Inhibition of oxidative phosphorylation

ATP SYNTHASE COMPLEX

 ATP synthase is embedded in the inner membrane, together with the respiratory chain complexes.

• Several subunits of the protein form a ball-like shape arranged around an axis known as F₁, which projects into the matrix and contains the phosphorylation mechanism.
• F₁ is attached to a membrane protein complex known as F₀, which also consists of several protein subunits (Figure-1).
• F₀ spans the membrane and forms a proton channel.
• The flow of protons through F₀ causes it to rotate, driving the production of ATP in the F₁ complex.

Figure-1-The enzyme complex consists of an F₀ subcomplex which is a disk of “C” protein subunits. Attached is a Υ subunit in the form of a “bent axle.” Protons passing through the disk of “C” units cause it and the attached Υ subunit to rotate. The Υ subunit fits inside the F1 subcomplex of three α and three β subunits, which are fixed to the membrane and do not rotate.

OXIDATIVE PHOSPHORYLATION

Chemiosmosis

• As the electrons are transferred, some electron energy is lost with each transfer.
• This energy is used to pump protons (H⁺) across the membrane from the matrix to the inner membrane space. A proton gradient is established (Figure-2)
• The higher negative charge in the matrix attracts the protons (H⁺) back from the intermembrane space to the matrix.
• The accumulation of protons in the intermembrane space drives protons into the matrix via diffusion.
• Most protons move back to the matrix through ATP synthase.
• ATP synthase uses the energy of the proton gradient to synthesize ATP from ADP + P_i

Figure-2- Showing electrochemical gradient across the inner mitochondrial membrane. The chemiosmotic theory, proposed by Peter Mitchell in 1961, postulates that the two processes are coupled by a proton gradient across the inner mitochondrial membrane so that the proton motive force caused by the electrochemical potential difference (negative on the matrix side) drives the mechanism of ATP synthesis.

P: O Ratio

•  Defined as the number of inorganic phosphate molecules incorporated into ATP for every atom of oxygen consumed.
• Oxidation of NADH yields 3 ATP molecules(P: O ratio 3, Latest concept 2.5)
• Oxidation of FADH2 yields 2 ATP molecules (P: O ratio 2, Latest concept 1.5)

Inhibition of Oxidative phosphorylation

Oxidative phosphorylation is susceptible to inhibition at all stages of the process.  

A) Site-specific inhibitors -Specific inhibitors of the electron transport chain are (figure-3) for example-

1) Inhibitors of complex I– Rotenone and amobarbital block electron transfer in NADH-Q oxidoreductase and thereby prevent the utilization of NADH as a substrate. In contrast, electron flow resulting from the oxidation of succinate is unimpaired, because these electrons enter through QH2, beyond the block. The other inhibitors are  Chlorpromazine, Piericidin A and Guanethidine

2) Inhibitors of Complex II

Malonate is a competitive inhibitor of Complex II. The other inhibitors are Carboxin and TTFA

Figure-3- Site-specific inhibitors block the flow of electron at specific sites

3) Inhibitors of Complex III

BAL (British Anti Lewisite), Antimycin A, Naphthoquinone and hypoglycemic agents interfere with electron flow from cytochrome b in Q-cytochrome c oxidoreductase.

4) Inhibitors of Complex IV

Furthermore, electron flow in cytochrome c oxidase can be blocked by hydrogen sulphide (H₂S),  cyanide (CN-), azide (N3 -), and carbon monoxide (CO). Cyanide and azide react with the ferric form of heme a 3, whereas carbon monoxide inhibits the ferrous form. Inhibition of the electron-transport chain also inhibits ATP synthesis because the proton-motive force can no longer be generated.

B) Inhibitors of ATP synthase complex 

Oligomycin and dicyclohexyl carbodiimide(DCCD) prevent the influx of protons through ATP synthase. If actively respiring mitochondria are exposed to an inhibitor of ATP synthase, the electron transport chain ceases to operate. Indeed, this observation clearly illustrates that electron transport and ATP synthesis are normally tightly coupled.

C) Inhibition of ATP-ADP translocase

ATP-ADP translocase is specifically inhibited by very low concentrations of Atractyloside (a plant glycoside) or Bongregate (an antibiotic from a mold). The unavailability of ADP also inhibits the process of ATP formation. This is because oxidation and phosphorylation are tightly coupled; ie, oxidation cannot proceed via the respiratory chain without concomitant phosphorylation of ADP.

Figure-4- ADP moves into the mitochondrial matrix and newly synthesized ATP is transported out into the cytoplasm to be used for cellular processes

D) Uncouplers of Oxidative phosphorylation

The tight coupling of electron transport and phosphorylation in mitochondria can be disrupted (uncoupled) by agents called uncouplers of oxidative phosphorylation. These substances carry protons across the inner mitochondrial membrane. In the presence of these uncouplers, electron transport from NADH to O₂ proceeds in a normal fashion, but ATP is not formed by mitochondrial ATP synthase because of the proton-motive force across the inner mitochondrial membrane is dissipated. This loss of respiratory control leads to increased oxygen consumption and oxidation of NADH. Indeed, in the accidental ingestion of uncouplers, large amounts of metabolic fuels are consumed, but no energy is stored as ATP. Rather, energy is released as heat.

Examples

Physiological Uncouplers

• Long-chain fatty acids
• Thyroxin
• Brown Adipose tissue-Thermogenin (or the uncoupling protein) is a physiological uncoupler found in brown adipose tissue that functions to generate body heat, particularly for the newborn and during hibernation in animals
• Calcium ions.

The regulated uncoupling of oxidative phosphorylation is a biologically useful means of generating heat. The uncoupling of oxidative phosphorylation is a means of generating heat to maintain body temperature in hibernating animals, in some newborn animals (including human beings), and in mammals adapted to cold. Brown adipose tissue, which is very rich in mitochondria (often referred to as brown fat mitochondria), is specialized for this process of non-shivering thermogenesis. The inner mitochondrial membrane of these mitochondria contains a large amount of uncoupling protein (UCP), here UCP-1, or thermogenin, a dimer of 33-kd subunits that resembles ATP-ADP translocase. UCP-1 forms a pathway for the flow of protons from the cytosol to the matrix. In essence, UCP-1 generates heat by short-circuiting the mitochondrial proton battery(figure-5).

Thus, the manner in which biologic oxidative processes allow the free energy resulting from the oxidation of foodstuffs to become available and to be captured is stepwise, efficient, and controlled—rather than explosive, inefficient, and uncontrolled, as in many non -biologic processes. The remaining free energy that is not captured as high-energy phosphate is liberated as heat. This need not be considered “wasted,” since it ensures that the respiratory system as a whole is sufficiently exergonic, allowing continuous unidirectional flow and constant provision of ATP. It also contributes to the maintenance of body temperature.

Figure-5- UCP-1 forms a pathway for the flow of protons from the cytosol to the matrix. The proton gradient is dissipated, oxidation proceeds without phosphorylation.

Pathological uncouplers

These compounds are toxic in vivo, causing respiration to become uncontrolled since the rate is no longer limited by the concentration of ADP or P_i.

•  2,4-dinitrophenol
• 2, 4- dinitrocresol
• CCCP(chloro carbonyl cyanide phenyl hydrazone)
• FCCP
• Valinomycin
• High dose of Aspirin
• The antibiotic Oligomycin completely blocks oxidation and phosphorylation by blocking the flow of protons through ATP synthase.