Function
The ATP synthase enzymes have been remarkably conserved through evolution. The bacterial enzymes are essentially the same in structure and function as those from mitochondria of animals, plants and fungi, and the chloroplasts of plants. The early ancestory of the enzyme is seen in the fact that the Archaea have an enzyme which is clearly closely related, but has significant differences from the Eubacterial branch. The H+-ATP-ase found in vacuoles of the eukaryote cell cytoplasm is similar to the archaeal enzyme, and is thought to reflect the origin from an archaeal ancestor.
In most systems, the ATP synthase sits in the membrane (the "coupling" membrane), and catalyses the synthesis of ATP from ADP and phosphate driven by a flux of protons across the membrane down the proton gradient generated by electron transfer. The flux goes from the protochemically positive (P) side (high proton electrochemical potential) to the protochemically negative (N) side. The reaction catalyzed by ATP synthase is fully reversible, so ATP hydrolysis generates a proton gradient by a reversal of this flux. In some bacteria, the main function is to operate in the ATP hydrolysis direction, using ATP generated by fermentative metabolism to provide a proton gradient to drive substrate accumulation, and maintain ionic balance.
In mitochondria, the P side is the intermembrane space, and the N side the mitochondrial matrix; in bacteria, the P side is the outside (the periplasm in gram negative bacteria), the N side the cytoplasm; in chloroplasts, the P side is the lumen and the N side the stroma.
Subunit composition of the ATP synthase
There are minor differences between bacteria, mitochondria and chloroplasts in some of the smaller subunits, which leads to a confusing nomenclature. The simplest system is that from E. coli. The ATP synthase can be dissociated into two fractions by relatively mild salt treatments.
A soluble portion, the F1 ATP-ase, contains 5 subunits, in a stoichiometry of 3a:3b:1g:1d:1e. Three substrate binding sites are in the b-subunits. Additional adenine nucleotide binding site in the a-subunits are regulatory. The F1 portion catalyzes ATP hydrolysis, but not ATP-synthesis.
Dissociation of the the F1 ATP-ase from the membranes of bacteria or organelles leaves behind a membrane embedded portion called FO. This consists (in E. coli) of three subunits a, b and c, with relative stoichiometries of 1:2:9-12. The c-subunit is very hydrophobic, and forms a helix turn helix structure which spans the membrane twice, with a hydrophilic loop on the side of attachment of F1. There is a conserved acidic residue half-way across the membrane in the C-terminal helix.
After dissociation, the membranes are permeable to protons. The proton leak can be stopped by addition of inhibitors, which are also inhibitors of ATP synthesis in the functional complex. Two "classical" inhibtors are commonly used. Oligomycin binds at the interface between Fo and F1; dicyclohexylcarbodiimide (DCCD) binds covalently to the conserved acidic residue in the c-subunit of Fo. One DCCD per ATP-ase is sufficient to block turn-over, suggesting a cooperative mechanism. The action of these inhibitors indicates that the proton permeability of the Fo is a part of its functional mechanism.
The proton leak can be plugged, and a functional ATP synthase can be reconstituted, by adding back the F1 portion to membranes containing the Fo portion.
This image of the complete complex, using image averaging and cryo-electron microscopy, and the model derived from it, showing a second stalk, are from Rod Capaldi's homepage.
Structure of the F1 ATP-ase
The structure of the soluble (F1) portion of the ATP synthase from beef heart mitochondria has been solved by X-ray crystallography. The pictures below are from Abrahams, J.P., Leslie, A.G., Lutter, R. and Walker, J.E. (1994) Structure at 2.8 Å resolution of F1-ATPase from bovine heart mitochondria. Nature 370, 621-628.
The protein was crystallized in the presence of ADP, and an ATP analogue, AMP-PNP, in which the the two terminal phosphates of ATP were replaced by the non-hydrolysible imidodiphosphate group. The three a-subunits each contained an AMP-PNP. The three b-subunits contained either ADP (bDP), AMP-PNP (bTP), or no nucleotide (bE).
Click on images for large version.
Left: The structure of the F1 ATP-ase, viewed from the side. The a-subunits are shown in yellow, theb-subunits in red, and the g-subunit in blue. The cartoon at the top left shows the orientation. Note that the a- b-subunits alternate in a ring around the g-subunit, which forms a rod up the middle. The a- and b-subunits are differentiated by subscript indicating the occupancy of the active site of the b-subunit of each a- b-pair: E - empty; DP - ADP; TP - ATP analogue, AMP-PNP. Scale bar is 20 Å.
Right: A vertical slice through the complex across the a-TP/b-DP diagonal highlighted in the cartoon.
Left: A vertical slice through the complex across the a-E/b-TP diagonal highlighted in the cartoon.
Right: A vertical slice through the complex across the a-DP/b-E diagonal highlighted in the cartoon.
Note how the "jaw" of the nutcracker swings open when the site is empty (arrow in picture on right).
View of the complex from the top (N-phase).
Left: A horizontal slice through the complex across the top, showing the b-sheet structure which provides a cap over the catalytic domain. Scale bar is 20 Å.
Right: A horizontal slice through the complex across the catalytic domain, which is predominantly helical.
Left: View of the calculated electrostatic surface potential of the a, b-sleeve formed by the structure below the b-sheet cap, showing regions of negative (red) and positive charge, and a predominantly neutral (hydrophobic) "hole" in the dough-nut, through which the top part of the g-subunit protrudes. The view is from inside the protein.
Right: Similar surface, but viewed from the side, of the g-subunit, showing a hydrophobic surface for most of the rod, but a marked neatively charged polar region half-way down. The upper part of the rod slides into the sleeve shown in the Fig. to the left, as indicated in cross-section by the ball-and-stick structure.
Cross-section through the structure showing surfaces, and highlighting the fit of the g-subunit in the a, b-ring. Also shown is the location of the bound ATP analogue (AMP-PNP) in the bTP-subunit. Note how the bulge, introduced in the g-subunit by the horizontal helix, abutts against the bTP-subunit, and forces a change in conformation. It is suggested that rotation of the g-subunit in the a, b-ring induces conformational changes in successive a, b-pairs so as to bring about the binding changes expected from the binding-change mechnism (see below).
Mechanism of the F1 ATP-ase
The ATP synthase operates through a mechanism in which the three active sites undergo a change in binding affinity for the reactants of the ATP-ase reaction, ATP, ADP and phosphate, as originally predicted by Paul Boyer. The change in affinity accompanies a change in the position of the g-subunit relative to the a, b-ring, which involves a rotation of the one relative to the other. In the direction of ATP synthesis, the rotation is driven by a flux of H+ down the proton gradient, through a coupling between the g-subunit, and the c-subunit of FO. This rotation has now been demonstrated experimentally.
Click here for some nice animation movies of the F1 ATPase mechanism in action, by Hongyun Wang and George Oster, University of California, Berkeley.
Two of these movies are available locally.
A perspective view of a3, b3, g in cartoon display with stereo on.
A top view of a3, b3, g in cartoon display.
Experimental support for rotational model
Biophysical approach
This rotational motion has been captured in dramatic videos from the laboratory of Masasuke Yoshida. In this work, the F1-ATPase was tethered to a glass surface by the b-subunit, using a His-tag engineered into the protein at the N-terminus, and NTA-ligand on the glass (see illustration from Junge et al. TIBS article, below).
The motion was detected by attaching an actin filament to the g-subunit, which was tagged with fluorescent groups to make it visible, and recorded using a video camera attached to a microscope. The motion was seen only under conditions of ATP-hydrolysis, and the direction of motion was always counter-clockwise when viewed from the Fo portion, giving the sign of the catalytic mechanism.
Hiroyuki Noji, Ryohei Yasuda, Masasuke Yoshida & Kazuhiko Kinosita Jr. (1997) Direct observation of the rotation of F1-ATPase. Nature, 386, 299 - 302.
An alternative approach using photometric methods has been explored in Wolfgang Junge's lab. Use of small chromophores attached directly to the g-subunit has the advantage of a time resolution unconstrained by the large torque associated with the movement of the actin filament above. Two methods have been used to explore the dynamics of the system.
In [1], the authors used photoselection experiments with a small chromophore (eg. eosin, see above) attached to the g-subunit of the active F1-ATPase, and observed the relaxation of polarization anisotropy on activation of turnover. The behavior was compatible with that expected for a three-stepped rotatory device. In [2], they extended their kinetic analysis of the rotation, aided by a new theory for assessing continuous versus stepped, and Brownian versus unidirectional molecular motion. The observed relaxation of the absorption anisotropy was fully compatible with a unidirectional and stepping rotation of g over three equidistantly spaced angular positions in the hexagon formed by the alternating subunits a and b. The results strongly supported a rotational catalysis with equal participation of all three catalytic sites.
In [3], polarized confocal fluorometry (POCOF) was applied to single molecules of engineered, immobilized and load-free spinach-CF1 ATP-ase, and was used to investigate transition states of the rotatory drive. Hydrolysis of ATP caused the stepped and sequential progression of subunit g through three discrete angular positions, with the transition states of g being too shortlived for detection. The authors also observed the stepped motion of e, whereas d, a and b subunits were immobile.
Reference [4] is a brief and readable review of this work.
- Sabbert D. Junge W. (1997) Stepped versus continuous rotatory motors at the molecular scale. Proc. Natl. Acad. Sci. (U.S.) 94, 2312-2317.
- Sabbert D. Engelbrecht S. Junge W. (1997) Functional and idling rotatory motion within F-1-ATPase. Proc. Natl. Acad. Sci. (U.S.) 94, 4401-4405.
- Hasler K. Engelbrecht S. Junge W. (1998) Three-stepped rotation of subunits gamma and epsilon in single molecules of F1-ATPase revealed by polarized confocal fluorometry. FEBS Lett. 426, 301-304.
- Junge W. Lill H. Engelbrecht S. ATP synthase - an electrochemical transucer with rotatory mechanics. TIBS 22, 420-423.
Siggi Engelbrecht's homepage for some nice images, and for pdb files of homology models for spinach F1-Atp-ase (with models of d and e subunits, and E. coli F1, F0-ATPase (with models of d, e subunits, and c and b subunits from F0).
Biochemical approach
(The pictures below are from Duncan, T.M., Bulygin, V.V., Zhou, Y., Hutcheon, M.L. and Cross, R.L. (1995) Rotation of subunits during catalysis by E. coli F1-ATPase. Proc. Natl. Acad. Sci., USA 92, 10964-10968.)
In the above cartoon showing the binding-change mechanism of Paul Boyer, rotation of the g-subunit (yellow) relative to the a, b-ring (the three a, b-pairs are represented by different shades of green or blue) induces a change in the binding affinities of reactants, as represented here by a change in the conformation of the site on going from left to right in the diagram. In step 2, ATP forms spontaneously from tightly bound ADP and Pi. The mechanism was proposed before the structure was known, so the structure provides a nice confirmation of the model. The Open site corresponds to the Empty site of the structure, the Tight site to the ATP site, and the Loose site to the ADP site.
Experimental evidence for the model comes from an extensive history of research:
- Detailed measurement of isotopic exchanges of 32P between ATP, ADP and inorganic phosphate, and of 18O between H2O and ATP originally led Boyer to suggest that the mechanism involved an energy-linked change in affinity for reactants.
- Experiments on the occupancy of the catalytic site showed that the ATP-hydrolysis reaction was poised with ADP and ATP at a ratio close to 1.
- In experiments in which the enzyme was allowed to hydrolyse ATP in a reaction mixture with the [ATP] substantially lower than [enzyme], it was found by Penefsky that the rate of reaction was very slow, and that the kinetics and binding constants of partial reactions could be readily measured. Under these conditions (uni-site catalysis), turnover is constrained to a single site on each F1, and the normal cooperative cycle cannot occur. The slowed reaction kinetics made it possible to construct the following thermodynamic cycle of reactions, in which the DGo' values (or equilibrium constants) for unmeasured partial reactions could be calculated from measured values. This confirmed that the main changes in free energy in the reaction were associated with the binding and unbinding of reactants, rather than the hydrolysis of ATP.
Equilibrium (K) and kinetic (k) constants for hydrolysis of ATP by F1 under uni-site turnover conditions. Values for some of the constants are:
k1 = 6.4 x 106 M-1sec-1
k-1 = 7 x 10-6 sec-1
K1 = ~1012 M-1
k2 = 12 sec-1
K2 = 0.5
k3 = 2.7 x 10-3 sec-1
k4 = 3.6 x 10-4 sec-1
k-4 = 1.3 x 103 M-1sec-1
K4 = 0.3 x 10-6 M
K'4 = 80 x 10-6 M
K = 3.6 x 105 M
- In similar experiments in which the [ATP] was varied, the rate accelerated rapidly as the [ATP] approached that of the enzyme, suggesting that cooperation between several sites was necessary for rapid hydrolysis.
- Since the structure has become available, a lot of nice work has been done to test the rotational model discussed above. Among the most convincing experiments are those from the paper above, which are shown schematically in the figure below.
The experiments depend on the fact that cysteine can often be substituted into a protein in place of other amino acids in the sequence without the mutation effecting the function. When two cysteines are close enough together in a structure, addition of an oxidant (Aox below) will induce formation of a disulphide (R-S-S-R) cystine bond between them. The bond can be broken, and the cysteines reformed, by addition of a reducing agent.
Aox + 2 R-SH <==> AH2 + R-S-S-R
In addition, the F1 could be reversibly dissociated into subunits without breaking the disulphide bond, so that the b-subunit could be removed and added back.
In these experiments, a cysteine introduced into the g-subunit (C87S) and a cysteine introduced in place of D380 of the b-subunit by site-directed mutations (D380C), were used to set up the system so that a cross-bridge could be formed between g and b-subunits. Since there is only one g per complex, only one of the three potential cross-bridges was formed in each F1. It had previously been shown that formation of the cross-bridge inactivated the enzyme.
Side by side experiments were done with unlabelled and 35S-isotopically labelled F1-ATP-ase.
- DTNB was added as oxidant to form an -S-S- bond
- The F1 was dissociated into subunits, and the unlabelled mixture was mixed in a 1:1 molar ratio with labelled mixture.
On reassociation, mixed complexes were formed in which unlabelled g-S-S-b bridged pairs were in association with labelled unbridged g and b-subunits.
- The mixture was reduced to break the bridges, ATP was added to induce turn-over, and the bridges were reformed by adding oxidizing agent.
When the unbridged and bridged subunits were separated, it was found that new bridges had been formed between unlabelled b-subunits, and labelled g-subunits in the ratio expected for a rotational mechanism. Control experiments ± ATP, Mg2+, etc., showed that the rotation indicated by the transfer of the disulphide bond required turnover of the enzyme.
Cartoon showing the two parts of the ATP synthase, with a rotation of g-subunit driven by coupling to a "motor" consisting of the c-subunits of FO. The c-subunits form a complex which moves in the membrane with respect to the a-subunit of FO. The idea suggested by Wolgang Junge (click here to see a model ) is that the a-subunit provides a port for entry of protons from the P-phase, and a port for exit to the N-phase. When a proton enters through the P-phase port, it neutralizes the conserved acidic residue in the helical hairpin of the c-subunit. Only in this neutral form (animation from Hongyun Wang's Home Page) can the c-subunit rotate away from association with the a-subunit. Rotation brings a neutral c-subunit to the exit port, allowing it to lose the proton, and associate with the a-subunit complex. Successive protonations allow the c-subunit complex to rotate by 1/n x 360o for each proton, where n is the stoichiometry of the c-subunit per ATP synthase (9-12). Because a complete rotation drives ATP synthesis at each of the 3 catalytic sites, 3 or 4 H+ are required for each ATP,- the stoichiometry found. DCCD (see above) blocks the mechanism by acting as a covalent "spanner", jamming the works when bound to any single c-subunit.
Click here for an animation of the complete mechanism.
The attachment to FO.
Experiments from Capaldi's lab, using engineered placement of cysteine residues to explore the neighborliness of subunits through formation of disulphide bridges, suggest that the b-subunits, together with the d-subunit of F1 form a stator, attached near the "top" of a b-subunit, which prevents the a, b-ring from moving. The e-subunit can be attached to g-, c-, a- or b-subunits. Presumably it changes its attachment to the a, b-ring, in order to allow the rotation with respect to g which is now an established part of the mechanism.
A cartoon showing the arrangement of the subunits which join the F1 section to the FO section.
Note that the stalk of the ATP synthase has now become two stalks, one central, composed of the e- and g-subunits, linked to the c-subunit complex, and the other peripheral, composed of the d- and b-subunits. Why is this second stalk not seen in electron microscopy images? Capaldi suggests that the reason reflects the averaging which is necessary to get high quality images. Symetrical structures like the a, b-ring and the central stalk will contribute to the average, but asymetric structures like the peripheral stalk will be "averaged out" of the image, unless special care is taken to select images with such a feature in a fixed orientation.
©Copyright 1996,
Antony Crofts, University of Illinois at Urbana-Champaign,
a-crofts@uiuc.edu
