Molecular Biology of the Cell - Volume 1 - Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J. 1994

Molecular Organization of Cells
Energy Conversion: Mitochondria and Chloroplasts
Mitochondria

Mitochondria occupy a substantial portion of the Cytoplasm in almost all Eukaryotic Cells. Although mitochondria are large enough to be seen with a conventional Light Microscope and were first discovered in the nineteenth century, the real opportunity to understand their function only arose after 1948, when Methods for isolating intact mitochondria were developed. For technical reasons, most biochemical studies have been carried out on mitochondria isolated from Liver.

Mitochondria are usually depicted as stiff, elongated, Bacteria-like cylinders with a diameter of 0.5 to 1 μm. However, time-lapse microcinematography of living cells reveals that mitochondria are remarkably mobile and plastic Organelles that constantly change their shape (Fig. 7-2) and even fuse with each other and then divide again. The pathways of mitochondrial movement in the cytoplasm are often associated with microtubules (Fig. 7-3), which may determine the characteristic orientation of mitochondria and their distribution in various cells. In some cells, mitochondria form long, mobile filaments or chains, while in others they are fixed near sites of high ATP consumption—for example, in cardiac Muscle they are located between myofibrils, and in sperm cells they tightly wrap around the flagellum (Fig. 7-4).

7.1.1. Mitochondria have outer and inner membranes that form two internal compartments [2]

Each mitochondrion is surrounded by two highly specialized membranes that play a key role in its activity. These membranes form two isolated mitochondrial compartments: the internal matrix and a much narrower intermembrane space.

Class="center">Cell/cell.files/image448.jpg" width="139"/>

Figure 7-2. Rapid changes in mitochondrial shape observed in living cells.

Figure 7-3. A. Light micrograph of chains of elongated mitochondria in a living cell in mammalian tissue culture. The Cell is stained with a vital fluorescent dye (rhodamine 123) that specifically stains mitochondria. B. Immunofluorescence micrograph of the same cell treated (after fixation) with fluorescent Antibodies to microtubules. Note that mitochondria are aligned mainly along microtubules. Scale bar 10 µm. (Courtesy of Lan Bo Chen.)

If purified mitochondria are gently disrupted and then fractionated (Fig. 7-5), the Biochemical Composition of each of the two membranes and the spaces between them can be determined. As shown in Fig. 7-6, each fraction contains a unique set of Proteins.

The outer membrane contains many copies of a protein called porin, which forms wide hydrophilic channels in The Lipid Bilayer. Thus, this membrane resembles a sieve that is permeable to all molecules with a mass of 10,000 daltons or less, including small proteins. These molecules can penetrate into the intermembrane space, but most of them are unable to pass through the impermeable inner membrane. This means that while The chemical composition of the intermembrane space is equivalent to that of the Cytosol, at least with respect to small molecules, the matrix contains a much more restricted set of small molecules.

As will be described in more detail later, the main functional part of the mitochondrion is the matrix and the surrounding inner membrane. The inner membrane is highly specific; it contains a large amount of the 'double' phospholipid cardiolipin (Section 7.5.15), which is believed to make the membrane particularly impermeable to ions. The inner membrane also contains a variety of transport proteins that determine its selective permeability to those small molecules that are either metabolized by the numerous Enzymes concentrated in the matrix or are required for their activity. In particular, the matrix contains enzymes that convert Pyruvate and Fatty acids into acetyl-CoA and then oxidize the latter in The Citric Acid Cycle. The main End products of this oxidation are CO₂, which leaves the cell, and NADH, which serves as the primary source of electrons transferred by the Respiratory Chain—as the Mitochondrial Electron Transport chain is called. The Enzymes of the respiratory chain are embedded in The inner mitochondrial membrane and are essential for The process of Oxidative Phosphorylation, which generates most of the ATP in animal cells.

Figure 7-4. Localization of mitochondria near sites of high ATP consumption in cardiac muscle and the sperm tail. During sperm flagellum development, microtubules wrap around the axoneme, thereby ensuring the proper positioning of mitochondria.

Figure 7-5. Methods for separating mitochondria into individual components make it possible to study different proteins in each mitochondrial compartment. The method shown here, which allows the simultaneous Processing of A large number of mitochondria, is based on the fact that in a medium of low Ionic strength, Water enters the mitochondrion and causes severe Swelling of the matrix. As a result, the cristae of the inner membrane unfold, and the outer membrane, which has no folds, ruptures, releasing a Structure consisting only of the inner membrane and the matrix.

Figure 7-6. General structure of a mitochondrion. In liver mitochondria, 67% of all protein is located in the matrix, 21% in the outer membrane, 6% in the inner membrane, and 6% in the intermembrane space. Each of these four compartments contains a specific set of enzymes according to its function (see diagram below). (Micrograph courtesy of Daniel S. Friend.)

7.1.2. The inner membrane forms folds called cristae [3]

The inner membrane typically forms a complex system of folds in the matrix, called cristae. These folds significantly increase the surface area of the inner membrane; for example, in liver mitochondria, the inner membrane accounts for one-third of all cell membranes (see Table 8-2). In cardiac muscle mitochondria, the number of cristae is three times greater than in liver mitochondria, which is apparently due to the high demand of Heart cells for ATP. Furthermore, mitochondrial cristae in different cells exhibit striking morphological features, The Significance of which is unknown (Fig. 7-7).

In addition to morphological features, different cell types differ significantly in the composition of their mitochondrial enzymes. However, in this chapter, we will set aside these differences and consider only the enzymes and properties common to all mitochondria.

7.1.3. Oxidative processes in mitochondria begin after a sufficient amount of acetyl-CoA is formed in the matrix from pyruvate and fatty acids [4]

The 'fuel' for oxidative METABOLISM in mitochondria is mainly Fatty Acids and pyruvate, which is produced by Glycolysis in the cytosol. These substances are selectively transported from the cytosol into the mitochondrial matrix, where they are broken down into two-carbon groups attached to acetyl coenzyme A (acetyl-CoA, Fig. 7-8). As part of the acetyl-CoA molecule, each acetyl group then enters The Citric Acid cycle for further degradation. The process culminates in the transfer along the respiratory chain of high-energy electrons extracted from the acetyl group.

To ensure a continuous supply of 'fuel' for oxidative metabolism, animal cells store it as fats, which serve as a source of fatty acids, and Glycogen, a source of glucose that is subsequently broken down into pyruvate. Quantitatively, fats are far more important, if only because their oxidation releases more than six times as much energy as The oxidation of an equal amount of glycogen in its hydrated form. The glycogen stores in an average adult's body are sufficient for one day of normal activity, whereas fat stores can last for a month. If glycogen, rather than fat, were the primary fuel reserve in our bodies, body weight would increase by an average of 25 kg.

Fig. 7-7. Some morphological differences in The structure of mitochondrial cristae isolated from various rat Tissues. The significance of these differences for mitochondrial function remains unknown.

Fig. 7-8. Acetyl-CoA is the major intermediate product formed during The breakdown of nutrients in mitochondria. The spatial model of this molecule is shown (see also Fig. 2-19). S denotes the sulfur atom, which forms a thioester bond with the acetate group. Because this bond is high-energy, the acetate group can be readily transferred to another molecule, such as oxaloacetate (see Fig. 7-14).

The bulk of our fat reserves is stored in adipose tissue, from which fats are transported via the bloodstream to other cells as needed. The demand for fats increases after a period of fasting; even following a night's Sleep, fat mobilization occurs, so that in the morning hours, most of the acetyl-CoA entering the citric acid cycle is derived from fatty acids rather than glucose. However, after a meal, dietary glucose becomes the primary source of acetyl-CoA for the citric acid cycle. Excess glucose is used to replenish depleted glycogen stores or to synthesize fats. (It should be noted that while sugars in animal cells are readily converted into fats, the reverse conversion of fats into sugars is not possible.)

A fat molecule consists of three fatty acid residues attached by ester bonds to a glycerol molecule. Such triacylglycerols (triglycerides) are nonpolar and practically insoluble in water, forming fat droplets in the cytosol (Fig. 7-9). In adipocytes—the cells of adipose tissue—a single large fat droplet occupies almost the entire cell volume; large fat cells are specialized for fat storage. Smaller fat droplets are typical of cells such as heart muscle fibers, which utilize the energy derived from Fatty acid oxidation; in these cells, fat droplets are often closely associated with mitochondria (Fig. 7-10). In all cells, enzymes located in the outer and inner mitochondrial membranes participate in transporting fatty acids, extracted from fat molecules, into the mitochondrial matrix. Within the matrix, each fatty acid molecule (in the form of acyl-CoA) is completely degraded via a cycle of reactions, during each turn of which it is shortened by two carbon atoms from the carboxyl end, yielding one molecule of acetyl-CoA (Fig. 7-11). Further oxidation of acetyl-CoA takes place in the citric acid cycle.

Glycogen is a large, branched glucose polymer stored as granules in the cytoplasm (Fig. 7-12); the Synthesis and Breakdown of glycogen are exquisitely regulated to meet the metabolic needs of the Organism (see Section 12.4.1). When the demand for glucose rises, glycogen is degraded to yield glucose-1-phosphate. During glycolysis, a six-carbon glucose molecule (or a related sugar) is converted into two three-carbon pyruvate molecules (see Section 2.3.2), which still retain most of the energy that can be extracted by complete sugar oxidation. This energy is released only after pyruvate is transported from the cytosol into the mitochondrial matrix, where it is acted upon by a multienzyme complex larger than a ribosome—the pyruvate dehydrogenase complex. This complex, containing multiple copies of three enzymes, five Coenzymes, and two regulatory proteins, rapidly converts pyruvate into acetyl-CoA with the release of CO₂ as a byproduct (Fig. 7-13). This acetyl-CoA, like that produced by fatty acid oxidation, enters the citric acid cycle.

Fig. 7-9. A. Electron micrograph of a lipid droplet containing triacylglycerols, the principal form of reserve fat in the cytoplasm. B. Structure of a triacylglycerol; the glycerol moiety is highlighted in color. (Micrograph A courtesy of Daniel S. Friend.)

Fig. 7-10. In heart muscle cells, lipid droplets are surrounded by mitochondria, which mediate the Oxidation of Fatty acids derived from triacylglycerols.

Fig. 7-11. The fatty acid oxidation cycle, the steps of which are sequentially catalyzed by four enzymes in the mitochondrial matrix. With each turn of the cycle, the fatty acid molecule is shortened by two carbon atoms (highlighted in color), generating one molecule of acetyl-CoA along with one molecule each of NADH and FADH₂. NADH dissolves freely in the matrix, whereas FADH₂ remains tightly bound to the enzyme acyl-CoA dehydrogenase; the two electrons from FADH₂ are rapidly transferred to ubiquinone located in the inner mitochondrial membrane (Section 7.2.5), thereby regenerating NAD. The four-step pathway of fatty acid oxidation shown here is chemically identical to the Cleavage of many other carbon-carbon bonds (see, for example, Fig. 7-14).

Fig. 7-12. Electron micrograph and schematic diagram of glycogen granules, the primary carbohydrate storage form in vertebrate cells. Glycogen is a glucose polymer, and each granule represents a single highly branched molecule. The Synthesis and degradation of glycogen are catalyzed by enzymes associated with the granule surface, including Glycogen synthase and the Glycogen phosphorylase degrading enzyme. (Courtesy of Robert Fletterick and Daniel S. Friend.)

Fig. 7-13. Reactions carried out by the pyruvate dehydrogenase complex, which converts pyruvate to acetyl-CoA in the mitochondrial matrix; these reactions also generate NADH. A, B, and C represent three enzymes: pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase, whose Functions are coupled as shown in the figure. The STRUCTURE OF THE complex is depicted in Fig. 2-40; the complex also contains a protein kinase and a protein phosphatase that regulate pyruvate dehydrogenase activity by "switching it off" at high ATP concentrations.

7.1.4. Oxidation of the acetyl group to acetyl-CoA in the citric acid cycle leads to the generation of NADH and FADH₂ molecules for the respiratory chain [5]

As early as the 19th century, biologists observed that in the absence of air (under anaerobic conditions), cells produce lactic acid (or ethanol), whereas under aerobic conditions they consume oxygen to produce CO₂ and H₂O. Efforts to elucidate the pathways of aerobic metabolism eventually focused on pyruvate oxidation, culminating in the 1937 Discovery of the citric acid cycle, also known as The Tricarboxylic Acid Cycle or the Krebs cycle. In most cells, the citric acid cycle accounts for about two-thirds of all carbon oxidation reactions. The main end products of this cycle are CO₂ and NADH. CO₂ is released as a byproduct, while NADH molecules transfer their high-energy electrons to the respiratory chain, where these electrons are ultimately used to reduce O₂ to H₂O.

The citric acid cycle begins with the reaction between acetyl-CoA—derived from fatty acids or pyruvate—and the four-carbon compound oxaloacetate, yielding the six-carbon citric acid, which gives the cycle its name. Subsequently, through seven consecutive enzymatic reactions, two carbon atoms are removed as CO₂, and oxaloacetate is ultimately regenerated. Each turn of the cycle yields two molecules of CO₂, derived from the two carbon atoms introduced in preceding turns of the cycle (Fig. 7-14). The conversion of the acetyl group within acetyl-CoA can be represented by the following overall reaction:

CH₃COOH (as acetyl-CoA) + 2H₂O + 3NAD⁺ + protein-bound FAD → 2CO₂ + 3H⁺ + 3NADH + protein-bound FADH₂.

In addition, this reaction synthesizes one molecule of ATP (via GTP) through substrate-level phosphorylation, similar to the process in glycolysis (see Section 2.3.2).

The most important metabolic contribution of the citric acid cycle is the extraction of high-energy electrons via the oxidation of the two carbon atoms in the acetyl-CoA molecule. These electrons are captured by NADH and FADH₂ and then rapidly transferred to the respiratory chain in the inner mitochondrial membrane. FADH₂—a component of the inner membrane succinate dehydrogenase complex—passes its electrons directly into the respiratory chain. By contrast, NADH forms a soluble pool of reducing equivalents in the matrix and donates its electrons through random collisions with membrane-bound dehydrogenases. We now examine how the energy of these electrons is utilized for ATP synthesis.

Fig. 7-14. The citric acid cycle. Intermediates are shown as free fatty acids, although their carboxyl groups are actually ionized. Each reaction shown is catalyzed by a specific enzyme; all these enzymes reside in the mitochondrial matrix. The two carbon atoms brought in by acetyl-CoA are converted into CO₂ in subsequent turns of the cycle. The two carbon atoms highlighted in color are converted into CO₂ during the current cycle turn. In addition, three molecules of NADH are produced. The resulting GTP molecule can be converted to ATP via the exchange reaction GTP + ADP → GDP + ATP. The FADH₂ molecule remains part of the succinate dehydrogenase complex located in the inner mitochondrial membrane; this complex transfers electrons from FADH₂ directly to ubiquinone.

7.1.5. In the mitochondrial membrane, the energy of oxidative reactions is converted into ATP energy via a chemiosmotic process [6]

Although the citric acid cycle is part of aerobic metabolism, molecular oxygen does not participate directly in any of the reactions of this cycle that lead to The formation of NADH and FADH₂; this occurs only in the final series of catabolic reactions taking place on the inner membrane. Almost all the energy obtained during the Cytology/cytology/16.html">Early stages of oxidation from the burning of CARBOHYDRATES, fats, and other nutrients is initially stored in the form of high-energy electrons carried by NADH and FADH. These electrons then interact with molecular oxygen in the respiratory chain. Since a large amount of the released energy is used by enzymes of the inner membrane to synthesize ATP from ADP and P_i, these latter reactions are called oxidative phosphorylation (Fig. 715).

As already mentioned, ATP Synthesis in the oxidative phosphorylation reactions occurring in the respiratory chain depends on a chemiosmotic process. The Mechanism of this process, first proposed in 1961, solved a long-standing problem in cell biology. However, the idea was so novel that it took several years to gain general acceptance as experimental evidence accumulated. It was previously thought that the energy for ATP synthesis in the respiratory chain was provided by the same mechanism as in substrate-level phosphorylation: it was assumed that the energy of oxidation was used to form a high-energy bond between a phosphate group and some intermediate compound, and that the conversion of ADP to ATP was driven by The energy released when this bond was broken. However, despite intensive searches, the postulated intermediate was never found.

Fig. 7-15. The main outcome of energy conversion occurring in the mitochondrion. In this process, called oxidative phosphorylation, the inner mitochondrial membrane acts as an energy-converting device that converts part of the energy of NADH (and FADH₂) oxidation into the energy of phosphate bonds of ATP.

Table 7-1. Chemiosmotic Coupling

According to the chemiosmotic hypothesis, instead of energy-rich intermediates, There is a direct link between chemical ("chemi...") and transport (osmotic, from the Greek osmos - push, thrust) processes—chemiosmotic coupling (Table 7-1). As high-energy electrons, delivered by NADH and FADH₂, pass along the respiratory chain of the inner mitochondrial membrane from one carrier to the next, energy is released and used to pump protons (H⁺) across the inner membrane from the matrix into the intermembrane space. As a result, an electrochemical proton gradient is generated across the inner membrane; the energy of the reverse flow of protons "down" this gradient is used by the membrane-bound enzyme ATP synthase, which catalyzes the formation of ATP from ADP and P_i, i.e., the final step of oxidative phosphorylation (Fig. 7-16).

Fig. 7-16. Flows of the most important metabolites entering and leaving the mitochondrion. Pyruvate and fatty acids enter the mitochondrion and are metabolized in the citric acid cycle, yielding NADH. Then, during oxidative phosphorylation, the high-energy electrons of NADH are transferred to oxygen via the respiratory chain located in the inner membrane; ATP is generated in this process through a chemiosmotic mechanism. NADH produced in the cytosol during glycolysis also transfers its electrons to the respiratory chain (not shown). Since NADH cannot cross the inner membrane, its electron transfer occurs indirectly—via one of several shuttle systems that transport another reduced compound into the mitochondrion; after oxidation, this compound returns to the cytosol, where it is reduced again by NADH.

In the remainder of this section, we will briefly examine the type of reactions that make oxidative phosphorylation possible; details will be discussed later (Section 7.2).

7.1.6. Electrons are transferred from NADH to oxygen by three large respiratory enzyme complexes [7]

Although the mechanisms of energy extraction in the Respiratory Chain and other catabolic reactions differ, they are based on common principles. The reaction H₂ + ¹/₂ O₂→ H₂O is broken down into many small "steps" so that the released energy can be captured in coupled forms rather than dissipated as heat. As with the formation of ATP and NADH in glycolysis or the citric acid cycle, this involves an indirect pathway. However, the uniqueness of the respiratory chain lies in the fact that here, first of all, hydrogen atoms are split into electrons and protons. The electrons are passed through a series of carriers embedded in the inner mitochondrial membrane. When the electrons reach the end of this electron-transport chain, protons are there to neutralize the negative charge generated when the electrons are transferred to an oxygen molecule (Fig. 7-17).

Let us trace the oxidation process, starting with the formation of NADH—the main acceptor of reactive electrons extracted during the oxidation of nutrient molecules. Each hydrogen atom

Fig. 7-17. These diagrams show how most of the energy of hydrogen "burning" is not dissipated as heat (left) but is captured and stored in a form useful to the cell by means of the electron-transport chain located in the inner mitochondrial membrane (right). The remaining energy is released by the mitochondrion as heat. In reality, the electrons and protons shown here are stripped from hydrogen atoms covalently bound to NADH or FADH₂ molecules (see Fig. 7-18).

Fig. 7-18. Proposed mechanism for the Biological Oxidation of an alcohol to an aldehyde. The components of two complete hydrogen atoms are removed from the alcohol molecule, with a hydride ion being transferred to NAD⁺ and a proton entering the aqueous medium. Only the nicotinamide rings of NAD⁺ and NADH are shown here (see Fig. 2-22). The stages shown occur On the surface of the enzyme Alcohol dehydrogenase (not shown) with the participation of its specific groups. (Reprinted with permission from P. F. Cook, N. J. Oppenheimer, W. W. Cleland, Biochemistry, 20: 1817 1825, 1981. Copyright 1981, American Chem. Soc.)

(denoted as H) consists of one electron (e^-) and one proton (H⁺). The mechanism of electron addition to NADH was discussed earlier (Section 2.3.4) and is shown in more detail in Fig. 7-18. As is clear from this diagram, each NADH molecule carries a hydride ion (a hydrogen atom plus an extra electron, H:^-), rather than just a hydrogen atom. However, because of the presence of free protons in the surrounding aqueous solution, The transfer of a hydride ion as part of NADH is equivalent to the transfer of two hydrogen atoms or a hydrogen molecule (H:^- + H⁺→ H₂).

Electron transfer along the respiratory chain begins with the removal of a hydride ion (H:^-) from NADH; this regenerates NAD⁺, and the hydride ion is converted into a proton and two electrons (H:^-→ H⁺ + 2e). These electrons are transferred to the first of more than 15 different electron carriers in the respiratory chain. At this point, the electrons possess very high energy, which gradually decreases as they pass along the chain. Most often, electrons pass from one metal atom to another, with each of these atoms tightly bound to a protein molecule that influences its electron affinity. Various types of electron carriers in the respiratory chain will be discussed in detail later (Section 7.2.5). It is important to note that all electron-carrier proteins are grouped into three large respiratory enzyme complexes, each containing transmembrane proteins that firmly anchor the complex in the inner mitochondrial membrane (see Section 7.2.6). Each successive complex has a higher affinity for electrons than the preceding one. Electrons pass sequentially from one complex to another until they are finally transferred to oxygen, which has the highest affinity for electrons.

7.1.7. The energy released during electron transfer along the respiratory chain is stored as an electrochemical proton gradient across the inner mitochondrial membrane [8]

Oxidative phosphorylation is made possible by the close association of electron carriers with protein molecules. Proteins guide electrons along the respiratory chain so that they pass sequentially from one enzyme complex to another without "skipping" intermediate steps. Most importantly, electron transfer is coupled to allosteric changes in specific protein molecules, so that the energetically favorable flow of electrons drives the pumping of protons (H⁺) across the inner membrane from the matrix into the intermembrane space and thus out of the mitochondrion. This movement of protons has two major consequences: 1) a pH gradient is established across the inner membrane—the pH in the matrix is higher than in the cytosol, where the pH is normally close to 7.0 (since small molecules pass freely through the outer mitochondrial membrane, the pH in the intermembrane space is the same as in the cytosol); 2) a voltage gradient (Membrane Potential) is generated across the inner membrane, with the matrix side of the membrane becoming negatively charged and the cytosolic side positively charged.

Fig. 7-19. The two Components of the electrochemical proton gradient. The total proton-motive force generated across the inner mitochondrial membrane is composed of a large force due to the membrane potential (traditionally designated as ∆ψ, but in our text as ∆V) and a smaller force generated by the proton concentration gradient (∆pH). Both forces drive protons back into the matrix.

The pH gradient (∆pH) drives H⁺ ions back into the matrix and OH^- ions out of the matrix, which reinforces The Effect of the membrane potential (∆V), under which any positive charge is attracted into the matrix and any negative charge is repelled from it. The combined action of these two forces results in the electrochemical proton gradient (Fig. 7-19).

The electrochemical proton gradient generates a proton-motive force measured in millivolts (mV). Since a pH gradient (∆pH) of 1 pH unit is equivalent to a membrane potential of about 60 mV, the proton-motive force is equal to ∆V - 60(∆pH). In a typical cell, this force across the inner membrane of a respiring mitochondrion is about 220 mV, consisting of a membrane potential of approximately 160 mV and a pH gradient of close to -1 pH unit.

7.1.8. The energy of the electrochemical proton gradient is used to synthesize ATP and transport metabolites and inorganic ions into the matrix [9]

The inner mitochondrial membrane has an unusually high protein content, consisting of approximately 70% protein and 30% phospholipid by weight. Many of these proteins are components of the electron-transport chain, which maintains the proton gradient across the membrane. Another major component is the enzyme ATP synthase, which catalyzes ATP synthesis. This large protein complex acts as a turbine, allowing protons to flow back down their electrochemical gradient into the matrix. In doing so, ATP synthase converts one form of energy into another, synthesizing ATP from ADP and P_i within the mitochondrial matrix through a reaction coupled to the inward flow of protons (Fig. 7-20).

However, ATP synthesis is not the only process driven by the electrochemical gradient. The matrix, which houses the enzymes of the citric acid cycle and other metabolic pathways, must maintain high concentrations of various substrates; specifically, ATP synthase requires a steady supply of ADP and phosphate. Consequently, a variety of charged substrates must be transported across the inner membrane. This is accomplished by specific carrier proteins embedded in the membrane (see Section 6.4.4), many of which actively pump specific molecules against their electrochemical gradients—a process requiring an energy input. For most metabolites, this energy is derived by coupling transport to the movement of other molecules "down" their electrochemical gradients (see Section 6.4.9). For instance, ADP transport relies on the ADP-ATP antiport system: as one molecule of ADP enters the matrix, one molecule of ATP exits down its electrochemical gradient. Simultaneously, a symport system couples the influx of phosphate into the mitochondrion with the cotransport of H⁺, where protons enter the matrix down their gradient, effectively dragging phosphate along with them. Pyruvate is imported into the matrix in a similar manner (Fig. 7-21). The energy of the electrochemical proton gradient is also harnessed to import Ca²⁺ ions into the matrix. These ions appear to play a crucial role in regulating The activity of certain mitochondrial enzymes, and mitochondrial uptake may also serve as an important mechanism for clearing Ca²⁺ from the cytosol when its concentration reaches dangerously high levels (see Section 12.3.7).

The more electrochemical gradient energy is consumed to transport molecules and ions into the mitochondria, the less remains available for ATP synthesis. For example, if isolated mitochondria are placed in a medium with a high Ca²⁺ concentration, they completely halt ATP production, as all the gradient energy is diverted into transporting Ca²⁺ into the matrix. In certain specialized cells, the electrochemical proton gradient is "shunted" so that the mitochondria generate heat instead of ATP (see Section 7.2.12). Evidently, cells can finely regulate how they utilize the energy of the electrochemical proton gradient, directing it toward the processes most critical at any given moment.

7.1.9. Rapid conversion of ADP to ATP in mitochondria helps maintain a high cellular ATP/ADP concentration ratio [10]

Via a specialized transport protein embedded in the inner membrane, ADP is imported into the matrix in exchange for ATP through an antiport mechanism (Fig. 7-21). As a result, the ADP molecules released during cytosolic ATP hydrolysis are rapidly delivered to the mitochondria for "recharging," while the newly synthesized ATP molecules generated via oxidative phosphorylation in the matrix are swiftly exported to the cytosol where they are needed. In The Human Body, ATP molecules undergo turnover several thousand times a day, maintaining a cytosolic ATP concentration that exceeds that of ADP by more than 10-fold.

Fig. 7-20. General Mechanism of Oxidative phosphorylation. As high-energy electrons pass along the electron-transport chain, a fraction of the released energy is used to drive three respiratory enzyme complexes that pump protons out of the matrix. This generates an electrochemical proton gradient across the inner membrane, which drives protons back into the matrix through ATP synthase—a transmembrane protein complex that harnesses the energy of the proton flow to synthesize ATP from ADP and P_i in the matrix.

Fig. 7-21. Several Active Transport processes driven by the electrochemical proton gradient maintained across the inner membrane. The net charge of each transported molecule is indicated. The outer membrane is freely permeable to all of these compounds. Transport mechanisms such as symport and antiport are discussed in detail in Chapter 6.

As discussed in Chapter 2, biosynthetic enzymes in the cell direct the transformations of their substrates along specific metabolic pathways, often driving energetically unfavorable reactions by coupling them to the energetically favorable hydrolysis of ATP (see Fig. 2-27). Thus, a high-concentration pool of ATP supplies energy to intracellular processes much like a battery powering an electric motor: if mitochondria were to cease their activity, the cellular "battery" would begin to discharge, eventually reaching a point where energetically unfavorable reactions could no longer be sustained by ATP hydrolysis.

At first glance, it might seem that this critical state would not be reached until the ATP concentration drops to zero. In reality, however, this state occurs much earlier—at a specific threshold level of ATP that depends on the concentrations of ADP and P_i. To understand why this happens, we must turn to some elementary Principles of Thermodynamics.

7.1.10. The difference between ∆G° and ∆G. For a cell to utilize ATP hydrolysis, a large negative value of ∆G is required [11]

According to The Second Law of thermodynamics, Chemical Reactions proceed spontaneously only in the direction that increases the "disorder" of the Universe. As noted in Chapter 2, reactions in which released energy is dissipated as heat into the surroundings (such as ATP hydrolysis) contribute to this increase in disorder by enhancing the random thermal motion of molecules. In addition, chemical reactions can alter the degree of disorder by changing the concentrations of reactants and products. The overall change in the disorder of the Universe resulting from a given reaction is determined by The change in Free energy, ∆G, accompanying that reaction: the greater the decrease in free energy (i.e., the more negative the value of ∆G), the greater the increase in the disorder of the Universe and the more readily the reaction proceeds (see Panel 2-7).

Fig. 7-22. The fundamental relationship between free-energy changes and reaction equilibrium is illustrated here using ATP hydrolysis as an example. The Equilibrium Constant K is expressed in liters per mole. (THE CONCEPT OF free energy is illustrated in Panel 2-7, pp. 96–97; the Definition of the equilibrium constant is given in Fig. 3-7).

The magnitude of the free-energy change for the hydrolysis of ATP to ADP and inorganic phosphate under typical intracellular conditions ranges from —11 to —13 kcal/mol. However, such favorable conditions for this reaction are maintained because the cellular ATP concentration is kept very high relative to the concentrations of ADP and P_i. Under so-called "standard conditions," where the concentrations of ATP, ADP, and P_i are all equal to 1 mol/L, the ∆G for ATP hydrolysis is termed the standard free-energy change for the reaction, ∆G°; it equals —7.3 kcal/mol. At some even lower ATP concentration relative to ADP and P_i, the value of ∆G drops to zero. Under this condition, The rate of ATP formation from ADP and P_i equals the rate of ATP hydrolysis; in other words, when ∆G = 0, the reaction is at equilibrium (Fig. 7-22).

At a constant Temperature, ∆G° remains constant and depends solely on the Chemical Nature of the reactants, whereas ∆G varies with the concentrations of the reactants and products, indicating how far a given reaction is from equilibrium. Therefore, it is ∆G, rather than ∆G°, that determines whether a given reaction can serve as an energy source for other processes. The high cellular concentration of ATP (relative to ADP and P_i) maintained by active ATP synthesis in mitochondria results in a large negative ∆G for ATP hydrolysis, holding this reaction far from equilibrium. Otherwise, ATP hydrolysis could not be used by the cell to drive other processes, and many biosynthetic reactions would run in reverse.

7.1.11. Cellular Respiration is remarkably efficient

During oxidative phosphorylation, each pair of electrons from NADH provides enough energy to drive the formation of approximately three molecules of ATP. A pair of electrons from FADH₂, possessing lower energy, yields enough energy to synthesize only two ATP molecules. On average, each molecule of acetyl-CoA entering the citric acid cycle yields about 12 molecules of ATP. This means that the Complete oxidation of one molecule of glucose produces 24 ATP molecules, whereas the oxidation of one molecule of palmitate—a 16-carbon fatty acid—yields 96 ATP molecules. Taking into account the exothermic reactions preceding the Formation of Acetyl-CoA, the complete oxidation of a single glucose molecule yields about 36 ATP molecules, while the complete oxidation of palmitate generates approximately 129 ATP molecules. These are maximum theoretical values, as the actual amount of ATP synthesized in mitochondria depends on what fraction of the proton-gradient energy is channeled into ATP synthesis rather than other processes.

If we compare the free-energy change of burning fats and carbohydrates directly to CO₂ and H₂O with the total energy captured in the phosphate bonds of ATP during biological oxidation, the efficiency of converting oxidation energy into ATP energy often exceeds 50%. This is significantly higher than the efficiency of most human-made energy-conversion devices. If a cell operated at the efficiency of an electric motor or automobile engine (10–20%), the organism would require vastly more food to sustain life. Furthermore, because all unused energy is released as heat, large organisms would need much more sophisticated mechanisms for dissipating heat into the environment.

When studying cellular respiration, students are sometimes puzzled as to why metabolic interconversions within the cell follow such a convoluted path. It might seem that one could dispense with the citric acid cycle and many components of the respiratory chain, oxidizing sugars directly to CO₂ and H₂O in a more straightforward manner. Yet, while such a pathway might be easier to memorize, it would prove catastrophic for the cell. The enormous amount of free energy released by oxidation can only be efficiently harnessed in small increments. The complex oxidation process involves numerous intermediates, each differing only slightly from the preceding one. As a result, the released energy is broken down into manageable quantities that can be efficiently captured via coupled reactions into the high-energy bonds of ATP and NADH molecules (see Fig. 2-17).

Summary

Mitochondria carry out the vast majority of the cell's oxidative processes and generate nearly all the ATP in an animal cell. The mitochondrial matrix contains a wide array of enzymes, including those that oxidize pyruvate and fatty acids to acetyl-CoA, as well as the enzymes that oxidize this acetyl-CoA to CO₂ in the citric acid cycle. These oxidative reactions generate large amounts of NADH (and FADH₂). The energy released when reactive electrons carried by NADH and FADH₂ are transferred to oxygen is harnessed by the electron-transport chain, located in the inner mitochondrial membrane and known as the respiratory chain. The respiratory chain pumps protons out of the matrix, creating a transmembrane electrochemical proton gradient composed of a membrane potential and a pH difference. The energy of this transmembrane gradient is, in turn, used to synthesize ATP and actively transport essential metabolites across the inner mitochondrial membrane. The combination of these reactions ensures an efficient ATP-ADP exchange between the mitochondrion and the cytosol, enabling the cell to maintain a high level of ATP.