6.4. Uncoupling of oxidation from phosphorylation

Uncouplers are lipophilic substances that can accept protons, and carry them through the inner mitochondrial membrane, but not passing its proton channel.

Natural uncouplers are products of peroxidation of lipid, fatty acids with a long chain, and high doses of thyroid hormones.

Artificial uncouplers are dinitrophenol, ether, derivatives of vitamin K, anesthetics, antibiotics (gramicidin, valinomycin)

Consider the example of uncoupling mechanism in dinitrophenol. It easily diffuses across the mitochondrial membrane in both ionized and a non-ionized forms and can carry hydrogen ions across the membrane. Therefore, 2,4-dinitrophenol destroys ΔpH of mitochondrial membrane. Oxygen consumption and substrate oxidation are continuing, but ATP synthesis is impossible. Since the energy of oxidation in uncoupling is dissipated in the form of heat, the uncouplers increase body temperature (pyrogenic action).

There is a particular tissue, specialized in heat production by uncoupling of respiration from oxidative phosphorylation. This is brown adipose tissue. It has such name because of a large number of mitochondria (they are brown). About 10% of all proteins of these mitochondria accounts for the so-called uncoupling protein. Brown adipose tissue is involved in maintaining body temperature.

Biological oxidation which is not accompanied with the storage of energy is called the free (uncoupling) oxidation. It accounts for 5-10% of oxygen entering the body. It is out of mitochondria, mostly in the endoplasmic reticulum, so sometimes this process is called microsomal oxidation (microsomes are fragments of ER).

One of the functions of free oxidation is transformation of natural or unnatural substrates, called xenobiotics.

Free oxidation takes place with the participation of oxygenases. Oxygenases are enzymes of oxidoreductases class; they catalyze the oxidation of substrates by including one oxygen atom (monooxygenases) or two oxygen atoms (dioxygenases) in their molecules.

Oxygenases work in the multienzymatic complex, embedded in the membrane. Multienzymatic complex consists of 3 components: flavin dehydrogenases, iron-sulfur protein, cytochrome P450.

Cytochrome P450 is a group of enzymes related to hemoproteins. Cytochrome P450 system participates in the oxidation of both endogenous (steroids, bile acids, unsaturated fatty acids) and exogenous substances which are called xenobiotics (xeno - incompatible, bios - life), for example, medicines, poisons and drugs.

Oxygen and reduced respiratory carriers (usually NADPH) are also involved in reactions of free oxidation. Cytochrome P-450 is an electron acceptor.

RH + O₂ + NADPH + H⁺  ROH + NADP⁺ + H₂O

Hydroxylation of xenobiotic makes it more soluble, facilitates the following destruction and elimination from the body.

6.5. Generation of free radicals in cells

Reactive oxygen species (ROS) are compounds in which oxygen has an unpaired electron.

ROS are formed in the following cases: when the conditions of the respiratory chain action are changed (e.g., during hypoxia); under UV rays; during the interaction of oxygen with metal ions of variable valence (iron); during the spontaneous oxidation of some substances; during the participation of the enzymes xanthine oxidase or NADPH oxidase. Under these conditions, a superoxide anion of oxygen О₂^ is formed, then hydrogen peroxide Н₂О₂ and hydroxyl radical HO^. Reactive oxygen species are causing lipid peroxidation. It is a process leading to severe damage of membranes, proteins and DNA damage.

Inactivation of reactive oxygen species in cells is under the influence of the antioxidant system. It consists of several antioxidant enzymes and low molecular antioxidants (vitamin C, glutathione, vitamin E, etc.).

Superoxide dismutase (SOD) converts superoxide anion of oxygen into hydrogen peroxide Н₂О₂:

2 O₂^-.+ 2Н⁺ H₂O₂ + O₂.

Catalase is hemin enzyme, containing Fe³⁺. It catalyzes the reaction of hydrogen peroxide splitting. In this reaction water and oxygen are formed:

2 H₂O₂ O₂ + 2H₂О.

The highest catalase activity in the body is typical of the liver. There is a lot of catalase in erythrocytes. There it protects the heme of hemoglobin from oxidation.

Peroxidase is hemin enzyme which reduces hydrogen peroxide to water, while another substance is oxidized.

2 H₂O₂ 2H₂О + RO₂.

Peroxidase is able to split other peroxides, converting them to alcohols. Peroxidase activity is found in the liver, kidneys, and neutrophilic leukocytes.

Antioxidants are biologically active substances that interact with free radicals and prevent the process of free radical oxidation of organic substances in the body.

Vitamins which have antioxidant properties are C, E, A, P. Glutathione, taurine (2-aminoetansulfonic acid), dipeptide carnosine are also reveal antioxidant properties.

Complete suppression of peroxide processes in the tissues seems inappropriate. Free radicals induce apoptosis and are involved in the formation of cellular immunity, stimulate phospholipases, thereby participating in the synthesis of eicosanoids.

However, the enhanced generation of free radicals accompanies pathological conditions (Parkinson's disease, Alzheimer's disease) and the process of biological aging.