Fundamentals of Biochemistry - A. A. Anisimov 1986

Biological Oxidation and Bioenergetics
Regulatory Heat Production and Free Oxidation. Oxygenases and Oxidases

In the body of an average healthy adult weighing about 70 kg, 8000 kJ of energy is generated and released every 24 hours. Over this period, neither body mass nor its Structure and composition change significantly. Therefore, all of this energy, except for the amount required to perform physical work, is released as heat and irreversibly dissipated into the environment. This entire process provides the body with the heat necessary to maintain a core Temperature of around 37°C.

When warm-blooded animals are exposed to cold conditions, heat production in the Muscles increases through several mechanisms. The first mechanism is shivering thermogenesis. Actomyosin hydrolyzes ATP, skeletal muscles contract, and shivering begins. Thus, under certain conditions, skeletal muscles, along with their primary contractile function, can act as the body's "heating system."

In other instances, thermogenesis is achieved by dissipating respiratory energy without ATP involvement—that is, through Free Oxidation. As demonstrated by S. E. Severin and V. P. Skulachev (1960), this process acts as a biological adaptation to low temperatures. During free oxidation, Respiration proceeds at maximum rate, but no ATP is formed; in other words, Electron transport is uncoupled from phosphorylation. Free oxidation occurs in the membranes of The Endoplasmic reticulum, Peroxisomes, and within Cell/35.html">Mitochondria themselves. According to V. P. Skulachev's hypothesis, free oxidation in the outer mitochondrial membrane involves a shortened Electron Transport Chain lacking coupling sites: NADH → cytochrome b₅ → cytochrome c.

Cytochrome b5 is invariably present in the outer mitochondrial membrane. Cytochrome c is found exclusively in the inner membrane, but it can partially desorb into the intermembrane space. In this scenario, the electron flow is directed straight to the terminal point of coupled phosphorylation, resulting in The formation of a single ATP molecule. Energetically, this process is less efficient, yet ATP production does not cease. This occurs, for example, when Inhibitors of the first two oxidation-phosphorylation coupling sites appear and accumulate within Cells.

Free oxidation can also take place within The inner mitochondrial membrane, such as under METABOLISM/18.html">The Influence of specific agents known as uncouplers of Oxidative Phosphorylation. When added to a mitochondrial incubation medium, these substances allow oxidation (i.e., the uptake of substrates and oxygen, and the release of CO₂) to continue at an accelerated rate, whereas ATP synthesis halts. Consequently, the energy of oxidation is dissipated.

In biochemical practice, one such substance—2,4-dinitrophenol (DNP)—has long been utilized. Approximately 40 uncoupling compounds are currently known, which has helped elucidate their MECHANISM OF ACTION on oxidative phosphorylation. All these substances share hydrophobic properties, meaning they dissolve well in Membrane Lipids and contain mobile protons. Work by V. P. Skulachev and his colleagues has proven that uncouplers increase the proton permeability of the coupling membrane, thereby reducing the electrochemical proton gradient generated by the Respiratory Chain. For this reason, uncouplers are referred to as protonophores, or proton carriers.

Fig. 7.9 illustrates the mitochondrial coupling membrane, across which the electrochemical proton gradient is generated by the respiratory chain. DNP dissociates to yield a proton and an anion. The anion dissolves in the lipids and moves electrophoretically toward the outer surface of the membrane. Here, it captures a proton from the external medium and then travels down its concentration gradient to the inner side of the membrane. This increases the proton conductivity of the membrane, dissipating the energy of the electrochemical proton gradient as heat.

In living cells, endogenous uncouplers of oxidative phosphorylation include thyroxine, phenols, Unsaturated Fatty acids and their peroxides, and certain specific Proteins. Uncoupling of oxidation and phosphorylation is also observed under the influence of extreme temperatures, radiation, and—in plants—during inadequate Water and mineral nutrient supply.

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Fig. 7.9. Proton transport by the uncoupler 2,4-dinitrophenol

Free oxidation has also been detected in microsomes. This term refers to the fraction of morphologically closed vesicles formed when the endoplasmic reticulum is homogenized. Some microsomal vesicles may even exist within the intact cell as isolated cisternae and vacuoles. Preparatively, microsomes are isolated by ultracentrifugation at g < 50,000 (after mitochondria are first removed at g = 24,000). A. I. Archakov (1983) proposed the specific term microsomal oxidation to describe oxidative processes in microsomes. This phenomenon is particularly characteristic of microsomal fractions from Organs such as The Liver and Adrenal Glands.

Microsomes contain active oxygenases—Enzymes that directly incorporate oxygen into various substrates. These enzymes are divided into two groups. Dioxygenases catalyze the incorporation of both atoms of molecular oxygen into the oxidized substrate and contain a heme or non-heme iron prosthetic group. Monooxygenases (hydroxylases) incorporate only a single O atom to form an OH group on the substrate, while the other atom is reduced to water with the participation of NADH or NADPH.

Microsomal oxidation chains contain Flavoproteins and Cytochromes that differ from their mitochondrial counterparts. Cytochromes P-450 play a crucial role in these oxidation chains. The most probable pathways of electron transfer in the microsomal membrane are described by the following scheme:

In this scheme, FP₁ represents the NADPH-specific cytochrome P-450 reductase flavoprotein, which oxidizes NADPH and reduces cytochromes P-450; FP₂ is the NADH-specific flavoprotein; CCF is the cyanide-sensitive factor; and RH is the oxidized substrate. In hydroxylation reactions, cytochromes P-450 perform several Functions. One of these is oxygen activation, which is characteristic of all cytochromes P-450. Oxygen is activated, one of its atoms is directly inserted into the oxidized substance to form an OH group, and the second atom is consumed in the formation of water. The second function—substrate binding—is highly specific.

Different cytochromes P-450 bind different substrates, specifically compounds of a particular class. Because of this, the microsomal fraction oxidizes hundreds of substances of diverse chemical nature. These include endogenous substrates such as saturated and unsaturated fatty acids, Steroid Hormones, Cholesterol, Bile acids, and Prostaglandins. Concurrently, a vast array of foreign substances entering the bodies of humans and animals from the environment—xenobiotics (from the Greek xenos, meaning stranger)—are degraded, including pesticides, pharmaceutical drugs, and cosmetic products. By inserting an oxygen atom into the oxidized substrate to form an OH group, the hydrophobic compound is converted into a polar one. Polar products are less toxic because they are easily eliminated from the membranes into the aqueous phase of The Cell.

Thus, microsomal oxidation serves as the primary detoxifying system in the animal and human body. Cytochromes P-450, which play The Central Role in this system, are found in the endoplasmic reticulum membranes of liver cells, the mitochondria of the adrenal cortex, and the Plasma Membranes of various Bacteria. In liver cells, they are classified as inducible enzymes: their quantity can increase 2- to 5-fold upon the administration of a foreign compound, such as phenobarbital or DDT. Consequently, the Treatment of acute intoxication can be facilitated by administering a harmless cytochrome P-450 inducer.

In addition to their detoxifying function, oxygenases in humans and animals play a key role in certain biosynthetic reactions (such as those of steroid hormones, bile acids, and prostaglandins) and other metabolic processes (for instance, The conversion of cyclic Amino Acids).

In bacterial and Yeast cells, oxygenases facilitate the uptake and assimilation of building blocks by oxidizing carbon-containing substances and easing their entry into the cells. In plants, The oxidation of Organic compounds via monooxygenase reactions is utilized primarily for the synthesis of BIOREGULATORS.

Biological Oxidation occurring without the participation of pyridine NUCLEOTIDES and cytochromes can be catalyzed by oxidases—oxidoreductases that transfer electrons or hydrogen directly to oxygen. These include L- and D-amino acid oxidases, xanthine oxidase, ascorbate oxidase, phenol oxidases, catalase, peroxidases, and cytochrome c oxidase, which functions within The electron transport chain. During the action of oxidases, energy is dissipated, with the sole exception of the oxidation mediated by cytochrome c oxidase.

Many oxidases are flavoproteins composed of a protein moiety and a flavin nucleotide. During catalysis, the flavin residue accepts a pair of hydrogen atoms, and the reduced form spontaneously reduces O₂ to H₂O₂. Occasionally, however, the substrate (MH₂) donates only a single electron to the flavin, causing the flavin to oscillate between its oxidized and reduced semiquinone forms:

Reactions of this type are a major source of hydrogen peroxide in metabolic systems.

Molecular oxygen is paramagnetic and possesses two unpaired electrons. These electrons reside in different orbitals because two electrons cannot occupy the same orbital unless their spins are antiparallel (opposite). The reduction of O₂ via the direct addition of a pair of electrons to its partially filled orbitals is impossible without "flipping" the spin of one of them; consequently, There is a "spin restriction" on O₂ reduction. Thus, the interaction of O₂ with substances whose electrons are all paired is hindered. The spin restriction of O₂ reduction can be overcome by the sequential addition of single electrons. Therefore, O₂ readily reacts with substances containing single, unpaired electrons. Free-radical intermediates participate in single-electron oxygen reduction reactions. The complete reduction of O₂ to H₂O requires four electrons. Single-electron reduction generates intermediates such as the superoxide anion radical O^-₂-, hydrogen peroxide H₂O₂, and the hydroxyl radical OH^-. These products are highly reactive, toxic to living organisms, and possess mutagenic activity. The reactions proceed According to the following scheme:

The superoxide anion generated by the one-electron reduction of oxygen (1) can be protonated into a hydroperoxyl radical in a subsequent reaction (2). The HO₂^- radical spontaneously undergoes a reaction leading to the formation of hydrogen peroxide (3). Thus, any system producing the superoxide anion will soon contain H₂O₂.

Iron-containing compounds can catalyze the reaction between O₂^- and H₂O₂ to form OH^-:

The hydroxyl radical OH^- is a very powerful oxidizing agent capable of attacking all organic compounds. It is generated in cells upon exposure to ionizing radiation.

The spontaneous oxidation of Hemoglobin, ferredoxins, and hydroquinones gives rise to O₂^- and H₂O₂. These compounds are also formed during other oxidative processes in Mitochondria and Chloroplasts and are eliminated through the action of corresponding enzymes. To protect the cell against the O^-₂ anion, the enzyme superoxide dismutase is employed, converting the superoxide radical into hydrogen peroxide:

O^-₂ + O^-₂ + 2Н⁺ → H₂O₂ + O₂

Superoxide dismutases are found in all respiring cells as well as in facultatively anaerobic bacteria. These are metalloenzymes in which a metal ion, such as Cu²⁺, Mn³⁺, or Fe³⁺, undergoes reduction and oxidation during the catalytic cycle. Introduction/5.html">Eukaryotic Cell superoxide dismutase contains Zn²⁺ and Cu²⁺ in close proximity at its Active Site. Cu²⁺ also functions in the catalytic cycle. A superoxide dismutase containing Mn³⁺ has been found in bacteria and eukaryotic mitochondria. Dismutases containing Fe³⁺ are found in bacteria and blue-green Algae.

Hydrogen peroxide, formed from the superoxide anion and also released during the aerobic reduction of flavoproteins, is detoxified by catalase and peroxidase enzymes. These are Hemoproteins. Catalase acts according to the reaction

Н₂O₂ + Н₂O₂ → O₂ + 2Н₂O

The activity of this enzyme is high in almost all animal cells and organs. It is detected in all plant specimens and in the majority of microorganisms (except obligate anaerobes).

Peroxidases are specific only with respect to the hydrogen acceptor, i.e., hydrogen peroxide. They can utilize various compounds as hydrogen Donors. For example, they oxidize polyphenols and certain Aromatic Compounds according to the following scheme, yielding the corresponding quinone and water:

Particularly active peroxidases are found in the Tissues of higher plants. Peroxidases are also detected in animal tissues (Blood, liver, Kidneys). The heme iron present in catalase and peroxidase molecules participates in the formation of intermediate peroxide compounds. Enzymes catalyzing H₂O₂ metabolism are abundant in specialized Organelles of PLANT AND ANIMAL cells known as peroxisomes. Catalase accounts for about 40% of the total protein content in rat liver peroxisomes.

Phenoloxidases are copper proteins most commonly found in the Cytoplasm of plant cells. These enzymes oxidize monophenols to diphenols, and the latter to Quinones. The spontaneous darkening of cut plant tissues (potatoes, fruits, mushrooms) is explained by the action of phenoloxidases, driven by quinone formation followed by spontaneous reactions among the quinones.

Ascorbate oxidase, a copper protein also localized mainly in the Cell Cytoplasm, is widespread in plants. Plants contain respiratory chains in which phenoloxidases and ascorbate oxidase function as terminal oxidases. These oxidative systems are localized outside the mitochondria.

Virtually all Structural components of the cell are endowed with autonomous oxidative systems of varying degrees of complexity and efficiency. For instance, nuclei possess not only glycolytic enzymes, but also A number of enzymes associated with oxidative phosphorylation. Plant Plastids, primarily chloroplasts, possess a quite complete system of redox enzymes.