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INTRODUCTION |
ATP synthase, a major ATP supplier in the cell,
is a rotary machine found next to the bacterial flagella motor
in the biological world. This enzyme is composed of two motors,
F0 and F1, connected by a common rotor shaft to
exchange the energy of proton translocation and ATP
synthesis/hydrolysis through mechanical rotation. Rotation of the
isolated F1 motor driven by ATP hydrolysis was directly observed with an optical microscope, and its marvelous performance has
been revealed. The motor rotates with discrete 120° steps, each
driven by hydrolysis of one ATP molecule with nearly perfect energy
efficiency. Apparently a cooperative domain bending motion of the
catalytic
subunits initiated by ATP binding generates the torque.
In the F0 motor, which we know less about, it has been
proposed that torque may be generated by the large twist of one helix
of F0c subunits or by the change in
electrostatic forces between rigid subunits.
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ATP Synthase |
ATP synthase is a ubiquitous enzyme that is located in the inner
membranes of mitochondria, thylakoid membranes of chloroplasts, or the
plasma membranes of bacteria. As implicated by the binding change
mechanism proposed by Boyer (1), ATP synthase employs mechanical
rotation to convert the electrochemical potential energy of protons
across the membranes (
H+),
built up by respiration or a photoreaction, to the chemical energy of ATP synthesis. This enzyme is comprised of two motors sharing a common
rotor shaft (Fig. 1A). The
F1 motor, a subcomplex of the ATP synthase corresponding to
the protruding portion from the membrane, can generate rotary torque
using the energy of ATP hydrolysis (Fig. 1B). Its subunit
composition is
3
3
1
1
1
and the Mr is ~380,000. The F0
motor, a membrane-embedded subcomplex, generates torque coupled with
proton movement down (
H+) (Fig. 1C). Bacterial F0 has the simplest subunit
structure
(a1b2c10-14(?)) with an Mr of ~150,000. The eukaryotic
F0 contains several kinds of subunits. The
and
of
F1 constitute a rotor shaft and are attached to the
F0c subunits. A stator stalk, made up of
and F0b2, also connects F1
and F0 keeping the stators
(
3
3 and F0a) from
spinning with the rotor. Under physiological conditions where the
driving force for the F0 motor is larger than that for the F1 motor, the F0 motor rotates the common shaft
in its intrinsic direction so as to reverse the F1 motor
enforcing the ATP synthesis (Fig. 1A). When the driving
force for the F1 motor is larger, the F1 motor
reverses the F0 motor to pump protons to the opposite side
of a membrane.

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Fig. 1.
Schematic diagram of the ATP synthase.
A, side view of the ATP synthase. ATP synthase is composed
of the F1 and F0 motor sharing a common rotary
shaft (gray). A stator stalk connects two motors
(red) that do not slip. The F0 motor generates a
rotary torque powered by the proton flow-enforcing F1 motor
to synthesize ATP. The rotational direction is clockwise viewed from
the membrane side. B, cross-section and side view of
F1 motor. The 3 3 cylinder
hydrolyzing ATP makes an anti-clockwise rotation of the rotor part
composed of the and subunits. C, cross-section and
side view of the F0 motor. Proton flow accompanies a
clockwise rotation of the ring structure made of 10-14 copies of the c
subunit.
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Structure of F1 |
F1 can be easily and reversibly dissociated from
F0 as a soluble enzyme that only hydrolyzes ATP and is
often called F1-ATPase. The catalytic sites are mainly
located on the
subunit, but the minimum stable ATPase-active
complex is the
3
3
subcomplex (2). The
crystal structures of
3
3
of the bovine
mitochondrial F1 show that three
s and
s are
alternatively arranged in a hexamer ring forming a large central cavity
in which half of the long coiled-coil structure of
is inserted (3).
According to the recently reported structure of the
F1-F0c complex of yeast ATP synthase
(4), the other half of the coiled-coil of
extends to touch the
F0c subunits. The
subunit binds to the side
surface of the lowest part of the coiled-coil. In ATP synthase,
also has close contact with F0c. The
subunit, the last subunit whose atomic structure is not known, is
likely to sit on top of the
3
3 ring (5).
Three catalytic sites on the
s are different in nucleotide binding
states; the first is occupied by
Mg·AMP-PNP1 (an analog of
ATP), the second is occupied by Mg·ADP, and the third is empty (no
bound nucleotide); these sites are termed
T,
D, and
E, respectively. These structural
features are quite consistent with what the binding change mechanism
predicted; the three catalytic sites should be in three different
nucleotide states at a given moment, and cooperative interconversion of
the states causes the rotation of
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Observing the Rotation |
To visualize the rotation, F1 molecules from a
thermophilic bacterium (Bacillus strain PS3) were fixed on
the glass surface of a coverslip, and a large marker, a fluorescently
labeled actin filament, was attached to
(Fig.
2A) (6). Dependent on ATP, the
rotation of the actin filaments with a length of 1-4 µm at 0.2-10
revolutions per s was seen under an optical microscope. The rotation
continued for several minutes with hundreds of revolutions. The
direction of the rotation was always anti-clockwise viewed from the
F0 side, consistent with the crystal structure in which one
undergoes transition from
T to
D to
E. The F1s from Escherichia coli
(7, 8) and the chloroplast (9) are also shown to be a rotary motor by
applying the same technique. No obvious difference among the
rotations was observed. The mechanical properties of the F1
motor described below seem to be conserved among species.

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Fig. 2.
The direct observation of the
rotation in the F1 motor.
A, experimental system for the observation of the rotation using an optical microscope. The F1 motor tagged
with 10 His residues at the N terminus of the subunit was
immobilized upside down on a coverslip coated with
nickel-nitrilotriacetic acid (Ni-NTA). An actin filament
(green) labeled with fluorescent dyes and biotins was
attached to the biotinylated subunit (gray) through
streptavidin (blue). B, rotary movement of an
actin filament observed from the bottom, the membrane side, with an
epifluorescent microscope. Length from the axis to tip, 2.6 µm;
rotary rate, 0.5 revolution per s; time interval between images, 133 ms.
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One ATP Drives 120° Step Rotation |
Because of the hydrodynamic friction, at high ATP concentrations
the rotation of an actin filament is the slowest step in the catalytic
turnover. The rates of rotation of the filaments with the same length
were, therefore, leveled off above 2 µM ATP. At ATP
concentrations below 600 nM, the slowest step is the ATP binding and actin filaments showed a stepwise rotation; F1
waits for ATP at the fixed position, makes a 120° rotation upon
arrival of the ATP, and waits for the next ATP (10). Obviously, a
120° step is a reflection of the 3-fold arrangement of the catalytic
subunits in the
3
3 hexamer. The
histogram of the duration time between 120° steps obeys an
exponential function, and the estimated apparent rate constant of ATP
binding to F1 agrees well with the rate obtained in a bulk
F1 solution. This confirms that the hydrolysis of one ATP
molecule suffices for making one 120° step. Interestingly,
F1 occasionally makes a backward step as fast as forward
steps and too fast to be ascribed to a thermal fluctuation. Presumably,
the molecular machine makes a mistake in the order of ATP binding or
product release.
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Torque and Energy of Rotation |
The rotational rate became slower with an increasing filament
length because of the increased viscous friction. However, when the
rotary torque is calculated from the frictional drag coefficient and
the rotation rate, it becomes clear that the F1 motor
generates a constant torque of 40 pN·nm irrespective of the length of
the actin filament (10). If the torque is produced at the
-
interface at a radius of ~1 nm from the central axis of the
3
3 hexamer, the force would amount to 40 pN. This is the highest value among reported nucleotide-driven motor
proteins (3-5 pN for myosin/actin, 5 pN for kinesin/microtubule, and
14 pN for RNA polymerase/DNA) (11). The torque of 40 pN·nm × 2
/3 radians (120°) or 80 pN·nm is the work done in a step
against the viscous load. To define the free energy of the ATP
hydrolysis, we purposely included 10 µM ADP and 10 mM Pi in addition to 2 mM ATP and
measured the rotation rates (10). The free energy of the ATP hydrolysis
under the condition is 90 pN·nm per one ATP molecule, and the energy
for the observed rotation was 80 pN·nm per 120° step. Therefore,
F1 works with almost perfect efficiency. The high
efficiency accords with the fully reversible nature of this motor.
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Rotation without Load |
As another probe to visualize the F1 rotation, a
single fluorophore was attached to
, and its orientation was
monitored (12). With this small marker, F1 can rotate
almost without a load. Under this condition, F1 showed the
120° stepwise rotation at low ATP concentrations as seen in the
experiment using an actin filament. Furthermore, the apparent rate of
ATP binding is the same as that observed with actin filaments. This
suggests that the torque-generating step in the ATPase catalytic cycle
of F1 is not the ATP binding but step(s) after
it, including the interconversion of the
subunit that initially
accommodates ATP from the "loose" binding state to the "tight" one.
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Kinetic Framework; Bi-site or Tri-site? |
Knowledge of the exact kinetic sequence in the catalytic turnover
of F1 is the prerequisite for any models of the rotation mechanism. Three catalytic modes are recognized when F1
hydrolyzes ATP. At extremely low ATP (less than 1 nM) or a
stoichiometric amount of ATP, only one ATP binds to the first catalytic
site, and the hydrolyzed products are only released slowly (uni-site catalysis) (13). Uni-site catalysis is not inhibited by the cross-link
between
and
(14) and is probably, if not exclusively, a process
that does not couple with rotation. Binding of a second ATP to the next
catalytic site significantly promotes the release of the products at
the first site (13). The apparent Km for this
process is in the micromolar range (bi-site catalysis). Boyer's
binding change mechanism has adopted the bi-site catalysis. We observed
rotation in this ATP concentration range. It has been proposed that the
third catalytic sites bind ATP to attain the maximum hydrolysis rate
(tri-site catalysis). Actually, the ATPase activity of F1
is usually saturated above 100 µM ATP. Using the fluorescence of tryptophan introduced near the catalytic site of
E. coli F1 as the signal of nucleotide binding,
Senior's group (15) found that the change was saturated at an ATP
concentration above ~100 µM and suggested that all
three catalytic sites were filled by nucleotides. A similar observation
has been made for the thermophilic F1 (16). However, the
interpretation has been complicated by the "Mg-ADP-inhibited form,"
a state observed for F1s from any sources in which one of
the catalytic sites is stuck with a tightly bound Mg-ADP.
F1 exerting the steady state catalysis is a dynamic mixture
of the inhibited and active forms, and this equilibrium is dragged to
the active form by the Mg-ATP binding to the noncatalytic
subunit
of which the affinity is below 100 µM (17). Boyer
has raised a question of whether deviation from simple kinetics at high
ATP concentrations could be because of the Mg-ADP-inhibited form rather
than the tri-site catalysis (18). Contrary to this, a mutant whose
subunits lost the nucleotide binding ability still showed kinetics best
interpreted by tri-site catalysis (19). The current results favor the
tri-site catalysis as a physiological mode, but exclusive evidence is
still needed to settle the argument. Noticeably, no obvious shift in
the properties of the
rotation was observed from 2 µM
to 2 mM ATP where the transition from the bi- to tri-site
catalysis should occur (10).
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Bending Motion of May Drive the Rotation |
The source of energy for the
rotation is ATP hydrolysis on the
subunits. The conformation changes occurring in
during the
ATPase cycle should then be responsible for (or at least closely related to) the torque generation. In the crystal structure of the
mitochondrial F1, both
T and
D are in the closed conformation in which the C-terminal
domain is lifted to the nucleotide-binding domain (3). The
E employs the open conformation with a wide crevice
between the two domains. The crystal of the isolated
subunit takes
the open conformation,2 and
the addition of a nucleotide caused the transition from the open to
closed conformation (NMR) (20). The binding energy of ATP to the
subunit facilitates an energetically unfavorable transition from the
empty to closed conformation of the
subunit. When
in
F1 is fixed in the closed conformation by cross-linking, ATP hydrolysis stops (21). Thus,
appears to undergo a bending motion upon binding and the release of the nucleotide during catalysis. Like an automobile engine, the reciprocal motion of
in
F1 is converted to the rotary motion of
. For this to
occur, three
s in F1 coordinate the motion, pushing and
pulling the eccentric
(22). A real time recording of the motion of
the
s simultaneously with ATP hydrolysis and
rotation is a
challenge to prove the above contention. Residues playing key roles in
the torque generation have been sought by mutagenesis (23, 24).
However, the F1 motor seems fairly robust against the
mutations of the
subunit at the "hinge region" of the bending
motion (23) and the conserved "DELSEED region" that has a contact
surface with
in the closed conformation (24).
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Structure of F0 |
F0 conducts proton movement across a membrane.
F0a is embedded in the membrane by five
transmembrane helices. A dimer of F0b is
anchored to the membrane by a single transmembrane helix (25). F0c is a small hydrophobic protein with a
hairpin structure, two transmembrane helices connected by a short polar
loop (26). The F0c subunits are arranged in a
ring structure, but agreement has not been established for the number
of subunits in a ring; 10, 12, 14, and variable copies have been
proposed (4, 27-29). F0a and
F0b2 most likely exist outside of
the F0c ring. A carboxyl group located in the
middle of the C-terminal helix of F0c (glutamate in most cases but aspartate (Asp-61) in the case of E. coli)
is proven to be essential for proton translocation (30). This carboxyl group is specifically labeled with dicyclohexylcarbodiimide (DCCD), and
the labeled ATP synthase loses the activity of the ATP
hydrolysis/synthesis coupled with proton movement (31). Genetic studies
indicated that several charged residues of F0a
are also essential and assumed to be components of a putative proton
path of F0 (reviewed in Ref. 32).
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Is the F0c Ring a Rotor? |
Although the assumption is widely accepted that the
F0c ring rotates together with
and
, it
has not been proven yet by experiment. Actually, we observed the
ATP-driven rotation of the actin filaments attached to the
F0c ring of the immobilized ATP synthase (33).
However, the detergent used in the experiments impaired the integrity
of the enzyme, and the DCCD-labeled enzyme showed uninhibited rotation
and ATP hydrolysis. The F0c ring of the
detergent-impaired ATP synthase could simply rotate by being dragged by
the rotating
without regard to whether the
F0c ring works as a stator in the native enzyme.
Other groups also reported the same results using DCCD-insensitive
preparations, but they thought that the rotation of the
F0c ring was proven (34). The loss of structural
integrity of the ATP synthase in the detergent was unambiguously shown
by the structure of the yeast ATP synthase crystals grown in detergent;
the enzyme lost at least F0a and F0b2. Whether the
F0c ring is a rotor or stator will be decided by
demonstration of, for example, the DCCD-sensitive rotation of
F0c or by a clear biochemical result such as
DCCD-sensitive proton translocation by ATP synthase containing a
-
-F0c
cross-link.3
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Hints and Problems of F0 Motor |
A monomer structure of F0c in a
water-saturated organic solvent, which mimics well the native
structure, was determined by NMR (26). Using this method, a large
conformational change of F0c induced by
deprotonation of essential Asp-61 was detected; the C-terminal helix
rotates 140° as a unit with respect to the N-terminal helix, and the
conformation of the loop region between two helices significantly
changes (35). If a deprotonated F0c subunit is
sandwiched by protonated F0c subunits in the
F0c ring, the deprotonated Asp-61 comes close to
the protonated Asp-61 of the adjacent F0c
subunit, and proton transfer among the F0c
subunits and essential Arg of F0a will occur.
Although the details are unknown, the process by which the protons
drive the F0 motor may be more mechanical than a simple
rotational diffusion of the rigid F0c ring
driven by electrostatic forces.
Related to the above contention, evolutional variation of
F0c family is worth noting. Members of the
F0c family in V-type ATPases, found in membranes
of archaebacteria, some eubacteria, and inside-acidic vacuolar systems
in eukaryotic cells, are mostly a tandemly fused dimer of prototype
F0c units composed of four transmembrane helices
(reviewed in Ref. 36). Interestingly, the dimer contains only a
single essential carboxylate in the second helix. Because the ring
structure of these double-sized c subunits in V-type ATPases
is made most likely using two helices as a unit, the question arises as
to how these homologues make a rotary motion with essential
carboxylates having two times longer intervals. This places the
restraint on any models trying to explain the common function of the
ATP synthase and V-type ATPases.
The 
H+ value has two
components; the concentration difference (
pH) and the transmembrane
voltage (
). Although they are energetically equivalent, they can
be kinetically different. Each proton in F0 receives the
force by 
and can possibly drive the F0 motor. On the
contrary,
pH does not apply any force to each proton in
F0. Using the ATP synthase from Propionegenium modestum, which utilizes Na+ instead of H+
as a coupling ion, Dimroth's group (37, 38) indicated that a certain
magnitude of 
is always required for ATP synthesis even when
pNa is a major component of

Na+. They further
suggested that the ATP synthesis in the classic acid-base transition
experiment cannot exclude contribution of the induced 
(39). It
is possible to think that
subunits of F1 resist the
torque by the F0 motor as a strong spring, and therefore
only 
can wind up the strong spring of the
as quickly as observed.
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Perspectives |
ATP synthase is a rotary motor enzyme. The decisive evidence for
the F1 rotation has justified Boyer's prediction in the
past few years. This is not the goal but the start of new exciting studies. The central questions are how the motor generates force and
how the motor is regulated. Models have been proposed. However, we
think more facts are required to develop a vivid model. We know
relatively little about F0. The proton-driven
F0 motor remains a matter of unproved rational prediction.
The direct observation of proton-driven rotation in a membrane using
the lipid bilayer membrane will be a challenge but probably is not an
impossible task. Of course, more atomic structures are a prerequisite
to understanding the F1 and F0 motors.