ATP synthase is an enzyme that generates ATP from ADP and inorganic phosphate using energy from the proton gradient across the inner mitochondrial membrane. It consists of two main parts - F0, which forms a channel for protons to pass through, and F1, which contains the catalytic sites to synthesize ATP. The passage of protons through F0 powers the rotation of F1, which facilitates the formation of ATP from ADP and phosphate at three catalytic binding sites. The overall reaction catalyzed by ATP synthase couples proton translocation across the membrane to ATP synthesis, and is essential for energy production in cells.

1. Structure and Function of ATP synthase

2. ATP synthase, highly conserved among all domains of life, converts mechanical work into
chemical energy by producing ATP.
ATP synthase

3. It is said that ATP synthase is the oldest enzymes on
Earth, which appeared even before photosynthetic or
respiratory enzyme machinery.
It is the primary source of ATP generation in almost all
living species on Earth.
In humans, ~50 kg of ATP is generated on daily basis
and is subsequently used to provide energy for various
biochemical reactions, including DNA and protein
synthesis, muscle contraction, transport of nutrients and
neural activity, etc.
In plants and photosynthetic bacteria ATP synthase is
essential for solar energy conversion and carbon
fixation.
Introduction

4. Glycolysis: 2 ATP
Krebs Cycle: 2 ATP
Electron Transport Phosphorylation: 32 ATP
Net Energy Production: 36 ATP (38 ATP in Plants)
Overview of cellular respiration process
How many ATPs are generated in respiration process?

5. What is the basic requirement of glycolysis to proceed to ETC and phosphorylation?

6. ATP synthase location

7. The primary function of ATP synthase in most organisms is ATP synthesis.
Physiological role of ATP synthase
However, in some cases the reverse reaction, i.e. transmembrane proton pumping powered by
ATP hydrolysis is more important. A typical example: anaerobic bacteria produce ATP by
fermentation and ATP synthase uses ATP to generate proton motive force necessary for ion
transport and flagella motility.
Many bacteria can live both from fermentation and respiration or photosynthesis. In such case
ATP synthase functions in both ways.
What is the physiological role(s) of ATP synthase?

8. An important issue is to control ATP-driven proton pumping activity of ATP synthase in order
to avoid wasteful ATP hydrolysis under conditions when no proton motive force can be
generated (e.g. leaky damaged membrane, etc.).
Regulation of ATP hydrolysis
In plants (chloroplasts), where it is necessary to preserve ATP pool through the whole night,
the inhibition is very strong: the enzyme hardly has any ATPase activity.
In contrast, in anaerobic bacteria where ATP synthase is the main generator of proton motive
force, such inhibition is very weak.
Should the ATP hydrolysis process be regulated?
Mitochondrial ATP synthase is somewhere in between.

9. Is this ATP synthase or ATP synthetase?

10. The architecture of ATP synthase
ATP synthase is a large mushroom-shaped asymmetric protein complex.

11. The subunit composition of ATP synthase

12. The catalytic portion of ATP synthase (F1) is formed by α3β3 hexamer with γ subunit inside it
and ε attached to the γ. Subunit δ is bound to the "top" of the hexamer and to subunits b.
The hydrophobic transmembrane segment of subunit b is in contact with subunit a. Subunits
γ and ε of the catalytic domain are bound to the ring-shaped oligomer of c-subunits.
Proton translocation take place at the interface of subunits a and c.
The simplest bacterial enzyme is composed of 8 subunit types.
Five of them (α, β, γ, δ, ε) form the catalytic hydrophilic F1-portion-the "cap" of the
mushroom).
The proton translocating Fo portion is composed of subunits of three types (a, b and c).

13. The composition of ATP synthetases
Subunits in F1 region Eukaryotic (bovine) α3β3γδε
α 509 aa, 55164 Da
β 480, 51595
γ 272, 30141
δ 190, 20967
ε 146, 15652
Subunits in Fo region Prokaryotic (E. coli) a,b2,c9-14
a 271, 30285
b 156, 17202
c 79, 8264
The total mass of the F1Fo
ATPase from bovine
mitochondria is ~450 kDa with
the F1 unit having a mass of
~370 kDa.
The catalytic sites are found to
be on β subunit having
sequence 149Gly-Gly-Ala-Gly-
Val-Gly-Lys-Thr-Ala157.

14. The reaction catalyzed
The equation of the reaction catalyzed is
ADP3- + Pi
2- + nH+
P <=> ATP4- + H2O + (n-1)H+
N ( pH > 7.2 )
The "P" and "N" indices denote the Positively and the Negatively charged sides of the
coupling membrane.

15. ATP synthesis and pH dependency
12.67pKaPOHHPO
7.21pKaHPOHPOH
2.12pKaPOHHPOH
3
4
2
4
2
442
42
4
3
For a neutral pH as in the cytosol (pH=7.0)
So only H2PO4
− and HPO4
2− ions are present in significant amounts (62% and 38%
respectively). Note that in the extracellular fluid (pH=7.4), this proportion is inverted (61%
HPO4
2− and 39% H2PO4
−).

16. The pK value for Pi2- + H+ <=> Pi
- is 7.2, while the corresponding pK values for phosphate
in ADP and ATP are close to 6.9.
This means that in the pH interval of 6.9-7.2 the prevailing reaction will not include trapping
of protons:
ADP3- + Pi
- + nH+
P <=> ATP4- + H2O + nH+
N ( pH 6.9-7.2 )
However, below pH = 6.9, the prevailing reaction is again accompanied by proton trapping:
ADP2- + Pi
- + nH+
P <=> ATP3- + H2O + (n-1)H+
N ( pH < 6.9 )

17. Driving force for ATP synthesis catalyzed by ATP synthase
ATP synthesis catalyzed by ATP synthase is powered by the transmembrane electrochemical
proton potential difference, composed of two components: the chemical and the electrical one.

18. Rotary Motors
ATP synthesis is composed of two rotary motors
1. F0: an electric motor
2. F1: a chemical motor
The two motors are connected together by a stator
so that when F0 turns, F1 turns too.

19. So why have two motors connected together?
So that one motor can force the other motor to turn and change the motor into a generator. In
cells, the F0 motor uses the power from a proton gradient to force the F1 motor to generate
ATP.
Motor to generator

20. How many catalytic site does the enzyme have?
The answer is three.
However, the total number of the nucleotide-binding sites
is six, three of them being non-catalytic.
Each site is located on the interface between subunits α
and β.
Larger part of each catalytic site is composed from amino
acid residues of the respective β-subunit, while each non-
catalytic site is situated mostly on the respective α
subunit.

21. The role of the non-catalytic sites is probably regulatory, they are not necessary for the
catalysis.
Occupation of the non-catalytic sites by nucleotides was shown to increase the enzyme
activity.
It is also possible that binding of nucleotides to the non-catalytic sites facilitate the enzyme
assembly in the cell.
In Bacillus genera, there is strong evidence that the ε subunit also can bind one nucleotide, so
there are 7 nucleotide binding sites in these bacteria.

22. F0 motor

23. Proton translocation through FO
Although the Fo portion of the ATP synthase is often referred to as "proton(ic) channel", it is
NOT a channel.
The transfer rate is too slow for a channel (at voltage of 100 mV, a rate of about 106 ions per
second for an ion channel, more than 100-fold higher than the maximal corresponding values
reported for FO portion).
It differs significantly from ‘real’ proton
channels (e.g. gramicidin, M2 from influenza
virus, etc.).
Importantly, being in conducting state, a membrane channel does not require conformational
changes for proton translocation, while FO portion of ATP synthase does.

24. The synthesis of ATP requires the binding of ADP and phosphate, the formation of the new
phosphate-phosphate bond and release of ATP.
F1 motor

25. Models of the rotation by movement of ions through the Fo domain of ATP synthase

26. Each circle represents the chemical state of the catalytic sites on the b-subunit. The red arrow represents the angular position of the g-
subunit. O, C0 and C indicate the open, half-closed and closed forms, respectively. The green catalytic site retains the bound nucleotide as
ATP until the g-subunit rotates 200 from the binding angle (0). At 200, the catalytic site hydrolyses ATP into ADP and Pi, each of which is
released at 240 and 320, respectively. The conformation of the b-subunit changes from open to closed upon ATP binding and remains in the
closed form until the g-subunit rotates 240. At 240, this b-subunit moves to the half-closed form, and then it returns to the open form with
accompanying rotation of the g-subunit.
Mechanochemical coupling scheme of F1

27. 1. F1+ATP ↔ F1·ATP
2. F1·ATP ↔ F1·ADP·Pi
3. F1·ADP·Pi ↔ F1’·ADP·Pi
4. F1’·ADP·Pi ↔ F1’·ADP+Pi
5. F1’·ADP ↔ F1+ADP

28. Inhibitors of ATP synthase
Oligomycin
Dicyclohexylcarbodiimide
• Binds at the interface of
subunits a and c.
• Can also block F1 portion.
• Specific for mitochondrial ATP
synthase.
• Binds covalently with the
protonated carboxyl groups.
• At pH > 8, it reacts with
conserved acidic amino acids of
subunit c.
• It is irreversible and universal
inhibitor.
• At pH < 7, it modifies several
carboxyl groups in F1 portion.

29. • Binds at the interface of
subunits a and c.
• Inhibitor fro mitochondrial,
bacterial and chloroplast ATP
synthases.
• Binds at the cleft between α and β of
F1.
• Specifically inhibits chloroplast ATP
synthases.
• No effect on bacterial and
mitochondrial enzyme.
Tentoxin

30. From the early experiments:
H+/ATP ratio for ATP synthesis: estimated to be 3 (mitochondria)
4 (chloroplast).
Proton/ATP ratio
Because the energy required for ATP synthesis under physiological conditions is about 50 kJ
mol-1 (~520 meV), so at physiological proton motive force values (~120-200 mV), at least 3
protons should be transferred to get the energy necessary.
From the thermodynamic considerations:
< 3 protons per ATP is hardly feasible,

31. This ratio is expected to depend on the number of c-subunits in the FO: as there are 3 catalytic
sites on the enzyme. Thus,
H+/ATP = (number of c-subunits) / 3
It is also possible that c-subunit stoichiometry varies depending on the situation in the cell.
However, the experimentally determined numbers of the c-subunits in ATP synthases from
different organisms are 10, 11, 14 and 15, suggesting ratios of 3.33, 3.67, 4.67 and 5,
respectively.

32. How fast is ATP synthase?
What is the values of "micromoles of ATP per minute per mg protein" i.e. the number of ATP
molecules synthesized (or hydrolyzed) by one ATP synthase in one second.
In the living cell the enzyme most probably operates below the maximal possible rate, making
tens of ATP molecules per second.
For uncoupled or solubilized enzyme rates over 100 per second are also reported.
Maximal rates over 100 per second are reported for bacterial, mitochondrial and chloroplast
enzymes for ATP synthesis.