From the Research Laboratory of Resources Utilization, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226, Japan and the § Institute Curie, Section de recherche, UMR 168 and LCR-CEA 8, 11 rue Pierre et Marie Curie, 75231 Paris, France
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ABSTRACT |
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ATP hydrolyzing activity of a mutant
3
3
subcomplex of
F0F1-ATP synthase (
NC) from the thermophilic
Bacillus PS3, which lacked noncatalytic nucleotide binding
sites, was inactivated completely soon after starting the reaction
(Matsui, T., Muneyuki, E., Honda, M., Allison, W. S., Dou, C., and
Yoshida, M. (1997) J. Biol. Chem. 272, 8215-8221).
This inactivation is caused by rapid accumulation of the "MgADP
inhibited form" which, in the case of wild-type enzyme, would be
relieved by ATP binding to noncatalytic sites. We reconstituted
F0F1-ATP synthase into liposomes together with
bacteriorhodopsin and measured illumination-driven ATP synthesis.
Remarkably,
NC F0F1-ATP synthase catalyzed
continuous turnover of ATP synthesis while it could not promote
ATP-driven proton translocation. ATP synthesis by
NC
F0F1-ATP synthase, as well as wild-type enzyme,
proceeded even in the presence of azide, an inhibitor of ATP hydrolysis
that stabilizes the MgADP inhibited form. The time course of ATP
synthesis by
NC F0F1-ATP synthase was
linear, and gradual acceleration to the maximal rate, which was
observed for the wild-type enzyme, was not seen. Thus, ATP synthesis
can proceed without nucleotide binding to noncatalytic sites even
though the rate is sub-maximal. These results indicate that the MgADP
inhibited form is not produced in ATP synthesis reaction, and in this
regard, ATP synthesis may not be a simple reversal of ATP
hydrolysis.
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INTRODUCTION |
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The F0F1-ATP synthase is a ubiquitous
enzyme in plasma membranes of bacteria, inner membranes of
mitochondria, and thylakoid membranes of chloroplasts which utilizes a
transmembrane electrochemical potential difference of protons
(µH+)1 for
ATP synthesis (1, 2). It can be reversibly separated into a
hydrophilic, water-soluble F1 part and a hydrophobic,
membrane-embedded F0 part. F0 conducts protons
across the membrane. F1, also called F1-ATPase,
has a subunit composition of
3
3
and shows strong activity of ATP hydrolysis. According to the crystal
structure of the major part of beef heart mitochondrial
F1-ATPase (3),
and
subunits are alternatively
arranged to form a hexagonal cylinder with a central cavity through
which coiled-coil
helices of the
subunit penetrate.
F1-ATPase was recently proven to be a "rotary motor
enzyme", the first ever found in the biological world; the
subunit rotates within an
3
3 hexagon
during ATP hydrolysis (4-6).
The F1-ATPase has six nucleotide binding sites. Three of
them are catalytic and located mainly on the subunits. The other three, called noncatalytic sites, are mainly located on the
subunits. The function of the noncatalytic site had been obscure but
recent studies indicated its regulatory role as follows. During multiple turnover of ATP hydrolysis, MgADP is prone to be entrapped in
a catalytic site producing the MgADP inhibited form of the enzyme,
which is inactive in ATP hydrolysis. ATP binding to the noncatalytic
sites causes release of this inhibitory MgADP from the affected
catalytic site and relieves the enzyme from the MgADP inhibited form
(7-14). In steady-state catalysis of ATP hydrolysis, the relative
population of the MgADP inhibited form of F1-ATPase is in
an equilibrium determined between entrapping inhibitory MgADP in a
catalytic site and releasing inhibitory MgADP by ATP binding to
noncatalytic sites. Azide inhibits ATPase activity of
F1-ATPase by stabilizing the MgADP inhibited form (7, 8, 15).
Results obtained for a mutant (NC)
3
3
subcomplex from the thermophilic
Bacillus PS3 provided strong evidence for the proposed role
of noncatalytic sites (16). The
NC enzyme is a quadruple mutant
(
K175A/
T176A/
D261A/
D262A) and completely lacks the ability
to bind ATP (and ADP) to the noncatalytic sites. One may predict that,
with the noncatalytic sites being empty, this mutant cannot release
entrapped inhibitory MgADP from a catalytic site, and all enzyme
molecules fall into the MgADP inhibited form rapidly during catalytic
turnover. Indeed, the
NC enzyme hydrolyzed ATP at an initial rate
similar to that of the wild-type enzyme, but it decayed rapidly to an
inactivated form. In the presence of ATP, the
NC enzyme did not
release preloaded inhibitory [3H]ADP, whereas the
wild-type enzyme did (16). Thus, binding of nucleotide to noncatalytic
sites seems to be essential for continuous catalytic turnover of ATP
hydrolysis by F1-ATPase.
The occurrence of the MgADP inhibited form during ATP hydrolysis reaction has been taken as established but the occurrence of it during ATP synthesis reaction has been questioned by Syroeshkin et al. (17). Using beef heart submitochondrial particles in the presence of substoichiometric amounts of oligomycin, they tested the effect of azide on ATP synthesis and ATP hydrolysis. Interestingly, ATP synthesis was not inhibited by azide, whereas ATP hydrolysis was strongly inhibited. Different azide sensitivity of ATP synthesis and ATP hydrolysis was also reported for chloroplast thylakoid membranes (18). This indicates that generation of the MgADP inhibited form is avoided in ATP synthesis reaction, and Syroeshkin et al. (17) have suggested a possibility that ATP synthesis catalyzed by submitochondrial particles is not a simple reversal of ATP hydrolysis. Since they have used submitochondrial particles and monitored ATP hydrolysis and synthesis indirectly by pH change, questions have remained, such as is the reaction kinetics of azide-insensitive ATP synthesis the same as that in the absence of azide, are there special protein components that are responsible for the azide-insensitive ATP synthesis, and is this a phenomenon universal in any F0F1-ATP synthases? A direct measurement with purified enzyme is expected to lead to a more definite conclusion.
Continuous turnover of ATP synthesis by the purified
F0F1-ATP synthase was measured with
reconstituted proteoliposomes (19). In that experiment, thermophilic
F0F1-ATP synthase was incorporated into
membranes of liposomes together with bacteriorhodopsin. Under illumination, bacteriorhodopsin pumped protons into proteoliposomes and
established a stable µH+, and the
F0F1-ATP synthase catalyzed continuous
synthesis of ATP for several hours. Considerable improvement of this
assay system was made recently (20, 21), and a biphasic time course of
ATP synthesis by thermophilic F0F1-ATP synthase
under constant
µH+ was found; a first phase with a
basic reaction rate accelerated to a second phase with a maximum rate
(22). Labeling experiments with
2-N3-[
-32P]ATP revealed that
ATP binding to a noncatalytic site was responsible for the shift from
the first phase to the second phase during catalysis (22). Here, we
have further extended the versatility of the assay system to be
applicable for the F0F1-ATP synthase assembled
from subcomplexes and subunits. We assembled the
NC
3
3
subcomplex into
F0F1-ATP synthase and used it for the
preparation of bacteriorhodopsin-F0F1-ATP
synthase proteoliposomes (bR-F0F1 liposomes).
If the ATP synthesis reaction by F0F1-ATP
synthase is just the reverse reaction of ATP hydrolysis, the
NC
F0F1-ATP synthase cannot catalyze continuous
ATP synthesis because of rapid accumulation of the MgADP inhibited form
due to defective noncatalytic sites. However, the result showed that
the
NC F0F1-ATP synthase was capable of ATP
synthesis, whereas it could not mediate ATP-driven proton
translocation. ATP synthesis, either by the wild-type or the
NC
F0F1-ATP synthases, proceeded in the presence
of azide. These results indicate that the MgADP inhibited form is not
generated during multiple turnover of ATP synthesis, and in this sense, the reaction pathway of ATP synthesis is not just the reversal of that
of ATP hydrolysis.
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EXPERIMENTAL PROCEDURES |
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Chemicals--
Phosphatidylcholine and phosphatidic acid were
purchased from Avanti Polar Lipids, -octylglucopyranoside was from
Nacalai, Bio-Beads SM-2 were from Bio-Rad, and luciferin/luciferase kit was from Boehringer Mannheim.
Preparation of Enzymes--
Authentic
F0F1-ATP synthase was purified from the
thermophilic Bacillus PS3 as described previously (23).
F0 was obtained from urea-treated
F0F1-ATP synthase by centrifugation (24). The
wild-type (specific activity, about 1.8 µmol of ATP hydrolyzed/mg/min at 40 °C at pH 7.3) and the NC
3
3
complexes were purified from
overexpressed Escherichia coli cells strain JM103
(uncB-uncD) (25) carrying the plasmids pKABG1 (16) and
pKABG1
k175A/
T176A/
D261A/
D262A, respectively (16).
Over-expressed
and
subunits of Bacillus PS3
F1-ATPase were isolated from E. coli strain
JM109 carrying the pKD2 plasmid and strain BL23 carrying the pTE2
plasmid, respectively (26). Purified proteins were stored as ammonium
sulfate precipitates at 4 °C. F1-ATPase was assembled
from the wild-type or
NC
3
3
complex
and the isolated
and
subunits, and excess free subunits were
removed by centrifugal filtration (26). To assemble
F0F1-ATP synthase further, 85 µg of
F1-ATPase (a 2.5-fold molar excess to F0
assuming molecular mass ratio of F1 and F0,
2.7:1) was mixed with 12.5 µg of F0 suspended in 10 mM Tricine/NaOH (pH 8.0) and incubated on ice for 1 h.
This mixture was used directly for reconstitution into liposomes. In
the case of authentic F0F1-ATP synthase, 45 µg of F0F1-ATP synthase, an amount equivalent
to 12.5 µg of F0, was used for proteoliposome
reconstitution. Protein concentration was determined by the
bischinchonic acid (BCA) method (27).
Assay of ATP Synthesis--
F0F1-ATP
synthase and bacteriorhodopsin were incorporated into membranes of
liposomes according to the method described previously (20, 21) with
minor modifications. Briefly, liposomes consisting of 90%
phosphatidylcholine and 10% phosphatidic acid were formed by reversed
phase evaporation. Purple membranes from Halobacterium halobium were solubilized by overnight incubation with 2% Triton X-100. For reconstitution of proteoliposomes, 4 mg of liposomes (250 µl) were mixed with 600 µl of the reaction buffer (25 mM potassium phosphate buffer, pH 7.3, 50 mM
Na2SO4, 50 mM
K2SO4), 40 µl of 20% (w/v) Triton X-100, 50 µl of bacteriorhodopsin solution (4 mg/ml), and 15 µl of the
authentic F0F1-ATP synthase (3 mg/ml) in 20 mM Tris/SO4, pH 8.0, 1.5% (w/v)
-octylglucopyranoside or an equivalent amount of assembled
F0F1-ATP synthase. The resultant clear solution
was incubated at room temperature for 5 min, and then 40 µl of 14.8%
(w/v)
-octylglucopyranoside was added. After a 2-min incubation, 10 µl of 20 mM pyranine solution in ethanol and wet
Bio-Beads (80 mg) were added and incubated for 1 h at room
temperature while stirring. Addition of Bio-Beads and the 1-h
incubation with Bio-Beads was repeated three times. The proteoliposomes were washed twice with the reaction buffer by centrifugation (30 min,
150,000 × g), and the pellet was suspended in 1 ml of
the reaction buffer. A 200-µl aliquot of the prepared proteoliposomes (bR-F0F1 liposomes) was mixed with 600 µl of
the reaction buffer, and 2 mM (final concentration) ADP was
added. The reaction mixture was preilluminated by three 300 W slide
projectors for 15 min on a magnetic stirrer at 40 °C. When
indicated, different concentrations of ADP and potassium phosphate
buffer were used. The reaction was started by addition of 2 mM (final concentration) MgSO4. At 15-min
intervals, 50 µl-aliquots were taken, and the reaction was stopped by
addition of the same volume of 4% trichloroacetic acid. The amount of
ATP was determined by the luciferin-luciferase assay method using a
luminescence reader BLR201 (Aloka). When the effect of azide was
tested, bR-F0F1 liposomes were incubated with 5 mM (final concentration) sodium azide for 20 min at
40 °C prior to initiation of pre-illumination. When indicated,
carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)
(final concentration, 5 µM) was added directly before
initiation of the ATP synthesis assay. Activity of ATP synthesis was
expressed as amounts of synthesized ATP per mg of
F0F1-ATP synthase per min. When the dependence
of ATP synthesis activity on Pi and ADP concentrations was
analyzed, Km and Vmax values
were calculated from a Hanes-type plot.
Assay for ATP-driven Proton Translocation-- ATP-driven proton translocation into bR-F0F1 liposomes was detected by decrease of pyranine fluorescence (20, 22) with an FP 777 fluorometer (Jasco) equipped with a stirring facility. An aliquot of 150 µl of bR-F0F1 liposomes (about 5 µg of F0F1-ATP synthase) was preincubated at 40 °C in 2 ml of the reaction buffer supplemented with 7 mM MgCl2, and a base line was monitored for 2-3 min. The reaction was started by adding 5 mM ATP. Excitation and emission wavelengths were 460 and 510 nm, respectively. Excess fluorescence from pyranine molecules not entrapped into bR-F0F1 liposomes was quenched by addition of 20 mM p-xylene-bispyridinium bromide. When the effect of azide was tested, bR-F0F1 liposomes were preincubated at 40 °C for 20 min with 5 mM sodium azide.
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RESULTS |
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ATP Synthesis by Assembled F0F1-ATP
Synthase--
Since an assay for ATP synthesis by
F0F1-ATP synthase assembled from
F0- and F1-ATPase has not been reported
previously, we have optimized the assay procedures. An attempt to
reconstitute bR-F0F1 liposomes by adding
F1-ATPase to bacteriorhodopsin-F0 proteoliposomes did not give optimal results probably because of
incomplete binding of F1-ATPase to membrane-embedded
F0. We mixed F0 and F1-ATPase and
then reconstituted proteoliposomes as described under "Experimental
Procedures." As shown in Fig. 1, the
assembled wild-type F0F1-ATP synthase catalyzed
ATP synthesis efficiently, a rate of ~90% of the authentic enzyme.
ATP synthesis by the authentic F0F1-ATP
synthase proceeded in two phases (Fig. 1, closed circles)
(22), and the assembled wild-type F0F1-ATP synthase also showed a biphasic kinetic (Fig. 1, open
circles). A first phase with a basic rate in the initial 20 min
was accelerated 1.7-1.8-fold to a second phase with a maximum rate,
and it continued for 60 min without deceleration. In agreement with the
reported values (22), the activities of ATP synthesis of the authentic F0F1-ATP synthase were 130 nmol of ATP/mg/min
(first phase, 0-5 min) and 230 nmol of ATP/mg/min (second phase,
40-60 min). The activity of the assembled
F0F1-ATP synthase was 110 nmol of ATP/mg/min (first phase) and 190 nmol of ATP/mg/min (second phase). These values
(turnover rate, 1 to ~2 ATP/sec/enzyme) were about 10-20% of the
ATP hydrolysis activity by the same F0F1-ATP
synthase in bR-F0F1-liposomes (1.5-2.5 µmol
of ATP hydrolyzed/mg/min). This relatively low activity of ATP
synthesis is attributable to the limited µH+
attainable with bacteriorhodopsin as analyzed in a previous paper (20).
ATP synthesis by the assembled
NC (Fig. 1) and the assembled wild-type F0F1-ATP synthase (Fig.
2B) was completely suppressed by addition of the uncoupler FCCP. Hereafter, we describe only the
properties of the assembled enzyme.
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ATP Synthesis in the Presence of Azide--
The NC
3
3
subcomplex is unable to catalyze
steady-state ATP hydrolysis due to rapid accumulation of the MgADP
inhibited form during catalytic turnover (16). Nonetheless, the
NC
F0F1-ATP synthase can catalyze continuous ATP
synthesis as described above, indicating that the MgADP inhibited form
may not be generated during ATP synthesis. Then, it is interesting to
test the effect of azide on ATP synthesis. As shown in Fig.
2A (closed squares), ATP synthesis by the
NC
F0F1-ATP synthase was hardly inhibited by 5 mM sodium azide (85-90 nmol of ATP/mg/min). The wild-type F0F1-ATP synthase also synthesized ATP in the
presence of 5 mM azide (Fig. 2B, open
triangles). The initial rate of ATP synthesis is nearly the same
as the value obtained for the enzyme in the absence of azide (100 nmol
of ATP/mg/min). However, ATP synthesis in the presence of 5 mM azide did not show the typical second phase, and the
acceleration was only 1.2-fold. Higher concentrations of azide up to 10 mM did not change the kinetics and rate of ATP synthesis
(data not shown).
Km for ADP and Pi--
The dependence of
rates of ATP synthesis on the concentration of ADP and Pi
by the NC F0F1-ATP synthase was almost the
same as that of the first phase of the wild-type
F0F1-ATP synthase. They show a Michaelis-Menten
type kinetics with apparent
Km(Pi), 8.9 mM
(
NC) and 6.3 mM (wild-type) (Fig.
3A), and apparent
Km(ADP), 0.30 mM (
NC) and
0.41 mM (wild-type) (Fig. 3B). Similar
dependence of the ATP synthesis rates on the concentrations of ADP and
Pi was observed for the catalysis of the second maximum
phase by the wild-type F0F1-ATP synthase except
that the apparent Vmax value was 1.7-fold of
that of the first phase. Furthermore, the first phase of ATP synthesis
in the presence of 5 mM azide by the wild-type
F0F1-ATP synthase was characterized by similar
kinetic parameters, apparent
Km(Pi), 7.5 mM
and apparent Km(ADP), 0.31 mM (Fig. 3, A and B).
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ATP-driven Proton Translocation--
The same preparation of
bR-F0F1 liposomes used for ATP synthesis assay
of the NC F0F1-ATP synthase could not
promote ATP-driven proton translocation (Fig.
4), as expected from the fact that the
NC
3
3
subcomplex could not catalyze
steady-state ATP hydrolysis (16). The bR-F0F1
liposomes prepared with the same procedures using the wild-type
F0F1-ATP synthase could translocate protons driven by ATP hydrolysis. In the presence of 5 mM azide,
proton translocation by the wild-type F0F1-ATP
synthase started at a slow initial rate, and it was stopped completely
in a couple of min. Since it has been known that ATP hydrolyzing
activity of the wild-type F1-ATPase in the presence of
azide is also subjected to turnover-dependent inactivation
(8, 15), this observation is reasonably interpreted as the result of
azide-facilitated accumulation of the MgADP inhibited form during
turnover of ATP hydrolysis.
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DISCUSSION |
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Noncatalytic Sites Are Not Essential for ATP Synthesis--
The
mutant NC F0F1-ATP synthase, which lacks the
ability to bind adenine nucleotide to noncatalytic nucleotide binding
sites, can still synthesize ATP at a rate comparable with that of the wild-type enzyme (Fig. 1). Thus, nucleotide binding to noncatalytic sites is not a prerequisite for ATP synthesis. Related to this conclusion, Weber et al. (29) observed that E. coli cells carrying mutant F0F1-ATP
synthase (
D261N/
R365W) with impaired noncatalytic sites grew well
on succinate plates. Although their mutant showed normal ATP
hydrolyzing activity and hence the growth on succinate was not
surprising, their observation is consistent with our conclusion. An
optional role of noncatalytic sites in ATP synthesis could be related
to the rigid structure of the
subunit. In the crystal structure of
mitochondrial F1-ATPase (3), noncatalytic sites on the
three
subunits are all occupied by AMP-PNP while they are all empty
in the crystal structure of the
3
3
subcomplex of thermophilic F1-ATPase (30). Nonetheless, the
structure of the
subunit of the former is very similar to that of
the
3
3 subcomplex, indicating that the
structure of the
subunit is not changed significantly by the
binding of adenine nucleotide, which is always in the "closed"
structure (3). This is in sharp contrast to the structure of
subunits which changes from "open" to "closed" form by
nucleotide binding.
ATP Synthesis Is Not a Simple Reversal of ATP Hydrolysis--
One
may raise questions from observations reported here. Why can
ATPase-inactive NC F0F1-ATP synthase be
active in ATP synthesis, and why can F0F1-ATP
synthase synthesize ATP in the presence of azide, an inhibitor of
ATPase activity? In both instances, rapid accumulation of the MgADP
inhibited form during turnover of ATP hydrolysis is responsible for the
absence of steady-state ATPase activities (8, 16). But, in the ATP
synthesis reaction, generation of the MgADP inhibited form is
apparently avoided, and therefore, the reaction kinetics are obviously
different from those in ATP hydrolysis. Continuous ATP synthesis is
measurable only in the presence of
µH+, whereas ATP
hydrolysis is usually assayed in the absence of
µH+.
The straightforward explanation of our observation is that
µH+ somehow directly blocks the generation of the
MgADP inhibited form. Another possibility is that
µH+
increases binding affinity of Pi to
F0F1-ATP synthase. This assumption has some
experimental support (31-33) and constitutes a part of the binding
change mechanism (1). Because the binding order of ADP and
Pi to F0F1-ATP synthase is random
(34), there are two pathways between
F0F1(MgADP·Pi) and
F0F1. In the absence of
µH+
(Fig. 5A), a significant
fraction of the ATP hydrolysis reaction proceeds through the pathway
F0F1(MgADP·Pi)
F0F1(MgADP)
F0F1, and the MgADP inhibited form (F0F1*(MgADP)) is
produced from F0F1(MgADP). With increased
affinity of Pi to F0F1 in the
presence of
µH+ (Fig. 5B), the reaction
pathway F0F1
F0F1(Pi)
F0F1(MgADP·Pi) becomes dominant,
and the relative population of F0F1(MgADP)
decreases. In addition, binding affinity of Pi to
F0F1(MgADP) to form
F0F1(MgADP·Pi) may also be
increased, and the population of F0F1(MgADP)
further decreases. As a consequence, generation of
F0F1*(MgADP) is suppressed.
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Acceleration of ATP Synthesis by ATP Binding to Noncatalytic Sites
Is Prevented by Azide--
The NC F0F1-ATP
synthase synthesized ATP with a linear time course at a rate
corresponding to the first phase of ATP synthesis by the wild-type
F0F1-ATP synthase (Fig. 1). This observation means that nucleotide binding to noncatalytic sites is necessary for
the shift from the first phase to the second maximum phase of ATP
synthesis and provides other evidence for the previous conclusion made
by Richard et al. (22) that the shift from the first to the
second maximum phase is triggered by the ATP binding to noncatalytic
sites. ATP synthesis by wild-type F0F1-ATP
synthase in the presence of azide obeyed a nearly monophasic time
course similar to that by the
NC F0F1-ATP
synthase (Fig. 2B). Addition of azide to the
NC
F0F1-ATP synthase did not change the kinetics, that is the effect of azide and that of deficient noncatalytic sites
are not additive. This implies that azide blocks a step in sequential
events from ATP binding to the noncatalytic site to propagation of rate
acceleration. Based on a labeling experiment during preincubation with
2-azido-[
-32P]ADP, Richard et al. (22)
suggested that the rate acceleration of ATP synthesis by ATP binding to
noncatalytic site is not related to the release of inhibitory MgADP.
Therefore, azide possibly inhibits the shift from the first phase to
the second maximum phase in a manner that is probably different from
its previously known mechanism, stabilization of the MgADP inhibited
form. Actually, because Km values of the catalysis
of the second maximum phase of the wild-type
F0F1-ATP synthase are almost identical to that
of the first phase and only Vmax was increased
(Fig. 3), rate acceleration may occur at the step
F0F1(MgADP·Pi)
F0F1(MgATP).
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ACKNOWLEDGEMENT |
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We are indebted to H. Noji and Y. Kato for productive discussion.
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FOOTNOTES |
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* 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.
Recipient of a post-doctoral fellowship from the Japan Society for
the Promotion of Science.
¶ Recipient of a fellowship from the European Union/Japan Society for the Promotion of Science.
To whom correspondence should be addressed: Research
Laboratory of Resources Utilization, R-1, Tokyo Institute of
Technology, 4259 Nagatsuta, Yokohama 226, Japan. Tel.: 81-45-924-5233;
Fax: 81-45-924-5277; E-mail: myoshida{at}res.titech.ac.jp.
1
The abbreviations used are:
µH+, transmembrane electrochemical potential of
protons; bR-F0F1 liposomes, proteoliposomes
reconstituted from bacteriorhodopsin and
F0F1-ATP synthase; FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone;
NC,
F0F1-ATP synthase or its subcomplex containing
the mutant
subunits with the four point mutations
K175A/
T176A/
D261A/
D262A; Tricine,
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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REFERENCES |
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