BIOCHEMISTRY - V. V. Emelyanov - 2016

SECTION 2. BIOLOGICAL OXIDATION

2.1. Stages of METABOLISM/26.html">Energy Metabolism

An essential property of living systems is metabolism, which is a complex aggregate of diverse biochemical processes through which incoming nutrients are assimilated, energy is released, and compounds necessary for the Organism are synthesized. Metabolism consists of two components: anabolism (the synthesis of complex substances from simpler ones with the consumption of energy) and Catabolism (The breakdown of more complex substances into simpler ones with the release of energy). Anabolism and catabolism are intrinsically interconnected: anabolism supplies substances for catabolism, while catabolism provides energy for anabolism. However, under conditions of constant Temperature, energy transfer in the form of heat from one chemical process to another is impossible. Therefore, living systems transfer energy via specialized high-energy compounds that possess a significant energy reserve. The universal high-energy compound in all Cells is adenosine triphosphate (ATP).

Catabolism is often metaphorically described as "combustion," and nutrients are said to "burn." The analogy between combustion and catabolism is quite conventional. In both cases, we are dealing with the chemical conversion (oxidation) of organic substances into CO₂ and H₂O in the presence of oxygen. However, combustion is an instantaneous, unregulated, and inefficient process (all energy is dissipated as heat). Conversely, during catabolism, the energy of organic substances is released in portions at various stages, efficiently conserved during ATP synthesis, and the process is regulated by The Cell's energy demands. Research has established that the efficiency (coefficient of performance) of the cellular ATP synthesis system is 40–45%. The balance between ATP synthesis and consumption is strictly maintained; hence, The amount of ATP present in the cell at any given moment is relatively small. Nevertheless, the total mass of ATP produced and consumed by an adult human body over the course of a day is comparable to their body weight!

Let us examine the metabolic "budget" in more detail: what is The Essence of ATP synthesis processes, and for what cellular needs is ATP consumed? Two pathways of ATP synthesis are known: aerobic (Oxidative Phosphorylation) and anaerobic (substrate-level phosphorylation). Anaerobic catabolism represents the incomplete oxidation of organic substances, characteristic exclusively of CARBOHYDRATES (Glycolysis), accompanied by The formation of lactic acid and a low energy yield—2 molecules of ATP per 1 molecule of glucose. Under physiological conditions, anaerobic catabolism provides no more than 10% of all cellular ATP. Skeletal Muscle is an exception to this general rule: in white muscle fibers, the bulk of ATP is synthesized anaerobically. Furthermore, glycolysis becomes the sole viable pathway for ATP production in any cell experiencing oxygen deprivation, or Hypoxia. However, in most cases, anaerobic catabolism cannot sustain cell viability for long. For instance, it is well known that the cells most sensitive to hypoxia—Neurons of the Cerebral Cortex—can survive without oxygen for no longer than 5 minutes.

The majority of cells derive over 90% of their required ATP through aerobic catabolism. This is a highly efficient biochemical process involving the Complete oxidation of organic substances (carbohydrates, Lipids, and Proteins) into Inorganic Compounds, CO₂ and H₂O, in the presence of oxygen. The aerobic oxidation of 1 molecule of glucose makes it possible to synthesize 38 molecules of ATP; amino acid oxidation yields similar values, while the complete oxidation of lipids supplies the cell with hundreds of ATP molecules per single molecule of the substance. Most cells can utilize all three classes of nutrients as Energy Sources. Nevertheless, a specific order of utilization is observed: carbohydrates serve as the primary energetic "fuel," and when their reserves are depleted, cells switch to lipid catabolism. Proteins undergo catabolism last, typically in extreme situations such as prolonged starvation.

Cells expend their entire synthesized ATP pool on performing various types of useful work. First, there is chemical work—anabolic reactions, which by definition require ATP expenditure. This includes all cellular biosyntheses, particularly the most "expensive" ones: the synthesis of Biopolymers (proteins, DNA and RNA, Polysaccharides) and lipids. Second, there is mechanical work—the movement of cells and their Organelles in space, including Muscle contraction. Finally, there is osmotic work—processes of active Transport of substances across Introduction/36.html">Biological Membranes aimed at establishing concentration gradients of these substances inside the cell and in the extracellular fluid. When the participating particles are charged ions—K⁺, Na⁺, Ca²⁺, Cl^-—both a concentration gradient and a potential difference are generated. In this case, electrical work is said to be performed. However, it should be borne in mind that the efficiency of all these types of work is substantially lower than 100%. The remaining fraction of ATP energy is converted into heat. This highlights yet another important function of all ATP breakdown processes—heat production.

It is customary to distinguish the following Main stages in energy metabolism.

Preparatory stage. This includes Hydrolysis reactions in the gastrointestinal tract that break down nutrients into their monomers: proteins into Amino Acids, polysaccharides into Monosaccharides (primarily glucose), and lipids into Fatty acids and glycerol. This stage is necessary to make nutrients accessible to every cell, since large molecules cannot be absorbed into the bloodstream.

Intermediary Metabolism. This encompasses numerous biochemical reactions involving amino acids, glucose, fatty acids, and glycerol, aimed at converting them into a limited range of "small" molecules—pyruvic acid, acetyl-CoA, and several others. Intermediary metabolism does not require oxygen and proceeds with the synthesis of a small amount of ATP via Substrate-Level Phosphorylation Reactions.

Terminal (final) stage. This involves the complete oxidation of intermediary metabolism products into inorganic substances. This stage takes place in the cell Mitochondria, where oxygen is consumed and CO₂ and H₂O are formed, and the bulk of ATP is synthesized via oxidative phosphorylation.

2.2. The Krebs Cycle

The Krebs cycle (Tricarboxylic Acid Cycle, TCA cycle) is The process of oxidizing acetyl-coenzyme A—the universal product of carbohydrate, lipid, and Protein Catabolism. The TCA cycle takes place in the mitochondria with the participation of 8 Enzymes localized in the matrix either in a free state or on the inner surface of the inner membrane.

The primary function of the TCA cycle is the formation of reduced Coenzymes NADH₂ and FADH₂, which supply protons to the Respiratory Chain. In addition, TCA cycle intermediates can be used for Gluconeogenesis, Transamination, and the synthesis of heme and fatty acids. Thus, the TCA cycle integrates all Types of Metabolism.

2.2.1. Reactions of the TCA Cycle

1. Citrate synthase catalyzes The conversion of oxaloacetate and acetyl-coenzyme A into citric acid (citrate):

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2. Aconitase converts citrate into isocitric acid (isocitrate). First, citrate undergoes dehydration with the formation of an intermediate product—cis-aconitic acid (cis-aconitate). This compound does not leave the Active Site of the enzyme, which is why its formula is conventionally enclosed in square brackets. The reaction is completed by The addition of Water across the double bond to form isocitrate:

3. Isocitrate dehydrogenase (decarboxylating) catalyzes The oxidative decarboxylation of isocitrate. First, the hydroxyl group of isocitrate is oxidized to a ketone group, yielding oxalosuccinic acid (oxalosuccinate). Subsequently, oxalosuccinate loses a carboxyl group as CO₂, forming α-ketoglutarate:

4. The next stage of the TCA cycle is the Oxidative Decarboxylation of α-ketoglutarate. It is catalyzed by the α-ketoglutarate dehydrogenase complex, consisting of 3 enzymes and 5 coenzymes: Thiamine diphosphate, coenzyme A, lipoamide, NAD⁺, and FAD. The reaction product, succinyl-coenzyme A, is a high-energy compound:

5. The synthesis of succinic acid (succinate) by the enzyme succinyl-coenzyme A synthetase. The energy of the high-energy thioester bond in the succinyl-coenzyme A molecule is utilized to synthesize a GTP molecule from GDP and phosphate—this is the only substrate-level phosphorylation reaction in the TCA cycle:

6. Succinate dehydrogenase, a flavoprotein of The inner mitochondrial membrane, catalyzes The oxidation of succinate to fumaric acid (fumarate):

7. Fumarase stereospecifically adds water across the double bond of fumarate, yielding L-malic acid (L-malate):

8. Malate dehydrogenase completes the TCA cycle by oxidizing the hydroxyl group of L-malate to form oxaloacetate, which then reacts with a new molecule of acetyl-CoA, and the cycle repeats:

The overall scheme of TCA cycle reactions is shown in Fig. 11.

Fig. 11. General scheme of the Krebs cycle reactions

2.2.2. Energy yield of a single turn of the TCA cycle

In 4 redox reactions of the TCA cycle, 3 NADH₂ and 1 FADH₂ are produced, which are subsequently channeled into the respiratory chain of oxidative phosphorylation. During oxidative phosphorylation in the respiratory chain, 1 NADH₂ yields 3 ATP, and 1 FADH₂ yields 2 ATP. From 1 GTP produced in the TCA cycle via substrate-level phosphorylation, 1 ATP is synthesized. Thus, a single turn of the TCA cycle yields 12 ATP.

2.3. Tissue Respiration and oxidative phosphorylation

The final stage of energy metabolism involves tissue respiration and oxidative phosphorylation, which take place in the mitochondria. Tissue Respiration is the consumption of oxygen in the mitochondrial respiratory chain for energy purposes. This function is carried out by a complex of enzymes localized in the inner mitochondrial membrane. Oxidative phosphorylation is the synthesis of ATP on the inner mitochondrial membrane in the presence of oxygen, catalyzed by the enzyme ATP synthase. A brief structural Overview of the components required for mitochondrial ATP synthesis is presented in Table 4. Let us now examine The Mechanism of their action.

The Components of the respiratory chain are arranged in the membrane in order of increasing electron affinity (e). Consequently, e moves along the respiratory chain from substances with lower affinity to those with higher affinity. The oxidation of NADH₂ proceeds as follows:

a) A pair of e and protons (H⁺) are transferred from NADH₂ to a flavoprotein, NADH dehydrogenase, which directs the pair of H⁺ into the intermembrane space and the pair of e to the iron-sulfur centers (FeS centers) of the respiratory chain.

b) Ubiquinone accepts a pair of e from the iron-sulfur centers and a pair of H⁺ from the matrix, becoming reduced ubiquinone; it then transfers the pair of H⁺ into the intermembrane space and the pair of e to cytochrome b₅₆₆.

Table 4. Components of the mitochondrial ATP synthesis system

c) Electrons from cytochrome b₅₆₆ are passed to cytochrome b₅₈₂ and then to ubiquinone, which accepts a pair of H⁺ from the matrix and is converted into reduced ubiquinone. Subsequently, the reduced ubiquinone transfers another pair of H⁺ into the intermembrane space and directs the electrons to the iron-sulfur center.

d) From this point onward, the respiratory chain transports only electrons; H⁺ transport into the matrix is complete. The electrons received from the iron-sulfur center are transported via the cytochrome system (c₁, c, a, and a₃) to the final acceptor, oxygen.

e) Cytochromes a and a₃ are closely linked and collectively termed cytochrome c oxidase, as they directly interact with molecular oxygen. Cytochrome c oxidase contains not only heme iron but also copper. The active site of cytochrome c oxidase catalyzes the reaction:

4e + 4H⁺ + O₂ —> 2H₂O.

Thus, four-electron reduction of oxygen takes place in the mitochondrial respiratory chain, resulting in the formation of "metabolic water".

In the case of FADH₂ oxidation, The sequence of events is identical, but it begins at step (b): ubiquinone accepts a pair of electrons and H⁺ directly from FADH₂.

It has been established that the difference in redox potentials between the first component of the respiratory chain (NADH₂) and the last (oxygen) is approximately 1.14 V, which provides an energy reserve of about 220 kJ, sufficient for the synthesis of 3 ATP molecules. Therefore, the entire respiratory chain can be conditionally divided into 3 segments where the difference in redox potentials is sufficient for the synthesis of 1 ATP molecule—these are the 3 coupling sites of respiration and phosphorylation: the 1st is between NADH₂ and NADH dehydrogenase, the 2nd is between cytochromes b₅₈₂ and c, and the 3rd is between cytochromes a and a₃. Thus, the oxidation of 1 molecule of NADH₂ in the respiratory chain makes it possible to synthesize 3 molecules of ATP, whereas the oxidation of 1 molecule of FADH₂ yields only 2 molecules of ATP.

2.3.1. Chemiosmotic Theory

To explain the Mechanism of Oxidative phosphorylation, the chemiosmotic theory was proposed by P. Mitchell in 1961. In generalized terms, the essence of the chemiosmotic theory can be described as follows: the inner mitochondrial membrane separates the flows of e and H⁺—the former are transported along the membrane, while the latter are transported across it. This creates a transmembrane Electrochemical Potential driven by the difference in charges and proton concentrations between the mitochondrial matrix and the intermembrane space. The energy of this electrochemical potential is utilized by ATP synthase to synthesize ATP from ADP and phosphate, i.e., for phosphorylation. According to P. Mitchell's concept, tissue respiration and oxidative phosphorylation are coupled, with the electrochemical potential serving as the linking bridge between them: respiration generates it, and phosphorylation consumes it.

During the translocation of H⁺ from the matrix into the intermembrane space, an osmotic proton gradient (chemical potential) is established across the inner membrane:

∆pH = 60 mV (at ∆pH = 1) (pH is higher in the matrix than in the intermembrane space). Since each H⁺ carries a positive charge, an electrical potential difference ∆V = 160 mV also arises across the inner membrane, with the inner side of the membrane charging negatively and the outer side positively. Together, the osmotic proton gradient and the potential difference form an electrochemical potential of about 220 mV.

The energy of the electrochemical potential generated across the inner mitochondrial membrane can be utilized for the phosphorylation of ADP to ATP, The transport of substances across the mitochondrial membrane, and heat production.

Information regarding the Structure AND FUNCTIONS of the components of the Respiratory Chain and ATP synthase is summarized in Table 4.

2.3.2. Mechanism of Oxidative Phosphorylation

The phosphorylation process is carried out by ATP synthase (H⁺-ATPase), which consumes 40–45% of the Free energy released during oxidation. ATP synthase is an integral protein composed of two subunits, F₀ and F₁. The F₀ subunit forms a channel in the membrane designed for the transport of protons from the intermembrane space into the matrix. The F₁ subunit is catalytic—it is responsible for catalyzing the synthesis of ATP from ADP and phosphate. When the value of the transmembrane potential reaches a critical threshold of 200–250 mV, H⁺ ions rush from the intermembrane space into the matrix through the opened channel. The energy released As a result is utilized by the enzyme to form the high-energy bond of ATP from ADP and phosphate—this is oxidative phosphorylation. With each transfer of H⁺ through the F₀ channel, the energy of the electrochemical potential is expended on rotating the stalk of the F₁ subunit, which alters the conformation of the enzyme and facilitates the binding of ADP and phosphate.

2.4. REGULATION OF ENERGY Metabolism

The Krebs cycle, tissue respiration, and oxidative phosphorylation function in a coordinated manner within the cell. The primary principle of their coordinated regulation is that ATP synthesis must meet the cell's demand for it. When ATP is actively consumed in the cell, ADP accumulates. ADP serves as the primary activator of all stages of energy metabolism. The dependence of the cell respiration rate on ADP concentration is known as Respiratory Control.

Respiratory control is carried out with the participation of 4 regulatory Enzymes of the TCA cycle: citrate synthase, isocitrate dehydrogenase, the α-ketoglutarate dehydrogenase complex, and malate dehydrogenase. These TCA cycle enzymes are inhibited by NADH₂ and ATP, which are the End products of the TCA cycle and oxidative phosphorylation. NAD⁺ and ADP activate these TCA cycle enzymes. Furthermore, The rate of oxidative phosphorylation is directly limited by the availability of ADP, which enters the matrix in exchange for ATP.

Energy metabolism is also under hormonal control. Insulin, through a series of intermediate messengers, activates the TCA cycle at the level of the α-ketoglutarate dehydrogenase complex, as well as activates the oxidative decarboxylation of pyruvate—which supplies acetyl-CoA to the Krebs cycle—and glycolysis, which produces pyruvate. It is well established that in insulin deficiency, which develops in Diabetes Mellitus, these aerobic processes are inhibited, and pyruvate is diverted to lactate. THYROID Hormones—thyroxine and triiodothyronine—stimulate cellular energy metabolism. Under The Influence of thyroid hormones, the transport of ADP into the mitochondrial matrix is activated, tissue respiration and ATP synthesis are accelerated, and heat production is increased.

2.5. Other Pathways of Oxygen Utilization

Pathways of oxygen utilization for plastic purposes are also well known. Microsomal oxidation reactions, which proceed actively in The Liver and Adrenal Glands, lead to the incorporation of oxygen into the substrate being oxidized. Endogenous substrates for microsomal oxidation include Cholesterol, Steroid Hormones, and Unsaturated fatty acids. This process plays a crucial role in the detoxification of xenobiotics (compounds foreign to the organism).

A small amount of oxygen in cells is converted into reactive oxygen species—free radicals and their precursors—which are capable of disrupting The structure of lipids, proteins, and Nucleic Acids. To protect against such damage, cells possess an antioxidant system that includes A number of enzymes (catalase, superoxide dismutase, Glutathione peroxidase, glutathione reductase) as well as low-molecular-weight organic substances, including Vitamins A, C, and E, Lipoic Acid, and glutathione.

A comparative characterization of various pathways of oxygen utilization in the cell is presented in Table 5.

Questions for Material Comprehension Review

1. THE CONCEPT OF energy metabolism in living systems, Bioenergetics, and Biological Oxidation. High-energy bonds and high-energy compounds, their types and significance. ATP as a universal energy carrier. Pathways of ATP generation and utilization in the cell; comparative characteristics of oxidative and substrate-level phosphorylation.

2. Stages of Nutrient energy utilization: preparatory stage, intermediary metabolism, and mitochondrial stage; their essence and significance. Formation and excretion of CO₂ and H₂O as the End Products of Metabolism.

3. The Tricarboxylic Acid Cycle (Krebs cycle): its essence and biological significance. Reactions of the Krebs cycle: substrates, enzymes, coenzymes. Regulation of the Krebs cycle.

Table 5. Pathways of oxygen utilization in BIOLOGICAL OXIDATION REACTIONS

4. Mitochondrial respiratory chain: structure and functions, properties of components, biological significance. Structure and functions of ATP synthase. Mechanism of coupling between tissue respiration (TR) and oxidative phosphorylation (OP)—Mitchell's chemiosmotic theory. Uncoupling of TR and OP: essence and biological significance.

5. Mono- and dioxygenase reactions (microsomal oxidation). The Role of microsomal oxidation in the detoxification of xenobiotics. Free-radical pathway of oxygen utilization: reactive oxygen species and their biological significance. Cellular antioxidant defense: enzymatic and non-enzymatic mechanisms, and their significance.

Written homework assignment

Mandatory

1. Ethanol catabolism in the body mainly occurs in the liver According to the following pathway:

and concludes with the oxidation of acetyl-CoA in the Krebs cycle.

Calculate the Energy balance (number of ATP molecules) for the complete oxidation of ethanol to CO₂ and H₂O. What substances (enzymes, coenzymes, substrates) are required for proper ethanol catabolism?

2. Mitochondria were isolated from myocardial cells and incubated in a medium with sufficient oxygen and biological oxidation substrates. During the experiment, various substances known to affect tissue respiration and oxidative phosphorylation were added to the suspension: ADP, a respiratory chain inhibitor, and an uncoupler of respiration and phosphorylation. The following graphs show oxygen consumption and ATP production in this system before the addition of modulators (section A) and after it (section B):

Which of the listed modulators could account for the observed patterns in each case? Provide a rationale for your answer.

3. Naphthalene is a toxic compound. Upon entering the body through the gastrointestinal tract, naphthalene accumulates in the liver, where it is detoxified according to the following pathway:

What type of oxygen utilization pathway takes place in this case? Indicate the Enzymes and Coenzymes for each stage on the diagram. What is the Biological Significance of The change in water solubility of the metabolites compared to naphthalene?

4. Under normal mitochondrial function, oxygen is reduced to water according to the following scheme: О₂ + 4ё + 4Н⁺ —> Н₂О, yet a small amount of it is converted into the superoxide anion radical: О₂ + ё —> О₂^-. What are the positive and negative implications of this process? What cellular mechanisms exist to inactivate this compound? Under what conditions would mitochondria be expected to produce large amounts of superoxide?

Optional

1. Acetylsalicylic acid (aspirin) is widely used as an anti-inflammatory, antipyretic, and analgesic agent. In the gastrointestinal tract, acetylsalicylic acid is hydrolyzed into acetic and salicylic acids; the latter is metabolized in the liver according to the following pathway:

Determine what type of oxygen utilization pathway is involved here. Indicate the enzymes and coenzymes for each stage on the diagram. How does the water solubility of the metabolites change compared to salicylic acid? What is the biological significance of this phenomenon?

2. A phospholipid radiolabeled with ¹⁴C in the linolenic acid residue was added to a cell culture. The introduced label was rapidly detected in The Plasma Membrane of the cells. However, following ultraviolet irradiation of the culture, the amount of the label in the membranes decreased twofold. The experiment was repeated after pre-incubating the culture with vitamin E. Under these conditions, the label content decreased by only 10%. What is the reason for the observed differences?

Sample test for the topic "Biological Oxidation"

Instructions: Unless otherwise specified in the test question, select a single correct answer.

1. Choose the name of the enzyme that catalyzes the following reaction of the Krebs cycle:

a) fumarase;

б) isocitrate dehydrogenase;

в) malate dehydrogenase;

г) succinate dehydrogenase;

д) citrate synthase.

2. Which of the following processes occurs during the mitochondrial stage of energy metabolism:

а) oxidative phosphorylation;

б) dehydrogenation of carboxylic acids in the Krebs cycle;

в) breakdown of acetyl-CoA to CO₂;

г) reduction of oxygen to water;

д) all of the above.

3. How do cyanide respiratory inhibitors affect oxygen consumption and ATP Synthesis in mitochondria:

а) oxygen consumption increases, ATP synthesis increases;

б) oxygen consumption increases, ATP synthesis decreases;

в) oxygen consumption decreases, ATP synthesis increases;

г) oxygen consumption decreases, ATP synthesis decreases.

4. What is the function of vitamin E in the cell:

а) it is essential for the Krebs cycle;

б) it participates in substrate-level phosphorylation reactions;

в) it transfers electrons and protons in the mitochondrial respiratory chain;

г) it is the primary fat-soluble antioxidant;

д) it acts as an uncoupler of respiration and phosphorylation.

5. Which coenzyme is required for the functioning of cytochrome P₄₅₀:

а) NADH₂;

б) NADPH₂;

в) ascorbic acid;

г) lipoic acid;

д) thiamine pyrophosphate.

6. What is the energy efficiency of oxidation of the coenzymes NADH₂ and FADH₂ in the respiratory chain:

а) NADH₂ and FADH₂ — 3 ATP each;

б) NADH₂ and FADH₂ — 2 ATP each;

в) NADH₂ — 3 ATP, FADH₂ — 2 ATP;

г) NADH₂ — 2 ATP, FADH₂ — 3 ATP.

7. Indicate the role of glutathione (G-SH) in the cellular antioxidant defense system:

а) acts as a coenzyme for glutathione peroxidase;

б) acts as a coenzyme for glutathione reductase;

в) neutralizes the superoxide anion radical;

г) serves as the primary fat-soluble antioxidant;

д) all of the above.

8. Choose the characteristic feature of microsomal xenobiotic oxidation:

а) oxygen is consumed, and lipophilic substances become hydrophilic;

б) oxygen is consumed, and hydrophilic substances become lipophilic;

в) oxygen is not consumed, and lipophilic substances become hydrophilic;

г) oxygen is not consumed, and hydrophilic substances become lipophilic.

9. Fill in the blanks (3 Answers) in the sentence: "The movement of ... along the mitochondrial respiratory chain creates an energy reserve for the translocation of ... across the membrane, and the energy of the Membrane Potential is utilized for the synthesis of ..."

10. Provide the names and class numbers of the enzymes (6 answers) catalyzing reactions 1, 2, and 3: