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Suraj Prakash Sharma | Ekta Chotia



Let's Talk about the electron-transport chain cooperation with the ATP synthetase to bring about oxidative phosphorylation of ADP to ATP.

There are 3 principal hypothesis have been put forward to explain the coupling of oxidation and phosphorylation. These hypothesis explains how the energy transfers between electron transport and ATP synthesis takes place.

1.            Chemical Coupling Hypothesis

It is the oldest hypothes is it proposes that electron transport is coupled to ATP synthesis. During the electron transport chain high energy covalent intermediate are  formed and subsequently the covalent bond is cleaved and the energy released in this process is utilized to make ATP.

2.            Conformational Coupling Hypothesis

In the excess of ADP the inner membrane of mitochondria pulls away from the outer membrane and assumes a “condensed state”. The energy released during the transport of electrons causes the conformational changes in mitochondria and it assumes an energy rich condensed state.

In the absence of ADP, the mitochondria have the normal structure or the “swollen state”, in which the cristae project into the large matrix.

3.            Chemiosmotic Coupling Hypothesis

A novel mechanism of ATP generation was postulated by Peter Mitchell, a British biochemist, in 1961. He proposed that electron transport and ATP synthesis are coupled by a proton gradient, rather than by a covalent high-energy intermediate or an activated protein.

As the high-energy electrons from the hydrogens of NADH and FADH2, are transported down the respiratory chain in the mitochondrial inner membrane, the energy released as they pass from one carrier molecule to the next. This released energy is used to pump protons (H+) from the matrix side (M-side) to the  cytoplasmic side (C-side) of inner mitochondrial membrane. Pumping of H+ creates electrochemical gradient across the inner mitochondrial membrane. This gradient is consists of a chemical gradient  (difference in H+ ion concentration) and a voltage gradient or potential, which becomes positive on the cytoplasmic side.

When the H+ ions are ejected by electron transport, flow back from the peri mitochondrial space to the matrix through a specific H+ channel present in the FOF1  ATP synthase molecule ,  ATP is formed.

The model requires that the electron carriers in the respiratory chain and the ATP synthase be anisotropically (= vectorially) organized, i.e., they must be oriented with respect to the two faces of the coupling membrane (= inner mitochondrial membrane).

For ATP formation the inner mitochondrial membrane must be intact. If the inner mitochondrial membrane is not in intact form than an H+ gradient across the inner membrane could not exist.  A ‘leak’ of proton across the membrane is induced by uncouplers. This is resulted into  discharge of  the proton gradient and consequently energy-coupling would fail.

Here the ATP production is coupled with the transport (Osmosis) of a chemical (H+) therefore named as Chemiosmotic Coupling.

Evidences in Favour

Mitchell’s hypothesis is that oxidation and phosphorylation are coupled by a proton gradient is supported by many evidences :

  • No hypothetical ‘high-energy’ intermediates have been found to date.
  • Oxidative phosphorylation requires a sealed  compartment. i.e.,  an intact inner mitochondrial membrane  is able to generate ATP.
  • Breaks or holes( can be produce by sonication) in the inner membrane do not allow oxidative Phosphorylation, although electron transport from substrates to oxygen may still continue.  The damaged  membrane is not able to produce ATP.
  • Both the electron transport system  and the ATPase are vectorially organized in the inner mitochondrial  membrane.
  • A proton gradient across the mitochondrial inner membrane is generated due to the electron transport.
  • The pH of mitochondrial matrix is 1.4 units higher than perimitochondrial space and the value of electric potential (voltage gradient) is 0.14 V, the outside being positive.

The total electrochemical potential(gradient) \DeltaV (in volts) consists of a membrane potential contribution and an H+ concentration-gradient contribution (pH). It is given as

Taking R as the gas constant, T as the absolute temperature and F as the caloric equivalent of Faraday. This total proton-motive force of 0.224 V corresponds to a free energy of 5.2 kcal per mole of protons.

  • Mitochondria or chloroplasts are able to produce ATP in the absence of electron transport. When a pH gradient is imposed on it.
  • Oxidative phosphorylation can be checked by uncouplers and certain ionophores. Uncouplers such as 2, 4-dinitrophenol increase the permeability of mitochondria to protons, thus reducing the electrochemical potential and short-circuiting the vectorial ATP synthetase system for the production of ATP.
  • Addition of acid to the external medium, establishing a proton gradient, leads to the synthesis of ATP.

16.1.      Stucture of ATP synthase

ATP synthase is also named as F1-F0 ATPase  because of its subunits are named as F0 and F1. F0 is the water insoluble transmembrane protein whereas F1 is the water soluble peripheral protein which is easily dissociated from the membrane by treating with urea. F1 component is composed of \alpha3\beta3g\varsigmae.

Here the b subunit is responsible for the catalysis of ATP synthesis. A and b subunits are arranged alternatively in a circular form. F0 component is consists of three transmembrane subunits a, b and c. Which are 1, 1 and 12 in number respectively. C subunit have aspartate at its active site which binds with H+ ions and transports them across the membrane by the proton half channels.

Oligomycin is an inhibitor of proton translocation in ATP synthase, putatively binding the Fo subunits a and c. In normally respiring mitochondria, Δψm is high. The value is -150 to -180mV. This value favors ATP production. However when  the mitochondrial respiration rate decrease the Δψm falls below a threshold, F1-FO ATP synthase  can operate in reverse process. This cause hydrolysis of ATP to pump protons through the membrane. However an inhibitor protein, IF1 inhibits mitochondrial F1-ATPase activity in a pH-dependent manner. The IF1 acts as a homodimer and  binds to subunits β and γ of ATP synthase.

The active form predominates at pH values<6.5. The IF1 conserves ATP at the expense of Δψm, which has been shown to be protective to cells during ischemia.

16.2.      Process of ATP synthesis

The main catalysing subunit is P subunit. The rotation of c subunit of F0 subunit causes the different rotating position of the a-b subunit which works as a rotary engine and forms ATP.

The a-b subunit have three orientation-


These are chemicals which dissipate the proton gradient across the inner cell membrane which allows the electron transport but not the ATP generation. Protons regularly move with the transportation of electrones so cell faces an increase in H+ ions which lowers the pH of the cell and makes is more acidic. Some example of uncoupler is Dinitrophenol (DNP) and thermogenin.

16.3.      Roles Of Electron Transport Energy

The main function of electron transport in mitochondria is to provide energy for the synthesis of ATP during oxidative phosphorylation. But the energy generated during electron transport is also used for other biological purpose which are listed below :

16.3.1.   Heat Production

The proton gradient generated by electron transport can be used to generate heat. Heat in human infants and some hibernating animals is produced by brown fat. For example, human infants, other mammals born hairless and some hibernating animals have a special type of brown fat in the neck and upper back.

The brown fat is so named because it contains profuse mitochondria which contain red-brown cytochromes. The brown fat mitochondria has special proton channels in their inner membrane proton to flow back to matrix. These specialized  brown-fat mitochondria do not usually produce ATP, rather they dissipate the free energy of electron transport as heat in order to maintain the body temperature of the young ones.

This is because the brown-fat mitochondria have special proton pores in their inner mitochondrial membrane. The proton pores allow the protons, pumped on by electron transport, to flow back into the matrix, rather than through the F0F1 ATPase or ATP synthetase. Consequently, the free energy of electron transport is diverted from ATP synthesis into heat production.

16.3.2.   Transport of Ca+2

The electron transport energy is also used to transport Ca+2 from the cytosol into the matrix of animal mitochondria so as to maintain the concentration of Ca+2 low inside the cytoplasm. High Ca+2 concentrations promote many cell functions such as muscle concentration, glycogen breakdown and the oxidation of pyruvate.

16.3.3.   Bacterial Movement

In bacteria the rotation of  flagella is controlled by the proton gradient generated across the membrane.

16.3.4.   Other Function

  1. Transfer for electrons from NADH to NADPH is also powered by the proton gradient.
  2. The energy generated during electron transport also help in transport of some amino acids and sugars.

16.4.      Uncouplers of Oxidative Phosphorylation

The transport of electrons is coupled with the synthesis of ATP. Uncoupling agents dissociate (or ‘uncouple’) these two process.  This means that the electron transport continues to function, leading to oxygen consumption but phosphorylation of ADP is inhibited, and the energy release in the form of Heat. This can be done by increasing the permeability of the inner membrane to H+.

The chemical nature of uncoupler is lipophilic and they bind H+ from perimitochondrial space and transport them to matrix. As the uncouplers bind and carry protons, they are also called protonophores. In Mitchell’s hypothesis, uncouplers are agents that are capable of destroying the vectorial, anisotropic structure of the membrane, leading to elimination of the pH gradient.

16.4.1.   Some uncouplers are :

1.            2, 4-dinitrophenol (DNP).           

It does not effect the substrate-level phosphorylation of glycolysis. At pH 7.0 DNP  exists mainly as the anion which is not soluble in the lipids. In its protonated form, it is lipid-soluble and hence can pass through inner membrane, carrying a proton and transport the proton to other side. The proton (H+) carried by DNP is discharged on the other side of the membrane. The phenolate ion then diffuses back towards the cytosol side, where it picks up a proton to repeat the process. In this way, uncouplers prevent formation of H+ gradient across the membrane. Dinitrophenol also stimulates the activity of the enzyme ATPase., which is normally inactive as a hydrolytic enzyme in mitochondria. Actually, ATP is never formed in the presence of DNP, since the high-energy intermediate is attacked i.e., it acts prior to the step of ATP synthesis.

2.            Dicoumarol

It has an action identical to that of 2,4-dinitrophenol. Dicoumarol is also an antagonist of vitamin K function.

3.            m-chlorocarbonyl cyanide phenylhydrazone (CCCP)

Its action is also similar to that of 2, 4-dinitrophenol but it is more active than the DNP.

16.5.      Energy Balance

ATP obtained from a complete catabolism of glucose

1.  From Glycolysis (in cytoplasm)

     For each glucose 2 ATP’s used                 -2

    4 ATP’s formed                                            +4

The NADH produce in glycolysis can yield either 2.5 ATP or 1.5 ATP depending upon the shuttle system.

     2NADH (Glycolysis)                                    5 or 3

2.  2 Pyruvate → 2 acetyl-CoA

     2 NADH molecules formed (2.5 ATP)       +5

(This NADH is already in the mitochondria and no transport is necessary.)

3.  Citric Acid Cycle (and Electron transport chain)

From each acetyl-CoA we get 3 NADH, 1 FADH2 and 1 ATP. Two acetyl-CoA enter the cycle (if we started with 1 glucose).

      6 NADH (2.5 ATP)                                     +15

      2 FADH2 (1.5 ATP)                                    +3

      2 ATP                                                          +2

      Total  (for two)                                         30/32

16.6.      Shuttle system :

The glycerophosphate shuttle. The electrons of cytosolic NADH are transported to the mitochondrial electron-transport chain in three steps : (1) Cytosolic oxidation of NADH by dihydroxyacetone phosphate catalyzed by glycerol-3-phosphate dehydrogenase.

This enzyme is present in cytosolic (2) Oxidation of glycerol-3-phosphate by flavoprotein dehydrogenase with the reduction of FAD to FADH2. (3) Reoxidation of FADH2 with the passage of electrons into the electron-transport chain.

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