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Файл:Carbohydrates. Bioenergetics. Study manual
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Substrates of NAD-dependent dehydrogenases are alcohols, aldehydes, amines,
dicarboxylic and keto acids. All NAD-dependent dehydrogenases are anaerobic dehydrogenases, i.e. they send hydrogen atoms to the nearest other enzyme in the oxidative chain.
An example of NAD-dependent dehydrogenase is alcohol dehydrogenase from
animal liver. The enzyme consists of two subunits, each carrying the NAD+ molecule and the Zn atom. It catalyzes the reaction of alcohol oxidation into aldehyde.
2. FAD-dependent dehydrogenases.
Flavine dehydrogenases contain FAD or FMN as prosthetic groups. Vitamin B
is the working part of FAD and FMN and is joined by two protons of H+ and two
electrons from the oxidizing substrate.
Most of the FAD-dependent dehydrogenases are soluble proteins localized in
the mitochondria matrix. They are acceptors of H+ protons and electrons from substrates: acyl-CoA, glycerol-3-phosphate, etc.
The only exception is dehydrogenase succinate fumarate in the inner mitochondria membrane, which is the second complex in the ETC. It is the acceptor of H
protons and electrons from the substrate – succinate.
2
+
3. Ubiquinone (coenzyme Q10). Ubiquinone is a fat-soluble quinone with a long
isoprenoid lateral chain. It is found to be practical in all cells. The number of isoprenoid units is displayed in the name. Most animal cells and mammalian cells contain
ubiquinone with the number of isoprenoid units –10: Co Q10.
Human enzyme Q10 organism synthesizes from mevalonic acid and exchange
products of tyrosine and phenylalanine. Coenzyme Q is involved in oxidative phosphorylation reactions and is a component of the electron transfer chain in mitochondria. Ubiquinone inhibitors stop oxidative phosphorylation reactions.
The antioxidant activity of Q10 coenzyme in 2 times exceeds this standard set of
antioxidant vitamins.
4. Cytochromes are hemoproteins, proteins containing red or brown colored
gems with Fe+3 as a closely related prosthetic group. This change in the redox state
includes a reversible equilibrium between Fe+2 and Fe+3 hem states:
Cytochrome is a single-electron redox compound. They cannot accept or give
away hydrogen, therefore they are called electron carriers. Cytochrome is present in
many animal, plant and bacterial tissues. Cytochromes are subdivided into cytochrome a, b, c and d according to the position of their spectral absorption bands in
the reduced state.
Cytochrome hem structure
5. Cytochrome oxidase (a and a3) contain copper cation Cu+2 hem. Cytochrome
oxidase, cytochrome, and, a3, an enzyme of oxidoreductase class, the final
component of the chain of respiratory enzymes that carries electrons from
cytochrome to molecular oxygen. Cytochrome oxidase was discovered in 1926 by
the German scientist O. Varburg (so-called "respiratory enzyme of Varburg"). In
plant and animal cells it is localized in the internal membranemitochondria. By its
chemical nature, cytochrome oxidase is a complex protein whose molecule consists
of two hems, two copper atoms, and a 20-30% lipid component. Both hems are represented by hem a, but only part of the hem a is oxidized by oxygen and is indicated
by a3. Copper is bound to the protein through an S-containing ligand. When
separating copper, Cytochrome oxidase loses its activity. The molecular mass of
cytochrome oxidase (according to different data) is from 50000 to 240000 Da.
Cytochrome oxidase inhibitors are cyanide, azide, CO, hydroxylamine.
6. Iron sulphur proteins are Fe-S-proteins, oxidation-reduction systems that
carry electrons. Ferrous proteins (FeS-proteins) contain iron atoms bound, on the
one hand, to cysteine amino acid sulfur and, on the other hand, to inorganic sulfide
sulfur. ferrous proteins have a small molecular weight of about 10 kDa. The respiratory chains contain a large number of FeS centers. These proteins are involved in the
transport of protons and electrons and are believed to be at several stages. However,
the mechanism by which iron-sulfuric proteins undergo reversible oxidation-recovery
is still unclear. The most famous protein from this family is called ferredoxin.
Iron sulphur proteins are oxidation-reduction systems that carry electrons. They
contain iron atoms bound, on the one hand, to cysteine amino acid sulphur and, on
the other hand, to inorganic sulphide sulphide. The latter is very easily detachable in
the form of hydrogen sulfide during acidification. Cysteine residues are part of the
polypeptide chains of the Fe-S-center can be considered as prosthetic groups of
polypeptide.
Organization of the respiratory chain
Oxidation of substrates during respiration can be represented as the transfer of
electrons and protons (i.e. hydrogen atoms) from organic matter to oxygen. This
process involves a number of intermediate carriers that form the respiratory chain.
Respiratory chain (electrontransport chain, electron transfer chain)
is a system of trans-membrane 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 in the form of 4 enzyme and
ATP-ase complexes in accordance with
the increase of redox potential.
1 complex. The main role in electron transport in the mitochondria
membrane thickness is played by the
respiratory chain. The first complex of
the transport chain is taken over by
molecules NADH+ (in animals) or NADPH+ (in plants) with subsequent separation
of four hydrogen protons. I complex is also called NADH - dehydrogenase (by the
name of the central enzyme). The dehydrogenase complex consists of 3 types of
iron-sulfur proteins as well as flavinmonucleotides (FMN). Pyridine dependent dehydrogenases carry out the transfer of electrons and protons (hydrogen) from the
substrate. FADH2, formed in the Krebs cycle as a result of glycolysis and other redox processes, is oxidized by dehydrogenase (complex I), which takes away two
electrons and protons from it and transfers them to lipid-soluble ubiquinone (into the
inner mitochondria membrane). During this process, complex I pumps protons from
the matrix into the intermembrane space of the mitochondria.
2 complex. Flavindependence dehydrogenases as a coenzyme contain FAD+ or
FMN. Flavin-dependent dehydrogenases include succinate dehydrogenase. FADH
is oxidized by succinate dehydrogenase (complex II). In this process, protons are not
pumped through the membrane, but ubiquinone receives additional electrons and
protons.
3 complex. Restored ubiquinone (hydroquinone) sends electrons to the cytochrome complex (complex III), and it in turn transfers electrons to two water-soluble
cytochrome from the inner mitochondria membrane. This process also transports
ubiquinone protons, which are pumped by the complex.
4 complex. The final acceptor of electrons in the respiratory chain is oxygen.
Transmission of electrons to oxygen is carried out by cytochrome oxidase (complex). In this case, complex IV pumps protons into the intermembrane space. Cytochromeoxidase releases electrons to oxygen and activates it. As a result, when electrons and protons are transferred in the respiratory chain or electron transfer chain, a
proton potential is formed on the internal mitochondrial membrane. In the considered respiratory chain, the final acceptor of electrons and protons is oxygen. In some
microorganisms the final acceptor of protons and electrons is nitrate, which is re-
2
stored to nitrogen or even to ammonia.
5 complex. ATP synthesis. The universally recognized theory of ATP synthesis
in ETC is the theory of chemoosmotic conjugation advanced by P. Mitchell. Its essence is as follows: the process of oxidative phosphorylation is carried out by the
fifth complex of mitochondria respiratory chain – proton ATP-synthase. In the process of electron transfer along the respiratory chain the hydrogen ion gradient is
formed on different sides of the inner mitochondria membrane. The resulting concentration gradient causes protons to pass through the ATP synthase to the other side
of the membrane, where the concentration of protons is lower. ATP-synthase holds
ADP and Pi. Proton passage through ATP-synthase leads to its conformational
changes and, accordingly, the interaction between ADP and Pi. The reaction of synthesis is as follows:
ADP + Pi→ ATP + H2O
This is the most common method of substrate oxidation in cells, carried out by
dehydrogenation. The efficiency of this method is characterized by a high coefficient of efficiency. Approximately 40 % of the energy contained in the chemical
bonding of oxidized substances is concentrated in macroergic bonds of ATP, and the
rest of the energy is dissipated in the form of heat.
The membrane on which the electrochemical trans-membrane proton gradient is
created is called energetic. An energized membrane tends to discharge itself by
pumping protons from the intermembrane space back into the matrix. This process is
carried out with the help of proton-dependent ATP-ase.
H+-ATP-ase is built into the internal mitochondrial membrane. It is similar to a
fungus and consists of two protein factors, F0 and F1. The F0 factor penetrates the entire thickness of the internal mitochondrial membrane. The spherical part protruding
from the matrix of mitochondria is the factor F1. The structure, properties and functions of these protein factors are quite different. F0 consists of three hydrophobic
polypeptide chains of different structure. The F1 factor is a water-soluble part of the
H+-ATPase and represents a protein complex consisting of nine subunits of five
different types. One epimolecule of factor F1 contains 3α, 3β and one subunit γ, δ, ε
(α, β, γ, δ) each. F1 synthesizes ATP from ADP and phosphoric acid. ADP and ATP
binding centers are located in α and β subunits, each of which can hold one ADP or
ATP molecule at a time. According to X-ray analysis, the ADP and ATP binding
centers are located at the junction of α and β subunits. Subunit β performs a catalytic
function in the synthesis of ATP.
The structure of the proton-dependent ATP-ase
There are several concepts that explain the mechanism of ATP formation
through the H+-ATP-synthase. All concepts consider hydrogen protons coming
through the proton conducting channel to the factor F1 as activators of various processes leading to the formation of ATP from ADP and phosphoric acid. As soon as
hydrogen atoms or electrons reach a certain component of the respiratory chain,
2 hydrogen protons are ejected from the matrix into the intermembrane space, and
Δμ(H+) appears on the inner mitochondria membrane; protons pass through the proton conducting channel and reach the factor F1 H+-ATPhase, which catalyzes the
synthesis of ATP. If hydrogen atoms are supplied to the breathing circuit by NAD,
there are 3 interfaces between the breathing circuit and ATP synthesis, i.e. 3 ATP
molecules are synthesized. If hydrogen atoms are supplied by NAD into the respiratory chain, 2 ATP molecules are synthesized and there is a mitochondrial reticulum
in the muscle cell, with the help of which mitochondria are connected in a single
chain or represent one giant branched mitochondria.
Its energetic membrane Δμ(H+) can be transmitted over long distances, providing the necessary amount of ATP in the right place for muscle work.
And so, the whole complex of processes occurring in mitochondria can be represented by the following scheme:
Synthesis of ATP from ADP and phosphoric acid due to the energy released
during tissue respiration is called oxidative phosphorylation.
The processes of oxidation and phosphorylation are interrelated. Their ratio is
determined by the phosphorylation coefficient P/O - the amount of phosphorylated
ADP in ½ molars of oxygen.
The oxidative phosphorylation coefficient is the ratio of the amount of phosphoric acid (P) used for ADP phosphorylation to the oxygen atom (O) absorbed in
the process of breathing.
At oxidation of NADH molecule electrons along the respiratory chain pass
3 points of conjugation that provides synthesis of 3 ATP at the expense of 3 H3PO
and 3 ADP per 1 oxygen atom. Accordingly, for NADH P/O=3.
At oxidation of FADH2 molecule, electrons along the respiratory chain pass
only 2 points of conjugation that provides synthesis of 2 ATP at the expense
of 2 H3PO4 and 2 ADP per 1 oxygen atom. Correspondingly for FADH2P/O=2.
These values of P/O reflect the theoretical maximum of synthesis of ATP, in
fact, this value is less because of the cost of transport.
Dependence of breathing intensity of mitochondria on ADP concentration is called
breathing control. A person consumes an average of 27 mole of oxygen per day. About
25 mole is used in the respiratory chain. At an average value of P / O = 2.5 daily
formed 125 mole ATP or 62 kg. Normally, tissue respiration substrates and O2 are
sufficient and do not limit oxidative phosphorylation. Activity of oxidative phosphorylation limits only concentration of ADP, which is inversely proportional to
concentration of ATP. At rest, the amount of ATP increases, and ADP decreases,
which inhibits breathing and phosphorylation.
As a result of respiratory control the speed of ATP synthesis meets the energy
needs of the cell. Total ATP content in the body is 30-50 g, but each ATP molecule
4
in the cell "lives" for less than a minute. A person synthesizes 40-60 kg of ATP per
day and decomposes as much.
About 40–45 % of the total energy of electrons transported by ETC is spent on
the synthesis of ATP molecules, about 25 % is spent on the work on the transfer of
substances through the membrane. The rest of the energy is dissipated in the form of
heat and used by warm-blooded animals to maintain body temperature.
Damage to the internal mitochondrial membrane or increased permeability under
the influence of decomposers causes the disappearance of electrochemical potential,
separation of oxidation and phosphorylation processes, and is stopped ATP synthesis.
Dissociation of breathing and phosphorylation is called the phenomenon of disappearance of electrochemical potential on the membrane under the influence of
separators and termination of ATP synthesis.
Dissociators are substances that can carry protons (protonophores) or other ions
(ions) through the membrane bypassing the channels of ATP-synthetase. As a result
of separation, the amount of ATP decreases, ADP increases, the rate of consumption
of O2, oxidation of FADH2increases, and the resulting free energy is released in the
form of heat slot. For example, the substance 2.4-dinitrophenol (carries H+), the drug
- dicumarol, metabolite - bilirubin, thyroid hormone – thyroxine, antibiotics –
valinomycin and gramycin.
Separation of oxidative phosphorylation can be biologically useful. It allows to
generate heat to maintain body temperature in newborns, winter-sleeping animals
and all mammals in the process of adaptation to the cold. Newborns as well as winter-sleeping animals have a special tissue that specializes in heat production through
separation of respiration and phosphorylation – brown fat. Brown fat contains many
mitochondria. The mitochondria membrane has a large excess of breathing enzymes
compared to ATP synthase. About 10 % of all proteins are thermogenic, the socalled decomposing protein (DP-1). Brown fat is present in newborns, but it is practically absent in adults.
2.6. Free radical oxidation
As a result of redox reactions in mitochondria, about 400 ml of endogenous water is accumulated daily, 15–20 ml of hydrogen peroxide, which is a toxic substance
for cells and cellular organoids. Catalase and peroxidase enzymes function in cells
to protect against peroxidation. By their structure they are hem containing proteins
and participate in the reaction of hydrogen peroxide cleavage:
2 H2O2 =2H2O + O
2
Hydrogen peroxide is involved in lipid peroxidation processes leading to cell
damage. Lipid peroxidation (POL) reactions are free radical and constant in the
body. Free radical oxidation disrupts the structure of many molecules. Some amino
acids are oxidized in proteins. As a result, the structure of proteins is destroyed and
covalent "cross-links" are formed between them, all of which activate proteolytic
enzymes in the cell that hydrolyze the damaged proteins. Active oxygen forms easily disrupt the structure of DNA. The non-specific binding of Fe2+ to the DNA molecule facilitates the formation of hydroxyl radicals that destroy the structure of nitrogen bases. But the most exposed to the action of active oxygen species of fatty acids
containing double bonds, located interlaced methylene - CH2-group. It is from this –
CH2-group that the free radical (oxidation initiator) easily removes the electron,
turning the lipid containing this acid into a free radical.
POL – chain reactions providing the expanded reproduction of free radicals, particles having an unpaired electron, which initiate the further spread of peroxidation.
Active oxygen species damage the structure of DNA, proteins and various cell
membrane structures. As a result of the appearance of hydrophilic zones in the hydrophobic layer of membranes due to the formation of fatty acid hydroperoxides,
water, sodium and calcium ions can penetrate into the cells, which leads to swelling
of cells, organelles and their destruction. Activation of peroxidation is typical for
many diseases: muscle dystrophy (Dushenne's disease), Parkinson's disease, in
which POL destroys nerve cells in the brain stem, atherosclerosis, tumor development. Peroxidation is also activated in tissues that are first ischemic and then reoxygenated, e.g. coronary artery cramping and subsequent dilatation.
The same situation occurs with the formation of a blood clot in the vessel feeding the myocardium. The formation of a blood clot leads to the occlusion of the vessel lumen and the development of ischemia in the corresponding part of the myocardium (tissue hypoxia). If you take rapid therapeutic measures to destroy the blood
clot, the tissue is restored to oxygen supply (reoxygenation). It is shown that at the
moment of reoxygenation the formation of active oxygen species, which can damage
the cell, sharply increases. Thus, even in spite of the rapid recovery of blood circulation, in the corresponding part of the myocardium there is damage to cells by activating peroxidation.
Changes in tissue structure as a result of POL can be observed on the skin: with
age, the number of pigment spots on the skin, especially on the back of the palms increases. This pigment is called lipofuscin, which is a mixture of lipids and proteins
bound together by transverse covalent bonds and denatured as a result of interaction
with chemically active groups of POL products. This pigment is phagocyted, but not
hydrolyzed by the enzymes of the lysosomes, and therefore accumulates in cells,
disrupting their function.
The enzymes protecting cells from the action of active oxygen species include
superoxide dismutase, catalase and glutathione peroxidase; these enzymes are most
active in the liver, adrenal glands and kidneys, where the content of mitochondria,
cytochrome P450 and peroxisomes is particularly high. Superoxide dismutase
(SOD) transforms superoxide anions into hydrogen peroxide:
2 -O-+ 2H+→ H2O2 + O
2
SOD isoenzymes are found in both cytosol and mitochondria and are the first
line of defense, because superoxide anion is usually the first of the active oxygen
species to be formed when electrons are leaking from the respiratory chain.
SOD is an induced enzyme, i.e. its synthesis increases when peroxidation is activated in cells.
Hydrogen peroxide, which can initiate the formation of the most active form of
OH-, is destroyed by the enzyme catalase:
2H2O2→ 2H2O + O2.
Catalase is mainly found in peroxisomes, where the largest amount of hydrogen
peroxide is formed, as well as in leukocytes, where it protects cells from the effects
of "respiratory explosion" (a sharp increase in oxygen in the cells and, consequently,
the increase of active oxygen species).
Glutathione peroxidase is the most important enzyme providing inactivation
of active oxygen species, as it destroys both hydrogen peroxide and lipid hydroperoxides. It catalyzes peroxide recovery with the help of glutathione tripeptide
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