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Lecture 10

ATP synthase

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.
ADP + Pi + nH+P <=> ATP + nH+N
Because the structures seen in EM, the subunit composition, and the sequences of the subunits
appeared to be so similar, it had been assumed that the mechanisms, and hence the
stoichiometries, would be the same. In this context, the evidence suggesting that the
stoichiometry of H+/ATP (n above) varied depending on system was surprising. Values based on
measure ATP/2e- ratios, and H+/2e- ratios had suggested that n was 3 for mitochondria, and 4 for
chloroplasts, but these values were based on the assumption of integer stoichiometries. Although
all the F1F0-type ATP-synthases likely had a common origin, both the assumption that the
stoichiometries are the same, and that n is integer, are called into question by emerging structural
data (see below).
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
3α :3β :1γ :1δ :1ε . Three substrate binding sites are in the β -subunits. Additional adenine
nucleotide binding site in the α -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 E. coli complex, using image averaging and cryo-electron
microscopy, and the model derived from it, showing a second stalk, are from Rod Capaldi's
homepage. (Note: the lettering of the subunits differs in ATP synthases from different sources.)
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.
Currently available structures for ATP-synthase subunits.
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 α -subunits each contained an AMP-PNP. The three β -subunits contained
either ADP (β DP), AMP-PNP (β TP), or no nucleotide (β E).

Click on images for large version.

Left: The structure of the F1 ATP-ase, viewed from the side. The α -subunits are shown in
yellow, theβ -subunits in red, and the γ -subunit in blue. The cartoon at the top left shows the
orientation. Note that the α - β -subunits alternate in a ring around the γ -subunit, which forms
a rod up the middle. The α - and β -subunits are differentiated by subscript indicating the
occupancy of the active site of the β -subunit of each α - β -pair: E - empty; DP - ADP; TP -
ATP analogue, AMP-PNP. Scale bar is 20 Å.
Right: A vertical slice through the complex across the α -TP/β -DP diagonal highlighted in the

Left: A vertical slice through the complex across the α -E/β -TP diagonal highlighted in the
Right: A vertical slice through the complex across the α -DP/β -E diagonal highlighted in the
Note how the "jaw" of the nutcracker swings open when the site is empty (arrow in picture on
View of the complex from the membrane (looking out towards the N-phase).

Left: A horizontal slice through the complex across the top, showing the β -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 α , β -sleeve formed by the
structure below the β -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
γ -subunit protrudes. The view is from inside the protein.
Right: Similar surface, but viewed from the side, of the γ -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 γ -subunit in
the α , β -ring. Also shown is the location of the bound ATP analogue (AMP-PNP) in the β TP-
subunit. Note how the bulge, introduced in the γ -subunit by the horizontal helix, abutts against
the β TP-subunit, and forces a change in conformation. It is suggested that rotation of the γ -
subunit in the α , β -ring induces conformational changes in successive α , β -pairs so as to
bring about the binding changes expected from the binding-change mechnism (see below).
The F1-ATP ase in a Chime tutorial. (Tutorial allows exploration of files 1bmf (original
Abrahams et al. structure), and 1e79 (structure with DCCD, γ more completely resolved,
and δ and ε subunits included).)
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 γ -subunit relative to the α , β -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 γ -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 α 3, β 3, γ in cartoon display with stereo on.
A top view of α 3, β 3, γ 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 β -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 γ -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 γ -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 γ -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 γ over three equidistantly spaced
angular positions in the hexagon formed by the alternating subunits α and β . 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
γ through three discrete angular positions, with the transition states of γ being too shortlived
for detection. The authors also observed the stepped motion of ε , whereas δ , α and β
subunits were immobile.
Reference [4] is a brief and readable review of this work.
1. Sabbert D. Junge W. (1997) Stepped versus continuous rotatory motors at the molecular
scale. Proc. Natl. Acad. Sci. (U.S.) 94, 2312-2317.
2. 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.
3. 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.
4. 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 δ and ε subunits, and E. coli F1-ATPase.
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 γ -
subunit (yellow) relative to the α , β -ring (the three α , β -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 ∆ Go' 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 β -subunit could be removed and added back.
In these experiments, a cysteine introduced into the γ -subunit (C87S) and a cysteine
introduced in place of D380 of the β -subunit by site-directed mutations (D380C), were
used to set up the system so that a cross-bridge could be formed between γ and β -
subunits. Since there is only one γ 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
S-isotopically labelled F1-ATP-ase.
1. DTNB was added as oxidant to form an -S-S- bond
2. 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 γ -S-S-β
bridged pairs were in association with labelled unbridged γ and β -subunits.
3. 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 β -subunits, and labelled γ -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

Cartoon showing the two parts of the ATP synthase, with a rotation of γ -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 δ -subunit of F1 form a stator, attached near the
"top" of a β -subunit, which prevents the α , β -ring from moving. The ε -subunit can
be attached to γ −, c-, α − or β -subunits. Presumably it changes its attachment to the α ,
β -ring, in order to allow the rotation with respect to γ 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
Note that the stalk of the ATP synthase has now become two stalks, one central,
composed of the ε − and γ −subunits, linked to the c-subunit complex, and the other
peripheral, composed of the δ − 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 α , β -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.
Evolution of the F1 ATPase
Structure of the yeast F1FO complex
Walker and colleagues have recently solved a structure from crystals containing a more
complete ATP-synthase complex from yeast mitochondria. Although the protein
contained a full complement of subunits, some of these dissociated on crystallization, and
only the c-subunit of F0 was retained. Nevertheless, the model shows the organization of
the proteolipid, DCCD-binding subunits (corresponding to the c-subunits of E. coli).
These are arranged in a ring, as expeced from the Junge mechanism.
Abstract: Adenosine triphosphate (ATP) synthase contains a rotary motor involved in
biological energy conversion. Its membrane-embedded F0 sector has a rotation generator
fueled by the proton-motive force, which provides the energy required for the synthesis
of ATP by the F1 domain. An electron density map obtained from crystals of a
subcomplex of yeast mitochondrial ATP synthase shows a ring of 10 c subunits. Each c
subunit forms an α -helical hairpin. The interhelical loops of six to seven of the c
subunits are in close contact with the g and d subunits of the central stalk. The extensive
contact between the c ring and the stalk suggests that they may rotate as an ensemble
during catalysis.
A brief Chime tutorial on the yeast F1F0structure, based on a Cα -backbone model.
However, a major surprise comes from a count, which shows 10 subunits. In a rotatory
mechanism with integer stoichiometry for H+/ATP, it had been expected that the number
of c-subunits would be divisible by 3, the stoichiometry of α , β -pairs in F1, to give
either 9 (for n=3) or 12 (for n=4).
Another surprise has come from the work by Norbert Dencher and Andreas Engel who
have used atomic force microscopy (AFM) to study the structure of the subunits
equivalent to the c-subunit from chloroplast F0 (subunit-III) reconstituted into protein
arrays, which self-organize into ring structures. Here the count of c-subunits in a ring is
Legend: Subunit-III oligomers of chloroplast ATP synthase visualized in 25 mM MgCl2,
10 mM Tris-HCl, pH 7.8, at room temperature using atomic force microscopy
(Nanoscope III, Digital Instruments)11. Top, the distinct wide and narrow rings represent
the two surfaces of the subunit-IIIx oligomer; middle, wide oligomer ends, showing 14
subunits-III; bottom, narrow oligomer ends. The full grey-level range of these topographs
was 2 nm.
Taken at face value, these two sets of data suggest that:
4. The stoichiometry of H+/ATP is not fixed, but varies with system (as had been
suggested by the different stoichiometries found for chloroplast and mitochondria
by conventional measurements).
5. The stoichiometry is not integral.
It will be interesting to follow the further development of this area. The most recent
results from AFM studies by Engel's group in collaboration with Dimroth suggest that at
least one bacterial species has an intermediate c-subunit count, - 11 c-subunits in the F0
ring of Ilyobacter tartaricus.
Stock, D., Leslie, A.G.W. and Walker, J.E. (1999) Molecular architecture of the rotary
motor in ATP synthase. Science 286, 1700-1705
Seelert, H., Poetsch, A., Dencher, N.A., Engel, A., Stahlberg, H. and M�ller, D.J. (2000)
Proton-powered turbine of a plant motor. Nature (Lond.) 405, 418-419
Stahlberg, H., Muller, D.J., Suda, K., Fotiadis, D., Engel, A., Meier, T., Matthey, U. and
Dimroth, P. (2001) Bacterial Na+-ATP synthase has an undecameric rotor. EMBO
Reports 2, 229-233

©Copyright 1996, Antony Crofts, University of Illinois at Urbana-Champaign,