Oxidation of Pyruvate: The Citric Acid Cycle

October 29, 2008 by vikas

In higher organisms, the oxidation of pyruvate takes place in subcellular, membranous organelles known as mitochondria. Because mitochondria are responsible for the synthesis of most of the ATP in nonphotosynthetic tissue, they are often referred to as the powerhouses of cells. Mitochondrial ATP synthesis is called oxidative phosphorylation since it is linked indirectly to oxidative reactions. In the complete oxidation of pyruvate, there are five oxidation–reduction reactions. Three of these reactions are oxidative decarboxylations. The electron acceptor (oxidizing agent) for four of the reactions is NAD+; the oxidizing agent for the fifth is flavin adenine dinucleotide, or FAD. Knowing the oxidation–reduction potentials of the reactants in an oxidation–reduction reaction permits the ready calculation of the standard free energy change for the reaction. It may be shown that

G0 = ?nFE0 , (1)

where n is the number of electrons transferred in the reaction, F is Faraday’s constant (23,060 cal/V-equivalent), and E0 is the difference between the E0 value of the oxidizing agent and that of the reducing agent. The reduced form of NAD+, NADH, is a strong reducing agent. The E0 at pH 7.0 of the NAD+–NADH couple is ?340 mV, which is equivalent to that of molecular hydrogen. E0 is the potential when the concentrations of the oxidized and reduced species of an oxidation–reduction pair are equal. Reduced FAD, FADH2, is a weaker reductant than NADH, with an E0 (pH 7.0) of about 0 V. In contrast, molecular oxygen is a potent oxidizing agent and fully reduced oxygen,water, is a very poor reducing agent.
The E0 (pH 7.0) for the oxygen–water couple is+815mV. The oxidation of NADH and FADH2 results in the reduction of oxygen to water:
H+ + NADH + 12O2 ? NAD+ + H2O (2)
and
FADH2 + 12 O2 ? FAD + H2O. (3)
In both cases two electrons are transferred to oxygen, so that the n in Eq. (1) is equal to 2. Under standard conditions, the oxidation of 1 mol of NADH by oxygen liberates close to 53 kcal, whereas the G0 for that of FADH2 is?38 kcal/mol. These two strongly exergonic reactions provide the energy for the endergonic synthesis of ATP. The details of carbon metabolism in the citric acid cycle are beyond the scope of this article. In brief, pyruvate is first oxidatively decarboxylated to yield CO2, NADH, and an acetyl group attached in an ester linkage to a thiol on a large molecule, known as coenzyme A, or CoA. Acetyl CoA condenses with a four-carbon dicarboxylic acid to form the tricarboxylic acid citrate. Free CoA is also a product. A total of four oxidation–reduction reactions, two of which are oxidative decarboxylations, take place, which results in the generation of the three remaining NADH molecules and one molecule of FADH2. The citric acid cycle is a true cycle. For each two-carbon acetyl moiety oxidized in the cycle, two CO2 molecules are produced and the four-carbon dicarboxylic acid with which acetyl CoA condenses is regenerated. The mitochondrial inner membrane contains proteins that act in concert to catalyze NADH and FADH2 oxidation by molecular oxygen. These reactions are carried out in many small steps by proteins that are integral to the membrane and that undergo oxidation–reduction. These proteins make up what is called the mitochondrial electron transport chain. Components of the chain include iron proteins (cytochromes and iron–sulfur proteins), flavoproteins (proteins that contain flavin), copper, and quinone binding proteins. The oxidation of NADH and FADH2 by molecular oxygen is coupled in mitochondria to the endergonic synthesis of ATP from ADP and Pi. For many years the nature of the common intermediate between electron transport and ATP synthesis was elusive. Peter Mitchell, who received a Nobel Prize in chemistry in 1978 for his extraordinary insights, suggested that this common intermediate was the proton electrochemical potential. He proposed in the early 1960s that electron transport through the mitochondrial chain is obligatorily linked to the movement of protons across the inner membrane of the mitochondrion. In this way, part of the energy liberated by oxidative electron transfer is conserved in the form of the proton electrochemical potential. Electron transport from NADH and FADH2 to oxygen provides the energy for the generation of the electrochemical potential of the proton. The flow of protons down this potential is exergonic and is the immediate source of energy for ATP synthesis. The proton-linked synthesis of ATP is catalyzed by a complex enzyme called ATP synthase. Remarkably similar enzymes are located in the coupling membranes of bacteria, mitochondria, and chloroplasts, the intracellular sites of photosynthesis in higher plants. Even though the reaction that they catalyze seems relatively straightforward, the ATP synthases contain a minimum of 8 different proteins and a total of about 20 polypeptide chains.

ATP is formed in the aqueous space bounded by the mitochondrial inner membrane. This space is known as the matrix. Most of the ATP generated within mitochondria is exported to the cytoplasm where it is used to drive energy-dependent reactions. TheADPand Pi formed in the cytoplasm must then be taken up by the mitochondria. The inner membrane contains specific proteins that mediate the export of ATP and the import of ADP and Pi. One transporter catalyzes counterexchange transport of ATP out of the matrix with ADP in the cytoplasm into the matrix. At physiological pH, ATP bears four negative charges, and ADP, three. Thus, the one-to-one exchange transport of ATP with ADP creates a membrane potential that is opposite in sign of that created by electrontransport- driven proton translocation. ATP/ADP transport costs energy and the direction of transport is poised by the proton membrane potential. In addition, phosphate uptake into mitochondria is coupled to the electrochemical proton potential. The phosphate translocator catalyzes the counterexchange transport of H2PO2? 4 and hydroxide anion (OH?). The outward movement of OH? causes acidification of the matrix, whereas the direction of proton transport driven by electron transport is out of the mitochondrial matrix and results in an increase in the pH of the matrix.
In the total oxidation of glucose to CO2 and water, six CO2 are released and six O2 are reduced to water. For each pyruvate oxidized, four NADH and one FADH2 are generated. Since two molecules of pyruvate are derived by means of glycolysis from one molecule of glucose, a total of eight NADH and two FADH2 are formed by pyruvate oxidation. Four electrons are required for the reduction of O2 to two molecules of H2O. Thus, pyruvate oxidation accounts for the reduction of five of the six molecules of O2 in the complete oxidation of glucose. The sixth O2 is reduced to water by electrons from the NADH formed by the oxidation of triose phosphate in glycolysis. Fermentation, or anaerobic glycolysis, yields but 2 mol of ATP per 1 mol of glucose catabolized. In contrast, complete oxidation of glucose to CO2 and water yields about 15 times more ATP. Thus, it is understandable why yeasts and some bacteria consume more glucose under anaerobic conditions than when oxygen is present. In animals, glucose is normally completely oxidized.
During strenuous exercise, however, the demand for oxygen by muscle tissues can outstrip its supply and the tissue may become anaerobic. Muscle contraction requires ATP, and rapid breakdown of glucose and its storage polymer, glycogen, takes place under anaerobiosis. Glycolysis would stop quickly if the NADH produced by the oxidation of triose phosphate were not converted back toNAD+.

In muscle cells under O2-limited conditions, pyruvate is reduced by NADH to lactic acid, a source of muscle cramps during exercise. At rest, lactic acid is converted back to glucose in the liver and kidneys and returned to muscle tissues where it stored in the form of glycogen.

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