Under aerobic conditions, pyruvate from glycolysis is converted to acetyl-coenzyme A (acetyl-CoA) and oxidized to CO2 in the tricarboxylic acid (TCA) cycle (also called the citric acid cycle). The electrons liberated by this oxidative process are passed via NADH and FADH2 through an elaborate, membrane-associated electron-transport pathway to O2, the final electron acceptor. Electron transfer is coupled to creation of a proton gradient across the membrane. Such a gradient represents an energized state, and the energy stored in this gradient is used to drive the synthesis of many equivalents of ATP. ATP synthesis as a consequence of electron transport is termed oxidative phosphorylation
(a) Pyruvate produced in glycolysis is oxidized in (b) the tricarboxylic acid (TCA) cycle. (c) Electrons liberated in this oxidation flow through the electron-transport chain and drive the synthesis of ATP in oxidative phosphorylation. In eukaryotic cells, this overall process occurs in mitochondria.
The universal importance of the TCA cycle, which results in low carbon isotopic fractionation, indicates it may have evolved especially early making it perhaps the most likely carbon fixation pathway for the last common ancestor (LCA). 1
THE CELL Evolution of the First Organism, Joseph Panno, Ph.D.
Glycolysis, the Krebs cycle, and the respiratory chain are all run and assembled by protein enzymes. These metabolic pathways are used by all prokaryotes that are alive today. The glycolytic pathway and the Krebs cycle are located in the protoplasm, while the respiratory chain is located in the cell membrane.
prokaryotes developed two aerobic systems for extracting energy from food molecules: the Krebs cycle, which stores most of the energy as electrons, and the electron transport chain, which uses the energy to make ATP. In eukaryotes, both processes occur in an organelle called the mitochondrion. Consequently, these organelles are responsible for providing the cell with the ATP it needs to power all its biochemical reactions (although a small amount of ATP is provided by glycolysis, which is carried out in the cell’s cytoplasm).
Some cells obtain energy (ATP) by fermentation, breaking down glucose in the absence of oxygen. For most eukaryotic cells and many bacteria, which live under aerobic conditionsand oxidize their organic fuels to carbon dioxide and water, glycolysis is but the first stage in the complete oxidation of glucose. Rather than being reduced to lactate, ethanol, or some other fermentation product, the pyruvate produced by glycolysis is further oxidized to H2O and CO2. This aerobic phase of catabolism is called respiration. In the broader physiological or macroscopic sense, respiration refers to a multicellular organism’s uptake of O2 and release of CO2. Biochemists and cell biologists, however, use the term in a narrower sense to refer to the molecular processes by which cells consume O2 and produce CO2—processes more precisely termed cellular respiration.
Catabolism of proteins, fats, and carbohydrates in the three stages of cellular respiration.
Stage 1: oxidation of fatty acids, glucose, and some amino acids yields acetyl-CoA.
Stage 2: oxidation of acetyl groups in the citric acid cycle includes four steps in which electrons are abstracted.
Stage 3: electrons carried by NADH and FADH2 are funneled into a chain of mitochondrial (or, in bacteria, plasma membrane–bound) electron carriers—the respiratory chain—ultimately reducing O2 to H2O. This electron flow drives the production of ATP.
In the first, organic fuel molecules—glucose, fatty acids, and some amino acids—are oxidized to yield two-carbon fragments in the form of the acetyl group of acetyl-coenzyme A (acetyl-CoA). In the second stage, the acetyl groups are fed into the citric acid cycle, which enzymatically oxidizes them to CO2; the energy released is conserved in the reduced electron carriers NADH and FADH2. In the third stage of respiration, these reduced coenzymes are themselves oxidized, giving up protons (H) and electrons.
The electrons are transferred to O2—the final electron acceptor—via a chain of electron-carrying molecules known as the respiratory chain. In the course of electron transfer, the large amount of energy released is conserved in the form of ATP, by a process called oxidative phosphorylation . Respiration is more complex than glycolysis and is believed to have emerged much later, after the appearance of cyanobacteria. The metabolic activities of cyanobacteria account for the rise of oxygen levels in the earth’s atmosphere, a dramatic turning point in evolutionary history. We consider first the conversion of pyruvate to acetyl groups, then the entry of those groups into the citric acid cycle, also called the tricarboxylic acid (TCA) cycle or the Krebs cycle. We next examine the cycle reactions and the enzymes that catalyze them. Because intermediates of the citric acid cycle are also siphoned off as biosynthetic precursors, we go on to consider some ways in which these intermediates are replenished. The citric acid cycle is a hub in metabolism, with degradative pathways leading in and anabolic pathways leading out, and it is closely regulated in coordination with other pathways.
Production of Acetyl-CoA (Activated Acetate)
In aerobic organisms, glucose and other sugars, fatty acids, and most amino acids are ultimately oxidized to CO2 and H2O via the citric acid cycle and the respiratory chain. Before entering the citric acid cycle, the carbon skeletons of sugars and fatty acids are degraded to the acetyl group of acetyl-CoA, the form in which the cycle accepts most of its fuel input. Many amino acid carbons also enter the cycle this way, although several amino acids are degraded to other cycle intermediates. Here we focus on how pyruvate, derived from glucose and other sugars by glycolysis, is oxidized to acetyl-CoA and CO2 by the pyruvate dehydrogenase (PDH) complex, a cluster of enzymes—multiple copies of each of three enzymes— located in the mitochondria of eukaryotic cells and in the cytosol of bacteria. A careful examination of this enzyme complex is rewarding in several respects. The PDH complex is a classic, much-studied example of a multienzyme complex in which a series of chemical intermediates remain bound to the enzyme molecules as a substrate is transformed into the final product. Five cofactors, four derived from vitamins, participate in the reaction mechanism. The regulation of this enzyme complex also illustrates how a combination of covalent modification and allosteric regulation results in precisely regulated flux through a metabolic step. Finally, the PDH complex is the prototype for two other important enzyme complexes: -ketoglutarate dehydrogenase, of the citric acid cycle, and the branched-chain -keto acid dehydrogenase,
of the oxidative pathways of several amino acids. The remarkable similarity in the protein structure, cofactor requirements, and reaction mechanisms of these three complexes doubtless reflects a common
Pyruvate Is Oxidized to Acetyl-CoA and CO2
The overall reaction catalyzed by the pyruvate dehydrogenase complex is an oxidative decarboxylation, an irreversible oxidation process in which the carboxyl group is removed from pyruvate as a molecule of CO2 and the two remaining carbons become the acetyl group of acetyl-CoA. The NADH formed in this reaction gives up a hydride ion (:H) to the respiratory chain , which carries the two electrons to oxygen or, in anaerobic microorganisms, to an alternative electron acceptor such as nitrate or sulfate. The transfer of electrons from NADH to oxygen ultimately generates 2.5 molecules of ATP per pair of electrons. The irreversibility of the PDH complex reaction has been demonstrated by isotopic labeling experiments: the complex cannot reattach radioactively labeled CO2 to acetyl-CoA to yield carboxyl-labeled pyruvate.
The Pyruvate Dehydrogenase Complex Requires Five Coenzymes
The combined dehydrogenation and decarboxylation of pyruvate to the acetyl group of acetyl-CoA requires the sequential action of three different enzymes and five different coenzymes or prosthetic groups—thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD), coenzyme A (CoA, sometimes denoted CoA-SH, to emphasize the role of the —SH group), nicotinamide adenine dinucleotide (NAD), and lipoate. Four different vitamins required in human nutrition are vital components of this system: thiamine (in TPP), riboflavin (in FAD), niacin (in NAD), and pantothenate (in CoA). We have already described the roles of FAD and NAD as electron carriers, and we have encountered TPP as the coenzyme of pyruvate decarboxylase
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