We now continue our detailed discussion of glucose oxidation and ATP generation, exploring what happens to the pyruvate generated during glycolysis (stage I, see Figures 1 and 2) after it is transported into the mitochondrial matrix. The last three of the four stages of glucose oxidation (Figure 3) are
● Stage II. Stage II can be subdivided into two distinct parts: (1) the conversion of pyruvate to acetyl CoA, followed by (2) oxidation of acetyl CoA to CO2 in the citric acid cycle. These oxidations are coupled to reduction of NAD+ to NADH and of FAD to FADH2. These two carriers can be considered the sources of high-energy electrons. (Fatty acid oxidation follows a similar route, with conversion of fatty acyl CoA to acetyl CoA.) Most of the reactions occur in or on the inner membrane facing the matrix.
● Stage III. Electron transfer from NADH and FADH2 to O2 via an electron-transport chain within the inner mem brane converts the energy carried in those electrons into an electrochemical gradient across that membrane, called the proton-motive force.
● Stage IV. The energy of the proton-motive force is harnessed for ATP synthesis in the inner mitochondrial membrane. Stages III and IV are together called oxidative phosphorylation.
Fig1. Overview of aerobic oxidation and photosynthesis. Eukaryotic cells use two fundamental mechanisms to convert external sources of energy into ATP. (Top) In aerobic oxidation, “fuel” molecules [primarily sugars and fatty acids (lipids)] undergo preliminary processing in the cytosol, such as breakdown of glucose to pyruvate (stage I), and are then transferred into mitochondria, where they are converted by oxidation with O2 to CO2 and H2O (stages II and III) and ATP is generated (stage IV). (Bottom) In photosynthesis, which occurs in chloroplasts, the radiant energy of light is absorbed by specialized pigments (stage 1); the absorbed energy is used both to oxidize H2O to O2 and to establish conditions (stage 2) necessary for the generation of ATP (stage 3) and of carbohydrates from CO2 (carbon fixation, stage 4). Both mechanisms involve the production of reduced high-energy electron carriers (NADH, NADPH, FADH2) and the movement of electrons down an electric potential gradient in an electron-transport chain through specialized membranes. Energy released from these electrons is captured as a pro ton electrochemical gradient (proton-motive force) that is then used to drive ATP synthesis. Bacteria use comparable processes.
Fig2. The glycolytic pathway. A series of ten reactions degrades glucose to pyruvate. Two reactions con sume ATP, forming ADP and phosphorylated sugars (red), two generate ATP from ADP by substrate-level phosphoryla tion (green), and one yields NADH by reduction of NAD+ (yellow). Note that all the intermediates between glucose and pyruvate are phosphorylated compounds. Steps 1, 3 and 10, with single arrows, are essentially irreversible (have large negative ΔG values) under ordinary conditions in cells.
Fig3. Summary of aerobic oxidation of glucose and fatty acids. Stage I: In the cytosol, glucose is converted to pyruvate (glycolysis) and fatty acid to fatty acyl CoA. Pyruvate and fatty acyl CoA then move into the mitochondrion. Mitochondrial porins make the outer membrane permeable to these metabolites, but specific transport proteins (colored ovals) in the inner membrane are required to import pyruvate (yellow) and fatty acids (blue) into the matrix. Fatty acyl groups are transferred from fatty acyl CoA to an intermediate carrier, transported across the inner membrane, and then reattached to CoA on the matrix side. Stage II: In the mitochondrial matrix, pyruvate and fatty acyl CoA are converted to acetyl CoA and then oxidized, releasing CO2. Pyruvate is converted to acetyl CoA with the formation of NADH and CO2; two carbons from fatty acyl CoA are converted to acetyl CoA with the formation of FADH2 and NADH. Oxidation of acetyl CoA in the citric acid cycle generates NADH and FADH2, GTP, and CO2. Stage III: Electron transport reduces O2 to H2O and generates a proton motive force. Electrons (blue) from reduced coenzymes are transferred via electron-transport complexes (blue boxes) to O2 concomitant with transport of H+ ions (red) from the matrix to the intermembrane space, generating the proton-motive force. Electrons from NADH flow directly from complex I to complex III, bypassing complex II. Electrons from FADH2 flow directly from complex II to complex III, bypassing com plex I. Stage IV: ATP synthase, also called the F0F1 complex (orange), harnesses the proton-motive force to synthesize ATP in the matrix. Antiporter proteins (purple and green ovals) transport ADP and Pi into the matrix and export hydroxyl groups and ATP. NADH generated in the cytosol is not transported directly to the matrix because the inner membrane is impermeable to NAD+ and NADH; instead, a shuttle system (red) transports electrons from cytosolic NADH to NAD+ in the matrix. O2 diffuses into the matrix, and CO2 diffuses out.
In the First Part of Stage II, Pyruvate Is Converted to Acetyl CoA and High-Energy Electrons
Within the mitochondrial matrix, pyruvate reacts with coenzyme A, forming CO2, acetyl CoA, and NADH (Figure 3, stage II, left). This reaction, catalyzed by pyruvate dehydroge nase, is highly exergonic (ΔG°′ = −8.0 kcal/mol) and essentially irreversible. Influx of calcium from the MAM into the mitochondrion increases the activity of pyruvate dehydrogenase, driving the formation of acetyl CoA.
Acetyl CoA is a molecule consisting of a two-carbon acetyl group covalently linked to a longer molecule known as coenzyme A (CoA) (Figure 4). It plays a central role in the oxidation of pyruvate, fatty acids, and amino acids. In addition, it is an intermediate in numerous biosynthetic re actions, including the transfer of an acetyl group to histone and many other mammalian proteins and the synthesis of lipids such as cholesterol. In respiring mitochondria, however, the two-carbon acetyl group of acetyl CoA is almost always oxidized to CO2 via the citric acid cycle. Note that the two carbons in the acetyl group come from pyruvate; the third carbon of pyruvate is released as carbon dioxide.
Fig4. The structure of acetyl CoA. This compound, consisting of an acetyl group covalently linked to a coenzyme A (CoA) molecule, is an important intermediate in the aerobic oxidation of pyruvate, fatty acids, and many amino acids. It also contributes acetyl groups to many biosynthetic pathways.
In the Second Part of Stage II, the Citric Acid Cycle Oxidizes the Acetyl Group in Acetyl CoA to CO2 and Generates High-Energy Electrons
Nine sequential reactions operate in a cycle to oxidize the acetyl group of acetyl CoA to CO2 (Figure 3, stage II, right). This cycle is referred to by several names: the citric acid cycle, the tricarboxylic acid (TCA) cycle, and the Krebs cycle. The net result is that for each acetyl group entering the cycle as acetyl CoA, two molecules of CO2, three of NADH, and one each of FADH2 and GTP are produced. NADH and FADH2 are high-energy electron carriers that will play a major role in stage III of mitochondrial oxidation: electron transport.
As shown in Figure 5, the cycle begins with condensation of the two-carbon acetyl group from acetyl CoA and the four-carbon molecule oxaloacetate to yield the six-carbon citric acid (hence the name citric acid cycle). Reactions step 4 and step 5 each release a CO2 molecule and reduce NAD+ to NADH. The source of the oxygen for generating the CO2 molecules in these reactions is water (H2O), not molecular oxygen (O2), and the enzymatic activities of the enzymes catalyzing reactions step 4 and step 5 are increased by the influx of calcium into the mitochondrion from the MAM. Reduction of NAD+ to NADH also occurs during reaction step 9; thus three NADHs are generated per turn of the cycle. In re action step 7, two electrons and two protons are transferred to FAD, yielding the reduced form of this coenzyme, FADH2. Reaction step 7 is distinctive not only because it is an intrinsic part of the citric acid cycle (stage II), but also because it is catalyzed by a membrane-attached enzyme that, as we shall see, also plays an important role in stage III. In reaction step 6, hydrolysis of the high-energy thioester bond in succinyl CoA is coupled to synthesis of one GTP by substrate-level phosphorylation. Because GTP and ATP are interconvertible,
this can be considered an ATP-generating step. Reaction step 9 regenerates oxaloacetate, so the cycle can begin again. Note that molecular O2 does not participate in the citric acid cycle.
Fig5. The citric acid cycle. Acetyl CoA is metabolized to CO2 and the high-energy electron carriers NADH and FADH2. In reaction 1, a two-carbon acetyl residue from acetyl CoA condenses with the four-carbon molecule oxaloacetate to form the six-carbon citrate. In the remaining reactions ( 2–9), each molecule of citrate is eventually converted back to oxaloacetate, losing two CO2 molecules in the process. In each turn of the cycle, four pairs of electrons are removed from carbon atoms, forming three molecules of NADH, one molecule of FADH2, and one molecule of GTP. The two carbon atoms that enter the cycle with acetyl CoA are highlighted in blue through succinyl CoA. In succinate and fumarate, which are symmetric molecules, they can no longer be specifically denoted. Isotope-labeling studies have shown that these carbon atoms are not lost in the turn of the cycle in which they enter; on average, one will be lost as CO2 during the next turn of the cycle and the other in subsequent turns.
Most enzymes and small molecules involved in the citric acid cycle are soluble in the aqueous mitochondrial matrix. These include CoA, acetyl CoA, succinyl CoA, NAD+, and NADH, as well as most of the citric acid cycle enzymes. Succinate dehydrogenase (reaction step 7), however, is a component of an integral membrane protein in the inner membrane, with its active site facing the matrix. When mitochondria are disrupted by gentle ultrasonic vibration or by osmotic lysis, the non-membrane-bound enzymes of the citric acid cycle are released as very large multiprotein complexes. It is believed that within such complexes, the reaction product of one enzyme passes directly to the next enzyme without diffusing through the solution.
Because glycolysis of one glucose molecule generates two pyruvate molecules, and thus two acetyl CoA molecules, the reactions in the glycolytic pathway and citric acid cycle produce six CO2 molecules, ten NADH molecules, and two FADH2 molecules per glucose molecule (Table 1). Al though these reactions also generate four high-energy phosphoanhydride bonds in the form of two ATP and two GTP molecules, this represents only a small fraction of the available energy released in the complete aerobic oxidation of glucose. The remaining energy is stored as high-energy electrons in the reduced coenzymes NADH and FADH2, which can be thought of as high-energy electron carriers. The goal of stages III and IV is to recover this energy in the form of ATP.
Table1. Net Result of the Glycolytic Pathway and the Citric Acid Cycle
Transporters in the Inner Mitochondrial Membrane Help Maintain Appropriate Cytosolic and Matrix Concentrations of NAD+ and NADH
In the cytosol, NAD+ is required for step 6 of glycolysis (see Figure 2), and in the mitochondrial matrix, NAD+ is re quired for the conversion of pyruvate to acetyl CoA and for three steps in the citric acid cycle (step 4, step 5, and step 9 in Figure5). In each case, NADH is a product of the reaction. If glycolysis and oxidation of pyruvate are to continue, NAD+ must be regenerated by oxidation of NADH to ensure that this substrate is available. (Similarly, the FADH2 generated in stage II reactions must be reoxidized to FAD if FAD-dependent reactions are to continue.) the electron-transport chain within the inner mitochondrial mem brane converts NADH to NAD+ and FADH2 to FAD as it reduces O2 to water and converts the energy stored in the high energy electrons in the reduced forms of these molecules into a proton-motive force (stage III). Even though O2 is not involved in any reaction of the citric acid cycle, in the absence of O2 this cycle soon stops operating because in such anaerobic conditions, the mitochondria cannot regenerate the required NAD+ and FAD substrates. NAD+ and FAD dwindle due to the inability of the electron-transport chain within the mitochondrion to oxidize NADH and FADH2. These observations raise the question of how a supply of NAD+ in the cytosol is regenerated.
If the NADH from the cytosol could move into the mitochondrial matrix and be oxidized by the electron-transport chain, and if the NAD+ product could be transported back into the cytosol, regeneration of cytosolic NAD+ would be simple when O2 is available. However, the inner mitochondrial membrane is impermeable to NADH. To bypass this problem and permit the electrons from cytosolic NADH to be transferred indirectly to O2 via the mitochondrial electron-transport chain, cells use several electron shuttles to transfer electrons from NADH in the cytoplasm to NAD+ in the matrix. The operation of the most widespread shuttle— the malate-aspartate shuttle—is depicted in Figure 6.
Fig6. The malate-aspartate shuttle. This cyclical series of reactions transfers electrons from NADH in the cytosol (via the inter membrane space) across the inner mitochondrial membrane, which is impermeable to NADH itself, to NAD+ in the matrix. The net result is the replacement of cytosolic NADH with NAD+ and matrix NAD+ with NADH. Step 1: Cytosolic malate dehydrogenase transfers electrons from cytosolic NADH to oxaloacetate, forming malate. Step 2: An antiporter (blue oval) in the inner mitochondrial membrane transports malate into the matrix in exchange for α-ketoglutarate. Step 3: Mitochondrial malate dehydrogenase converts malate back to oxaloacetate, reducing NAD+ in the matrix to NADH in the process. Step 4: Oxaloacetate, which cannot directly cross the inner membrane, is converted to aspartate by addition of an amino group from glutamate. In this transaminase-catalyzed reaction in the matrix, glutamate is converted to α-ketoglutarate. Step 5: A second antiporter (red oval) exports aspartate to the cytosol in exchange for glutamate. Step 6 A cytosolic transaminase converts aspartate to oxaloacetate and α-ketoglutarate to glutamate, completing the cycle. The blue arrows reflect the movement of the α-ketoglutarate, the red arrows the movement of glutamate, and the black arrows that of aspartate/malate. It is noteworthy that as aspartate and malate cycle clockwise, glutamate and α-ketoglutarate cycle in the opposite direction.
If the NADH from the cytosol could move into the mitochondrial matrix and be oxidized by the electron-transport chain, and if the NAD+ product could be transported back into the cytosol, regeneration of cytosolic NAD+ would be simple when O2 is available. However, the inner mitochondrial membrane is impermeable to NADH. To bypass this problem and permit the electrons from cytosolic NADH to be transferred indirectly to O2 via the mitochondrial electron-transport chain, cells use several electron shuttles transfer electrons from NADH in the cytoplasm to NAD+ in the matrix. The operation of the most widespread shuttle— the malate-aspartate shuttle—is depicted in Figure6.
For every complete cycle of the shuttle, there is no overall change in the numbers of NADH and NAD+ molecules or the intermediates aspartate or malate. In the cytosol, however, NADH is oxidized to NAD+, which can be used for glycolysis, and in the matrix, NAD+ is reduced to NADH, which can be used for electron transport:
Mitochondrial Oxidation of Fatty Acids Generates ATP
Up to now, we have focused mainly on the oxidation of carbohydrates, namely glucose, for ATP generation. Fatty acids are another important source of cellular energy. Cells can take up either glucose or fatty acids from the extracellular space with the help of specific transporter proteins. Should a cell not need to burn these molecules immediately, it can store them as a polymer of glucose called glycogen (especially in muscle or liver) or as a trimer of fatty acids covalently linked to glycerol, called a triacylglycerol or triglyceride. In some cells, excess glucose is converted into fatty acids and then triacylglycerols for storage. However, unlike microorganisms, animals are unable to convert fatty acids to glucose. When the cells need to burn these energy stores to make ATP (e.g., when a resting muscle begins to do work and needs to burn glucose or fatty acids as fuel), enzymes break down glycogen to glucose or hydrolyze triacylglycerols to fatty acids, which are then oxidized to generate ATP:
Fatty acids are the major energy source for some tissues, particularly adult heart muscle. In humans, in fact, more ATP is generated by the oxidation of fats than by the oxidation of glucose. The oxidation of 1 g of triacylglycerol to CO2 generates about six times as much ATP as does the oxidation of 1 g of hydrated glycogen. Thus, considering the mass of stored fuel an organism must carry, triglycerides are more efficient than carbohydrates for storage of energy, in part because they are stored in anhydrous form and can yield more energy when oxidized, and in part because they are intrinsically more reduced (have more hydrogens) than carbohydrates. In mammals, the primary site of storage of triacylglycerol is fat (adipose) tissue, whereas the primary sites for glycogen storage are muscle and the liver. In animals, when tissues need to generate a lot of ATP, as in exercising muscle, signals are sent to adipose tissue to hydrolyze triacylglycerols and to release the fatty acids into the circulatory system so that they can move to and be transported into the ATP-requiring tissues.
Just as there are four stages in the oxidation of glucose, there are four stages in the oxidation of fatty acids. To optimize the efficiency of ATP generation, part of stage II (citric acid cycle oxidation of acetyl CoA) and all of stages III and IV of fatty acid oxidation are identical to those of glucose oxidation. The differences lie in cytosolic stage I and in the first part of mitochondrial stage II. In stage I, fatty acids are converted to a fatty acyl CoA in the cytosol in a reaction coupled to the hydrolysis of ATP to AMP and PPi (inorganic pyrophosphate):
Subsequent hydrolysis of PPi to two molecules of Pi releases energy that drives this reaction to completion. To enter the mitochondrial matrix, the fatty acyl group must be covalently transferred to a molecule called carnitine and moved across the inner mitochondrial membrane by an acylcarnitine transporter protein (see Figure 3, blue oval); then, on the matrix side, the fatty acyl group is released from carnitine and reattached to another CoA molecule. The activity of the acylcarnitine transporter is regulated to prevent oxidation of fatty acids when cells have adequate energy (ATP) supplies.
In the first part of stage II, each molecule of a fatty acyl CoA in the mitochondrion is oxidized in a cyclical sequence of four reactions in which all the carbon atoms are converted, two at a time, to acetyl CoA with generation of FADH2 and NADH (Figure 7a). For example, mitochondrial oxidation of each molecule of the 18-carbon stearic acid, CH3(CH2)16COOH, yields nine molecules of acetyl CoA and eight molecules each of NADH and FADH2. In the second part of stage II, as with acetyl CoA generated from pyruvate, these acetyl groups enter the citric acid cycle and are oxidized to CO2. As will be described in detail in the next section, the reduced NADH and FADH2 with their high-energy electrons will be used in stage III to generate a proton-motive force, which in turn is used in stage IV to power ATP synthesis.
Fig7. Oxidation of fatty acids in mitochondria and in peroxisomes. In both mitochondrial oxidation (a) and peroxisomal oxidation (b), fatty acids are converted to acetyl CoA by a series of four enzyme-catalyzed reactions (shown down the center of the figure). A fatty acyl CoA molecule is converted to acetyl CoA and a fatty acyl CoA shortened by two car bon atoms. Concomitantly, one FAD molecule is reduced to FADH2 and one NAD+ molecule is reduced to NADH. The cycle is repeated on the shortened acyl CoA until fatty acids with an even number of carbon atoms are completely converted to acetyl CoA. In mitochondria, electrons from FADH2 and NADH enter the electron-transport chain and are ultimately used to generate ATP; the acetyl CoA generated is oxidized in the citric acid cycle, resulting in the release of CO2 and ultimately the synthesis of additional ATP. Because peroxisomes lack the protein complexes composing the electron transport chain and the enzymes of the citric acid cycle, oxidation of fatty acids in these organelles yields no ATP.
Peroxisomal Oxidation of Fatty Acids Generates No ATP
Mitochondrial oxidation of fatty acids is the major source of ATP in mammalian liver cells, and biochemists at one time believed this was true in all cell types. However, rats treated with clofibrate, a drug that affects many features of lipid metabolism, were found to exhibit an increased rate of fatty acid oxidation and a large increase in the number of peroxisomes in their liver cells. This finding suggested that peroxisomes, as well as mitochondria, can oxidize fatty acids. These small organelles, 0.2–1 μm in diameter, are lined by a single membrane. They are present in all mammalian cells except erythrocytes and are also found in plant cells, yeasts, and probably most other eukaryotic cells.
Mitochondria preferentially oxidize short-chain [fewer than 8 carbons ( <C8 )], medium-chain (C8–C12), and long chain (C14–C20) fatty acids, whereas peroxisomes preferentially oxidize very long chain fatty acids (VLCFAs, >C20), which cannot be oxidized by mitochondria. Most dietary fatty acids have long chains, which means that they are oxidized mostly in mitochondria. In contrast to mitochondrial oxidation of fatty acids, which is coupled to generation of ATP, peroxisomal oxidation of fatty acids is not linked to ATP formation, and energy is released as heat.
The reaction pathway by which fatty acids are degraded to acetyl CoA in peroxisomes is similar to that used in mitochondria (Figure 7b). However, peroxisomes lack an electron transport chain, and electrons from the FADH2 produced during the oxidation of fatty acids are immediately transferred to O2 by oxidases, regenerating FAD and forming hydrogen peroxide (H2O2). In addition to oxidases, peroxisomes contain abundant catalase, which quickly decomposes the H2O2, a highly cytotoxic metabolite. NADH produced during peroxisomal oxidation of fatty acids is exported and reoxidized in the cytosol; there is no need for a malate-aspartate shuttle here. Peroxisomes also lack the citric acid cycle, so acetyl CoA generated during peroxisomal degradation of fatty acids cannot be oxidized further; instead, it is transported into the cytosol for use in the synthesis of cholesterol and other metabolites.
