanism (10) suggested that ATP synthesis and hydrolysis involves
transitions between such different conformational states via rotation
of the
-subunit relative to an
3
3
subassembly. So this structural information initiated a drive to
demonstrate that such rotation is possible (11-13).
These important observations do not explain how rotation drives
catalytic site transitions, which must involve intricate
protein-protein interactions within the /
catalytic interfaces as
well as between them and various domains on the
subunit. Full
elucidation of the unique mechanism of action of the ATP synthases will
therefore require clear definition of the role of amino acid residues
that are involved in catalysis, as well as identification and detailed characterization of all interacting subdomains on the
,
, and
subunits, and their relation to the catalytic sites.
Genetic/biochemical assay systems that are crucial for such
studies have been developed in respiratory bacteria and yeast mitochondria (see Refs. 1, 2, and 6), but the application of this
approach to photosynthetic systems lagged behind the respiratory ones.
Although the conserved structure of all isolated F1-ATPases suggests a common catalytic mechanism, there are clear differences in
various properties between the respiratory and photosynthetic F0F1 and F1-complexes, for instance
in regulation of activity and sensitivity to some inhibitors (3-5). It
is, therefore, important to develop recombinant molecular techniques
and direct in vivo and/or in vitro assays of
activity for photosynthetic complexes containing mutated
F1-subunits. Two such systems have recently been reported
for Chlamydomonas reinhardtii and spinach chloroplast F1- subunits (14-17).
The F1- subunit of the photosynthetic bacterium
Rhodospirillum rubrum
(RrF1
)1
provides an especially suitable system for mutational analysis, based
on the available MF1 crystal structure. Thus, the published sequence of the RrF1 operon (18) revealed that
RrF1
is most closely related to MF1
.
Their amino acid sequences show >76% identity, as compared with only
72% for EcF1
, 69% for tobacco CF1
(18),
and 68% for TF1
(19). The very high similarity between
RrF1
and MF1
extends also to the R. rubrum and mitochondrial F0F1 and
F1 which, unlike the chloroplast and Escherichia
coli enzymes, exhibit an identical sensitivity to inhibition by
oligomycin and efrapeptin (20-22). Furthermore, a large number of
in vitro assays of activity have been developed for the
RrF1
subunit that was isolated in functional form from
the chromatophore membrane-bound RrF0F1. They
include the ability of the isolated RrF1
to bind ATP,
ADP, and Pi (23, 24) as well as to rebind to the
-depleted enzyme and restore its lost ATP synthesis and hydrolysis
activities (25, 26).
Baltscheffsky et al. (27) have cloned the
RrF1 gene and expressed it in E. coli as a
fusion protein with glutathione S-transferase but did not
carry out any assays of activity. We have recently cloned this gene and
have expressed the RrF1
subunit in E. coli lacking the whole unc operon as a soluble protein that could
restore ATP synthesis to
-depleted chromatophores (28). Here we
describe the expression and full purification of RrF1
mutated in Glu-195. A detailed comparison of the activities of these
mutants with those of the expressed WT
subunit revealed that
RrF1
-E195 plays an important role in divalent
cation-dependent ATP synthesis and hydrolysis.
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EXPERIMENTAL PROCEDURES |
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Bacterial Strains--
R. rubrum cells were grown as
described previously (25). Three unc operon-deleted E. coli strains, LM2800, LM3115 (29), and DK8 (30), were used as
hosts for recombinant plasmids (28) carrying the WT and Glu-195 mutated
RrF1 genes. The transformed cells were grown in LB
medium supplemented with thymine (50 µg/ml), thiamine (2 µg/ml),
0.2 mM isoleucine, 0.2 mM valine, 0.2%
glucose, and 5% glycerol.
Cloning and Site-directed Mutagenesis--
The
RrF1 gene was amplified from R. rubrum
genomic DNA by PCR and cloned as described in (28). Since the PCR is
known to cause mutations, two PCR products, one obtained with Vent and the second with Taq DNA polymerase, were cloned and fully
sequenced by the Taq dye deoxy chain termination method,
using a 373A DNA Sequenase. Both clones were found to contain one
mutated nucleotide. In the first clone, nucleotide 222 was changed,
altering the published ACC codon of threonine 74 (18) into an ACA one
that also codes for threonine. So this clone encodes the WT
RrF1
polypeptide. But the second clone had a mutation in
nucleotide 584, which changed the GAG codon of glutamic acid 195 into
the GGG codon of glycine. This clone thus encodes an
RrF1
-E195G mutant polypeptide.
Identification of Expressed RrF1--
Cultures of
the various transformed unc-depleted E. coli
strains were centrifuged and resuspended in buffer containing 50 mM Tricine-NaOH, pH 8.0; 4 mM MgATP, and 10%
glycerol, which is optimal for isolating active native
RrF1
(25). The protease inhibitors benzamidine,
phenylmethanesulfonyl fluoride, and
N
-p-tosyl-L-lysine
chloromethyl ketone were added at 1.5 mM, 1 mM,
and 30 µM, respectively, and the cells were disrupted
under argon in a Yeda press (26). The soluble cytoplasmic and insoluble membrane fractions were analyzed for the presence of expressed RrF1
by SDS-PAGE (31, 32) and Western immunoblotting
(33).
Preparation of -less R. rubrum Chromatophores and Their
Reconstitution with RrF1
--
-less chromatophores
were obtained as described in (26). This technique releases all their
RrF1
(Refs. 25 and 34; see Fig. 4, lane 6)
together with trace amounts of RrF1
(28, 35), leading to
loss of their ATP synthesis and hydrolysis activity. Reconstitution was
carried out by incubating
-less chromatophores, at 5 µg of Bchl,
for 1 h at 35 °C in 0.2 ml of a reaction mixture containing 50 mM Tricine-NaOH (pH 8.0), 25 mM
MgCl2, 4 mM ATP, 1 mM
dithiothreitol, and unless otherwise stated, 1 µg of
RrF1
and saturating amounts of RrF1
at
various stages of purification.
Assays of Restored ATP Synthesis and Hydrolysis--
ATP
synthesis was usually assayed by a 5-fold dilution of the 0.2-ml
mixture of reconstituted chromatophores into an assay mixture which
contained in 1 ml a final concentration of 50 mM Tricine-NaOH (pH 8.0), 5 mM MgCl2, 4 mM sodium phosphate (containing 0.4-0.8 × 106 cpm of 32Pi), 2 mM
ADP, 15 mM glucose, 24 units of hexokinase, and 66 µM N-methylphenazonium methosulfate. When
assaying the Mg2+ requirement of the restored ATP synthesis
(and hydrolysis), about 3 ml of the reconstituted chromatophores were
subjected to two rounds of centrifugation and resuspension in 50 mM Tricine-NaOH (pH 8.0) and 10% glycerol to remove the 25 mM MgCl2, which are essential for the
reconstitution (25, 26). These washed chromatophores, at 3-5 µg of
Bchl, were added to the synthesis assay mixture, together with the
indicated concentrations of MgCl2, and preequilibrated for
5 min at 35 °C in the dark. ATP synthesis was started by
illumination and stopped after 3 min by turning off the lights and
adding 0.1 ml of 2 M trichloroacetic acid. The synthesized
[-32P]ATP was determined as described by Avron
(36).
Other Procedures--
For measurement of ATP binding, purified
RrF1 preparations were depleted of bound MgATP by
elution-centrifugation through Sephadex G-50 columns (38)
preequilibrated with TGN buffer containing 50 mM
Tricine-NaOH (pH 8.0), 20% glycerol, and 50 mM NaCl.
Binding was assayed by incubating 10 µM depleted
RrF1
for 90 min at 23 °C in TG buffer with the
indicated concentrations of MgCl2 and ATP, containing
1-2 × 106 cpm of [2,8-3H]ATP.
Incubation was started by addition of RrF1
and stopped by subjecting 50-µl samples to elution-centrifugation, and the effluent was assayed for protein and radioactivity as described by
Gromet-Elhanan and Kananshvili (23).
Materials-- E. coli DK8 was a gift of Dr. M. Futai, Institute of Scientific and Industrial Research, Osaka University. E. coli LM2800 and LM3115 were a gift of Dr. P. R. Jensen, The Netherlands Cancer Institute, Amsterdam.
Oligonucleotides were synthesized by Dr. Ora Goldberg, Biological Services, The Weizmann Institute of Science. Restriction enzymes, Vent DNA polymerase, and T4 ligase were from New England Biolabs. Taq DNA polymerase, and dNTP were purchased from Boehringer Mannheim. Plasmids pBSKS+ and pBTacI were from Stratagene and Boehringer Mannheim, respectively. The U.S.E. Mutagenesis Kit was obtained from Pharmacia Biotech Inc.. [2,8-3H]ATP was purchased from NEN Life Science Products. All other reagents were of the highest purity available. ![]() |
RESULTS |
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Construction of RrF1-E195 Mutants--
The
-E195G mutation was obtained as a mistake, which changed the GAG
codon of glutamic acid to the GGG codon of glycine, during
amplification of the RrF1
gene from genomic R. rubrum DNA by PCR. Since the equivalent MF1
-E199 is
described in the crystal structure as pointing into the conical tunnel
leading to the catalytic nucleotide binding site (8), it was
interesting to check whether the E195G mutation affects activity. Using
the system described previously (28), the expressed, partially purified mutated
subunit was found to restore a much lower rate of ATP synthesis in
-less chromatophores than the native or expressed WT
RrF1
. We have therefore prepared two additional
RrF1
-E195 mutants by oligonucleotide-directed
mutagenesis: from glutamic acid to lysine (
-E195K), carrying a
positive charge, and to glutamine (
-E195Q), carrying no charge.
Expression and Purification of WT and Mutant RrF1
Subunits--
Cloned WT and mutant
genes were ligated into the
expression vector pBTacI, and the recombinant plasmid was transformed
into E. coli LM3115 (28). This strain was found to express
larger amounts of RrF1
than two other
unc-deleted strains, LM2800 (29) and DK8 (30), under all
tested growth conditions (not shown). Optimal conditions for expression
of large amounts of RrF1
as a soluble protein include
growth of E. coli LM3115 at 22 °C in the presence of 5%
glycerol to about A0.65. Growth at higher
temperatures or to a higher optical density increased the fraction of
expressed
subunit appearing in inclusion bodies (42).
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Activities of the Highly Purified Native, Expressed WT and E195G
RrF1--
All three types of RrF1
subunits were found to bind, in the presence of Mg2+, up to
2 mol of ATP/mol of
. But, whereas the binding of ATP to the
expressed WT as to pure native RrF1
(Fig.
2), saturated at 200 µM ATP
and showed a pronounced cooperativity, with a Hill coefficient
(nH) of 2.2, the RrF1
-E195G
mutant required a higher concentration of ATP for saturation and showed
a much lower cooperativity with a Hill coefficient of only 1.5. The
native RrF1
, which contained at least 5% of
RrF1
(28, 35), was earlier shown to bind up to 2 mol of
ATP/mol (23) but with somewhat different properties than those reported
here for the highly purified native
subunit.
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Divalent Cation-dependent Restoration of ATP Hydrolysis
and Synthesis by WT and Several RrF1-E195
Mutants--
The similar binding of WT and the E195G mutant
RrF1
to
-less chromatophores, but the much lower
ability of the mutant to restore ATP synthesis, suggested that
-E195
plays an important role in the catalytic activity of the ATP synthase.
To test this possibility, we compared the capacity of WT and all three
-E195 mutants to restore ATP hydrolysis (Fig.
5). ATP hydrolysis, unlike its synthesis,
was shown to occur in control R. rubrum chromatophores in
the presence of Ca2+ as well as Mg2+ and
Mn2+ but was coupled to proton translocation only in the
presence of the last two divalent cations (44). Reconstitution of
-less chromatophores with WT RrF1
restored their
capacity to hydrolyze ATP with all three divalent cations (Fig. 5,
A-C). As in control chromatophores (44), hydrolysis in the
presence of Mg2+ and Mn2+ showed a narrow range
of dependence on the cation concentration, resulting in maximal
activity at cation/ATP ratios around 0.5 followed by a drastic
inhibition at a cation/ATP ratio of 2 (Fig. 5, A and
B). The CaATPase activity in control (44), as well as WT
RrF1
reconstituted, chromatophores (Fig. 5C)
was much less sensitive to inhibition by an excess of free
Ca2+-ions, remaining optimal at cation/ATP ratios between
0.5 and 2.0. Even with 32 mM CaCl2, at a
cation/ATP ratio of 8, this restored ATPase activity was inhibited by
only 35%.
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DISCUSSION |
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Glutamic acid 195 of RrF1 is equivalent to
MF1
-Glu-199, which in the bovine heart crystal structure
(8) is described as pointing into the conical tunnel leading to the
nucleotide binding sites. This fully conserved glutamic acid residue
was shown to bind the inhibitor DCCD in MF1
(46) and
EcF1
(47). But a mutation in the equivalent
EcF1
-E192 to Gln was found to grow on succinate and
showed normal assembly of F0F1, with only 6-8-fold inhibition of its membrane ATPase activity and small perturbations in rate constants and equilibrium constants of unisite catalysis (48). It was, therefore, suggested that the full inhibition of F0F1 by DCCD could be due to the presence of
the two bulky cyclohexyl moieties in the conical tunnel (8).
The system developed in R. rubrum chromatophores enables
reconstitution of -less chromatophores with RrF1
and
detailed characterization of ATP hydrolysis as well as synthesis
restored to the same preparation of reconstituted chromatophores (25,
26). This system was used here for a direct comparison of the
activities restored to
-less chromatophores reconstituted with WT
and three Glu-195 mutants of RrF1
and provided rather
unexpected results. ATP synthesis was restored to about 60% by
-E195G and
-E195Q and to 30% by
-E195K (Fig. 6), whereas the
Mg- and Mn-dependent ATP hydrolysis was not restored by
E195Q and E195K mutants, even when assayed in the presence of
saturating concentrations of sulfite (Fig. 7, A and
B). The E195G mutant could restore some MgATPase and MnATPase activities in the presence of 100 mM sulfite, but
they showed different divalent cation dependences and reached a much lower maximal rate than the activities restored by WT
RrF1
.
Similar properties were recently reported for the MgATPase activity of
S-carboxymethyl-185 EcF1 (49). This residue
is equivalent to MF1
-E192, which is located at the
catalytic nucleotide binding sites of F1, whereas the
RrF1
-E195 residue is pointing into the tunnel leading to
these sites (8). Our results indicate that residues within the tunnel
might also affect divalent cation and nucleotide binding, suggesting
their participation in the interconversion of the catalytic sites
between different conformational states. Interestingly, two earlier
reports describing mutations in EcF1
-E192 to V (50) and
TF1
-E201 to Q (51), which are equivalent to RrF1
-E195, have observed some type of interaction
between this glutamic acid and EcF1-G149 or
TF1
-E190, which are located in the catalytic site (8).
It would be worthwhile to check whether other amino acid residues that
are located in the tunnel show similar effects.
The strange results obtained with the RrF1-E195 mutants,
which enable restoration of ATP synthesis but not hydrolysis, could be
explained by differences in affinity of the catalytic sites to MgADP as
compared with MgATP. These differences are especially important during
ATP hydrolysis in photosynthetic organisms, which is highly regulated
to limit wasteful hydrolysis of low concentrations of ATP in the dark
(3-5). One aspect of this regulation involves the onset of inhibition
of ATPase activity, and of exchange of ADP tightly bound at a catalytic
site with medium nucleotides, by added free Mg2+ (52).
Sulfite has been shown to overcome the Mg2+-induced
inhibition of both ATPase activity and release of the tightly bound
MgADP (53, 54). Addition of a saturating sulfite concentration did
indeed enable low MgATPase and MnATPase activities in the
-E195G
mutant, but not in
-E195Q or
-E195K (Fig. 7, A and
B). These mutations might impede more severely the release of the inhibitory MgADP as compared with the release of the more loosely bound MgATP.
The overall higher activities restored by -E195G (Figs.
5C, 6, and 7) can be explained by the capacity of glycine to
adopt a much wider range of conformations than the other residues and thus allow unusual main chain conformations in proteins (55). In
RrF1
-E195G, it could enable conformational changes that
compensate the change of charge and size, and lead to the higher
activity of this mutant.
An additional interesting effect observed with the
RrF1-E195G and -E195Q, is their capacity to restore
CaATPase, but not MgATPase and MnATPase activities (compare Fig. 5,
A, B, and C). CaATPase, unlike the MgATPase and
MnATPase activity, is not coupled to proton translocation and not
subject to the tight regulation observed with MgATP and MnATP
hydrolysis (44). Moreover CaCl2 does not enable ATP
synthesis. So the binding of Ca2+ to the catalytic sites on
RrF1
might convert it to a different conformational
state that is less impeded by the Glu-195 mutations. Work with
RrF1
-T159 mutants has provided direct experimental support for this conclusion.2
It would be most interesting to crystallize F1 in the
presence of CaCl2 since any differences between the
CaCl2- and MgCl2-containing crystals might
illuminate the domains, and/or specific amino acid residues, that are
involved in the coupling of proton translocation to ATP synthesis and
hydrolysis. This information is essential for the understanding of the
mechanism of ATP synthesis.
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FOOTNOTES |
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* This work was supported by Grants from the Basic Research Foundation administered by the Israel Academy of Sciences and Humanities and by the Avron-Wilstätter Minerva Center for Research in Photosynthesis, Rehovot, Israel.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Biochemistry and Molecular Biology,
University of Miami School of Medicine, Miami, FL 33101-6129.
§ To whom correspondence should be addressed. Tel.: 972 8 9342729; Fax: 972 8 9344118.
1
The abbreviations used are: RrF1,
RrF1
, CF1
, CF1
,
EcF1
, MF1
, and TF1
,
and
subunits of the F1-ATPase of R. rubrum, chloroplasts, E. coli, mitochondria, and thermophilic
Bacillus PS3, respectively; Bchl, bacteriochlorophyll; PCR,
polymerase chain reaction; Tricine,
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; WT, wild
type; DCCD, dicyclohexylcarbodiimide.
2 L. Nathanson and Z. Gromet-Elhanan, manuscript in preparation.
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REFERENCES |
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