published online November 15 2000J Biol Chem 101074jbcR000021200Access the most updated version of this article at doi Alerts When a correction for this article is posted149 ID: 820756
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Hiroyuki Noji and Masasuke YoshidaThe ro
Hiroyuki Noji and Masasuke YoshidaThe rotary mechine in the cell; ATP Synthase published online November 15, 2000J. Biol. Chem. 10.1074/jbc.R000021200Access the most updated version of this article at doi: Alerts: When a correction for this article is posted• When this article is cited• to choose from all of JBC's e-mail alertsClick hereSupplemental material: http://www.jbc.org/content/suppl/2000/11/16/R000021200.DC1 by guest on November 20, 2020http://www.jbc.org/Downloaded from \f\r\f\r\b\f\r\f\r\r\f by guest on November 20, 2020http://www.jbc.org/Downloaded from \b\t\n\b \b\t by guest on November 20, 2020http://www.jbc.org/Downloaded from Figure Legends Fig. 1. Schematic diagram of the ATP synthase. A side view of the ATP synthase (a). ATP synthase is composed of F motor sharing a common rotary shaft (). A stator stalk connects two motors () that do not slip. The F motor generates a rotary torque powered by the proton-flow enforcing F motor to synthesize ATP. The rotational direction is clockwise viewed from the membrane side. A cross-section and a side view of F motor (b). cylinder hydrolyzing ATP makes an anticlockwise rotation of the rotor part composed of the subunits. A cross-section and a side view of the F motor (c). Proton-flow accompanies a clockwise rotation of the ring structure made of 10~14 copies of the c subunit. Fi
g. 2. motor. The experimental system fo
g. 2. motor. The experimental system for the observation of the optical microscope (a). The F motor tagged with 10 His residues at the N-terminus of the subunit was immobilized upside-down on a cover slip coated with Ni-NTA. An actin filament (with fluorescent dyes and biotins was attached to the biotinylated ) through streptavidin (). The rotary movement of an actin filament was observed from the bottom, the membrane side, with an epifluorescent microscope (b). Length from the axis to tip, 2.6 m; rotary rate, 0.5 r.p.s; time interval between images, 133 ms. by guest on November 20, 2020http://www.jbc.org/Downloaded from \f \r+,()11 \f/\f\b7\r&) '\f8 !"\n,& 3\f%\f F) /\f 4\n11\n\r"$ /\f3\f \r+, )$\n1@ 4\f8\f%\f\b/\f\r & \n1\f '\f8.\r1, 4\f (\r !1\n,& 4\f \r+,4\r !& 6\f\b \f7\r&1\f% \r,\f8 \n\fA\f8\f%\f '\f "1\n+ /\f \r+,\t\n11\n+ \r" 3\f\f\b/\f\n1\f:!"\f '\f 5 $'
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\n& 2\f 2 \n /\f 8&\r1D 4\f \r+,%1&+, B =\f\b\n !\n"\f\n#!$\f% &\r\f '\f*$)+,\r 8\f \f % 1 3\f 7;\n \f =\n+$\n&\r =\f / \f -$!\n,\r (\f \r+,:\r#\r1,\n 3\f%\f\b '\f8\r".+ \n -\f ?5 -\f *\r+\r. (\f &#x-390;&#x.3 0;"& \f ?@\r"&'=\n!\r \r %\f A,\r ?\f -\r+\r \n,\r *\f 4\r,\r -\f \r+,\t)&\r\n (\f\b '\f3\r$& \n G\f=\f \r+,2\n C\n+ (\f6\f\b7\r&) '\f\t \r (\f\b '\f=\r\n" 2\f \r+, \n" &! \f\b &#
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18; '\f \n" &! \f =\r\n" 2\f \r+,(\r&&! A\f\b/\f6E#\f\n1\f '\f=\r\n" 2\f \r+, \n" &! \f\b ' by guest on November 20, 2020http://www.jbc.org/Downloaded from \f2\r \n\r /\f/\f \r+,:\r#\r1,\n 3\f%\f\b/\f\n1\f:!"\f '\f4. /\f \r+,8+\n %\f6\f\b\n !\n"\f\n#!$\f% &\r\f '\f ) :\f \t &$ \f%\f \r+,%11\n$+ 4\f8\f\b\n !"\n$& '\f/\r)1& /\f(\f (\r&$)\n *\f /\r)1& \t\f(\f =\r\n.\r \r :\f ()+)5\n 6\f -$!\n,\r (\f =\r \r@\r -\f \r+,%11\n$+ 4\f8\f\b\n !"\n$&
&
'\f(\n1 " -\f(\f () \r&\r1\nC (\f\f \r+, \f \f\b\n !"\f/\f '\f(\r&$)\n *\f ()+)5\n 6\f +,\r (\f %11\n$+ 4\f8\f ) :\f \r+,-$!\n,\r (\f\b '\f-\r \n \f *D\r@\r =\f 85\n+ 7\f ?@\r.) !\n *\f -$!\n,\r (\f \r+,%5)&$) \f\b\n#!$\f/\f '\f3+ \f ) :\f 8&1D (\f8\f \r+,%11\n$+ 4\f8\f\b\n1\f:!"\f '\f4\r+ \f \r+,.3 ;$& 2\f\b7\r&) '\f(\r$\r\n5 *\f (\n&" 7\f 7;\n
;\f ()+)5\n 6\
;\f ()+)5\n 6\f -\r$),\r 3\f =\n+$\n&\r =\f \r+,-$!\n,\r (\f\b/\f6E#\f\n1\f '\f\r \r =\f 7;\n \f -\r$),\r 3\f =\n+$\n&\r =\f / \f \r+,-$!\n,\r (\f\b '\f )++ 8\f \f ( 0\r !1\n+ \f*\f \r+,3C\n+ &+ (\f\b\n !\n"\f\n#!$\f% &\r\f '\f2\n C\n+ (\f6\f \r+,\t\n11\n+ \r" 3\f\f\b\n !"\n$& '\f/+$ \f:\f \r+,\t\n11\n+ \r" 3\f\f\b/\f\n1\f:!"\f '\f81 & \f &$ ! %\f + ! 7\f%\f 6+ 1 %\f 8&\r!1. by guest on November 20, 2020http://www.jbc.org/Downloaded from \f
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; \f \f\b\n !\n"\f\n#!$\f% &\r\f '\f(\r&$)\n *\f \r+,-$!\n,\r (\f\b\n !\n"\f\n#!$\f% &\r\f '\f%. \r!\r"$ /\f \f 0$1\n %\f2\f 0)&& 3\f \r+,4\r15 /\f6\f\b7\r&) '\f8& 5 \f 0$1\n %\f2\f \r+,4\r15 /\f6\f\b '\f4\n15+$ 8\f 9!) /\f 7\r5\r\r"\r 3\f )++ 8\f \f \r+,:\r#\r1,\n 3\f%\f\b '\f7;\n \f -\r$),\r 3\f -$!\n,\r (\f \r+,=\n+$\n&\r =\f / \f\b7\r&) '\f7;\n \f \r$1 =\f /)+ 4\f
=\n+$\n&\r =\f
=\n+$\n&\r =\f / \f -$!\n,\r (\f \r+,6+ 1. !& 8\f\b\n !"\f\n#!$\f3$\f:"")+\f '\f.2 ;"& \f 8\r".+"\r&$) 7\f 8\r\n& =\f 8\r".+ \n -\f ?@\r"&'=\n!\r \r %\f -\r+\r \n,\r *\f 4\r,\r -\f \r+,\t)&\r\n (\f\b \f7\r&1\f% \r,\f8 \n\fA\f8\f%\f '\f\n$\r. \n *\f =+,! %\f \r+,-$!\n,\r (\f\b\t680&& '\f-\r$),\r 3\f 7;\n \f =\n+$\n&\r =\f / \f \r+,-$!\n,\r (\f\b:11 '\f=\n+$\n&\r =\f / \f -\r$),\r 3\f 7;\n \f ?$!\n@\r&\r 8\f \r+,-$!\n,\r (\f\b:11 '\f%,\r !\n =\f -\r$),\r 3\f 7;\n \f ?&! \f \r \r,\
r -\f -$!\n,\r
r -\f -$!\n,\r (\f \r+,=\n+$\n&\r =\f / \f\b \f7\r&1\f% \r,\f8 \n\fA\f8\f '\f2 )." :\f : $$ 3\f0\f \r+, +B$5 \f8\f\b\n1\f:!"\f ' by guest on November 20, 2020http://www.jbc.org/Downloaded from \b. It is possible to think that resists the torque by the F motor as a strong spring, and, therefore, only can wind up the strong spring of the as quickly as observed. ATP synthase is a rotary motor enzyme. The decisive evidence for the Frotation has justified Boyers 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, more facts, we feel, are required to develop a vivid model. We know relatively little about F. The proton-driven Fmotor 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 not impossible. Of course, more atomic structures are a prerequisite to understanding the Fmotors. by guest on November 20, 2020http://www.jbc.org/Downloaded from subunit is sandwiched by protonated F ring, the deprotonated Asp61 comes close to the protonated Asp61 of subunit and proton transfer among the Fessential Arg of F will occu
r. No matter if details of this speculat
r. No matter if details of this speculation are really the case, the process how the protons drive the F motor could be more mechanical than a simple rotational diffusion of the rigid Fdriven by electrostatic forces. Related to the above contention, evolutional variation of F family is worth noting. Members of the F 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 units, composed of four transmembrane helices (reviewed in Ref. (36)). Interestingly, it contains only a single essential carboxylate in the second helix. Since the ring structure of these double-sized subunits in V-type ATPases is made most likely using two helices as a unit, the question arises as to how these homologues makes 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 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 receives the force by and is possible to drive the F motor. On the contrary, pH does not apply any force to each proton in F. Using the ATP synthase from Propionegenium modestum, which utilizes Na as a coupling ion, Dimroths group indicated that a certain magnitude is always required for ATP synthesis even when pNa is a major component of (37,38). They further suggested that the ATP synthesis in the classic acid-base transition experiment cannot exclude by guest on November 20, 2020http://www.jbc.org/Downloaded from are also essential and assumed to be components (reviewed in Ref. (32)). Although the assumption is widely accepted tha
t the Ftogether with , it has been yet u
t the Ftogether with , it has been yet unproved by experiment. Actually, we observed the ATP-driven rotation of the actin filaments attached to the Fring of the immobilized ATP synthase (33). However, the detergent used in the experiments impaired the integrity of the enzyme and DCCD-labeled enzyme showed uninhibited rotation and ATP hydrolysis. The Fthe detergent-impaired ATP synthase could simply rotate by being dragged by the rotating without regard of whether the F 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 Fring was proved (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 F. Whether the F ring is a rotor or stator will be decided by demonstration of, for example, the DCCD sensitive rotation of Fbiochemical result such as DCCD sensitive proton translocation by ATP synthase containing a cross-link. Hints and problems of Fmotor A monomer structure of Fwhich well mimics the native structure, was determined by NMR (26). Using this method, a large conformational change of F induced by deprotonation of essential Asp61 was detected; the C-terminal helix rotates 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 by guest on November 20, 2020http://www.jbc.org/Downloaded from to closed conformation of the subunit. When in Fclosed-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 is converted to the rotary motion of coordin
ate the motion, pushing and pulling the
ate 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 has been sought by mutagensis (23,24). However, the F motor seems fairly robust against the mutations of the of the bending motion (23) DELSEED regionthe closed-conformation (24). Structure of F conducts proton movement across a membrane. F is embedded in the membrane by five transmembrane helices. A dimer of Fthe membrane by a single transmembrane helix (25). F is a small hydrophobic protein with a hairpin structure, two transmembrane helices connected by a short polar loop (26). The Fring 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)]. F most likely exist outside of the F ring. A carboxyl group located in the middle of the C-terminal helix of F(glutamate in most cases but aspartate (Asp61) in case of E. colito 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 by guest on November 20, 2020http://www.jbc.org/Downloaded from concentration above ~100 M and suggested that all of three catalytic sites were filled by nucleotides (15). A similar observation has been made for the thermophilic F(16). However, the interpretation has been complicated by the ADPMg inhibited-forms from any sources in which one of the catalytic sites is stuck with a tightly bound ADPMg. Fexerting the steady state catalysis is a dynamic mixture of the inhibited and active forms, and this equilibrium is drag
ged to the active form by the ATPMg bind
ged to the active form by the ATPMg binding to the noncatalytic subunit of which the affinity is in the order of 100 M (17). Boyer has raised a question whether deviation from simple kinetics at high ATP concentrations could be due to the ADPMg 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 rotation was observed from 2 M to 2 mM ATP where the transition from the bi- to tri-site catalysis should occur (10). Bending motion of \b\bmay drive the rotationThe 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, or at least closely related, to the torque generation. In the crystal structure of the mitochondrial F, both are in the closed-conformation in which the C-terminal domain is lifted to the nucleotide-binding domain (3). The employs the open-conformation with a wide crevice between the two domains. The crystal of the isolated subunit takes the open conformation (Miki and Yoshida, unpublished result) 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 by guest on November 20, 2020http://www.jbc.org/Downloaded from As another probe to visualize the F rotation, a single fluorophore and its orientation was monitored (12). With this small marker, F can rotate almost without a load. Under this condition, Fshowed the 120 step-wise rotation at
low ATP concentrations as seen in the e
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 Fis not the ATP binding but step(s) it, including the interconversion of subunit that initially accommodates ATP from the one. Kinetic framework; bi-site or tri-site? Knowledge of the exact kinetic sequence in the catalytic turnover of Fthe prerequisite for any models of the rotation mechanism. Three catalytic modes are recognized when F 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 released only slowly (uni-site catalysis) (13). Uni-site catalysis is not inhibited by the cross-link , 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 Kcatalysis). Boyers 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 Fusually saturated above 100 M ATP. Using the fluorescence of tryptophan introduced near catalytic site of E. coli as signal of nucleotide binding, s group found that the change was saturated at an ATP by guest on November 20, 2020http://www.jbc.org/Downloaded from obeys an exponetial function, and the estimated apparent rate constant of ATP binding to F agrees well with the rate obtained in a bulk F solution. This confirms that the hydrolysis of one ATP molecule suffices for making step. Int
erestingly, F occasionally makes a backw
erestingly, F 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. Torque and energy of rotation The rotational rate became slower with an increasing filament length due to 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 F motor generates a constant torque of 40 nm irrespective of the length of the actin filament (10). If the torque is interface at a radius of ~1nm from the central axis of 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 pNnm times 2nm is the work done in a step against the viscous load. To define the free energy of the ATP hydrolysis, we purposely included 10 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 nm per one ATP molecule and the energy for the observed rotation was nm per 120 step. Therefore, F works with almost perfect efficiency. The high efficiency accords with the fully reversible nature of this motor. by guest on November 20, 2020http://www.jbc.org/Downloaded from predicted; the three catalytic sites should be in three different nucleotide states at a given moment and cooperative interconversion of the states Observing the rotation To visualize the rotation, F molecules from a thermophilic bacterium Bacilluslarge marker, a fluorescently labeled actin filament, was attached to \r\b. Dependent on ATP, the rotation of the actin filaments with a length of 1
~4 m at 0.2-10 revolutions per sec were
~4 m at 0.2-10 revolutions per sec were seen under an optical microscope. The rotation continued for several min, with hundreds of revolutions. The direction of the rotation was always anticlockwise viewed from the F side, consistent with the crystal structure in which one undergoes transition from \b. The Fs from (7,8) and the chloroplast (9) are also shown to be a rotary motor by applying the same technique. No obvious difference among the was observed. The mechanical properties of the F motor described below seem to be conserved among species.One ATP drives 120 step rotation At high ATP concentrations, due to the hydrodynamic friction, 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 M ATP. At ATP concentrations below 600 nM, the slowest step is the ATP binding and actin filaments showed a step-wise waits for ATP at the fixed position, makes a 120arrival of the ATP, and wait for the next ATP (10). Obviously, a 120is a reflection of the threefold arrangement of the catalytic hexamer. The histogram of the duration time between 120 by guest on November 20, 2020http://www.jbc.org/Downloaded from has the simplest subunit structure 10~14(?) with an Mr of ~150 k. The Eukaryotic F contains several kinds of subunits. The subunits. A stator stalk, made up of keeping the stators () from spinning with the rotor. Under physiological conditions motor is larger than that for the F motor, motor rotates the common shaft in its intrinsic direction so as to motor enforcing the ATP synthesis (Fig 1a). When the motor is larger, the F motor reverses the F motor to pump protons to the opposite side of a membrane. can be easily and reversibly dissociated from F as a soluble enzyme that only hydrolyzes ATP and
is often called FTheatalytic sites are
is often called FTheatalytic sites are mainly located on the subunit but the minimum stable ATPase-active complex is the subcomplex (2). The Crystal structures of the bovine mitochondrial F’s and’s are alternatively arranged in a hexamer ring forming a large central cavity in which half of is inserted (3). According to the recently reported structure of the F complex of yeast ATP synthase (4), the other half of the coiled-coil of subunits. The subunit binds to the side surface of the lowest part of the coiled-coil. In ATP synthase, also has a close contact with F. The subunit, the last subunit whose atomic structure is not known, is likely to sit on top of the ring (5). Three catalytic sites on the ’s are different in nucleotide-binding states; the first is occupied by Mgof ATP), the second by MgADP, and the third is empty (no bound nucleotide), and are termed , and , respectively. These structural features are quite consistent with what the binding-change mechanism by guest on November 20, 2020http://www.jbc.org/Downloaded from ATP synthase, a major ATP supplier in the cell, is a rotary machine found second to the bacterial flagella motor in the biological world. This enzyme is composed of two motors, F, connected by a common rotor shaft to exchange the energy of proton translocation and ATP synthesis/hydrolysis through mechanical rotation. Rotation of the isolated 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 F motor, which we know less about, it has been proposed tha
t torque may be generated by the large t
t torque may be generated by the large twist of one helix of F subunits or by the change in electrostatic forces between rigid subunits. 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 P. Boyer (1), ATP synthase employs mechanical rotation to convert the electrochemical potential energy of protons across the membranes (), built up by respiration or a photo-reaction, to chemical energy of the ATP synthesis. This enzyme is comprised of two motors sharing a common rotor shaft (Fig. 1a). The F motor, a subcomplex of the ATP synthase corresponding to the protruding portion from the membrane, can generate rotary torque using the energy of the ATP hydrolysis (Fig. 1b). Its subunit composition is and the Mr is ~380 k. The F motor, a membrane-embedded subcomplex, generates torque coupled with proton movement down by guest on November 20, 2020http://www.jbc.org/Downloaded from Classification: Minireview The rotary machine in the cell; ATP synthase Hiroyuki Noji & Masasuke YoshidaCREST (Core Research for Evolutional Science and Technology) ProgrammingTeikyo University Biotechnology Research Center 3F, Nogawa 907, Miyamae-ku, Research Laboratory of Resource Utilization, Tokyo Institute of Technology, Yokohama 226-8503, Japan To whom correspondence should be addressed: Masasuke Yoshida Research Laboratory of Resource Utilization, Tokyo Institute of Technology, Yokohama 226-8503, Japan Tel. +81 45 924 5232; Fax. +81 45 924 5277 E-mail: myoshida@res.titech.ac.jp Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.JBC Papers in Press. Published on November 15, 2000 as Manuscript R000021200 by guest on November 20, 2020http://www.jbc.org/Down