ATP SYNTHASE STRUCTURE ATP SYNTHESIS.pptx

ATP Synthesis
• Mechanism of ATP Synthesis
• Substrate level phosphorylation
• Chemiosmotic mechanism (oxidative and photophosphorylation)
• ATP Synthase
• Boyer’s conformational model
• Racker’s experiment
• Jagendorf’s experiment
• Role of uncouplers
10-04-2024 ATP SYNTHASE CHARU KALRA

Mechanism of ATP Synthesis
1. Oxidative phosphorylation-It is the chemiosmotic
synthesis of ATP associated with the transfer of
electrons through the electron transport chain
(from NADH / FADH 2 to O 2 by a series of electron
carriers) and the accompanying consumption of
oxygen. ATP is formed as a result of the transfer of
electrons This process, which takes place in
mitochondria, is the major source of ATP in
aerobic organisms.
2. Photophosphorylation- The light-dependent
chemiosmotic synthesis of ATPin the chloroplast
in the presence of light. ATP is formed as a result
of the transfer of electrons which takes place in
chloroplast, is the major source of ATP in
photosynthetic organisms
3. Substrate level phosphorylation-ATP synthesis by
the transfer from a high-energy compound
(phosphate group) ADP, without involvement of
any electron transport is called substrate-level
phosphorylation.

Photophosphorylation
Light-dependent ATPsynthesis- coupled reaction
• This process was discovered by Daniel Arnon and co-workers in
1950s
• Electron flow without accompanying phosphorylation is said to
be uncoupled.
• Photophosphorylation works via the chemiosmotic mechanism,
first proposed in the 1960s by Peter Mitchell.
• The total energy available for ATP synthesis, called the proton
motive force (∆p), is the sum of a proton chemical potential and
a transmembrane electric potential, ∆p = ∆E − 59(pHi − pHο)
• A transmembrane pH difference of 1 pH unit is equivalent to a
membrane potential of 59 mV.

Substrate-level phosphorylation
• ATP synthesis by the transfer from a high-energy compound
(phosphate group) ADP, without involvement of any electron
transport is called substrate-level phosphorylation.
• In simple terms, it is the production of ATP by the transfer of a
phosphoryl group from the substrate of a reaction to ADP.
• Substrate-level phosphorylation differs from the other two ways of
ATP synthesis i.e., oxidative-phosphorylation and photo-
phosphorylation, in that the ATP synthesis is not coupled to any
electron transport.
• Oxidative-phosphorylation and photo-phosphorylation involves ATP
synthesis coupled with electron transport.

2 Steps in Glycolysis where substrate
level phosphorylation occurs

• In the first reaction, energy is harvested in the form
of ATP. The enzyme phosphoglycerate kinase
catalyzes the transfer of the phosphoryl group of
1,3-bisphosphoglycerate to ADP. This is the first
substrate-level phosphorylation of glycolysis, and
it produces ATP and 3-phosphoglycerate. It is a
coupled reaction in which the high energy bond is
hydrolyzed and the energy released is used to drive
the synthesis of ATP.
• The final substrate-level phosphorylation in the
pathway is catalyzed by pyruvate kinase where
Phosphoenolpyruvate is coverted to Pyruvate.
Phosphoenolpyruvate serves as a donor of the
phosphoryl group that is transferred to ADP to
produce ATP. This is another coupled reaction in
which hydrolysis of the phosphoester bond in
phosphoenolpyruvate provides energy for the
formation of the phospho-anhydride bond of ATP.

ATP synthase
• The ATP is synthesized by a large (400 kDa) enzyme complex known by several names: ATP
synthase, ATP ase (afterthe reverse reaction of ATP hydrolysis), and CFo–C1 (Boyer,1997).
• This enzyme consists of two parts: a hydrophobic membrane-bound portion called CFo and
a portion that sticks out into the stroma called CF1.
• CF1 is the portion of the complex that synthesizes ATP. CF1 is made up of several peptides,
including three copies of each of the αand βpeptides arranged alternately much like the
sections of an orange. Whereas the catalytic sites are located largely on the βpolypeptide,
many of the other peptides are thought to have primarily regulatory functions.
• CFo appears to form a channel across the membrane through which protons can pass.
• The molecular structure of the mitochondrial ATP synthase has been determined by X-ray
crystallography (Stock et al. 1999). Although there are significant differences between the
chloroplast and mitochondrial enzymes, they have the same overall architecture and
probably nearly identical catalytic sites. In fact, there are remarkable similarities in the way
electron flow is coupled to proton translocation in chloroplasts, mitochondria, and purple
bacteria .
• Another remarkable aspect of the mechanism of the ATP synthase is that the internal stalk
and probably much of the CFo portion of the enzyme rotate during catalysis (Yasuda et al.
2001). The enzyme is actually a tiny molecular moto

Structure of ATP synthase

ATP synthase- A tiny molecular motor
ATPase or CFo–CF1
• CF1 is the portion of the complex that
synthesizes ATP.
• CFo appears to form a channel across the
membrane through which protons can pass.
• It is a large multi-subunit complex, CF1, attached
on the stromal side of the membrane to an
integral membrane portion, known as CFo.
• CF1 consists of five different polypeptides, with
a stoichiometry of α3, β3, γ, δ, ε. Cfo contains
probably four different polypeptides, with a
stoichiometry of a, b, b‘, c12.

• Model of the FoF1-ATPase, showing the attachment of the
catalytic complex to the membrane via the β subunit and the
δ subunit.
• When the reaction runs in reverse (ATP synthesis), protons
diffuse through the Fo complex down their electrochemical
gradient. The movement of protons through the channel
drives the rotation of the entire Fo complex within the
membrane.
• The Y subunit, which is attached to the Fo complex, then
turns within the catalytic complex, causing the
conformational changes that are required for ATP synthesis.
• It is assumed that the catalytic complex itself does not
rotate, but is anchored to the membrane. The δ subunit is
located on the outside of the β subunit and serves as the site
of attachment of the β subunit, which anchors the catalytic
complex to the membrane and prevents it from spinning.
• In mechanical terms, the F1 complex and its membrane
anchor act as a stationary housing, or “stator,” while Y
subunit (and possibly the Fo complex) serves as the “rotor.”
(From Junge et al. 1997.)

Uncouplers / Protonophores
• Uncouplers are amphiphilic compounds(which are soluble both in water and lipids). They are
agents with conjugated double bonds which allow them to diffuse across the membrane in
both the protonated form and the unprotonated form, and thus dissipate the electrochemical
proton gradient.
• Uncouplers which transfer protons across the membrane are known as protonophores.
• They disrupts phosphorylation by dissociating the reactions of ATP synthesis from the electron
transport chain. They directly bypasses the ATP synthase by allowing passive proton influx,
without affecting electron flow, but ATP synthesis does not occur.
• The result is that the cell or mitochondrion expends energy to generate a proton motive force,
but the proton motive force is dissipated before the ATP synthase can recapture this energy and
use it to make ATP.
• Uncouplers increases the proton permeability of the inner mitochondrial membrane and
dissipates the proton gradient. Uncouplers are capable of transporting protons through
mitochondrial and chloroplast membranes.
• Both mammalian and plant mitochondria contain uncoupling protein (UCP). This protein
facilitates the movement of protons across the inner membrane and therefore partially
uncouples electron transport and decreases the ATP yield of respiration. Electron flow without
accompanying phosphorylation is said to be uncoupled.
• Uncoupling proteins (UCPs) occur in the inner mitochondrial membrane and dissipate the
proton gradient across this membrane that is normally used for ATP synthesis.

Role of uncoupling agents
• Addition of uncouplers results in continuation of electron transport and
proton pumping, without generation of any proton gradient. ATP synthesis
does not occur without affecting uptake of oxygen. In the absence of
proton gradient, however, protons are transported in reverse direction
through ATP synthase at the expense of ATP. Protonated DNP (a weak acid)
diffuses from high proton concentration side of the membrane to low
proton concentration side where it gets dissociated to generate protons
resulting in dissipation of proton gradient. Membrane is permeable to both
protonated and anionic forms of these.
• E.g. FCCP (trifluoromethoxy carbonyl cyanide phenylhydrazone), a very
efficient mitochondrial uncoupler. Other exampes of uncouplers- Carbonyl
cyanide phenylhydrazone (CCP) 2,4-dinitrophenol (DNP), Carbonyl
cyanide m-chlorophenyl hydrazine (CCCP)

Dr. Roshni Rajamohan
The mechanism of action of uncouplers
• (B) In the presence of an electrochemical proton gradient across the
membrane, FCCP will become protonated and thus pick up a proton on
the positive side of the membrane, move across the membrane in the
neutral form, and lose the proton on the negative side. This is driven by
the electrochemical proton gradient. In the negative form, FCCP will be
driven back out across the membrane by the membrane potential. The
net result is the movement of one proton from the positive to the
negative side of the membrane, which results in the dissipation of the
electrochemical proton gradient, and the movement of one electrical
charge from the negative to the positive side of the membrane, which
results in the dissipation of the electrical gradient. The shaded area
represents the system of conjugated double bonds (u-orbital system).
(Modified from Nicholls and Ferguson 1992.)
(A) The protonation / deprotonation
of FCCP which is a weak acid.

CHEMIOSMOTIC THEORY
• Peter Mitchell in 1961 proposed CHEMIOSMOTIC THEORY
• Nobel Prize for Chemistry in 1978
• Membrane potential with high negative charges and positive charges operating on the
opposite surfaces of the membranes can generate energy rich bond between ADP and
Pi to synthesize ATPs.
• The basic principle of chemiosmosis is that ion concentration differences and electric-
potential differences across membranes are a source of free energy that can be utilized
by the cell
• Early evidence supporting a chemi-osmotic mechanism of photosynthetic ATP formation
was provided by an experiment carried out by André Jagendorf and co-workers.

Jagendorf’s Experiment
• Andre T. Jogendorf & Earnest Uribe Placed
chloroplasts extracted from cells in darkness,
thereby eliminating light absorption & electron
transfer as a source of energy for
photosynthesis.
• In the dark, thylakoids were first incubated in a
medium of pH 4 until both the exterior and
interior of the vesicles had ph4.
• Then, the thylakoid vesicles were quickly
transferred to a medium with pH 8. At this
point, there was a pH gradient, with the
interior of the thylakoid (pH 4) having a higher
H+ concentration than the exterior (pH 8).
• When ADP was added, ATP was made, even in
the dark. This is convincing evidence linking a
pH gradient to ATP synthesis.

Efraim Racker
• Efraim Racker- identified and purified
Factor-1 (F1), the first part of the ATP
synthase enzyme to be characterised.
• F1 is only a part of a larger ATP synthase
complex. It is a peripheral membrane
protein attached to component Fo, which is
integral to the membrane.
• Racker was able to confirm
Mitchell's hypothesis that contrary
Peter D.
to
popular opinion, ATP synthesis was not
coupled to respiration through a high-
energy intermediate but instead by a
transmembrane proton gradient.

Can a proton gradient drive the synthesis of ATP?
Peter Mitchell’s hypothesis that a proton gradient can drive the
synthesis of ATP was proposed before experimental evidence
supported it and was therefore met with skepticism. In the 1970s,
biochemist Efraim Racker and his collaborator Walther Stoeckenius
tested the hypothesis.
RACKER’S EXPERIMENT Racker and Stoeckenius built an artificial
system consisting of a membrane, a bacterial proton pump activated by
light, and ATP synthase.They measured the concentration of protons in
the external medium and the amount of ATP produced in the presence
and absence of light.
RESULTS In the presence of light, the concentration of protons
increased inside the vesicles, suggesting that protons were taken up by
the vesicles.
In the dark, the concentration of protons returned to the starting level.
ATP was generated in the light, but not in the dark.
INTERPRETATION In the presence of light, the proton pump was
activated and protons were pumped to one side of the membrane,
leading to the formation of a proton gradient. The proton gradient, in
RACKER’S EXPERIMENT
CONCLUSION: A membrane, proton
gradient, and ATP synthase are sufficient
to synthesize ATP. This result provided
experimental evidence for Mitchell’s
hypothesis.

Boyer’s conformational model
(Binding change model)
Our understanding of how ATP is synthesized was advanced by Paul Boyer (1997) at the
University of California, Los Angeles, who proposed the binding-change mechanism for
catalysis by F-ATPases. From the detailed understanding of how the ATPases function, he
proposed that the binding-change mechanism for ATP synthesis contains three important
components:
1. The major energy-requiring step is not the synthesis of ATP from ADP and Pi, but the
release of ATP from the enzyme.
2. Substrate is bound and products are released at three separate but interacting
catalytic sites, corresponding to the three catalytic subunits (β subunits).
3. Each catalytic site can exist in one of three conformations: tight, loose, or open.
The binding changes are coupled to proton transport by rotation of the γ subunit. That is,
the flow of protons down their electrochemical gradient through the Fo complex causes
the γ subunit to rotate.
Rotation of the γ subunit then brings about the conformational changes in the catalytic
complex that allow the release of ATP from the enzyme, and the reaction is driven
forward. The reverse occurs when the enzyme functions as an ATP-driven proton pump

Binding change model
• In this model, the F1 subunit exists in three states:
1. an O - open - state with very low affinity for
substrates and has no catalytic activity;
2. a L - loose - state with low affinity for substrates and
also no catalytic activity, and
3. a T - tight - tight state with high affinity for substrates
and with catalytic activity.
• The F1 subunit consist of three α and three β subunits,
which can cycle between three conformations, bind
substrate, and have catalytic activity.
• The collapse of the proton gradient (i.e. the proton-
motive force) causes the γ subunit to rotate like a
crankshaft relative to the F1 subunit, forcing the β
subunit to change conformation from the T to the O
(releasing ATP) and then the L (binding ADP and Pi).
• The γ subunit does not appear to undergo any
significant conformational change on ATP hydrolysis as
evidenced by tritium exchange studies of amide
protons.
Top view

The binding-change mechanism as seen from the top of the F1 complex. There are three catalytic sites
in three different conformations: loose, open, and tight. (For clarity, only the three β subunits are
shown.) Substrate (ADP + Pi) initially binds to the open site and is converted to ATP at the tight site. In
step 1, rotation of the γ subunit causes a conformational change, resulting in a change in the formation
of the sites. As a result, ATP is released from the enzyme. In step 2, substrate again binds to the open
site, and another ATP is synthesized at the tight site. (After Duncan et al. 1995.)
http://6e.plantphys.net/topic12.04.html

The model of Boyer's binding change mechanism. The α and β
subunits configure three nucleotide binding sites: O, which provides the
early binding site for ADP and inorganic phosphate, L, to which the ADP
and inorganic phosphate bind after migrating from O, and T, which tightly
binds ATP. Energy ensuing from the movement of protons from the
chloroplast lumen to the stroma drives the rotation of the Y subunit of CF1,
and the interconversion of the binding sites and the release of an ATP
molecule.

Rotary motion of the Y subunit
• To visualize the rotation of the enzyme, Masasuke Yoshida
and his colleagues at the Tokyo Institute of Technology
attached an actin filament labeled with a fluorescent dye to
the base of the γ subunit using another protein as a "glue."
• They then attached the F1 complex upside down to a glass
surface. If the γ subunit rotates with respect to the catalytic
complex, the actin filament should swing around with it.
Since the filament is very long compared to the ATP synthase
(about 1 μm), its rotation should be visible in a fluorescence
microscope.
• In other words, the fluorescently tagged actin filament,
which is large enough to visualize in a light microscope,
reports the rotation of the γ subunit.
• When ATP was added to the modified enzyme, the actin
filaments were seen to swing around in a circle at as much as
4 revolutions per second in a fluorescence microscope .
Demonstration of the rotary motion of the γ subunit made it
possible to put together a model of how the ATP synthase
works

The γ subunit is inserted like a shaft through the center of the
catalytic complex, which consists of three α subunits and
three β subunits arranged alternately in a doughnut-like
structure. The interface between the γ subunit and the α and
β subunits is highly hydrophobic. The hydrophobicity of the
interface minimizes the interactions between the subunits,
consistent with the rotation of the γ subunit within the hole
formed by the catalytic complex. In other words, the γ
subunit looks like a molecular bearing lubricated by a
hydrophobic interface.
A method for visualizing rotation of the γ subunit. A
fluorescently labeled actin filament was attached to one
protruding end of the γ subunit. The F1 complex was then
attached upside down to a coverslip. When ATP was added to
the coverslip, the actin filament rotated. (After Noji et al.
1997.)
Sequential images of the rotating actin filament attached to the Y
subunit, as viewed in a fluorescence microscope. There is 133 ms
between the images and the rotation rate is 0.5 r.p.s. (From Noji et al.
1997, courtesy of S. Noji.)
http://docencia.izt.uam.mx/docencia/alva/atpaseyoshida.htm.

Paul D. Boyer
Proposed passage of protons through
the channel of the basal unit makes
the stalk & headpiece spin like a top
just as the flow of water makes a
water wheel turn. The turn cycles
each of 3
headpiece.
catalytic sites on the
Through sequential
conformational changes that pickup
ADP & phosphate, combine them, &
release the ATP product. Nobel prize
w/ walker for research into the
mechanisms by which ATP synthase
makes ATP.
John E. Walker
Used X-ray diffraction to create a
3 dimensional
synthase that
picture of ATP
clearly verified
Boyers model by showing the
different
head in
positions as
rotational
ATP synthesis
proceeds. Nobel prize w/ Boyer
for research into the mechanisms
by which ATP synthase makes
ATP
For their contributions to elucidation of the mechanism of ATP synthesis, Paul Boyer and John
Walker shared half the Nobel prize in physiology or medicine in 1997. The other half went to
Jens Skou for his pioneering work on the K+,Na+-ATP synthase, the mammalian counterpart
of the plant plasma membrane H+-ATP synthase

Suggested Reading:
1. Taiz, L. and Zeiger, E. (2010) Plant Physiology. 5th Edition, Sinauer
Associates.
2. William G. Hopkins, Norman P
. A. Hüner (2008) Introduction to
Plant Physiology, 4th Edition, Wiley.
3. Bhatla, Satish C, Lal Manju A. (2018) Plant Physiology, Development
and Metabolism, Springer.

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