Harper's Biochemistry Chapter 13 - The Respiratory Chain & Oxidative Phosphorylation.PDF

C H A P T E R The Respiratory Chain & Oxidative Phosphorylation Kathleen M. Botham, PhD, DSc, & Peter A. Mayes, PhD, DSc 13 OBJ E C TI VE S Describe the double membrane structur...

C H A P T E R The Respiratory Chain & Oxidative Phosphorylation Kathleen M. Botham, PhD, DSc, & Peter A. Mayes, PhD, DSc 13 OBJ E C TI VE S Describe the double membrane structure of mitochondria and indicate the location of various enzymes. After studying this chapter, Appreciate that energy from the oxidation of fuel substrates (fats, you should be able to: carbohydrates, amino acids) is almost all generated in mitochondria via a process termed electron transport in which electrons pass through a series of complexes (the respiratory chain) until they are finally reacted with oxygen to form water. Describe the four protein complexes involved in the transfer of electrons through the respiratory chain and explain the roles of flavoproteins, iron-sulfur proteins, and coenzyme Q. Explain how coenzyme Q accepts electrons from NADH via Complex I and from FADH2 via Complex II. Indicate how electrons are passed from reduced coenzyme Q to cytochrome c via Complex III in the Q cycle. Explain the process by which reduced cytochrome c is oxidized and oxygen is reduced to water via Complex IV. Describe how electron transport generates a proton gradient across the inner mitochondrial membrane, leading to the buildup of a proton motive force that generates ATP by the process of oxidative phosphorylation. Describe the structure of the ATP synthase enzyme and explain how it works as a rotary motor to produce ATP from ADP and Pi. Explain that oxidation of reducing equivalents via the respiratory chain and oxidative phosphorylation are tightly coupled in most circumstances, so that one cannot proceed unless the other is functioning. Indicate examples of common poisons that block respiration or oxidative phosphorylation and identify their site of action. Explain, with examples, how uncouplers may act as poisons by dissociating oxidation via the respiratory chain from oxidative phosphorylation, but may also have a physiologic role in generating body heat. Explain the role of exchange transporters present in the inner mitochondrial membrane in allowing ions and metabolites to pass through while preserving electrochemical and osmotic equilibrium. 121 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 + + + H H Cyt c 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 Complex II FAD Succinate Fe-S – Carboxin TTFA H2S CO BAL – CN– Antimycin A – Complex IV Complex I Complex III heme a heme a3 NADH FMN, Fe-S Q Cyt b, Fe-S, Cyt c1 Cyt c O2 Cu Cu Piericidin A – – Uncouplers – Amobarbital – Uncouplers – Rotenone Oligomycin – – Oligomycin – ADP + Pi ATP ADP + P i ATP ADP + P i ATP FIGURE 13–9 Sites of inhibition () of the respiratory chain by specific drugs, chemicals, and antibiotics. (BAL, dimercaprol; TTFA, an Fe-chelating agent. Other abbreviations as in Figure 13–5.) 128 SECTION III Bioenergetics THE SELECTIVE PERMEABILITY OF THE INNER MITOCHONDRIAL MEMBRANE NECESSITATES EXCHANGE TRANSPORTERS Exchange diffusion systems involving transporter proteins that span the membrane are present in the membrane for exchange of anions against OH− ions and cations against H+ ions. Such sys- tems are necessary for uptake and output of ionized metabolites while preserving electrical and osmotic equilibrium. The inner mitochondrial membrane is freely permeable to uncharged small molecules, such as oxygen, water, CO2, NH3, and to mono- carboxylic acids, such as 3-hydroxybutyric, acetoacetic, and acetic, especially in their undissociated, more lipid soluble form. Long-chain fatty acids are transported into mitochondria via the carnitine system (see Figure 22–1), and there is also a special car- rier for pyruvate involving a symport that utilizes the H+ gradi- ent from outside to inside the mitochondrion (see Figure 13–10). However, dicarboxylate and tricarboxylate anions (eg, malate, citrate) and amino acids require specific transporter or carrier systems to facilitate their passage across the membrane. The transport of di- and tricarboxylate anions is closely linked to that of inorganic phosphate, which penetrates read- ily as the H2PO4− ion in exchange for OH−. The net uptake of malate by the dicarboxylate transporter requires inorganic FIGURE 13–10 Transporter systems in the inner mito- phosphate for exchange in the opposite direction. The net chondrial membrane. ➀ Phosphate transporter, ➁ pyruvate uptake of citrate, isocitrate, or cis-aconitate by the tricarboxyl- symport, ➂ dicarboxylate transporter, ➃ tricarboxylate transporter, ➄ α-ketoglutarate transporter, ➅ adenine nucleotide transporter. ate transporter requires malate in exchange. α-Ketoglutarate N-Ethylmaleimide, hydroxycinnamate, and atractyloside inhibit () transport also requires an exchange with malate. The adenine the indicated systems. Also present (but not shown) are transporter nucleotide transporter allows the exchange of ATP and ADP, systems for glutamate/aspartate (Figure 13–13), glutamine, ornithine, but not AMP. It is vital for ATP to exit from mitochondria to the neutral amino acids, and carnitine (see Figure 22–1). sites of extramitochondrial utilization and for the return of ADP for ATP production within the mitochondrion (Figure 13–11). particularly for the newborn and during hibernation in ani- Since in this translocation four negative charges are removed mals (see Chapter 25). from the matrix for every three taken in, the electrochemi- cal gradient across the membrane (the proton motive force) THE CHEMIOSMOTIC THEORY Inner CAN ACCOUNT FOR RESPIRATORY Outside mitochondrial membrane Inside CONTROL AND THE ACTION F1 ATP Synthase OF UNCOUPLERS 3H+ The electrochemical potential difference across the mem- brane, once established as a result of proton translocation, inhibits further transport of reducing equivalents through the respiratory chain unless discharged by back-translocation of ATP4– protons across the membrane through the ATP synthase. This ATP4– in turn depends on availability of ADP and Pi. 2 ADP3– ADP3– Uncouplers such as dinitrophenol are amphipathic (see Chapter 21) and increase the permeability of the lipoid inner P i– 1 mitochondrial membrane to protons, while physiologic uncou- H+ H+ plers such as UCP1 are membrane-spanning proteins which have a similar effect, thus the electrochemical potential is reduced and FIGURE 13–11 Combination of phosphate transporter with ATP synthase is short-circuited (see Figure 13–7). In these cir- the adenine nucleotide transporter in ATP synthesis. The H+/Pi sym- cumstances, oxidation can proceed without phosphorylation. port shown is equivalent to the Pi/OH− antiport shown in Figure 13–10.  CHAPTER 13 The Respiratory Chain & Oxidative Phosphorylation 129 Outer membrane Inner membrane NAD+ Glycerol-3-phosphate Glycerol-3-phosphate FAD Glycerol-3-phosphate Glycerol-3-phosphate dehydrogenase dehydrogenase (Cytosolic) (Mitochondrial) NADH + H+ Dihydroxyacetone Dihydroxyacetone FADH2 phosphate phosphate Cytosol Mitochondrion Respiratory chain FIGURE 13–12 Glycerophosphate shuttle for transfer of reducing equivalents from the cytosol into the mitochondrion. favors the export of ATP. Na+ can be exchanged for H+, driven glutamate dehydrogenase and hydroxylases involved in steroid by the proton gradient. It is believed that active uptake of synthesis. Ca2+ by mitochondria occurs with a net charge transfer of 1 (Ca+ uniport), possibly through a Ca2+/H+ antiport. Calcium release from mitochondria is facilitated by exchange with Na+. Oxidation of Extramitochondrial NADH Is Mediated by Substrate Shuttles Ionophores Permit Specific Cations to NADH cannot penetrate the mitochondrial membrane, but Penetrate Membranes it is produced continuously in the cytosol by glyceraldehyde- Ionophores are lipophilic molecules that complex specific 3-phosphate dehydrogenase, an enzyme in the glycolysis cations and facilitate their transport through biologic mem- sequence (see Figure 17–2). However, under aerobic conditions, branes, for example, valinomycin (K+). The classic uncouplers extramitochondrial NADH does not accumulate and is pre- such as dinitrophenol are, in fact, proton ionophores. sumed to be oxidized by the respiratory chain in mitochondria. The transfer of reducing equivalents through the mitochondrial membrane requires substrate pairs, linked by suitable dehydro- Proton-Translocating Transhydrogenase genases on each side of the mitochondrial membrane. The mech- Is a Source of Intramitochondrial NADPH anism of transfer using the glycerophosphate shuttle is shown in Proton-translocating transhydrogenase (also called NAD(P) Figure 13–12. Since the mitochondrial enzyme is linked to the transhydrogenase), a protein in the inner mitochondrial mem- respiratory chain via a flavoprotein rather than NAD, only 1.5 mol brane, couples the passage of protons down the electrochemi- rather than 2.5 mol of ATP are formed per atom of oxygen con- cal gradient from outside to inside the mitochondrion with sumed. Although this shuttle is present in some tissues (eg, brain, the transfer of H from intramitochondrial NADH to NADP white muscle), in others (eg, heart muscle) it is deficient. It is forming NADPH for intramitochondrial enzymes such as therefore believed that the malate shuttle system (Figure 13–13) FIGURE 13–13 Malate shuttle for transfer of reducing equivalents from the cytosol into the mitochondrion. α-Ketoglutarate trans- porter and glutamate/aspartate transporter (note the proton symport with glutamate). α-KG, α-ketoglutarate. NAD+ is generated from NADH outside the mitochondrion via the formation of malate from oxaloacetate. Malate crossed the inner membrane in exchange for α-KG and is converted back to oxaloacetate, releasing NADH inside the matrix. (Asp) and α-KG are then produced from oxaloacetate and glutamate by a transaminase enzyme and are transported into the cytosol, where oxaloacetate is reformed by a second transaminase and may be used to generate another NAD+ from NADH. 130 SECTION III Bioenergetics is of more universal utility. The complexity of this system is due the transfer of high-energy phosphate to creatine from ATP to the impermeability of the mitochondrial membrane to oxa- emerging from the adenine nucleotide transporter. In turn, loacetate. This is overcome by a transamination reaction with the creatine phosphate is transported into the cytosol via pro- glutamate forming aspartate and α-ketoglutarate which can tein pores in the outer mitochondrial membrane, becoming then cross the membrane via specific transporters and reform available for generation of extramitochondrial ATP. oxaloacetate in the cytosol. CLINICAL ASPECTS The Creatine Phosphate Shuttle The condition known as fatal infantile mitochondrial Facilitates Transport of High-Energy myopathy and renal dysfunction involves severe diminution Phosphate From Mitochondria or absence of most oxidoreductases of the respiratory chain. MELAS (mitochondrial encephalopathy, lactic acidosis, and The creatine phosphate shuttle (Figure 13–14) augments stroke) is an inherited condition due to NADH-Q oxidoreductase the functions of creatine phosphate as an energy buffer by (Complex I) or cytochrome oxidase (Complex IV) deficiency. It acting as a dynamic system for transfer of high-energy phos- is caused by a mutation in mitochondrial DNA and may be phate from mitochondria in active tissues such as heart and involved in Alzheimer disease and diabetes mellitus. A num- skeletal muscle. An isoenzyme of creatine kinase (CKm) is ber of drugs and poisons act by inhibition of oxidative phos- found in the mitochondrial intermembrane space, catalyzing phorylation (see earlier). Energy-requiring processes SUMMARY (eg, muscle contraction) Virtually all energy released from the oxidation of ATP ADP carbohydrate, fat, and protein is made available in mitochondria as reducing equivalents (—H or e−). These are CKa funneled into the respiratory chain, where they are passed down a redox gradient of carriers to their final reaction with ATP ADP oxygen to form water. Creatine Creatine-P The redox carriers are grouped into four respiratory chain CKc complexes in the inner mitochondrial membrane. Three of the four complexes are able to use the energy released in the redox CKg gradient to pump protons to the outside of the membrane, ATP ADP creating an electrochemical potential between the matrix and Glycolysis the inner membrane space. Outer mitochondrial Cytosol ATP synthase spans the membrane and acts like a rotary motor membrane P using the potential energy of the proton gradient or proton P motive force to synthesize ATP from ADP and Pi. In this way, CKm oxidation is closely coupled to phosphorylation to meet the energy needs of the cell. Inter-membrane Since the inner mitochondrial membrane is impermeable to ATP ADP space protons and other ions, special exchange transporters span Adenine the membrane to allow ions such as OH−, ATP4−, ADP3−, nucleotide and metabolites to pass through without discharging the transporter mi I t electrochemical gradient across the membrane. me oc nn ond ne m er rial h ra Many well-known poisons such as cyanide arrest respiration by Oxidative b phosphorylation inhibition of the respiratory chain. Matrix FIGURE 13–14 The creatine phosphate shuttle of heart and REFERENCES skeletal muscle. The shuttle allows rapid transport of high-energy Kocherginsky N: Acidic lipids, H(+)-ATPases, and mechanism of phosphate from the mitochondrial matrix into the cytosol. (CKa, cre- oxidative phosphorylation. Physico-chemical ideas 30 years after atine kinase concerned with large requirements for ATP, eg, muscular P. Mitchell’s Nobel Prize award. Prog Biophys Mol Biol 2009;99:20. contraction; CKc, creatine kinase for maintaining equilibrium between creatine and creatine phosphate and ATP/ADP; CKg, creatine kinase Mitchell P: Keilin’s respiratory chain concept and its chemiosmotic coupling glycolysis to creatine phosphate synthesis; CKm, mitochon- consequences. Science 1979;206:1148. drial creatine kinase mediating creatine phosphate production from Nakamoto RK, Baylis Scanlon JA, Al-Shawi MK: The rotary ATP formed in oxidative phosphorylation; P, pore protein in outer mechanism of the ATP synthase. Arch Biochem Biophys mitochondrial membrane.) 2008;476:43.