Plant Physiology - Musienko M. M. 2001

Respiration
Tricarboxylic Acid Cycle

The Tricarboxylic acid cycle (Krebs Cycle): The Second Stage of Respiration

The sequence of reactions converting glucose into pyruvic acid is remarkably similar across all organisms and Cell types. However, this is not the case for Pyruvate, which undergoes utilization via diverse pathways.

In Yeast and certain other microorganisms under anaerobic conditions, pyruvate is converted through Alcoholic Fermentation:

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This reaction is catalyzed by pyruvate decarboxylase. Under normal conditions, many microorganisms convert pyruvic acid into lactate in a similar environment; under anoxia, this process also occurs in higher plants:

The reduction of pyruvate coupled with NADH oxidation to yield lactate is catalyzed by Lactate dehydrogenase.

Notably, the regeneration of NAD⁺ during the reduction of pyruvate to lactate sustains an uninterrupted glycolytic process under anaerobic conditions.

Thus, under anaerobic conditions, pyruvate enters fermentation pathways that yield no significant additional ATP synthesis. Consequently, in waterlogged soils, restricted aerobic respiration in The ROOT System diminishes the likelihood of ATP synthesis, which is essential for mineral ion uptake. As a result, plants growing in such soils exhibit a severe deficiency in various mineral elements.

Therefore, only a minor fraction of energy is released during fermentation. Considerably more energy is liberated under aerobic conditions within the tricarboxylic acid cycle and The final stage of respiration along the Electron Transport Chain. The aerobic pathway drives the Complete oxidation of glucose, generating significantly more ATP molecules than Glycolysis. All of these reactions take place within eukaryotic Mitochondria in two distinct stages: first in the tricarboxylic acid cycle, and subsequently in the Respiratory Electron Transport chain (ETC). The initial preparatory stage (A) involves the incorporation of Acetyl-CoA into the cycle, while the second stage (B) entails energy release stored in the form of NADH, FADH₂, and ATP (Fig. 90).

Fig. 90. Two Stages of the Krebs cycle (A — preparatory stage, B — energy release)

Mitochondria are known to be bounded by a double membrane, with the inner membrane forming numerous folds known as cristae. Their internal matrix is packed with Enzymes, Coenzymes, and phosphates involved in respiration. The outer membrane is permeable to most molecules, whereas the inner membrane is freely permeable only to pyruvic acid and ATP. The Enzymes of the tricarboxylic acid cycle reside in the matrix, while the Components of the respiratory ETC are embedded in the cristae membranes. The tricarboxylic acid cycle represents the final common pathway for The oxidation of respiratory substrates—Amino Acids, Fatty acids, and CARBOHYDRATES. It is also referred to as the Citric Acid Cycle or the Krebs cycle, named after Hans Krebs, who discovered and described it in 1937 and was awarded the Nobel Prize for this discovery. The operation of this cycle in plant Cells was first investigated by A. Chibnall (1939). The entry of pyruvate into this cycle occurs at the level of acetyl-coenzyme A (acetyl-CoA), which is formed in mitochondria via The oxidative decarboxylation of pyruvic acid mediated by the multi-enzyme pyruvate dehydrogenase complex (Fig. 91 a, b):

Fig. 91. a — model of acetyl-CoA, b — structural formula of acetyl-CoA

This reaction serves as the connecting link between glycolysis and the tricarboxylic acid cycle. The structural integration of Three types of enzymes and five coenzymes enables the multi-enzyme com-

plex to coordinate catalysis during this reaction. All intermediate products of the coupled pyruvate decarboxylation remain tightly bound to this complex.

During this reaction, NAD is reduced to NADH₂, and two carbon residues from the initial glucose molecule (pyruvic acid) form two acetyl groups (CH₃CO^-) with the simultaneous release of CO₂. Notably, fats and Amino acids can also be converted into acetyl-CoA, thereby entering THE RESPIRATORY PROCESS through the corresponding Reactions of the Krebs cycle.

The cycle begins with the aldol Condensation of oxaloacetate (OAA) and acetyl-CoA to yield citrate and CoA, a reaction catalyzed by citrate synthase:

Next, citrate is isomerized into isocitrate through a dehydration step followed by Hydration. This results in the reciprocal transfer of H⁺ and OH^-. This reaction is catalyzed by the enzyme aconitase, with cis-aconitate acting as the intermediate product:

Finally, the first of the four redox reactions of the tricarboxylic acid cycle takes place, in which isocitrate is oxidized and decarboxylated to a-ketoglutarate with the participation of isocitrate dehydrogenase:

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The Oxidative Decarboxylation of α-ketoglutarate yields succinyl-CoA, which contains a high-energy bond with enough energy to drive ATP synthesis. The Cleavage of the thioester bond in succinyl-CoA is coupled with the phosphorylation of guanosine diphosphate (GDP):

This reversible reaction is catalyzed by succinyl-CoA synthetase.

During the reaction, the phosphate group from GTP is transferred to ADP by nucleoside diphosphate kinase, yielding ATP:

This is another classic example of substrate-level phosphorylation. Remember that this is the only reaction in the tricarboxylic acid cycle that directly results in The formation of a high-energy phosphate bond. In glycolysis, we previously noted substrate-level phosphorylation during the oxidation of glyceraldehyde 3-phosphate and The conversion of phosphoenolpyruvate to pyruvate.

The final stage of the TCA cycle ensures the regeneration of oxaloacetate through the oxidation of succinate:

Thus, succinate is converted into oxaloacetate step-by-step via oxidation, hydration, and subsequent reoxidation.

Each turn of the cycle regenerates one molecule of oxaloacetate and generates energy in the form of FADH₂ and NADH (Fig. 92).

Succinate is oxidized by succinate dehydrogenase. Unlike the previous three oxidative steps in the cycle, FAD rather than NAD⁺ serves as the proton acceptor here. This is no coincidence; the Free energy change in this reaction is insufficient to reduce NAD⁺, which is why FAD acts as the acceptor in such reactions. In addition to a flavin moiety, succinate dehydrogenase contains four iron-sulfur (Fe-S) Proteins. Unlike most cycle enzymes and much like aconitase, it is an integral protein of The inner mitochondrial membrane and is directly linked to the Mitochondrial Electron Transport chain. Consequently, without dissociating from the enzyme, FADH₂ transfers electrons from succinate oxidation to the Fe³⁺ ions of its two Fe-S clusters and further through intermediate carriers to O₂.

Fig. 92. The Krebs cycle

Fumarase catalyzes the hydration of fumarate to form malate, which is then oxidized to oxaloacetate by malate dehydrogenase:

Thus, the Krebs cycle completes the oxidative pathway for respiratory substrate molecules as a source of energy. Its overall equation can be written as follows:

As shown, two carbons enter the cycle (via the condensation of the acetyl group with oxaloacetate) and two carbon atoms leave it as CO₂ during decarboxylation (of isocitrate and α-ketoglutarate).

In the four redox reactions of the cycle, three pairs of electrons are transferred to NAD⁺ and one to FAD. NAD⁺ molecules are reduced during the oxidative decarboxylation of isocitrate and α-ketoglutarate, as well as during the oxidation of malate. An additional NADH is formed during the oxidative decarboxylation of pyruvate.

Molecular oxygen (O₂) does not directly participate in any reaction of the cycle, although the cycle Functions exclusively under aerobic conditions. This is because NAD and FAD in mitochondria are regenerated subsequently when electrons are transferred to molecular oxygen during respiration in The electron transport chain. Two ATP molecules are formed per molecule of NADH. Only one high-energy ATP bond (per acetyl residue) is generated directly within the Krebs cycle from succinyl-CoA, while the rest (14 ATP) are synthesized in the electron transport chain.

Two molecules of H₂O are also consumed, which Supports V.I. Palladin's theory that during respiration, the oxygen from Water is incorporated into the oxidized substrate, while hydrogen is transferred to O₂ via "respiratory pigments" (dehydrogenases).

Consequently, the oxidation of a single pyruvate molecule yields 15 ATP molecules. Since the initial glucose molecule breaks down during glycolysis into two pyruvate molecules, the oxidation of two residues produces 30 ATP molecules.

It should be noted that The breakdown of a single pyruvic acid molecule in the aerobic phase of respiration (pyruvate decarboxylation and the Krebs cycle) releases 3 molecules of CO₂, 4 NADH₂, and 2 FADH₂. Thus, 5 pairs of H₂ (which can be viewed as 2H⁺ + 2e^-) derived from pyruvic acid and water enter the Respiratory Chain.

Intermediates of the Krebs Cycle. Regulation of Its Reactions

So far, we have examined the energy contribution of the Krebs cycle to plant cell METABOLISM, whereas it also plays a crucial role as a source of various intermediates for metabolic processes. Specifically, most of the carbon atoms for porphyrin Biosynthesis are supplied by succinyl-CoA, while the amination of oxaloacetic and α-ketoglutaric acids yields a series of amino acids. The utilization of intermediates must be accompanied by their replenishment, because the continuation of the cycle requires the condensation reaction of acetyl-CoA with oxaloacetic acid. The organic acids of the cycle play an important role in Nitrogen metabolism, and acetyl-CoA is used for the synthesis of Lipids and carbohydrates.

Thus, the functioning of the tricarboxylic acid cycle serves as a linking hub for the metabolism of such vital compounds as proteins, fats, and carbohydrates.

Overall, the Regulation of the rate at which the tricarboxylic acid cycle operates depends on The Cell's demand for ATP (Fig. 93).

An important regulatory reaction of the cycle is the synthesis of citrate from oxaloacetate and acetyl-CoA. The point is that ATP acts as an inhibitor of citrate synthase; an increase in its level reduces the saturation of the enzyme with acetyl-CoA.

The second regulatory step is the reaction catalyzed by isocitrate dehydrogenase. The enzyme is stimulated by the presence of ADP, whereas NADH inhibits enzyme activity by displacing NAD.

The third regulatory step is the reaction catalyzed by α-ketoglutarate dehydrogenase, which is inhibited by the products of this very reaction—succinyl-CoA and NADH×H. As a rule, a high energy charge decreases The activity of all three aforementioned enzymes. In such cases, an alternative electron transport pathway via alternative oxidase may form, maintaining a low level of ATP and a low NADH×H/NAD⁺ ratio.