Bioenergetics: The Role of ATP PDF

S E C T I O N Bioenergetics III C H A P T E R Bioenergetics: The Role of ATP Kathleen M. Botham, PhD, DSc, & Peter A. M...

S E C T I O N Bioenergetics III C H A P T E R Bioenergetics: The Role of ATP Kathleen M. Botham, PhD, DSc, & Peter A. Mayes, PhD, DSc 11 OBJ E C TI VE S State the first and second laws of thermodynamics and understand how they apply to biologic systems. After studying this chapter, Explain what is meant by the terms free energy, entropy, enthalpy, exergonic, you should be able to: and endergonic. Appreciate how reactions that are endergonic may be driven by coupling to those that are exergonic in biologic systems. Explain the role of group transfer potential, adenosine triphosphate (ATP), and other nucleotide triphosphates in the transfer of free energy from exergonic to endergonic processes, enabling them to act as the “energy currency” of cells. BIOMEDICAL IMPORTANCE FREE ENERGY IS THE USEFUL Bioenergetics, or biochemical thermodynamics, is the study ENERGY IN A SYSTEM of the energy changes accompanying biochemical reactions. Gibbs change in free energy (ΔG) is that portion of the Biologic systems are essentially isothermic and use chemical total energy change in a system that is available for doing energy to power living processes. The way in which an ani- work—that is, the useful energy, also known as the chemical mal obtains suitable fuel from its food to provide this energy potential. is basic to the understanding of normal nutrition and metab- olism. Death from starvation occurs when available energy reserves are depleted, and certain forms of malnutrition are Biologic Systems Conform to the associated with energy imbalance (marasmus). Thyroid hor- General Laws of Thermodynamics mones control the metabolic rate (rate of energy release), and The first law of thermodynamics states that the total energy disease results if they malfunction. Excess storage of surplus of a system, including its surroundings, remains constant. energy causes obesity, an increasingly common disease of It implies that within the total system, energy is neither lost Western society which predisposes to many diseases, includ- nor gained during any change. However, energy may be ing cardiovascular disease and diabetes mellitus type 2, and transferred from one part of the system to another, or may be lowers life expectancy. transformed into another form of energy. In living systems, 109 110 SECTION III Bioenergetics chemical energy may be transformed into heat or into electri- A cal, radiant, or mechanical energy. The second law of thermodynamics states that the total Heat Ex entropy of a system must increase if a process is to occur er go D spontaneously. Entropy is the extent of disorder or random- ni c ness of the system and becomes maximum as equilibrium is Free energy approached. Under conditions of constant temperature and pressure, the relationship between the free-energy change (ΔG) of a reacting system and the change in entropy (ΔS) is Chemical expressed by the following equation, which combines the two nic energy laws of thermodynamics: r go de En ∆G = ∆H −T∆S where ΔH is the change in enthalpy (heat) and T is the abso- C B lute temperature. A+C B + D + Heat In biochemical reactions, since ΔH is approximately equal to the total change in internal energy of the reaction or ΔE, FIGURE 11–1 Coupling of an exergonic to an endergonic the above relationship may be expressed in the following way: reaction. ∆G = ∆E −T∆S If ΔG is negative, the reaction proceeds spontaneously with may be represented as shown in Figure 11–1. The conversion loss of free energy, that is, it is exergonic. If, in addition, ΔG is of metabolite A to metabolite B occurs with release of free of great magnitude, the reaction goes virtually to completion energy and is coupled to another reaction in which free energy and is essentially irreversible. On the other hand, if ΔG is posi- is required to convert metabolite C to metabolite D. The terms tive, the reaction proceeds only if free energy can be gained, exergonic and endergonic, rather than the normal chemical that is, it is endergonic. If, in addition, the magnitude of ΔG is terms “exothermic” and “endothermic,” are used to indicate great, the system is stable, with little or no tendency for a reac- that a process is accompanied by loss or gain, respectively, of tion to occur. If ΔG is zero, the system is at equilibrium and no free energy in any form, not necessarily as heat. In practice, an net change takes place. endergonic process cannot exist independently, but must be a When the reactants are present in concentrations of component of a coupled exergonic–endergonic system where 1.0 mol/L, ΔG0 is the standard free-energy change. For bio- the overall net change is exergonic. The exergonic reactions chemical reactions, a standard state is defined as having a pH are termed catabolism (generally, the breakdown or oxidation of 7.0. The standard free-energy change at this standard state of fuel molecules), whereas the synthetic reactions that build is denoted by ΔG0′. up substances are termed anabolism. The combined catabolic The standard free-energy change can be calculated from and anabolic processes constitute metabolism. the equilibrium constant Keq, If the reaction shown in Figure 11–1 is to go from left to right, then the overall process must be accompanied by loss ∆G 0′ = −RT ln Keq of free energy as heat. One possible mechanism of coupling could be envisaged if a common obligatory intermediate (I) where R is the gas constant and T is the absolute temperature took part in both reactions, that is, (see Chapter 8). It is important to note that the actual ΔG may be larger or smaller than ΔG0′ depending on the concentra- A+C→I→B+D tions of the various reactants, including the solvent, various ions, and proteins. Some exergonic and endergonic reactions in biologic systems In a biochemical system, an enzyme only speeds up the are coupled in this way. This type of system has a built-in mech- attainment of equilibrium; it never alters the final concentra- anism for biologic control of the rate of oxidative processes tions of the reactants at equilibrium. since the common obligatory intermediate allows the rate of utilization of the product of the synthetic path (D) to deter- mine by mass action the rate at which A is oxidized. Indeed, ENDERGONIC PROCESSES these relationships supply a basis for the concept of respiratory PROCEED BY COUPLING TO control, the process that prevents an organism from burning out of control. An extension of the coupling concept is pro- EXERGONIC PROCESSES vided by dehydrogenation reactions, which are coupled to The vital processes—for example, biosynthetic reactions, hydrogenations by an intermediate carrier (Figure 11–2). muscular contraction, nerve impulse conduction, and active An alternative method of coupling an exergonic to an end- transport—obtain energy by chemical linkage, or coupling, to ergonic process is to synthesize a compound of high-energy oxidative reactions. In its simplest form, this type of coupling potential in the exergonic reaction and to incorporate this  CHAPTER 11 Bioenergetics: The Role of ATP 111 AH2 Carrier BH2 The Intermediate Value for the Free Energy of Hydrolysis of ATP Has A Carrier H2 B Important Bioenergetic Significance FIGURE 11–2 Coupling of dehydrogenation and hydrogena- The standard free energy of hydrolysis of a number of bio- tion reactions by an intermediate carrier. chemically important phosphates is shown in Table 11–1. An estimate of the comparative tendency of each of the phosphate new compound into the endergonic reaction, thus effecting groups to transfer to a suitable acceptor may be obtained from a transference of free energy from the exergonic to the ender- the ΔG0′ of hydrolysis at 37°C. This is termed the group trans- gonic pathway. The biologic advantage of this mechanism is fer potential. The value for the hydrolysis of the terminal phos- that the compound of high potential energy, ~ , unlike I in phate of ATP (when ATP is converted to ADP + Pi) divides the previous system, need not be structurally related to A, B, the list into two groups. Low-energy phosphates, having a low C, or D, allowing to serve as a transducer of energy from a group transfer potential, exemplified by the ester phosphates wide range of exergonic reactions to an equally wide range of found in the intermediates of glycolysis, have G0′ values smaller endergonic reactions or processes, such as biosynthesis, mus- than that of ATP, while in high-energy phosphates, with a cular contraction, nervous excitation, and active transport. In more negative G0′, the value is higher than that of ATP. The the living cell, the principal high-energy intermediate or car- components of this latter group, including ATP, are usually rier compound is ATP (Figure 11–3). anhydrides (eg, the 1-phosphate of 1,3-bisphosphoglycerate), enol phosphates (eg, phosphoenolpyruvate), and phosphogua- nidines (eg, creatine phosphate, arginine phosphate). HIGH-ENERGY PHOSPHATES The symbol ~ P indicates that the group attached to the PLAY A CENTRAL ROLE IN ENERGY bond, on transfer to an appropriate acceptor, results in trans- fer of the larger quantity of free energy. Thus, ATP has a high CAPTURE & TRANSFER group transfer potential, whereas the phosphate in adenosine In order to maintain living processes, all organisms must obtain monophosphate (AMP) is of the low-energy type since it is a supplies of free energy from their environment. Autotrophic normal ester link (Figure 11–4). In energy transfer reactions, organisms utilize simple exergonic processes; for example, the ATP may be converted to ADP and Pi or, in reactions requir- energy of sunlight (green plants), the reaction Fe2+ → Fe3+ ing a greater energy input, to AMP + PPi (see Table 11–1). (some bacteria). On the other hand, heterotrophic organ- isms obtain free energy by coupling their metabolism to the TABLE 11–1 Standard Free Energy of Hydrolysis of breakdown of complex organic molecules in their environ- Some Organophosphates of Biochemical Importance ment. In all these organisms, ATP plays a central role in the transference of free energy from the exergonic to the ender- DG0 gonic processes. ATP is a nucleotide consisting of the nucleo- Compound kJ/mol kcal/mol side adenosine (adenine linked to ribose) and three phosphate groups (see Chapter 32). In its reactions in the cell, it functions Phosphoenolpyruvate −61.9 −14.8 as the Mg2+ complex (see Figure 11–3). Carbamoyl phosphate −51.4 −12.3 The importance of phosphates in intermediary metabo- 1,3-Bisphosphoglycerate −49.3 −11.8 lism became evident with the discovery of the role of ATP, (to 3-phosphoglycerate) adenosine diphosphate (ADP), and inorganic phosphate (Pi) Creatine phosphate −43.1 −10.3 in glycolysis (see Chapter 17). ATP → AMP + PPi −32.2 −7.7 NH2 ATP → ADP + Pi −30.5 −7.3 N N Glucose-1-phosphate −20.9 −5.0 Mg2+ N PPi −19.2 −4.6 N Fructose-6-phosphate −15.9 −3.8 O– O– O– –O P O P O P O CH2 O Glucose-6-phosphate −13.8 −3.3 O O O C C Glycerol-3-phosphate −9.2 −2.2 H H Abbreviations: PPi, pyrophosphate; Pi, inorganic orthophosphate. ATP H H Note: All values are taken from Jencks WP: Free energies of hydrolysis and decarboxylation. In: Handbook of Biochemistry and Molecular Biology, vol 1. Physical and Chemical Data. OH OH Fasman GD (editor). CRC Press, 1976:296-304, except that for PPi which is from Frey PA, Arabshahi A: Standard free-energy change for the hydrolysis of the alpha, beta- phosphoanhydride bridge in ATP. Biochemistry 1995;34:11307. Values differ between FIGURE 11–3 Adenosine triphosphate (ATP) is shown as the investigators, depending on the precise conditions under which the measurements magnesium complex. were made. 112 SECTION III Bioenergetics O– O– O– products of hydrolysis, ADP and orthophosphate, are more stable, and so lower in energy, than ATP (Figure 11–5). Other Adenosine O P O P O P O– “high-energy compounds” are thiol esters involving coenzyme O O O A (eg, acetyl-CoA), acyl carrier protein, amino acid esters involved in protein synthesis, S-adenosylmethionine (active or Adenosine P P P methionine), uridine diphosphate glucose (UDPGlc), and Adenosine triphosphate (ATP) 5-phosphoribosyl-1-pyrophosphate (PRPP). Adenosine P P Adenosine P Adenosine diphosphate (ADP) Adenosine monophosphate (AMP) ATP ACTS AS THE “ENERGY CURRENCY” OF THE CELL FIGURE 11–4 Structure of ATP, ADP, and AMP showing the The high group transfer potential of ATP enables it to act as position and the number of high-energy phosphates (~ ). a donor of high-energy phosphate to form those compounds below it in Table 11–1. Likewise, with the necessary enzymes, The intermediate position of ATP allows it to play an ADP can accept phosphate groups to form ATP from those important role in energy transfer. The high free-energy change compounds above ATP in the table. In effect, an ATP/ADP on hydrolysis of ATP is not in itself caused by the breaking cycle connects those processes that generate ~ to those pro- of the P-O bond linking the terminal phosphate to the mol- cesses that utilize ~ (Figure 11–6), continuously consuming ecule (see Figure 11–4), in fact, energy is needed to bring and regenerating ATP. This occurs at a very rapid rate since this about. It is the consequences of this bond breakage that the total ATP/ADP pool is extremely small and sufficient to cause net energy to be released. Firstly, there is strong electro- maintain an active tissue for only a few seconds. static repulsion between the negatively charged oxygen atoms There are three major sources of ~ taking part in energy in the adjacent phosphate groups of ATP (see Figure 11–4), conservation or energy capture: which destabilizes the molecule and makes the removal of one phosphate group energetically favorable. Secondly, the ortho- 1. Oxidative phosphorylation is the greatest quantitative phosphate produced is greatly stabilized by the formation of source of ~ in aerobic organisms. ATP is generated in the resonance hybrids in which the three negative charges are mitochondrial matrix as O2 is reduced to H2O by electrons shared between the four oxygen atoms. Overall, therefore, the passing down the respiratory chain (see Chapter 13). FIGURE 11–5 The free-energy change on hydrolysis of ATP to ADP. Initially, energy input is required to break the terminal P-O bond. However, the breaking of the bond relieves the strong electrostatic repulsion between the negatively charged oxygen atoms in the adjacent phosphate groups of ATP, making the removal of one phosphate group energetically favorable. In addition, the orthophosphate released is greatly stabilized by the formation of resonance hybrids in which the three negative charges are shared between the four oxygen atoms. These effects more than compensate for the initial energy input and result in the high free-energy change seen when ATP is hydrolyzed to ADP.  CHAPTER 11 Bioenergetics: The Role of ATP 113 Phosphoenolpyruvate 1,3-Bisphosphoglycerate ATP Allows the Coupling of Succinyl- CoA Oxidative phosphorylation Thermodynamically Unfavorable Creatine P Reactions to Favorable Ones P Endergonic reactions cannot proceed without an input of free (Store of P ) energy. For example, the phosphorylation of glucose to glucose- ATP Creatine 6-phosphate, the first reaction of glycolysis (see Figure 17–2): Glucose + Pi → Glucose-6-phosphate + H2O ATP/ADP cycle (DG0) = +13.8 kJ/mol (1) P Glucose-6-phosphate ADP Other phosphorylations, is highly endergonic and cannot proceed under physiologic activations, conditions. Thus, in order to take place, the reaction must be Glucose-1,6- and endergonic Glycerol-3-phosphate bisphosphate processes coupled with another—more exergonic—reaction such as the hydrolysis of the terminal phosphate of ATP. FIGURE 11–6 Role of ATP/ADP cycle in transfer of high- energy phosphate. ATP → ADP + Pi (ΔG0′ = −30.5 kJ/mol) (2) When (1) and (2) are coupled in a reaction catalyzed by 2. Glycolysis. A net formation of two ~ results from the for- hexokinase, phosphorylation of glucose readily proceeds in a mation of lactate from one molecule of glucose, generated highly exergonic reaction that under physiologic conditions in two reactions catalyzed by phosphoglycerate kinase and is irreversible. Many “activation” reactions follow this pattern. pyruvate kinase, respectively (see Chapter 17). 3. The citric acid cycle. One ~ is generated directly in the Adenylyl Kinase (Myokinase) cycle at the succinate thiokinase step (see Figure 16–3). Interconverts Adenine Nucleotides Phosphagens act as storage forms of group transfer poten- This enzyme is present in most cells. It catalyzes the following tial and include creatine phosphate, which occurs in vertebrate reaction: skeletal muscle, heart, spermatozoa, and brain, and arginine phosphate, which occurs in invertebrate muscle. When ATP is rapidly being utilized as a source of energy for muscular contrac- tion, phosphagens permit its concentrations to be maintained, but when the ATP/ADP ratio is high, their concentration can increase to act as an energy store (Figure 11–7). Adenylyl kinase is important for the maintenance of energy When ATP acts as a phosphate donor to form com- homeostasis in cells because it allows: pounds of lower free energy of hydrolysis (see Table 11–1), 1. The group transfer potential in ADP to be used in the syn- the phosphate group is invariably converted to one of low thesis of ATP. energy. For example, the phosphorylation of glycerol to form glycerol-3-phosphate: 2. The AMP formed as a consequence of activating reactions involving ATP to be rephosphorylated to ADP. GLYCEROL KINASE 3. AMP to increase in concentration when ATP becomes Glycerol + Adenosine P P P depleted so that it is able to act as a metabolic (allosteric) signal to increase the rate of catabolic reactions, which in Glycerol P + Adenosine P P turn lead to the generation of more ATP (see Chapter 14). When ATP Forms AMP, Inorganic Pyrophosphate (PPi) Is Produced ATP can also be hydrolyzed directly to AMP, with the release of PPi (see Table 11–1). This occurs, for example, in the activa- tion of long-chain fatty acids (see Figure 22–3). This reaction is accompanied by loss of free energy as heat, which ensures that the activation reaction will go to the right, and is further aided by the hydrolytic splitting of PPi, FIGURE 11–7 Transfer of high-energy phosphate between catalyzed by inorganic pyrophosphatase, a reaction that itself ATP and creatine. has a large ΔG0′ of −19.2 kJ/mol. Note that activations via the 116 SECTION III Bioenergetics TABLE 12–1 Some Redox Potentials of Special Interest AH2 1 /2O2 AH2 O2 in Mammalian Oxidation Systems (Red) Oxidase Oxidase System E′0 Volts H+/H2 −.0.42 A H2O A H2O2 NAD+/NADH −0.32 (Ox) A B Lipoate; ox/red −0.29 Acetoacetate/3-hydroxybutyrate −0.27 FIGURE 12–1 Oxidation of a metabolite catalyzed by an oxidase (A) forming H2O and (B) forming H2O2. Pyruvate/lactate −0.19 Oxaloacetate/malate −0.17 oxidase enzyme compex comprises heme a3 combined with Fumarate/succinate +0.03 another heme, heme a, in a singe protein and so is aso termed cytochrome aa3. It contains two moecues of heme, each hav- Cytochrome b; Fe3+/Fe2+ +0.08 ing one Fe atom that osciates between Fe3+ and Fe2+ during Ubiquinone; ox/red +0.10 oxidation and reduction. Furthermore, two atoms of copper Cytochrome c1; Fe3+/Fe2+ +0.22 are present, one associated with each heme unit. Cytochrome a; Fe3+/Fe2+ +0.29 Other Oxidases Are Flavoproteins Oxygen/water +0.82 Favoprotein enzymes contain flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) as prosthetic groups. FMN and FAD are formed in the body from the vitamin ribo- (E′0) is normay expressed at pH 7.0, at which pH of the flavin (see Chapter 44). FMN and FAD are usuay tighty— eectrode potentia of the hydrogen eectrode is −0.42 V. The but not covaenty—bound to their respective apoenzyme redox potentias of some redox systems of specia interest in proteins. Metalloflavoproteins contain one or more metas as mammaian biochemistry are shown in Table 12–1. The rea- essentia cofactors. Exampes of favoprotein oxidases incude tive positions of redox systems in the tabe aow prediction l-amino acid oxidase, an enzyme found in kidney with gen- of the direction of fow of eectrons from one redox coupe to era specificity for the oxidative deamination of the naturay another. occurring l-amino acids; xanthine oxidase, a moybdenum- Enzymes invoved in oxidation and reduction are caed containing enzyme which pays an important roe in the con- oxidoreductases and are cassified into four groups: oxidases, version of purine bases to uric acid (see Chapter 33), and is of dehydrogenases, hydroperoxidases, and oxygenases. particuar significance in uricoteic animas (see Chapter 28); and aldehyde dehydrogenase, an FAD-inked enzyme pres- ent in mammaian ivers, which contains moybdenum and OXIDASES USE OXYGEN AS nonheme iron and acts on adehydes and N-heterocycic sub- A HYDROGEN ACCEPTOR strates. The mechanisms of oxidation and reduction of these Oxidases catayze the remova of hydrogen from a substrate enzymes are compex. Evidence suggests a two-step reaction using oxygen as a hydrogen acceptor.* They form water or as shown in Figure 12–2. hydrogen peroxide as a reaction product (Figure 12–1). DEHYDROGENASES PERFORM Cytochrome Oxidase Is a Hemoprotein TWO MAIN FUNCTIONS There are a arge number of enzymes in the dehydrogenase Cytochrome oxidase is a hemoprotein widey distributed in cass. Their two main functions are as foows: many tissues, having the typica heme prosthetic group pres- ent in myogobin, hemogobin, and other cytochromes (see 1. Transfer of hydrogen from one substrate to another in a Chapter 6). It is the termina component of the chain of respi- couped oxidation–reduction reaction (Figure 12–3). These ratory carriers found in mitochondria (see Chapter 13) and dehydrogenases often utiize common coenzymes or hydro- it functions to transfer eectrons resuting from the oxidation gen carriers, for exampe, nicotinamide adenine dinuceotide of substrate moecues by dehydrogenases to their fina accep- (NAD+). This type of reaction in which one substrate is oxi- tor, oxygen. The action of the enzyme is bocked by carbon dized/reduced at the expense of another is freey reversibe, monoxide, cyanide, and hydrogen sulfide, and this causes enabing reducing equivaents to be transferred within the ce poisoning by preventing ceuar respiration. The cytochrome and oxidative processes to occur in the absence of oxygen, such as during the anaerobic phase of gycoysis (see Figure 17–2). * The term “oxidase” is sometimes used coectivey to denote a 2. Transfer of eectrons from substrate to oxygen in the respi- enzymes that catayze reactions invoving moecuar oxygen. ratory chain eectron transport system (see Figure 13–3).  CHAPTER 12 Biologic Oxidation 117 R R R H H H3C N N O H3C N N O H3C N N O Oxidized Substrate + + NH NH NH substrate H3C N H 3C N H 3C N O O H O H H Oxidized flavin Semiquinone (H+ + e–) (H+ + e –) Reduced flavin (FAD) intermediate (FADH2) FIGURE 12–2 Oxidoreduction of isoalloxazine ring in flavin nucleotides via a semiquinone intermediate. In oxidation reactions, the flavin (eg, FAD) accepts two electrons and two H+ in two steps, forming the semiquinone intermediate followed by the reduced flavin (eg, FADH2) and the substrate is oxidized. In the reverse (reduction) reaction, the reduced flavin gives up two electrons and two H+ so that it becomes oxi- dized (eg, to FAD) and the substrate is reduced. Many Dehydrogenases Depend on NADP-linked dehydrogenases are found characteristicay in biosynthetic pathways where reductive reactions are required, Nicotinamide Coenzymes as in the extramitochondria pathway of fatty acid synthesis These dehydrogenases use NAD+ or nicotinamide adenine (see Chapter 23) and steroid synthesis (see Chapter 26)—and dinucleotide phosphate (NADP+)—or both—which are aso in the pentose phosphate pathway (see Chapter 20). formed in the body from the vitamin niacin (see Chapter 44). The structure of NAD+ is shown in Figure 12–4. NADP+ has a H O H O phosphate group esterified to the 2′ hydroxy of its adenosine H moiety, but otherwise is identica to NAD+. The oxidized forms NH2 NH2 O– of both nuceotides have a positive charge on the nitrogen + atom of the nicotinamide moiety as indicated in Figure 12–4. O P O N N The coenzymes are reduced by the specific substrate of the O H+ dehydrogenase and reoxidized by a suitabe eectron accep- R tor. They are abe to freey and reversiby dissociate from their respective apoenzymes. O OH OH NH2 Generay, NAD-linked dehydrogenases catayze oxido- reduction reactions of the type: N N OH O O P O N C + NAD+ C + NADH + H+ N O– O H When a substrate is oxidized, it oses two hydrogen atoms and two eectrons. One H+ and both eectrons are accepted by NAD+ OH OH to form NADH and the other H+ is reeased (see Figure 12–4). Many such reactions occur in the oxidative pathways of Oxidized metaboism, particuary in gycoysis (see Chapter 17) and substrate/product the citric acid cyce (see Chapter 16). NADH is generated in OH O these pathways via the oxidation of fue moecues, and NAD+ NAD+ + + NADH + H+ C C is regenerated by the oxidation of NADH as it transfers the eectrons to O2 via the respiratory chain in mitochondria, a H process which eads to the formation of ATP (see Chapter 13). Reduced substrate/product FIGURE 12–4 Oxidation and reduction of nicotinamide AH2 Carrier BH2 coenzymes. Nicotinamide coenzymes consist of a nicotinamide ring (Red) (Ox) (Red) linked to an adenosine via a ribose and a phosphate group, forming a dinucleotide. NAD+/NADH are shown, but NADP+/NADPH are identi- cal except that they have a phosphate group esterified to the 2′ OH A Carrier–H2 B of the adenosine. An oxidation reaction involves the transfer of two (Ox) (Red) (Ox) electrons and one H+ from the substrate to the nicotinamide ring of Dehydrogenase Dehydrogenase NAD+ forming NADH and the oxidized product. The remaining hydro- specific for A specific for B gen of the hydrogen pair removed from the substrate remains free as a hydrogen ion. NADH is oxidized to NAD+ by the reverse reaction. FIGURE 12–3 Oxidation of a metabolite catalyzed by cou- R, the part of the molecule unchanged in the oxidation/reduction pled dehydrogenases. reaction. 118 SECTION III Bioenergetics Other Dehydrogenases Depend on species (ROS). ROS are highy reactive oxygen-containing moecues such as peroxides, which are formed during norma Riboflavin metaboism, but can be damaging if they accumuate. They are The flavin groups such as FMN and FAD are associated with beieved to contribute to the causation of diseases such as can- dehydrogenases as we as with oxidases as described earier. cer and atheroscerosis, as we as the aging process in genera FAD is the eectron acceptor in reactions of the type: (see Chapters 21, 44, 54). H C C + FAD C C + FADH2 Peroxidases Reduce Peroxides Using H H H Various Electron Acceptors Peroxidases are found in mik as we as in eukocytes, pate- FAD accepts two eectrons and two H + in the reaction (see ets, and other tissues invoved in eicosanoid metaboism (see Figure 12–2), forming FADH2. Favin groups are generay Chapter 23). Their prosthetic group is protoheme. In the reac- more tighty bound to their apoenzymes than are the nicotin- tion catayzed by peroxidase, hydrogen peroxide is reduced at amide coenzymes. Most of the riboflavin-linked dehydro- the expense of severa substances that act as eectron acceptors, genases are concerned with eectron transport in (or to) the such as ascorbate (vitamin C), quinones, and cytochrome c. respiratory chain (see Chapter 13). NADH dehydrogenase acts The reaction catayzed by peroxidase is compex, but the over- as a carrier of eectrons between NADH and the components of a reaction is as foows: higher redox potentia (see Figure 13–3). Other dehydrogenases such as succinate dehydrogenase, acyl-CoA dehydrogenase, PEROXIDASE and mitochondrial glycerol-3-phosphate dehydrogenase H2O2 + AH2 2H2O + A transfer reducing equivaents directy from the substrate to the respiratory chain (see Figure 13–5). Another roe of the In erythrocytes and other tissues, the enzyme glutathione per- favin-dependent dehydrogenases is in the dehydrogenation oxidase, containing selenium as a prosthetic group, catayzes (by dihydrolipoyl dehydrogenase) of reduced ipoate, an the destruction of H2O2 and ipid hydroperoxides through the intermediate in the oxidative decarboxyation of pyruvate and conversion of reduced gutathione to its oxidized form, pro- α-ketogutarate (see Figures 13–5 and 17–5). The electron- tecting membrane ipids and hemogobin against oxidation by transferring flavoprotein (ETF) is an intermediary carrier of peroxides (see Chapter 21). eectrons between acy-CoA dehydrogenase and the respiratory chain (see Figure 13–5). Catalase Uses Hydrogen Peroxide as Cytochromes May Also Be Regarded Electron Donor & Electron Acceptor as Dehydrogenases Catalase is a hemoprotein containing four heme groups. It The cytochromes are iron-containing hemoproteins in which can act as a peroxidase, catayzing reactions of the type shown the iron atom osciates between Fe3+ and Fe2+ during oxida- earier, but it is aso abe to catayze the breakdown of H2O2 tion and reduction. Except for cytochrome oxidase (described formed by the action of oxygenases to water and oxygen: earier), they are cassified as dehydrogenases. In the respi- ratory chain, they are invoved as carriers of eectrons from CATALASE favoproteins on the one hand to cytochrome oxidase on the 2H2O2 2H2O + O2 other (see Figure 13–5). Severa identifiabe cytochromes occur in the respiratory chain, they are; cytochromes b, c1, c, This reaction uses one moecue of H2O2 as a substrate eec- and cytochrome oxidase (aa3). Cytochromes are aso found in tron donor and another moecue of H2O2 as an oxidant or other ocations, for exampe, the endopasmic reticuum (cyto- eectron acceptor. It is one of the fastest enzyme reactions chromes P450 and b5), and in pant ces, bacteria, and yeasts. known, destroying miions of potentiay damaging H 2O2 moecues per second. Under most conditions in vivo, the peroxidase activity of cataase seems to be favored. Cata- HYDROPEROXIDASES USE ase is found in bood, bone marrow, mucous membranes, HYDROGEN PEROXIDE OR AN kidney, and iver. Peroxisomes are membrane-bound ORGANIC PEROXIDE organees (see Chapter 49) found in many tissues, incud- ing iver. They are rich in oxidases and in cataase. Thus, AS SUBSTRATE enzymes that produce and breakdown H 2O2 are contained Two types of enzymes found both in animas and pants fa within the same subceuar compartment. However, mito- into the hydroperoxidase category: peroxidases and catalase. chondria and microsoma eectron transport systems as Hydroperoxidases pay an important roe in protect- we as xanthine oxidase must be considered as additiona ing the body against the harmfu effects of reactive oxygen sources of H 2O2.  CHAPTER 12 Biologic Oxidation 119 OXYGENASES CATALYZE Cytochromes P450 Are THE DIRECT TRANSFER & Monooxygenases Important in Steroid INCORPORATION OF OXYGEN Metabolism & for the Detoxification of INTO A SUBSTRATE MOLECULE Many Drugs Oxygenases are concerned with the synthesis or degrada- Cytochromes P450 are an important superfamiy of heme- tion of many different types of metaboites. They catayze the containing monooxygenases, and more than 50 such enzymes incorporation of oxygen into a substrate moecue in two steps: have been found in the human genome. They are ocated (1) oxygen is bound to the enzyme at the active site and mainy in the endopasmic reticuum in the iver and intes- (2) the bound oxygen is reduced or transferred to the sub- tine, but are aso found in the mitochondria in some tissues. strate. Oxygenases may be divided into two subgroups, dioxy- The cytochromes participate in an eectron transport chain in genases and monooxygenases. which both NADH and NADPH may donate reducing equiva- ents. Eectrons are passed to cytochrome P450 in two types of reaction invoving FAD or FMN. Cass I systems consist of an Dioxygenases Incorporate Both Atoms FAD-containing reductase enzyme, an iron sufur (Fe2S2) pro- of Molecular Oxygen Into the Substrate tein, and the P450 heme protein, whie cass II systems con- The basic reaction catayzed by dioxygenases is as foows: tain cytochrome P450 reductase, which passes eectrons from FADH2 to FMN (Figure 12–5). Cass I and II systems are we A + O2 → AO2 characterized, but in recent years, other cytochromes P450, Exampes incude the iver enzymes, homogentisate dioxy- which do not fit into either category, have been identified. In genase (oxidase) and 3-hydroxyanthranilate dioxygenase the fina step, oxygen accepts the eectrons from cytochrome (oxidase), which contain iron; and l-tryptophan dioxygenase P450 and is reduced, with one atom being incorporated into (tryptophan pyrroase) (see Chapter 29), which utiizes heme. H2O and the other into the substrate, usuay resuting in its hydroxyation. This series of enzymatic reactions, known as the hydroxylase cycle, is iustrated in Figure 12–6. In the Monooxygenases (Mixed-Function endopasmic reticuum of the iver, cytochromes P450 are Oxidases, Hydroxylases) Incorporate found together with another heme-containing protein, cyto- Only One Atom of Molecular Oxygen chrome b5 (see Figure 12–5) and together they have a major Into the Substrate roe in drug metaboism and detoxification. Cytochrome b5 aso has an important roe as a fatty acid desaturase. Together, The other oxygen atom is reduced to water, an additiona eec- cytochromes P450 and b5 are responsibe for about 75% of the tron donor or cosubstrate (Z) being necessary for this purpose: modification and degradation of drugs which occurs in the A — H + O2 + ZH2 → A — OH + H2O + Z body. The rate of detoxification of many medicina drugs by Class I P450 REDUCTASE Fe2S2 P450 NAD(P)H FAD FADH2 Fe3+ Fe2+ O2+RH H2O+ROH Hydroxylation Class II P450 P450 REDUCTASE P450 NAD(P)H Hydroxylation FAD FMN FMNH2 O2+RH H2O+ROH Cytochrome b5 O2+Oleoyl CoA b5 REDUCTASE NADH b5 FAD FADH2 Stearoyl CoA + H2O Stearoyl CoA desaturase P450 REDUCTASE P450 Hydroxylation FAD FMN FMNH2 O2+RH H2O+ROH FIGURE 12–5 Cytochromes P450 and b5 in the endoplasmic reticulum. Most cytochromes P450 are class I or class II. In addition to cytochrome P450, class I systems contain a small FAD-containing reductase and an iron sulfur protein, and class II contains cytochrome P450 reductase, which incorporates FAD and FMN. Cytochromes P450 catalyze many steroid hydroxylation reactions and drug detoxification steps. Cytochrome b5 acts in conjunction with the FAD-containing cytochrome b5 reductase in the fatty acyl-CoA desaturase (eg, stearoyl-CoA desaturase) reaction and also works together with cytochromes P450 in drug detoxification. It is able to accept electrons from cytochrome P450 reductase via cytochrome b5 reductase and donate them to cytochrome P450. 120 SECTION III Bioenergetics Substrate A-H P450-A-H Fe3+ e– P450 P450-A-H NADPH-Cyt P450 reductase Fe3+ Fe2+ NADP+ FADH2 2Fe2S23+ O2 e– – NADPH + H+ FAD 2Fe2S22+ CO 2H+ P450-A-H Fe2+ O2 H2O P450-A-H Fe2+ O2 – A-OH FIGURE 12–6 Cytochrome P450 hydroxylase cycle. The system shown is typical of steroid hydroxylases of the adrenal cortex. Liver microsomal cytochrome P450 hydroxylase does not require the iron-sulfur protein Fe2S2. Carbon monoxide (CO) inhibits the indicated step. cytochromes P450 determines the duration of their action. Superoxide can reduce oxidized cytochrome c Benzpyrene, aminopyrine, aniine, morphine, and benzphet- O2−. + Cytc(Fe3+) → O2 + Cytc(Fe2+) amine are hydroxyated, increasing their soubiity and aiding their excretion. Many drugs such as phenobarbita have the or be removed by SOD, which catayzes the conversion of abiity to induce the synthesis of cytochromes P450. superoxide to moecuar oxygen and hydrogen peroxide. Mitochondria cytochrome P450 systems are found in In this reaction, superoxide acts as both oxidant and steroidogenic tissues such as adrena cortex, testis, ovary, and reductant. Thus, SOD protects aerobic organisms against the pacenta and are concerned with the biosynthesis of steroid potentia deeterious effects of superoxide. The enzyme occurs hormones from choestero (hydroxyation at C22 and C20 in in a major aerobic tissues in the mitochondria and the cyto- side chain ceavage and at the 11β and 18 positions). In addi- so. Athough exposure of animas to an atmosphere of 100% tion, rena systems catayzing 1α- and 24-hydroxyations of oxygen causes an adaptive increase in SOD, particuary in the 25-hydroxychoecacifero in vitamin D metaboism—and ungs, proonged exposure eads to ung damage and death. choestero 7α-hydroxyase and stero 27-hydroxyase invoved Antioxidants, for exampe, α-tocophero (vitamin E), act as in bie acid biosynthesis from choestero in the iver (see scavengers of free radicas and reduce the toxicity of oxygen Chapters 26, 41)—are P450 enzymes. (see Chapter 44). SUMMARY SUPEROXIDE DISMUTASE In bioogic systems, as in chemica systems, oxidation (oss of PROTECTS AEROBIC ORGANISMS eectrons) is aways accompanied by reduction of an eectron acceptor. AGAINST OXYGEN TOXICITY Oxidoreductases have a variety of functions in metaboism; Transfer of a singe eectron to O2 generates the potentiay oxidases and dehydrogenases pay major roes in respiration; damaging superoxide anion-free radical (O2−.), which gives hydroperoxidases protect the body against damage by free rise to free-radica chain reactions (see Chapter 21), ampi- radicas; and oxygenases mediate the hydroxyation of drugs fying its destructive effects. The ease with which superoxide and steroids. can be formed from oxygen in tissues and the occurrence of Tissues are protected from oxygen toxicity caused by the superoxide dismutase (SOD), the enzyme responsibe for superoxide free radica by the specific enzyme superoxide its remova in a aerobic organisms (athough not in obigate dismutase. anaerobes), indicate that the potentia toxicity of oxygen is due to its conversion to superoxide. Superoxide is formed when reduced favins—present, for REFERENCES exampe, in xanthine oxidase—are reoxidized univaenty by Neson DL, Cox MM: Lehninger Principles of Biochemistry, 7th ed. moecuar oxygen: Macmian Education, 2017. Nichos DG, Ferguson SJ: Bioenergetics, 4th ed. Academic Press, Enz − Favin − H2 + O2 → Enz − Favin − H + O2−. + H+ 2013. 122 SECTION III Bioenergetics BIOMEDICAL IMPORTANCE enzymes of the respiratory chain, ATP synthase, and various membrane transporters. Aerobic organisms are able to capture a far greater propor- tion of the available free energy of respiratory substrates than anaerobic organisms. Most of this takes place inside THE RESPIRATORY CHAIN mitochondria, which have been termed the “powerhouses” of the cell. Respiration is coupled to the generation of the OXIDIZES REDUCING high-energy intermediate, ATP (see Chapter 11), by oxida- EQUIVALENTS & ACTS tive phosphorylation. A number of drugs (eg, amobarbital) AS A PROTON PUMP and poisons (eg, cyanide, carbon monoxide) inhibit oxida- tive phosphorylation, usually with fatal consequences. Sev- Most of the energy liberated during the oxidation of carbo- eral inherited defects of mitochondria involving components hydrate, fatty acids, and amino acids is made available within of the respiratory chain and oxidative phosphorylation have mitochondria as reducing equivalents (—H or electrons) been reported. Patients present with myopathy and encepha- (Figure 13–2). The enzymes of the citric acid cycle and lopathy and often have lactic acidosis. β-oxidation (see Chapters 22 and 16), the respiratory chain complexes, and the machinery for oxidative phosphorylation are all found in mitochondria. The respiratory chain collects SPECIFIC ENZYMES and transports reducing equivalents, directing them to their final reaction with oxygen to form water, and oxidative phos- ARE ASSOCIATED WITH phorylation is the process by which the liberated free energy is COMPARTMENTS SEPARATED trapped as high-energy phosphate (ATP). BY THE MITOCHONDRIAL MEMBRANES Components of the Respiratory Chain The mitochondrialmatrix(the internal compartment) is enclosed Are Contained in Four Large Protein by a double membrane. The outer membrane is permeable to Complexes Embedded in the Inner most metabolites and the inner membrane is selectively perme- Mitochondrial Membrane able (Figure 13–1). The outer membrane is characterized by Electrons flow through the respiratory chain through a redox span the presence of various enzymes, including acyl-CoA synthetase of 1.1 V from NAD+/NADH to O2/2H2O (see Table 12–1), passing (see Chapter 22) and glycerol phosphate acyltransferase (see through three large protein complexes: NADH-Q oxidoreduc- Chapter 24). Other enzymes, including adenylyl kinase tase (Complex I), where electrons are transferred from NADH (see Chapter 11) and creatine kinase (see Chapter 51) are to coenzyme Q (Q) (also called ubiquinone); Q-cytochrome c found in the intermembrane space. The phospholipid cardio- oxidoreductase (Complex III), which passes the electrons on lipin is concentrated in the inner membrane together with the to cytochrome c; and cytochrome c oxidase (Complex IV), which completes the chain, passing the electrons to O2 and caus- ing it to be reduced to H2O (Figure 13–3). Some substrates with more positive redox potentials than NAD+/NADH (eg, succinate) pass electrons to Q via a fourth complex, succinate-Q reduc- tase (Complex II), rather than Complex I. The four complexes are embedded in the inner mitochondrial membrane, but Q and cytochrome c are mobile. Q diffuses rapidly within the membrane, while cytochrome c is a soluble protein. Flavoproteins & Iron-Sulfur Proteins (Fe-S) Are Components of the Respiratory Chain Complexes Flavoproteins (see Chapter 12) are important components of Complexes I and II. The oxidized flavin nucleotide (flavin mononucleotide [FMN] or flavin adenine dinucleotide [FAD]) can be reduced in reactions involving the transfer of two elec- trons (to form FMNH2 or FADH2), but they can also accept one electron to form the semiquinone (see Figure 12–2). Iron- sulfur proteins (nonheme iron proteins, Fe-S) are found in Complexes I, II, and III. These may contain one, two, or four FIGURE 13–1 Structure of the mitochondrial membranes. Fe atoms linked to inorganic sulfur atoms and/or via cysteine- Note that the inner membrane contains many folds or cristae. SH groups to the protein (Figure 13–4). The Fe-S take part in  CHAPTER 13 The Respiratory Chain & Oxidative Phosphorylation 123 FIGURE 13–2 Role of the respiratory chain of mitochondria in the conversion of food energy to ATP. Oxidation of the major foodstuffs leads to the generation of reducing equivalents (2H) that are collected by the respiratory chain for oxidation and coupled generation of ATP. Succinate Fumarate Complex II succinate-Q reductase NADH + H+ 1/ O 2 2 + 2H+ Q Cyt c NAD H2O Complex I Complex III Complex IV NADH-Q Q-cyt c Cyt c oxidoreductase oxidoreductase oxidase FIGURE 13–3 Overview of electron flow through the respiratory chain. (cyt, cytochrome; Q, coenzyme Q or ubiquinone.) Pr Pr Cys Cys S S Fe Pr S S Cys Cys Cys S Pr Pr S Fe A Pr Cys S Fe S Pr Pr Cys Cys Fe S S S S S S Fe Fe Fe Cys S S S S Cys Pr Cys Cys Pr C Pr Pr B FIGURE 13–4 Iron-sulfur proteins (Fe-S). (A) The simplest Fe-S with one Fe bound by four cysteines. (B) 2Fe-2S center. (C) 4Fe-4S center. (Cys, cysteine; Pr, apoprotein; , inorganic sulfur.) 124 SECTION III Bioenergetics single electron transfer reactions in which one Fe atom under- Fe atoms is linked to two histidine residues rather than two goes oxidoreduction between Fe2+ and Fe3+. cysteine residues) (see Figure 13–4) and is known as the Q cycle (Figure 13–6). Q may exist in three forms: the oxidized quinone, Q Accepts Electrons via Complexes I & II the reduced quinol, or the semiquinone (see Figure 13–6). The semiquinone is formed transiently during the cycle, one NADH-Q oxidoreductase or Complex I is a large L-shaped turn of which results in the oxidation of 2QH2 to Q, releas- multisubunit protein that catalyzes electron transfer from ing 4H+ into the intermembrane space, and the reduction of NADH to Q, and during the process four H+ are transferred one Q to QH2, causing 2H+ to be taken up from the matrix across the membrane into the intermembrane space: (see Figure 13–6). Note that while Q carries two electrons, the NADH + Q + 5H+matrix → NAD + QH2 + 4H+intermembrance space cytochromes carry only one, thus the oxidation of one QH2 is coupled to the reduction of two molecules of cytochrome c via Electrons are transferred from NADH to FMN initially, then the Q cycle. to a series of Fe-S centers, and finally to Q (Figure 13–5). In Complex II (succinate-Q reductase), FADH2 is formed during Molecular Oxygen Is Reduced the conversion of succinate to fumarate in the citric acid cycle (see Figure 16–3) and electrons are then passed via several Fe-S to Water via Complex IV centers to Q (see Figure 13–5). Glycerol-3-phosphate (generated Reduced cytochrome c is oxidized by Complex IV (cytochrome c in the breakdown of triacylglycerols or from glycolysis; see oxidase), with the concomitant reduction of O2 to two molecules Figure 17–2) and acyl-CoA also pass electrons to Q via different of water: pathways involving flavoproteins (see Figure 13–5). 4Cyt creduced + O2 + 8H+matrix → The Q Cycle Couples Electron Transfer 4Cyt coxidized + 2H2O + 4H+intermembrane space to Proton Transport in Complex III Four electrons are transferred from cytochrome c to O2 via two Electrons are passed from QH2 to cytochrome c via Complex III heme groups, a and a3, and Cu (see Figure 13–5). Electrons are (Q-cytochrome c oxidoreductase): passed initially to a Cu center (CuA), which contains 2Cu atoms QH2 + 2Cyt coxidized + 2H+matrix → linked to two protein cysteine-SH groups (resembling an Fe-S), then in sequence to heme a, heme a3, a second Cu cen- Q + 2Cyt creduced + 4H+intermembrane space ter, CuB, which is linked to heme a3, and finally to O2. Eight H+ are removed from the matrix, of which four are used to form The process is believed to involve cytochromes c1, bL, and two water molecules and four are pumped into the intermem- bH and a Rieske Fe-S (an unusual Fe-S in which one of the brane space. Thus, for every pair of electrons passing down Glycerol-3-phosphate + + + + 4H 4H 2H 4H Intermembrane FAD space Cyt c Cyt c Complex I Complex II Inner Fe-S Q Cyt b Cyt b Q Fe-S mitochondrial Heme a + a3 membrane FMN Cyt c1 CuACuB Cyt c1 FAD Fe-S Complex III Complex IV Complex III Complex III Mitochondrial matrix NADH + H+ NAD ETF Fumarate Succinate 1/ O 2 2 + 2H+ H2 O Pyruvate Citric acid cycle FAD Ketone bodies Acyl CoA FIGURE 13–5 Flow of electrons through the respiratory chain complexes, showing the entry points for reducing equivalents from important substrates. Q and cyt c are mobile components of the system as indicated by the dotted arrows. The flow through Complex III (the Q cycle) is shown in more detail in Figure 13–6. (cyt, cytochrome; ETF, electron transferring flavoprotein; Fe-S, iron-sulfur protein; Q, coenzyme Q or ubiquinone.)  CHAPTER 13 The Respiratory Chain & Oxidative Phosphorylation 125 FIGURE 13–6 The Q cycle. During the oxidation of QH2 to Q, one electron is donated to cyt c via a Rieske Fe-S and cyt c1 and the second to a Q to form the semiquinone via cyt bL and cyt bH, with 2H+ being released into the intermembrane space. A similar process then occurs with a second QH2, but in this case the second electron is donated to the semiquinone, reducing it to QH2, and 2H+ are taken up from the matrix. (cyt, cytochrome; Fe-S, iron-sulfur protein; Q, coenzyme Q or ubiquinone.) the chain from NADH or FADH2, 2H+ are pumped across the A Membrane-Located ATP Synthase membrane by Complex IV. The O2 remains tightly bound to Complex IV until it is fully reduced, and this minimizes the Functions as a Rotary Motor to Form ATP release of potentially damaging intermediates such as super- The proton motive force drives a membrane-located ATP syn- oxide anions or peroxide which are formed when O2 accepts thase that forms ATP in the presence of Pi + ADP. ATP synthase one or two electrons, respectively (see Chapter 12). is embedded in the inner membrane, together with the respira- tory chain complexes (Figure 13–7). Several subunits of the pro- tein form a ball-like shape arranged around an axis known as F1, which projects into the matrix and contains the phosphorylation ELECTRON TRANSPORT VIA mechanism (Figure 13–8). F1 is attached to a membrane pro- THE RESPIRATORY CHAIN tein complex known as F0, which also consists of several protein subunits. F0 spans the membrane and forms a proton channel. CREATES A PROTON GRADIENT As protons flow through F0 driven by the proton gradient across WHICH DRIVES THE SYNTHESIS the membrane it rotates, driving the production of ATP in the F1 OF ATP complex (see Figures 13–7 and 13–8). This is thought to occur by a binding change mechanism in which the conformation of The flow of electrons through the respiratory chain gener- the β subunits in F1 is changed as the axis rotates from one that ates ATP by the process of oxidative phosphorylation. The binds ATP tightly to one that releases ATP and binds ADP and Pi chemiosmotic theory, proposed by Peter Mitchell in 1961, so that the next ATP can be formed. As indicated earlier, for each postulates that the two processes are coupled by a proton gra- NADH oxidized, Complexes I and III translocate four protons dient across the inner mitochondrial membrane so that the each and Complex IV translocates two. proton motive force caused by the electrochemical potential difference (negative on the matrix side) drives the mechanism of ATP synthesis. As we have seen, Complexes I, III, and IV act THE RESPIRATORY CHAIN as proton pumps, moving H+ from the mitochondrial matrix PROVIDES MOST OF THE ENERGY to the intermembrane space. Since the inner mitochondrial membrane is impermeable to ions in general and particularly CAPTURED DURING CATABOLISM to protons, these accumulate in the intermembrane space, cre- ADP captures, in the form of high-energy phosphate, a sig- ating the proton motive force predicted by the chemiosmotic nificant proportion of the free energy released by catabolic theory. processes. The resulting ATP has been called the energy 126 SECTION III Bioenergetics + 4H 4H+ 2H+ H+ H+ Uncouplers + Cyt c H H+ H+ Intermembrane space + + + + + + + + + + + + +++ Complex Complex Complex F0 Inner I III IV mitochondrial QQ membrane F1 H+ H+ H+ Mitochondrial matrix NADH + H+ NAD 1/ O 2 2 + 2H + H2O ADP + Pi ATP Complex II ATP synthase Succinate Fumarate FIGURE 13–7 The chemiosmotic theory of oxidative phosphorylation. Complexes I, III, and IV act as proton pumps creating a proton gradient across the membrane, which is negative on the matrix side. The proton motive force generated drives the synthesis of ATP as the pro- tons flow back into the matrix through the ATP synthase enzyme (Figure 13–8). Uncouplers increase the permeability of the membrane to ions, collapsing the proton gradient by allowing the H+ to pass across without going through the ATP synthase, and thus uncouple electron flow through the respiratory complexes from ATP synthesis. (cyt, cytochrome; Q, coenzyme Q or ubiquinone.) “currency” of the cell because it passes on this free energy to drive those processes requiring energy (see Figure 11–5). There is a net direct capture of two high-energy phosphate groups in the glycolytic reactions (see Table 17–1). Two more high-energy phosphates per mole of glucose are captured in the citric acid cycle during the conversion of succinyl-CoA to succinate (see Chapter 16). All of these phosphorylations occur at the substrate level. For each mol of substrate oxidized via Complexes I, III, and IV in the respiratory chain (ie, via NADH), 2.5 mol of ATP are formed per 0.5 mol of O2 con- sumed, that is, the P:O ratio = 2.5 (see Figure 13–7). On the other hand, when 1 mol of substrate (eg, succinate or 3-phophoglycerate) is oxidized via Complexes II, III, and IV, only 1.5 mol of ATP are formed, that is, P:O = 1.5. These reactions are known as oxidative phosphorylation at the respiratory chain level. Taking these values into account, it can be estimated that nearly 90% of the high-energy phos- phates produced from the complete oxidation of 1 mol glu- cose is obtained via oxidative phosphorylation coupled to the respiratory chain (see Table 17–1). FIGURE 13–8 Mechanism of ATP production by ATP synthase. The enzyme complex consists of an F0 subcomplex which is a disk of “C” protein subunits. Attached is a γ subunit in the form of a “bent Respiratory Control Ensures axle.” Protons passing through the disk of “C” units cause it and the attached γ subunit to rotate. The γ subunit fits inside the F1 subcom- a Constant Supply of ATP plex of three α and three β subunits, which are fixed to the mem- The rate of respiration of mitochondria can be controlled by brane and do not rotate. ADP and Pi are taken up sequentially by the the availability of ADP. This is because oxidation and phos- β subunits to form ATP, which is expelled as the rotating γ subunit squeezes each β subunit in turn and changes its conformation. Thus, phorylation are tightly coupled; that is, oxidation cannot three ATP molecules are generated per revolution. For clarity, not all proceed via the respiratory chain without concomitant phos- the subunits that have been identified are shown—eg, the “axle” also phorylation of ADP. Table 13–1 shows the five conditions contains an ε subunit. controlling the rate of respiration in mitochondria. Most cells  CHAPTER 13 The Respiratory Chain & Oxidative Phosphorylation 127 TABLE 13–1 States of Respiratory Control allowing continuous unidirectional flow and constant pro- vision of ATP. It also contributes to maintenance of body Conditions Limiting the Rate of Respiration temperature. State 1 Availability of ADP and substrate State 2 Availability of substrate only State 3 The capacity of the respiratory chain itself, when MANY POISONS INHIBIT all substrates and components are present in THE RESPIRATORY CHAIN saturating amounts Much information about the respiratory chain has been State 4 Availability of ADP only obtained by the use of inhibitors, and, conversely, this has State 5 Availability of oxygen only provided knowledge about the mechanism of action of several poisons (Figure 13–9). They may be classified as inhibitors of the respiratory chain, inhibitors of oxidative phosphorylation, or uncouplers of oxidative phosphorylation. in the resting state are in state 4, and respiration is controlled Barbiturates such as amobarbital inhibit electron trans- by the availability of ADP. When work is performed, ATP is port via Complex I by blocking the transfer from Fe-S to Q. converted to ADP, allowing more respiration to occur, which At sufficient dosage, they are fatal. Antimycin A and dimer- in turn replenishes the store of ATP. Under certain conditions, caprol inhibit the respiratory chain at Complex III. The classic the concentration of inorganic phosphate can also affect the poisons H2S, carbon monoxide, and cyanide inhibit Complex IV rate of functioning of the respiratory chain. As respiration and can therefore totally arrest respiration. Malonate is a com- increases (as in exercise), the cell approaches states 3 or 5 petitive inhibitor of Complex II. when either the capacity of the respiratory chain becomes sat- Atractyloside inhibits oxidative phosphorylation by urated or the PO2 decreases below the Km for heme a3. There inhibiting the transporter of ADP into and ATP out of the is also the possibility that the ADP/ATP transporter, which mitochondrion (Figure 13–10). The antibiotic oligomycin facilitates entry of cytosolic ADP into and ATP out of the completely blocks oxidation and phosphorylation by blocking mitochondrion, becomes rate limiting. the flow of protons through ATP synthase (see Figure 13–8). Thus, the manner in which biologic oxidative processes Uncouplers dissociate oxidation in the respiratory chain allow the free energy resulting from the oxidation of food- from phosphorylation (see Figure 13–7). These compounds stuffs to become available and to be captured is stepwise, are toxic, causing respiration to become uncontrolled, since efficient, and controlled—rather than explosive, inefficient, the rate is no longer limited by the concentration of ADP or Pi. and uncontrolled, as in many nonbiologic processes. The The uncoupler that has been used most frequently in studies remaining free energy that is not captured as high-energy of the respiratory chain is 2,4-dinitrophenol, but other com- phosphate is liberated as heat. This need not be considered pounds act in a similar manner. Thermogenin (or uncou- “wasted” since it ensures that the respiratory system as a whole pling protein 1 [UCP1]) is a physiologic uncoupler found is sufficiently exergonic to be removed from equilibrium, in brown adipose tissue that functions to generate body heat, Malonate