MECHANISMS OF OXIDATIVE PHOSPHORYLATION UNCOUPLERS OF OXIDATIVE PHOSPHORYLATION

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.