6.3. Organization and operation of the respiratory chain

Oxidation of substrates in the process of respiration can be represented as the transfer of electrons and protons (i.e. hydrogen atoms) of organic substances to oxygen. This process involves a series of intermediate transporters which form the respiratory chain.

Respiratory chain (electron transport chain) is the system of transmembrane proteins and electron carriers that transfer electrons from substrates to oxygen. In eukaryotic cells the respiratory chain is located in the inner membrane of mitochondria.

Reduced NAD is a universal donor of hydrogen atoms to the respiratory chain. The interaction of NAD and NADP with hydrogen atoms is a reversible addition of hydrogen atoms. Two electrons and one proton are included in the molecule of NAD (NADP); and the second proton remains in the environment.

Another primary source of hydrogen atoms and electrons is reduced flavoprotein (FAD or FMN).

Reduced forms of these cofactors are able to carry hydrogen and electrons to the mitochondrial respiratory chain.

The components of the respiratory chain are embedded in the mitochondrial membrane in the form of four protein- lipid complexes (Fig. 19).

Fig. 19. Mitochondria lrespiratory chain

Complex I (NADH dehydrogenase, NADH-CoQ reductase) contains FMN and iron-sulfur protein FeS (non-heme iron). Fe can be in Fe²⁺ and Fe³⁺-form. Iron-sulfur protein is involved in the redox process. Complex I oxidizes NADH and transfers 2 electrons from it to KoQ. Complex I also pumps four protons from the matrix into the intermembrane space of mitochondria.

KoQ (ubiquinone) is a derivative of benzoquinone. It is not large lipophilic molecule. Ubiquinone moves into the lipid bilayer of the membrane and provides transfer of electrons between complexes I - III and II - III.

Complex II (succinate dehydrogenase, succinate-CoQ reductase) contains FAD and iron-sulfur protein. It provides input of additional electrons into the chain from succinate oxidation.

Complex III (QH₂ dehydrogenase, CoQ-cytochrome C reductase) contains cytochrome b and с₁ and iron-sulfur protein. Cytochromes are hemoproteins which prosthetic groups are close to heme of hemoglobin (in the cytochrome b it is identical). Complex III transfers electrons from ubiquinone to cytochrome c and pumps 2 protons into the intermembrane space.

Complex IV (cytochrome oxidase) is composed of cytochromes a and a₃, which, in addition to heme, contain copper ions. Cytochrome а₃is the terminal component of respiratory chain. Complex IV catalyzes the transfer of electrons from molecules of cytochrome to O₂ and pumps four protons into the intermembrane space. Cytochrome oxidase carries out the oxidation of cytochrome c and formation of water.

300-400 ml of water per day (metabolic water) are formed in the humans mitochondrial respiratory chain.

The components of the mitochondrial respiratory chain are in descending order of redox potential. Electron transport in the respiratory chain occurs on a gradient of redox potential and is a source of energy for proton transfer. As a result, difference in the concentrations of protonsoccurs on the sides of the membrane; while the difference in electrical potential from the "plus" sign - on the outer surface. Electrochemical potential of protons makes protons move in the opposite direction - from the outer surface inward. However, the membrane is impermeable to them, except in areas where the enzyme is a proton ATP synthase (Fig. 20). It consists of two parts - stator and rotor.

The stator consists of three α-subunits and three β-subunits - they participate directly in the synthesis of ATP from ADP and phosphate. δ- subunit joins them, and together they form the F1- subunit.

The rotor consists of gamma and epsilon subunits.

The stator is held in the membrane, and the rotor is rotated by the energy of the protons.

In the stator there is a proton channel (F0). It consists of two parts which are shifted relative to one another. Proton passes one half of the channel, and then it enters into the second half of the channel on the rotating rotor. The difference of electrochemical potentials resulting during the movement of protons through the channel activates the ATP synthase, which catalyzes reaction:

ADP + H₃PO₄  ATP + H₂O

Chemiosmotic hypothesis of energy transformation in living cells was proposed by P. Mitchell in 1960 to explain the molecular mechanism of coupling of electron transport and ATP formation in respiratory chain. For the development of research in the field of bioenergetics P. Mitchell was awarded the Nobel Prize in 1978.

Fig. 20. The structure of the proton ATP-synthase.

There are only three sites in the respiratory chain where the transfer of electrons is connected with energy storage, sufficient for the formation of ATP.

The coefficient of phosphorylation is the ratio of ATP formed by the absorbed oxygen: ATP/O, or ratio refers to the number of inorganic phosphate molecules utilized for ATP generation for every atom of oxygen consumed: P/O. The maximum value of the coefficient of phosphorylation is 3 (if the oxidation reaction goes with the participation of NAD), and 2 (if the oxidation of substrate goes with the participation of FAD). Actually obtained value is smaller (2.5 and 1.5). That is the process of respiration is not completely coupled with phosphorylation. The degree of coupling depends mainly on the integrity of the mitochondrial membrane.

Formed ATP is transported from the matrix on the outer side of the membrane with the participation of the ADP-ATP-translocase, and then goes into the cytosol. At the same time, the same translocase carries ADP in the reverse direction, from the cytosol into the mitochondria matrix.

The total content of ATP is 30-50 grams in the body, but the average life expectancy of ATP molecule is less than 1 minute. A person synthesizes 40-60 kg of ATP and the same amount splits per day.

At each contraction of the heart muscle, about 2% of the available ATP in it are consumed. All ATP would be expended over 1 minute, if there was no its regeneration. In the formation of a blood clot in the coronary artery, the supply of oxygen to the cells is ceased; and the regeneration of ATP is respectively blocked and cells die (myocardial infarction).

Increase of concentrations of ADP leads to acceleration of respiration and phosphorylation. The intensity of respiration of mitochondria on the concentration of ADP is called respiratory control.

We use cell energy charge (CEC) to assess the impact of adenine nucleotide over the processes of metabolism:

Normally CEC = 0.7-0.8: the rate of ATP formation equals the rate of its use and adenylic system is full of energy.

When CEC<0.7 the formation of ATP is accelerated by increasing the rate of reactions of common way catabolism.

If CEC = 1, then the processes of ATP synthesis are inhibited, and its use is accelerated.

Hypoenergy states are divided into:

- alimentary (starvation, vitamin deficiency);

- hypoxic:

- disorder of oxygen entry in the blood (pulmonary hypoxia),

- disorder of oxygen transport to tissues (hemodynamic (blood loss, shock, heart defects) and hemoglobin hypoxia (Hb pathology, blocking it with poisons));

- mitochondrial (difficulties of oxygen use in cells). This is disruption of mitochondria function by inhibitors of respiratory chain enzymes, releasers of oxidation and phosphorylation, membrane-acting agent.

At complete starvation, food reserves of the body are enough for several weeks, and oxygen is enough only for 2-3 minutes. Therefore, hypoxia is the most common cause of hypoenergy states, and hypoxia of the brain is the direct cause of death. Therefore, the measures for restoring the oxygen supply take the leading place among the resuscitation procedures.