Structure and Function of ATP synthase

ATP synthase, highly conserved among all domains of life, converts mechanical work into

chemical energy by producing ATP.

ATP synthase

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

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?

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

ATP synthase location

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?

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.

Is this ATP synthase or ATP synthetase?

The architecture of ATP synthase

ATP synthase is a large mushroom-shaped asymmetric protein complex.

The subunit composition of ATP synthase

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).

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.

The reaction catalyzed

The equation of the reaction catalyzed is

ADP3- + Pi2- + 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.

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%

HPO42− and 39% H2PO4

−).

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 )

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.

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.

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

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.

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.

F0 motor

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.

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

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

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

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

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.

• 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

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,

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.

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.