8885_c19_690-7503/1/0411:32 AM Page690mac76mac76:385-z: chaptar 19 OXIDATIVE PHOSPHORYLATION AND PHOTOPHOSPHORYLATION OXIDATIVE PHOSPHORYLATION 19.1 Electron-Transfer Reactions in Mitochondria 691 yielding metabolism in aerobic organisms. All oxi 19.2 ATP Synthesis 704 dative steps in the degradatic hydrates, fats, and amino acids conve llul 19.3 Regulation of Oxidative Phosphorylation 716 respiration, in which th es the 19.4 Mitochondrial Genes: Their Origin and the Effects synthesis of ATP. P of Mutations 719 by which phot 19.5 The Role of Mitochondria in Apoptosis and of sunlight- Oxidative Stress 721 sphere-and h tive phosp PHOTOSYNTHESIS: HARVESTING LIGHT ENERGY for most of t of the time 19.6 General Features of Photophosphorylation 723 occurs in 19.7 Light Absorption 725 iIn euka plasts Oxidative 19.8 The Central Photochemical Event: Light-Driven Electron Flow 730 19.9 ATP Synthesis by Photophosphorylation 740 ylation in NADP* as ult olutely differ- If an idea presents itself to us, we must not reject it simply because it does not agree with the logical deductions of a reigning theory. -Claude Bemard, An Introduction to the The aspect of the present position find most remarkable and admirable, is is the altruism and generosity with which former opponents of the chemiosmotic hypothesis have not only come to acce but have actively promoted it to the status of a theory. -Peter Mitchell, Nobel Address, 1978 espects. (1)Both 690
chapter Oxidative phosphorylation is the culmination of energyyielding metabolism in aerobic organisms. All oxidative steps in the degradation of carbohydrates, fats, and amino acids converge at this final stage of cellular respiration, in which the energy of oxidation drives the synthesis of ATP. Photophosphorylation is the means by which photosynthetic organisms capture the energy of sunlight—the ultimate source of energy in the biosphere—and harness it to make ATP. Together, oxidative phosphorylation and photophosphorylation account for most of the ATP synthesized by most organisms most of the time. In eukaryotes, oxidative phosphorylation occurs in mitochondria, photophosphorylation in chloroplasts. Oxidative phosphorylation involves the reduction of O2 to H2O with electrons donated by NADH and FADH2; it occurs equally well in light or darkness. Photophosphorylation involves the oxidation of H2O to O2, with NADP as ultimate electron acceptor; it is absolutely dependent on the energy of light. Despite their differences, these two highly efficient energy-converting processes have fundamentally similar mechanisms. Our current understanding of ATP synthesis in mitochondria and chloroplasts is based on the hypothesis, introduced by Peter Mitchell in 1961, that transmembrane differences in proton concentration are the reservoir for the energy extracted from biological oxidation reactions. This chemiosmotic theory has been accepted as one of the great unifying principles of twentieth century biology. It provides insight into the processes of oxidative phosphorylation and photophosphorylation, and into such apparently disparate energy transductions as active transport across membranes and the motion of bacterial flagella. Oxidative phosphorylation and photophosphorylation are mechanistically similar in three respects. (1) Both 19 690 OXIDATIVE PHOSPHORYLATION AND PHOTOPHOSPHORYLATION If an idea presents itself to us, we must not reject it simply because it does not agree with the logical deductions of a reigning theory. —Claude Bernard, An Introduction to the Study of Experimental Medicine, 1813 The aspect of the present position of consensus that I find most remarkable and admirable, is the altruism and generosity with which former opponents of the chemiosmotic hypothesis have not only come to accept it, but have actively promoted it to the status of a theory. —Peter Mitchell, Nobel Address, 1978 OXIDATIVE PHOSPHORYLATION 19.1 Electron-Transfer Reactions in Mitochondria 691 19.2 ATP Synthesis 704 19.3 Regulation of Oxidative Phosphorylation 716 19.4 Mitochondrial Genes: Their Origin and the Effects of Mutations 719 19.5 The Role of Mitochondria in Apoptosis and Oxidative Stress 721 PHOTOSYNTHESIS: HARVESTING LIGHT ENERGY 19.6 General Features of Photophosphorylation 723 19.7 Light Absorption 725 19.8 The Central Photochemical Event: Light-Driven Electron Flow 730 19.9 ATP Synthesis by Photophosphorylation 740 8885d_c19_690-750 3/1/04 11:32 AM Page 690 mac76 mac76:385_reb:
8885ac19690-7503/1/0411:32 AM Page691mac76mac76:385 19.1 Electron-Transfer Reactions in Mitochondria processes involve the flow of electrons through a chain ATP synthase Outer membrane of membrane-bound carriers. (2) The free energy made available by this"downhill"(exergonic) electron flow Freely permeable to Cristae small molecules and ions is coupled to the "uphill"transport of protons across a proton-impermeable membrane, conserving the free energy of fuel oxidation as a transmembrane electro- chemical potential (p. 391).3)The transmembrane brane rmeable to most flow of protons down their concentration gradient all molecules and ions through specific protein channels provides the free including H energy for synthesis of ATP, catalyzed by a membrane protein complex(ATP synthase) that couples proton Respiratory electron flow to phosphorylation of ADP. carriers( Complexes I-IV) we begin this chapter with oxidative phosphoryla- ADP-atP translocase tion. We first describe the components of the electron- ATP synthase(FoF1 transfer chain, their organization into large functional Other membrane complexes in the inner mitochondrial membrane, the path of electron flow through them, and the proton movements that accompany this flow. we then consider Matrix the remarkable enzyme complex that, by " rotational Contains: atalysis, captures the energy of proton flow in ATP, nd the regulatory mechanisms that coordinate oxida- tive phosphorylation with the many catabolic pathways complex by which fuels are oxidized. with this understanding of mitochondrial oxidative phosphorylation, we turn to photophosphorylation, looking first at the absorption of Fatty acid light by photosynthetic pigments, then at the light- driven flow of electrons from h,o to nadp and the Amino acid molecular basis for coupling electron and proton flow We also consider the similarities of structure and mech anism between the AtP synthases of chloroplasts and DNA. ribosomes mitochondria, and the evolutionary basis for this con- Porin channels servation of mechanism ATP,ADP, P, Mg2+, Ca2+,K+ Many soluble metabolic OXIDATIVE PHOSPHORYLATION 19. 1 Electron-Transfer Reactions FIGURE 19-1 Biochemical anatomy of a mitochondrion. The convo- lutions(cristae) of the inner membrane provide a very large surfac in Mitochondria area. The inner membrane of a single liver mitochondrion may have more than 10,000 sets of electron-transfer systems(respiratory chains) The discovery in 1948 by Eugene Kennedy and Albert and ATP synthase molecules, distributed over the membrane surface Lehninger that mitochondria are the site of oxidative Heart mitochondria, which have more profuse cristae and thus a much phosphorylation in eukaryotes marked the beginning larger area of inner membrane, contain more than three times as many f the modern phase of studies sets of electron-transfer systems as liver mitochondria. The mitochon- in biological energy transduc- drial pool of coenzymes and intermediates is functionally separate from tions Mitochondria, like gram- the cytosolic pool. The mitochondria of invertebrates, plants, and mi- negative bacteria, have two crobial eukaryotes are similar to those shown here, but with much vari- membranes (Fig. 19-1). The ation in size, shape, and degree of convolution of the inner membrane. outer mitochondrial membrane is readily permeable to small molecules (M <5, 000) and molecules and ions, including protons (; the only ions, which move freely species that cross this membrane do so through specific through transmembrane chan- transporters. The inner membrane bears the compo- nels formed by a family of inte- nents of the respiratory chain and the ATP synthase gral membrane proteins called The mitochondrial matrix, enclosed by the inner porins. The inner membrane is membrane, contains the pyruvate dehydrogenase com- 1917-1986 impermeable to most small plex and the enzymes of the citric acid cycle, the fatty
processes involve the flow of electrons through a chain of membrane-bound carriers. (2) The free energy made available by this “downhill” (exergonic) electron flow is coupled to the “uphill” transport of protons across a proton-impermeable membrane, conserving the free energy of fuel oxidation as a transmembrane electrochemical potential (p. 391). (3) The transmembrane flow of protons down their concentration gradient through specific protein channels provides the free energy for synthesis of ATP, catalyzed by a membrane protein complex (ATP synthase) that couples proton flow to phosphorylation of ADP. We begin this chapter with oxidative phosphorylation. We first describe the components of the electrontransfer chain, their organization into large functional complexes in the inner mitochondrial membrane, the path of electron flow through them, and the proton movements that accompany this flow. We then consider the remarkable enzyme complex that, by “rotational catalysis,” captures the energy of proton flow in ATP, and the regulatory mechanisms that coordinate oxidative phosphorylation with the many catabolic pathways by which fuels are oxidized. With this understanding of mitochondrial oxidative phosphorylation, we turn to photophosphorylation, looking first at the absorption of light by photosynthetic pigments, then at the lightdriven flow of electrons from H2O to NADP and the molecular basis for coupling electron and proton flow. We also consider the similarities of structure and mechanism between the ATP synthases of chloroplasts and mitochondria, and the evolutionary basis for this conservation of mechanism. OXIDATIVE PHOSPHORYLATION 19.1 Electron-Transfer Reactions in Mitochondria The discovery in 1948 by Eugene Kennedy and Albert Lehninger that mitochondria are the site of oxidative phosphorylation in eukaryotes marked the beginning of the modern phase of studies in biological energy transductions. Mitochondria, like gramnegative bacteria, have two membranes (Fig. 19–1). The outer mitochondrial membrane is readily permeable to small molecules (Mr 5,000) and ions, which move freely through transmembrane channels formed by a family of integral membrane proteins called porins. The inner membrane is impermeable to most small molecules and ions, including protons (H); the only species that cross this membrane do so through specific transporters. The inner membrane bears the components of the respiratory chain and the ATP synthase. The mitochondrial matrix, enclosed by the inner membrane, contains the pyruvate dehydrogenase complex and the enzymes of the citric acid cycle, the fatty 19.1 Electron-Transfer Reactions in Mitochondria 691 Outer membrane Freely permeable to small molecules and ions ATP synthase (FoF1) Cristae Impermeable to most small molecules and ions, including H Contains: Contains: Ribosomes Porin channels • Respiratory electron carriers (Complexes I–IV) • ADP-ATP translocase • ATP synthase (FoF1) • Other membrane transporters • Pyruvate dehydrogenase complex • Citric acid cycle enzymes • Amino acid oxidation enzymes • DNA, ribosomes • Many other enzymes • ATP, ADP, Pi , Mg2, Ca2, K • Many soluble metabolic intermediates Inner membrane Matrix • Fatty acid -oxidation enzymes Albert L. Lehninger, 1917–1986 FIGURE 19–1 Biochemical anatomy of a mitochondrion. The convolutions (cristae) of the inner membrane provide a very large surface area. The inner membrane of a single liver mitochondrion may have more than 10,000 sets of electron-transfer systems (respiratory chains) and ATP synthase molecules, distributed over the membrane surface. Heart mitochondria, which have more profuse cristae and thus a much larger area of inner membrane, contain more than three times as many sets of electron-transfer systems as liver mitochondria. The mitochondrial pool of coenzymes and intermediates is functionally separate from the cytosolic pool. The mitochondria of invertebrates, plants, and microbial eukaryotes are similar to those shown here, but with much variation in size, shape, and degree of convolution of the inner membrane. 8885d_c19_690-750 3/1/04 11:32 AM Page 691 mac76 mac76:385_reb:
8885dc19690-7503/1/0411:32 AM Page692mac76mac76:385 Chapter 19 Oxidative Phosphorylation and Photophosphorylation acid B-oxidation pathway, and the pathways of amino in the cytosol, others are in mitochondria, and still oth acid oxidation--all the pathways of fuel oxidation ex- ers have mitochondrial and cytosolic isozymes cept glycolysis, which takes place in the cytosol. The NAD-linked dehydrogenases remove two hydrogen selectively permeable inner membrane segregates the atoms from their substrates. One of these is transferred intermediates and enzymes of cytosolic metabolic path- as a hydride ion ( H) to NAD; the other is released ways from those of metabolic processes occurring in the as H in the medium(see Fig. 13-15). NADH and matrix. However, specific transporters carry pyruvate, NADPH are water-soluble electron carriers that associ- fatty acids, and amino acids or their a-keto derivatives ate reversibly with dehydrogenases NADH carries elec into the matrix for access to the machinery of the citric trons from catabolic reactions to their point of entry int acid cycle. ADP and Pi are specifically transported into the respiratory chain, the nadh dehydrogenase com- the matrix as newly synthesized aTP is transported out. plex described below. NADPH generally supplies elec- trons to anabolic reactions. Cells maintain separate Electrons Are funneled to universal pools of NADPH and NADH, with different redox po Electron Acceptors tentials. This is accomplished by holding the ratios of [reduced formyoxidized form relatively high for Oxidative phosphorylation begins with the entry of elec- NADPH and relatively low for NADH. Neither NADHnor trons into the respiratory chain. Most of these electrons NADPH can cross the inner mitochondrial membrane arise from the action of dehydrogenases that collect but the electrons they carry can be shuttled across in- electrons from catabolic pathways and funnel them into directly, as we shall see universal electron acceptors--nicotinamide nucleotides Flavoproteins contain a very tightly, sometimes (NAD* or NADP)or flavin nucleotides (FMN or FAD). covalently, bound flavin nucleotide, either FMN or FAD Nicotinamide nucleotide-linked dehydroge-(see Fig 13-18). The oxidized flavin nucleotide can ac- nases catalyze reversible reactions of the following gen- cept either one electron (yielding the semiquinone eral types form) or two (yielding FADH, or FMNH2). Electron Reduced substrate nad= transfer occurs because the flavoprotein has a higher reduction potential than the compound oxidized. The oxidized substrate + nadh +H+ standard reduction potential of a flavin nucleotide, Reduced substrate NADP+= like that of NAD or NADP, depends on the protein with oxidized substrate NAdPH +H+ which it is associated. Local interactions with functional groups in the protein distort the electron orbitals in the Most dehydrogenases that act in catabolism are spe flavin ring, changing the relative stabilities of oxidized for NAD as electron acceptor (Table 19-1). Some and reduced forms The relevant standard reduction TABLE 19-1 Some Important Reactions Catalyzed by NAD(P)H-Linked Dehydrogen Reaction Location NAD-linked ar-Ketoglutarate CoA +NAD= succinyl-CoA+ CO2+ NADH+ H L-Malate NAd- oxaloacetate NAdh+ H M and C Pyruvate CoA NAd= acetyl-CoA CO 2 NADH +H Glyceraldehyde 3-phosphate Pi+ NAD 1, bisphosphoglycerate NADH+ H Lactate t NAD= pyruvate NADH+ H B-Hydroxyacyl-CoA + NAD+ B-ketoacyl-CoA NADH+ H NADP-linked Glucose 6-phosphate t NADP= 6-phosphogluconate NADPH H NAD- or NADP-linked Glutamate H20+ NAD(P)= a-ketoglutarate NHA NAD(P)H Isocitrate NAD(P)= a-ketoglutarate CO2+ NAD(P)H+ H M and C These reactions and their enzymes are discussed in Chapters 14 through 18
acid -oxidation pathway, and the pathways of amino acid oxidation—all the pathways of fuel oxidation except glycolysis, which takes place in the cytosol. The selectively permeable inner membrane segregates the intermediates and enzymes of cytosolic metabolic pathways from those of metabolic processes occurring in the matrix. However, specific transporters carry pyruvate, fatty acids, and amino acids or their -keto derivatives into the matrix for access to the machinery of the citric acid cycle. ADP and Pi are specifically transported into the matrix as newly synthesized ATP is transported out. Electrons Are Funneled to Universal Electron Acceptors Oxidative phosphorylation begins with the entry of electrons into the respiratory chain. Most of these electrons arise from the action of dehydrogenases that collect electrons from catabolic pathways and funnel them into universal electron acceptors—nicotinamide nucleotides (NAD or NADP) or flavin nucleotides (FMN or FAD). Nicotinamide nucleotide–linked dehydrogenases catalyze reversible reactions of the following general types: Reduced substrate NAD oxidized substrate NADH H Reduced substrate NADP oxidized substrate NADPH H Most dehydrogenases that act in catabolism are specific for NAD as electron acceptor (Table 19–1). Some are yz yz in the cytosol, others are in mitochondria, and still others have mitochondrial and cytosolic isozymes. NAD-linked dehydrogenases remove two hydrogen atoms from their substrates. One of these is transferred as a hydride ion (: H) to NAD; the other is released as H in the medium (see Fig. 13–15). NADH and NADPH are water-soluble electron carriers that associate reversibly with dehydrogenases. NADH carries electrons from catabolic reactions to their point of entry into the respiratory chain, the NADH dehydrogenase complex described below. NADPH generally supplies electrons to anabolic reactions. Cells maintain separate pools of NADPH and NADH, with different redox potentials. This is accomplished by holding the ratios of [reduced form]/[oxidized form] relatively high for NADPH and relatively low for NADH. Neither NADH nor NADPH can cross the inner mitochondrial membrane, but the electrons they carry can be shuttled across indirectly, as we shall see. Flavoproteins contain a very tightly, sometimes covalently, bound flavin nucleotide, either FMN or FAD (see Fig. 13–18). The oxidized flavin nucleotide can accept either one electron (yielding the semiquinone form) or two (yielding FADH2 or FMNH2). Electron transfer occurs because the flavoprotein has a higher reduction potential than the compound oxidized. The standard reduction potential of a flavin nucleotide, unlike that of NAD or NADP, depends on the protein with which it is associated. Local interactions with functional groups in the protein distort the electron orbitals in the flavin ring, changing the relative stabilities of oxidized and reduced forms. The relevant standard reduction 692 Chapter 19 Oxidative Phosphorylation and Photophosphorylation TABLE 19–1 Some Important Reactions Catalyzed by NAD(P)H-Linked Dehydrogenases Reaction* Location† NAD-linked -Ketoglutarate CoA NAD succinyl-CoA CO2 NADH H M L-Malate NAD oxaloacetate NADH H M and C Pyruvate CoA NAD acetyl-CoA CO2 NADH H M Glyceraldehyde 3-phosphate Pi NAD 1,3-bisphosphoglycerate NADH H C Lactate NAD pyruvate NADH H C -Hydroxyacyl-CoA NAD -ketoacyl-CoA NADH H M NADP-linked Glucose 6-phosphate NADP 6-phosphogluconate NADPH H C NAD- or NADP-linked L-Glutamate H2O NAD(P) -ketoglutarate NH4 NAD(P)H M Isocitrate NAD(P) -ketoglutarate CO2 NAD(P)H H yz M and C yz yz yz yz yz yz yz yz * These reactions and their enzymes are discussed in Chapters 14 through 18. † M designates mitochondria; C, cytosol. 8885d_c19_690-750 3/1/04 11:32 AM Page 692 mac76 mac76:385_reb:
8885dc19690-7503/1/0411:32 AM Page693mac76mac76:385 19.1 Electron-Transfer Reactions in Mitochondria potential is therefore that of the particular flavoprotein not that of isolated fad or fmn. The flavin nucleotide should be considered part of the flavoproteins active CHO CH2-CH=C—CH210-H site rather than a reactant or product in the electron- (fully oxidize transfer reaction. Because flavoproteins can participate CH. in either one- or two-electron transfers, they can serve as intermediates between reactions in which two elec H++e trons are donated (as in dehydrogenations)and those in which only one electron is accepted (as in the reduction O° of a quinone to a hydroquinone, described below) CHO Electrons Pass through a Series CH&O of Membrane- Bound carriers OH The mitochondrial respiratory chain consists of a series H +e of sequentially acting electron carriers, most of which are integral proteins with prosthetic groups capable of accepting and donating either one or two electrons CHo Three types of electron transfers occur in oxidative phosphorylation: (1) direct transfer of electrons, as in (fully reduced the reduction of Fe to Fe;(2) transfer as a hydro- gen atom(H +e and 3) transfer as a hydride ion CH), which bears two electrons. The term reducing FIGURE 19-2 Ubiquinone (Q, or coenzyme Q). Complete reductio equivalent is used to designate a single electron equiv- of ubiquinone requires two electrons and two protons, and occurs in alent transferred in an oxidation-reduction reaction two steps through the semiquinone radical intermediate In addition to NAD and flavoproteins, three types of electron-carrying molecules function in the res- piratory chain: a hydrophobic quinone (ubiquinone) and near 560 nm in type b, and near 550 nm in type c. To two different types of iron-containing proteins(cyto- distinguish among closely related cytochromes of one chromes and iron-sulfur proteins ). Ubiquinone (also type, the exact absorption maximum is sometimes used called coenzyme Q, or simply Q) is a lipid-soluble ben- in the names, as in cytochrome b56 coquinone with a long isoprenoid side chain (Fig. 19-2) The heme cofactors of a and b cytochromes are The closely related compounds plastoquinone (of plant tightly, but not covalently, bound to their associated pro chloroplasts) and menaquinone (of bacteria) play roles teins; the hemes of c-type cytochromes are covalently analogous to that of ubiquinone, carrying electrons in attached through Cys residues(Fig. 19-3). As with the membrane-associated electron-transfer chains. Ubiqui- flavoproteins, the standard reduction potential of the none can accept one electron to become the semi- heme iron atom of a cytochrome depends on its inter quinone radical (Qh) or two electrons to form ubiquinol action with protein side chains and is therefore differ (QH2)(Fig. 19-2) and, like flavoprotein carriers, it can ent for each cytochrome. The cytochromes of type a act at the junction between a two-electron donor and a and b and some of type c are integral proteins of the one-electron acceptor Because ubiquinone is both small inner mitochondrial membrane. One striking exception and hydrophobic, it is freely diffusible within the lipid is the cytochrome c of mitochondria, a soluble protein bilayer of the inner mitochondrial membrane and can that associates through electrostatic interactions with huttle reducing equivalents between other, less mobile the outer surface of the inner electron carriers in the membrane. and because it car- membrane. We encountered ries both electrons and protons, it plays a central role cytochrome c in earlier dis in coupling electron flow to proton movement. cussions of protein structure The cytochromes are proteins with characteristic (see Fig. 4-18) strong absorption of visible light, due to their iron- In iron-sulfur proteins, containing heme prosthetic groups (Fig. 19-3). Mito- first discovered by Helmut chondria contain three classes of cytochromes, desig- Beinert, the iron is present not nated a, b, and c, which are distinguished by differences in heme but in association in their light-absorption spectra. Each type of cyto- with inorganic sulfur atoms or chrome in its reduced (Fe) state has three absorp- with the sulfur atoms of Cys tion bands in the visible range(Fig. 19-4). The longest- residues in the protein, or wavelength band is near 600 nm in type a cytochromes, both. These iron-sulfur(Fe-s) Helmut Beinert
potential is therefore that of the particular flavoprotein, not that of isolated FAD or FMN. The flavin nucleotide should be considered part of the flavoprotein’s active site rather than a reactant or product in the electrontransfer reaction. Because flavoproteins can participate in either one- or two-electron transfers, they can serve as intermediates between reactions in which two electrons are donated (as in dehydrogenations) and those in which only one electron is accepted (as in the reduction of a quinone to a hydroquinone, described below). Electrons Pass through a Series of Membrane-Bound Carriers The mitochondrial respiratory chain consists of a series of sequentially acting electron carriers, most of which are integral proteins with prosthetic groups capable of accepting and donating either one or two electrons. Three types of electron transfers occur in oxidative phosphorylation: (1) direct transfer of electrons, as in the reduction of Fe3 to Fe2; (2) transfer as a hydrogen atom (H e); and (3) transfer as a hydride ion (:H), which bears two electrons. The term reducing equivalent is used to designate a single electron equivalent transferred in an oxidation-reduction reaction. In addition to NAD and flavoproteins, three other types of electron-carrying molecules function in the respiratory chain: a hydrophobic quinone (ubiquinone) and two different types of iron-containing proteins (cytochromes and iron-sulfur proteins). Ubiquinone (also called coenzyme Q, or simply Q) is a lipid-soluble benzoquinone with a long isoprenoid side chain (Fig. 19–2). The closely related compounds plastoquinone (of plant chloroplasts) and menaquinone (of bacteria) play roles analogous to that of ubiquinone, carrying electrons in membrane-associated electron-transfer chains. Ubiquinone can accept one electron to become the semiquinone radical (QH) or two electrons to form ubiquinol (QH2) (Fig. 19–2) and, like flavoprotein carriers, it can act at the junction between a two-electron donor and a one-electron acceptor. Because ubiquinone is both small and hydrophobic, it is freely diffusible within the lipid bilayer of the inner mitochondrial membrane and can shuttle reducing equivalents between other, less mobile electron carriers in the membrane. And because it carries both electrons and protons, it plays a central role in coupling electron flow to proton movement. The cytochromes are proteins with characteristic strong absorption of visible light, due to their ironcontaining heme prosthetic groups (Fig. 19–3). Mitochondria contain three classes of cytochromes, designated a, b, and c, which are distinguished by differences in their light-absorption spectra. Each type of cytochrome in its reduced (Fe2) state has three absorption bands in the visible range (Fig. 19–4). The longestwavelength band is near 600 nm in type a cytochromes, near 560 nm in type b, and near 550 nm in type c. To distinguish among closely related cytochromes of one type, the exact absorption maximum is sometimes used in the names, as in cytochrome b562. The heme cofactors of a and b cytochromes are tightly, but not covalently, bound to their associated proteins; the hemes of c-type cytochromes are covalently attached through Cys residues (Fig. 19–3). As with the flavoproteins, the standard reduction potential of the heme iron atom of a cytochrome depends on its interaction with protein side chains and is therefore different for each cytochrome. The cytochromes of type a and b and some of type c are integral proteins of the inner mitochondrial membrane. One striking exception is the cytochrome c of mitochondria, a soluble protein that associates through electrostatic interactions with the outer surface of the inner membrane. We encountered cytochrome c in earlier discussions of protein structure (see Fig. 4–18). In iron-sulfur proteins, first discovered by Helmut Beinert, the iron is present not in heme but in association with inorganic sulfur atoms or with the sulfur atoms of Cys residues in the protein, or both. These iron-sulfur (Fe-S) 19.1 Electron-Transfer Reactions in Mitochondria 693 O• CH C H R OH CH3 CH3O (CH2 O CH3O CH3 CH2)10 Ubiquinone (Q) (fully oxidized) Semiquinone radical ( •QH) Ubiquinol (QH2) (fully reduced) H e O CH3O CH3 CH3O H e OH OH R CH3O CH3 CH3O FIGURE 19–2 Ubiquinone (Q, or coenzyme Q). Complete reduction of ubiquinone requires two electrons and two protons, and occurs in two steps through the semiquinone radical intermediate. Helmut Beinert 8885d_c19_690-750 3/1/04 11:32 AM Page 693 mac76 mac76:385_reb:
8885dc19690-7503/1/0411:32 AM Page694mac76mac76:385 Chapter 19 Oxidative Phosphorylation and Photophosphorylation CHs CHECH CHs CHCH C -Fe- CH. CH2CO0 CH2CH2CO0 CH2CH2CO0 CH2 CHCO0 on protoporphyrin IX CHeCH FIGURE 19-3 Prosthetic groups of cytochromes. Each group consists of four five-membered CH。CH called a porphyrin. The four nitrogen atoms are CH3 CHs CHS ordinated with a central Fe ion either Ch, CH, Coo- cytochromes and in hemoglobin and myoglobin (see Fig. 4-17). Heme c is covalently bound he protein of cytochrome c through thioether CHO CHOCH.COO bonds to two Cys residues. Heme a, found in the a-type cytochromes, has a long isoprenoid tail attached to one of the five-membered rings. The conjugated double-bond system(shaded pink) of visible light by these hemes centers range from simple structures with a single Fe atom coordinated to four Cys-SH groups to more com- Reduced plex Fe-S centers with two or four Fe atoms (Fig. 19-5) Rieske iron-sulfur proteins (named after their dis coverer,John S Rieske) are a variation on this theme Oxidized in which one fe atom is coordinated to two his residues rather than two Cys residues. All iron-sulfur proteins participate in one-electron transfers in which one iron atom of the iron-sulfur cluster is oxidized or reduced At least eight Fe-s proteins function in mitochondrial electron transfer. The reduction potential of Fe-s pro- teins varies from -0.65 V to +0.45 V, depending on the microenvironment of the iron within the protein. In the overall reaction catalyzed by the mitochon drial respiratory chain, electrons move from NADH,suc- cinate, or some other primary electron donor through flavoproteins, ubiquinone, iron-sulfur proteins, and cy- Wavelength (nm) chromes, and finally to O. A look at the methods used to determine the sequence in which the carriers act is of cytochrome c (cyt c)in its ox instructive, as the same general approaches have been dized(red) and reduced(bl ns. Also labeled are the character- used to study other electron- transfer chains, such as stic a, B, and y bands of the reduced form. those of chloroplasts
centers range from simple structures with a single Fe atom coordinated to four Cys OSH groups to more complex Fe-S centers with two or four Fe atoms (Fig. 19–5). Rieske iron-sulfur proteins (named after their discoverer, John S. Rieske) are a variation on this theme, in which one Fe atom is coordinated to two His residues rather than two Cys residues. All iron-sulfur proteins participate in one-electron transfers in which one iron atom of the iron-sulfur cluster is oxidized or reduced. At least eight Fe-S proteins function in mitochondrial electron transfer. The reduction potential of Fe-S proteins varies from 0.65 V to 0.45 V, depending on the microenvironment of the iron within the protein. In the overall reaction catalyzed by the mitochondrial respiratory chain, electrons move from NADH, succinate, or some other primary electron donor through flavoproteins, ubiquinone, iron-sulfur proteins, and cytochromes, and finally to O2. A look at the methods used to determine the sequence in which the carriers act is instructive, as the same general approaches have been used to study other electron-transfer chains, such as those of chloroplasts. 694 Chapter 19 Oxidative Phosphorylation and Photophosphorylation Fe N N N N CH3 CH3 CH2CH2COO CH2 CH CH2 CH CH3 Heme A (in a-type cytochromes) Fe N N N N CH3 CH3 CH3 CH3 CH2CH2COO CH2 CH2 CHO CH2 CH2 CH Iron protoporphyrin IX (in b-type cytochromes) COO CH3 OH Cys S Cys Fe N N N N CH3 CH3 CH3 CH3 CH2CH2COO CH2CH2 Heme C (in c-type cytochromes) COO CH3 CH CH2 S CH CH CH2 CH3 CH3 CH3 CH3 COO FIGURE 19–3 Prosthetic groups of cytochromes. Each group consists of four five-membered, nitrogen-containing rings in a cyclic structure called a porphyrin. The four nitrogen atoms are coordinated with a central Fe ion, either Fe2 or Fe3. Iron protoporphyrin IX is found in b-type cytochromes and in hemoglobin and myoglobin (see Fig. 4–17). Heme c is covalently bound to the protein of cytochrome c through thioether bonds to two Cys residues. Heme a, found in the a-type cytochromes, has a long isoprenoid tail attached to one of the five-membered rings. The conjugated double-bond system (shaded pink) of the porphyrin ring accounts for the absorption of visible light by these hemes. 100 Relative light absorption (%) 50 300 400 500 600 Wavelength (nm) Oxidized cyt c Reduced cyt c 0 FIGURE 19–4 Absorption spectra of cytochrome c (cyt c) in its oxidized (red) and reduced (blue) forms. Also labeled are the characteristic , , and bands of the reduced form. 8885d_c19_690-750 3/1/04 11:32 AM Page 694 mac76 mac76:385_reb:
8885dc19690-7503/1/0411:32 AM Page695mac76mac76:385 19.1 Electron-Transfer Reactions in Mitochondria Protein FIGURE 19-5 Iron-sulfur centers. The Fes centers of iron-sulfur proteins may be as simple as (a), with a single Fe ion surrounded by the S atoms of four Cys residues. Other centers include both and Cys S atoms, as in(b)2Fe-2S or (c) d) The ferredoxin of the cyanobacterium Anabaena 7120 has one Fe-25 center(PDB ID 1FRD) Fe is red, inorganic S2 is yellow, and the S of Cys is orange. (Note that in these designations only the inorganic S atoms are counted. For example, in the 2Fe-2S center (b), each Fe ion is actually surrounded by four S atoms. )The exact standard reduction potential of the iron in these centers depends on the type of center and its interaction with the associated protein First, the standard reduction potentials of the in- Q- cytochrome b - cytochrome C1 - cytochrome dividual electron carriers have been determined ex- C- cytochrome a -)cytochrome a3-O2. Note, how- perimentally (Table 19-2). We would expect the carri- ever, that the order of standard reduction potentials is ers to function in order of increasing reduction not necessarily the same as the order of actual reduc potential, because electrons tend to flow spontaneously tion potentials under cellular conditions, which depend from carriers of lower e to carriers of higher E. The on the concentration of reduced and oxidized forms order of carriers deduced by this method is NADH-)(p 510). A second method for determining the sequence TABLE 19-2 Standard Reduction Potentials of Respiratory Chain and Related Electron Carriers Redox reaction(half-reaction) -0.414 NAD++H++2 NADH 0.320 NADPH -0.324 NADH dehydrogenase(FMN)+ 2H+ 2e -NADH dehydrogenase(FMNH2) Ubiquinone 2HT 2e ubiquinol Cytochrome b(Fea+)+ 0.077 Cytochrome C,(Fe +)+e- cytochrome c,(Fe) Cytochrome c(Fe)+e-cytochrome c(Fe4) 0.254 Cytochrome a(Fe)+e- cytochrome a(Fe") Cytochrome a3(Fe)+e- cytochrome a3(Fe 0.35 202+2H++2e一→H20 0.8166
First, the standard reduction potentials of the individual electron carriers have been determined experimentally (Table 19–2). We would expect the carriers to function in order of increasing reduction potential, because electrons tend to flow spontaneously from carriers of lower E to carriers of higher E . The order of carriers deduced by this method is NADH → Q → cytochrome b → cytochrome c1 → cytochrome c → cytochrome a → cytochrome a3 → O2. Note, however, that the order of standard reduction potentials is not necessarily the same as the order of actual reduction potentials under cellular conditions, which depend on the concentration of reduced and oxidized forms (p. 510). A second method for determining the sequence 19.1 Electron-Transfer Reactions in Mitochondria 695 S (c) Cys Cys Fe S S S S S S S Fe Fe Fe Cys S Cys (b) Cys C S ys Fe S Fe S S Cys Cys S Cys S S S S Cys (a) Cys Cys Fe Protein (d) FIGURE 19–5 Iron-sulfur centers. The Fe-S centers of iron-sulfur proteins may be as simple as (a), with a single Fe ion surrounded by the S atoms of four Cys residues. Other centers include both inorganic and Cys S atoms, as in (b) 2Fe-2S or (c) 4Fe-4S centers. (d) The ferredoxin of the cyanobacterium Anabaena 7120 has one 2Fe-2S center (PDB ID 1FRD); Fe is red, inorganic S2 is yellow, and the S of Cys is orange. (Note that in these designations only the inorganic S atoms are counted. For example, in the 2Fe-2S center (b), each Fe ion is actually surrounded by four S atoms.) The exact standard reduction potential of the iron in these centers depends on the type of center and its interaction with the associated protein. TABLE 19–2 Standard Reduction Potentials of Respiratory Chain and Related Electron Carriers Redox reaction (half-reaction) E (V) 2H 2e 8n H2 0.414 NAD H 2e 8n NADH 0.320 NADP H 2e 8n NADPH 0.324 NADH dehydrogenase (FMN) 2H 2e 8n NADH dehydrogenase (FMNH2) 0.30 Ubiquinone 2H 2e 8n ubiquinol 0.045 Cytochrome b (Fe3) e 8n cytochrome b (Fe2) 0.077 Cytochrome c1 (Fe3) e 8n cytochrome c1 (Fe2) 0.22 Cytochrome c (Fe3) e 8n cytochrome c (Fe2) 0.254 Cytochrome a (Fe3) e 8n cytochrome a (Fe2) 0.29 Cytochrome a3 (Fe3) e 8n cytochrome a3 (Fe2) 0.35 1 2 O2 2H 2e 8n H2O 0.8166 8885d_c19_690-750 3/1/04 11:32 AM Page 695 mac76 mac76:385_reb:
8885dc196963/1/041:58 PM Page696mac76mac76:385reb Chapter 19 Oxidative Phosphorylation and Photophosphorylation FIGURE 19-6 Method for determining the sequence of electron carriers. This method NADH Q→cytb→)cytc1→)cytc→Cyt(a+a3)→>O2 measures the effects of inhibitors of electron ransfer on the oxidation state of each carrier. In the presence of an electron donor and O2, each inhibitor causes a characteristic pattern of oxidized/reduced carriers: those before the NADH 2→cytb→ Cyt cI→)Cytc t(a+ as) block become reduced(blue), and those after the block become oxidized(pink) cN or CO NADH—Q→cytb→Cytc1→}cytc→Cyt(a+a3) of electron carriers involves reducing the entire chain complexes that can be physically separated. Gentle of carriers experimentally by providing an electron treatment of the inner mitochondrial membrane with source but no electron acceptor (no O,). When Oe is detergents allows the resolution of four unique electron suddenly introduced into the system, the rate at which carrier complexes, each capable of catalyzing electron each electron carrier becomes oxidized (measured transfer through a portion of the chain (Table 19-3; Fig spectroscopically) reveals the order in which the car- 19-7. Complexes I and Il catalyze electron transfer to riers function. The carrier nearest O2 (at the end of the ubiquinone from two different electron donors: NADH chain) gives up its electrons first, the second carrier (Complex D and succinate(Complex ID). Complex Ill from the end is oxidized next, and so on. Such exper- carries electrons from reduced ubiquinone to cyto- iments have confirmed the sequence deduced from chrome c, and Complex Iv completes standard reduction potentials transferring electrons from cytochrome c to O In a final confirmation, agents that inhibit the flow We now look in more detail at the structure and of electrons through the chain have been used in com- function of each complex of the mitochondrial respira bination with measurements of the degree of oxidation tory chain of each carrier. In the presence of O, and an electron donor, carriers that function before the inhibited step Complex I: NADH to Ubiquinone Figure 19-8 illustrates the become fully reduced, and those that function after this relationship between Complexes I and II and ubiquinone Complex I, also called NADH: ubiquinone oxidore- eral inhibitors that block different steps in the chain, in- ductase or NADH dehydrogenase, is a large enzyme vestigators have determined the entire sequence; it is composed of 42 different polypeptide chains, including the same as deduced in the first two approaches an FMN-containing flavoprotein and at least six iron- ulfur centers. High-resolution electron microscopy Electron Carriers Function in Multienzyme Complexes shows Complex I to be L-shaped, with one arm of the L in the membrane and the other extending into the ma- The electron carriers of the respiratory chain are or- trix. As shown in Figure 19-9, Complex I catalyzes two ganized into membrane-embedded supramolecular simultaneous and obligately coupled processes: (1) the TABLE 19-3 The Protein Components of the Mitochondrial Electron-Transfer Chain Enzyme complex/protein Mass(kDa) Number of subunits Prosthetic group(s) I NADH dehydrogenase 850 43(14) MN. Fe-S lI Succinate dehydrogenase 140 4 FAD. Fe-S Ill Ubiquinone cytochrome C oxidoreductase mes Fe-s Cytochrome ct Heme Iv Cytochrome oxidase 13(3-4) Hemes: CUA, CuB Numbers of subunits in the bacterial equivalents in parentheses. Cytochrome c is not part of an enzyme complex, it moves between Complexes ll and iv as a freely soluble protein
of electron carriers involves reducing the entire chain of carriers experimentally by providing an electron source but no electron acceptor (no O2). When O2 is suddenly introduced into the system, the rate at which each electron carrier becomes oxidized (measured spectroscopically) reveals the order in which the carriers function. The carrier nearest O2 (at the end of the chain) gives up its electrons first, the second carrier from the end is oxidized next, and so on. Such experiments have confirmed the sequence deduced from standard reduction potentials. In a final confirmation, agents that inhibit the flow of electrons through the chain have been used in combination with measurements of the degree of oxidation of each carrier. In the presence of O2 and an electron donor, carriers that function before the inhibited step become fully reduced, and those that function after this step are completely oxidized (Fig. 19–6). By using several inhibitors that block different steps in the chain, investigators have determined the entire sequence; it is the same as deduced in the first two approaches. Electron Carriers Function in Multienzyme Complexes The electron carriers of the respiratory chain are organized into membrane-embedded supramolecular complexes that can be physically separated. Gentle treatment of the inner mitochondrial membrane with detergents allows the resolution of four unique electroncarrier complexes, each capable of catalyzing electron transfer through a portion of the chain (Table 19–3; Fig. 19–7). Complexes I and II catalyze electron transfer to ubiquinone from two different electron donors: NADH (Complex I) and succinate (Complex II). Complex III carries electrons from reduced ubiquinone to cytochrome c, and Complex IV completes the sequence by transferring electrons from cytochrome c to O2. We now look in more detail at the structure and function of each complex of the mitochondrial respiratory chain. Complex I: NADH to Ubiquinone Figure 19–8 illustrates the relationship between Complexes I and II and ubiquinone. Complex I, also called NADH:ubiquinone oxidoreductase or NADH dehydrogenase, is a large enzyme composed of 42 different polypeptide chains, including an FMN-containing flavoprotein and at least six ironsulfur centers. High-resolution electron microscopy shows Complex I to be L-shaped, with one arm of the L in the membrane and the other extending into the matrix. As shown in Figure 19–9, Complex I catalyzes two simultaneous and obligately coupled processes: (1) the 696 Chapter 19 Oxidative Phosphorylation and Photophosphorylation NADH Q Cyt c1 Cyt (a a3) O2 rotenone antimycin A CN or CO NADH Q O Cyt b Cyt c 2 NADH Q O2 Cyt b Cyt c1 Cyt c Cyt b Cyt c1 Cyt Cyt c Cyt (a a3) a a3 ( ) FIGURE 19–6 Method for determining the sequence of electron carriers. This method measures the effects of inhibitors of electron transfer on the oxidation state of each carrier. In the presence of an electron donor and O2, each inhibitor causes a characteristic pattern of oxidized/reduced carriers: those before the block become reduced (blue), and those after the block become oxidized (pink). TABLE 19–3 The Protein Components of the Mitochondrial Electron-Transfer Chain Enzyme complex/protein Mass (kDa) Number of subunits* Prosthetic group(s) I NADH dehydrogenase 850 43 (14) FMN, Fe-S II Succinate dehydrogenase 140 4 FAD, Fe-S III Ubiquinone cytochrome c oxidoreductase 250 11 Hemes, Fe-S Cytochrome c † 13 1 Heme IV Cytochrome oxidase 160 13 (3–4) Hemes; CuA, CuB * Numbers of subunits in the bacterial equivalents in parentheses. † Cytochrome c is not part of an enzyme complex; it moves between Complexes III and IV as a freely soluble protein. 8885d_c19_696 3/1/04 1:58 PM Page 696 mac76 mac76:385_reb:
8885dc19690-7503/1/0411:32 AM Page697 6mac76:385 19.1 Electron-Transfer Reactions in Mitochondria Treatment with digitonin Intermembrane phosphate FAD FeS I FMN Osmotic rupture (FAD) NADH NAD+ ETF: Q Inner ETF fragments (FAD) Outer membrane Matrix II ATP FIGURE 19-8 Path of electrons from NADH, succinate, fatty Solubilization with detergent acyl-CoA, and glycerol 3-phosphate to ubiquinone. Electrons from followed by ion-exchange chromatography NADH pass through a flavoprotein to a series of iron-sulfur proteins (in Complex I)and then to Q. Electrons from succinate pass through centers(in Complex In)on the ATP Q. Glycerol 3-phosphate donates electrons to a flavoprotein(glycerol 3-phosphate dehydrogenase) on the outer face of the inner mito chondrial membrane, from which they pass to Q Acyl-CoA dehydro- genase(the first enzyme of B oxidation) transfers electrons to electron- transferring flavoprotein (ETF), from which they pass to Q ne OxI exergonic transfer to ubiquinone of a hydride ion from NADH Q Suc- QQ Cytc Cytc O2 ATP ADP NADH and a proton from the matrix, expressed by NADH+H++Q→→NAD++QH2(19-1) Reactions catalyzed by isolated vitro and (2)the endergonic transfer of four protons from the matrix to the intermembrane space Complex I is there- FIGURE 19-7 Separation of functional complexes of the respiratory fore a proton pump driven by the energy of electron chain. The outer mitochondrial membrane is first removed by treat- transfer, and the reaction it catalyzes is vectorial: it ment with the detergent digitonin. Fragments of inner membrane are moves protons in a specific direction from one location then obtained by osmotic rupture of the mitochondria, and the frag-(the matrix, which becomes negatively charged with the ments are gently dissolved in a second detergent. The resulting mix- departure of protons) to another (the intermembrane ture of inner membrane proteins is resolved by ion-exchange chro- space, which becomes positively charged). To empha matography into different complexes(I through IV) of the respiratory size the vectorial nature of the process, the overall re- chain, each with its unique protein composition(see Table 19-3),an action is often written with subscripts that indicate the the enzyme ATP synthase(sometimes called Complex V). The isolated Complexes I through IV catalyze transfers between donors (NADH location of the protons: P for the positive side of the in- and succinate), intermediate carriers(Q and cytochrome c), and O2, ner membrane(the intermembrane space), N for the as shown. In vitro, isolated ATP synthase has only ATP-hydrolyzing negative side(the matrix): (ATPase), not ATP-synthesizing, activity. NADH+5H+Q→→NAD++QH2+4H(19-2)
exergonic transfer to ubiquinone of a hydride ion from NADH and a proton from the matrix, expressed by NADH H Q On NAD QH2 (19–1) and (2) the endergonic transfer of four protons from the matrix to the intermembrane space. Complex I is therefore a proton pump driven by the energy of electron transfer, and the reaction it catalyzes is vectorial: it moves protons in a specific direction from one location (the matrix, which becomes negatively charged with the departure of protons) to another (the intermembrane space, which becomes positively charged). To emphasize the vectorial nature of the process, the overall reaction is often written with subscripts that indicate the location of the protons: P for the positive side of the inner membrane (the intermembrane space), N for the negative side (the matrix): NADH 5H N Q On NAD QH2 4H P (19–2) 19.1 Electron-Transfer Reactions in Mitochondria 697 Osmotic rupture Inner membrane fragments Outer membrane fragments discarded ATP synthase IV III II I I II III IV ATP synthase NADH Q Succinate Q Q Cyt c Cyt c O2 ATP ADP Pi Reactions catalyzed by isolated fractions in vitro Solubilization with detergent followed by ion-exchange chromatography Treatment with digitonin FIGURE 19–7 Separation of functional complexes of the respiratory chain. The outer mitochondrial membrane is first removed by treatment with the detergent digitonin. Fragments of inner membrane are then obtained by osmotic rupture of the mitochondria, and the fragments are gently dissolved in a second detergent. The resulting mixture of inner membrane proteins is resolved by ion-exchange chromatography into different complexes (I through IV) of the respiratory chain, each with its unique protein composition (see Table 19–3), and the enzyme ATP synthase (sometimes called Complex V). The isolated Complexes I through IV catalyze transfers between donors (NADH and succinate), intermediate carriers (Q and cytochrome c), and O2, as shown. In vitro, isolated ATP synthase has only ATP-hydrolyzing (ATPase), not ATP-synthesizing, activity. I II Intermembrane space Matrix Fe-S Fe-S FAD Glycerol 3-phosphate (cytosolic) glycerol 3-phosphate dehydrogenase FAD FMN NADH NAD+ Succinate ETF:Q oxidoreductase acyl-CoA dehydrogenase ETF (FAD) Fe-S (FAD) Fatty acyl–CoA FAD Q FIGURE 19–8 Path of electrons from NADH, succinate, fatty acyl–CoA, and glycerol 3-phosphate to ubiquinone. Electrons from NADH pass through a flavoprotein to a series of iron-sulfur proteins (in Complex I) and then to Q. Electrons from succinate pass through a flavoprotein and several Fe-S centers (in Complex II) on the way to Q. Glycerol 3-phosphate donates electrons to a flavoprotein (glycerol 3-phosphate dehydrogenase) on the outer face of the inner mitochondrial membrane, from which they pass to Q. Acyl-CoA dehydrogenase (the first enzyme of oxidation) transfers electrons to electrontransferring flavoprotein (ETF), from which they pass to Q via ETF:ubiquinone oxidoreductase. 8885d_c19_690-750 3/1/04 11:32 AM Page 697 mac76 mac76:385_reb:
8885dc19690-7503/1/0411:32 AM Page698mac76mac76:385 Chapter 19 Oxidative Phosphorylation and Photophosphorylation Amytal (a barbiturate drug), rotenone (a plant product commonly used as an insecticide), and piericidin A(an Complex I Intermembrane antibiotic) inhibit electron flow from the Fe-s centers space(P side of Complex I to ubiquinone (table 19-4) and therefore block the overall process of oxidative phosphorylation Ubiquinol (QH2, the fully reduced form; Fig. 19-2) - QH diffuses in the inner mitochondrial membrane from Fe-S Complex I to Complex Ill, where it is oxidized to Q in a process that also involves the outward movement of H Matrix FMN Matrix(N side) Complex Il: Succinate to Ubiquinone We encountered Complex II in Chapter 16 as succinate dehydroge nase, the only membrane-bound enzyme in the citric acid cycle(p. 612). Although smaller and simpler than NAD++h+ Complex I, it contains five prosthetic groups of two types and four different protein subunits(Fig. 19-10) FIGURE 19-9 NADH: ubiquinone oxidoreductase(Complex D. Com- Subunits C and D are integral membrane proteins, each plex I catalyzes the transfer of a hydride ion from NADH to FMN, from which two electrons pass through a series of Fe-S centers to the iron- with three transmembrane helices. They contain a heme ulfur protein N-2 in the matrix arm of the complex. Electron transfer group, heme b, and a binding site for ubiquinone, the from N-2 to ubiquinone on the membrane arm forms QH2, which dif- final electron acceptor in the reaction catalyzed by fuses into the lipid bilayer. This electron transfer also drives the ex- Complex I Subunits A and B extend into the matrix(or pulsion from the matrix of four protons per pair of electrons. The de. the cytosol of a bacterium); they contain three 2Fe-2S tailed mechanism that couples electron and proton transfer in centers, bound FAD, and a binding site for the substrate I is not yet known, but probably involves a Q cycle similar succinate. The path of electron transfer from the Complex Ill in which QH2 participates twice per electron succinate-binding site to FAD, then through the Fe-s -12)Proton flux produces an electrochemical potential across centers to the Q-binding site, is more than 40 A long, nner mitochondrial membrane(N side negative, P side positive), but none of the individual electron-transfer distances conserves some of the energy released by the electron-transfer exceeds about 11 A-a reasonable distance for rapid reactions. This electrochemical pe electron transfer (Fig. 19-10) TABLE 19-4 Agents That Interfere with Oxidative Phosphorylation or Photophosphorylation type of interference Compound larget / mode of action Inhibition of electron transfer Cyanide Inhibit cytochrome oxidase from cytochrome b to cytochrome c1 Rotenone Amita Prevent electron transfer from Fe-s center to ubiquinone ompetes with QB for binding site in PSIl Inhibition of ATP synthase Aurovertin Inhibits F1 Inhibit Fo and CF Blocks proton flow through Fo and CFo Hydrophobic proton carrie In brown fat, forms proton-conducting pores in inner mitochondrial Inhibition of ATP-ADP exchange Atractyloside Inhibits adenine nucleotide translocase
Amytal (a barbiturate drug), rotenone (a plant product commonly used as an insecticide), and piericidin A (an antibiotic) inhibit electron flow from the Fe-S centers of Complex I to ubiquinone (Table 19–4) and therefore block the overall process of oxidative phosphorylation. Ubiquinol (QH2, the fully reduced form; Fig. 19–2) diffuses in the inner mitochondrial membrane from Complex I to Complex III, where it is oxidized to Q in a process that also involves the outward movement of H. Complex II: Succinate to Ubiquinone We encountered Complex II in Chapter 16 as succinate dehydrogenase, the only membrane-bound enzyme in the citric acid cycle (p. 612). Although smaller and simpler than Complex I, it contains five prosthetic groups of two types and four different protein subunits (Fig. 19–10). Subunits C and D are integral membrane proteins, each with three transmembrane helices. They contain a heme group, heme b, and a binding site for ubiquinone, the final electron acceptor in the reaction catalyzed by Complex II. Subunits A and B extend into the matrix (or the cytosol of a bacterium); they contain three 2Fe-2S centers, bound FAD, and a binding site for the substrate, succinate. The path of electron transfer from the succinate-binding site to FAD, then through the Fe-S centers to the Q-binding site, is more than 40 Å long, but none of the individual electron-transfer distances exceeds about 11 Å—a reasonable distance for rapid electron transfer (Fig. 19–10). 698 Chapter 19 Oxidative Phosphorylation and Photophosphorylation Complex I Intermembrane space (P side) Matrix (N side) 2H+ 4H+ Fe-S FMN NADH NAD+ H+ 2e– 2e– N-2 Q QH2 Matrix arm Membrane arm FIGURE 19–9 NADH:ubiquinone oxidoreductase (Complex I). Complex I catalyzes the transfer of a hydride ion from NADH to FMN, from which two electrons pass through a series of Fe-S centers to the ironsulfur protein N-2 in the matrix arm of the complex. Electron transfer from N-2 to ubiquinone on the membrane arm forms QH2, which diffuses into the lipid bilayer. This electron transfer also drives the expulsion from the matrix of four protons per pair of electrons. The detailed mechanism that couples electron and proton transfer in Complex I is not yet known, but probably involves a Q cycle similar to that in Complex III in which QH2 participates twice per electron pair (see Fig. 19–12). Proton flux produces an electrochemical potential across the inner mitochondrial membrane (N side negative, P side positive), which conserves some of the energy released by the electron-transfer reactions. This electrochemical potential drives ATP synthesis. TABLE 19–4 Agents That Interfere with Oxidative Phosphorylation or Photophosphorylation Type of interference Compound* Target/mode of action Inhibition of electron transfer Cyanide Carbon monoxide Antimycin A Blocks electron transfer from cytochrome b to cytochrome c1 Myxothiazol Rotenone Amytal Piericidin A DCMU Competes with QB for binding site in PSII Inhibition of ATP synthase Aurovertin Inhibits F1 Oligomycin Venturicidin DCCD Blocks proton flow through Fo and CFo Uncoupling of phosphorylation FCCP from electron transfer DNP Hydrophobic proton carriers Valinomycin K ionophore Thermogenin In brown fat, forms proton-conducting pores in inner mitochondrial membrane Inhibition of ATP-ADP exchange Atractyloside Inhibits adenine nucleotide translocase * DCMU is 3-(3,4-dichlorophenyl)-1,1-dimethylurea; DCCD, dicyclohexylcarbodiimide; FCCP, cyanide-p-trifluoromethoxyphenylhydrazone; DNP, 2,4-dinitrophenol. Inhibit cytochrome oxidase Prevent electron transfer from Fe-S center to ubiquinone Inhibit Fo and CFo 8885d_c19_690-750 3/1/04 11:32 AM Page 698 mac76 mac76:385_reb:
8885dc19690-7503/1/0411:32 AM Page699 6mac76:385 19.1 Electron-Transfer Reactions in Mitochondria FIGURE 19-10 Structure of Complex ll(succinate dehydrogenase of E coli(PDB ID 1NEK) zyme has two transmembrane sub- units, C(green)and D(blue); the cytoplasmic extensions contain sub- units B(orange)and A (purple). Just behind the FAD in subunit A (gold) the binding site for succinate(occupied in this crystal structure by the inhibitor oxaloacetate, green). Subunit B has three sets of Fe-S cen- ters(yellow and red); ubiquinone (yellow)is bound to subunit C; and heme b(purple) is sandwiched between subunits C and D. A cardi- FAD lipin molecule is so tightly bound to subunit C that it shows up in the crystal structure (gray spacefilling). Electrons move(blue arrows) from succinate to FAD, then through the three Fe-S centers to ubiquinone. The heme b is not on the main path of electron transfer but protects against the formation of reactive oxygen species(ROS)by electrons that go astray. B Cytoplasm flavoprotein acyl-CoA dehydrogenase(see Fig 17-8) (N side) involves transfer of electrons from the substrate to the FAD of the dehydrogenase, then to electron-transferring flavoprotein (Etf), which in turn passes its electrons to e Ubiquinone enzyme transfers electrons into the respiratory chain by reducing ubiquinone Glycerol 3-phosphate, formed ei- Heme b ther from glycerol released by triacylglycerol breakdown or by the reduction of dihydroxyacetone phosphate from glycolysis, is oxidized by glycerol 3-phosphate dehydrogenase(see Fig 17-4). This enzyme is a flavo- protein located on the outer face of the inner mito- chondrial membrane, and like succinate dehydrogenase and acyl-CoA dehydrogenase it channels electrons into the respiratory chain by reducing ubiquinone (Fig B.The of glycerol hydrogenase in shuttling reducing equivalents from cytosolic NADH into the mitochondrial matrix is de- scribed in Section 19. 2(see Fig. 19-28). The effect of each of these electron-transferring enzymes is to con- The heme b of Complex I is apparenty not in tribute to the pool of reduced ubiquinone QH, from all the direct path of electron transfer; it may ser these reactions is reoxidized by Complex II instead to reduce the frequency with which electrons leak"out of the system, moving from succinate to mo- Complex II: Ubiquinone to Cytochrome c The next respi- lecular oxygen to produce the reactive oxygen species ratory complex, Complex Ill, also called cytochrome (ROS) hydrogen peroxide(h202)and the superoxide bci complex or ubiquinone: cytochrome c oxidore- radical (O2)described in Section 19.5. Humans with ductase, couples the transfer of electrons fror point mutations in Complex II subunits near heme b or ubiquinol(QH2) to cytochrome c with the vectorial the quinone-binding site suffer from hereditary para- transport of protons from the matrix to the intermem- ganglioma. This inherited condition is characterized by brane space. The determination of the complete struc benign tumors of the head and neck, commonly in the ture of this huge complex (Fig. 19-11) and of Complex carotid body, an organ that senses O2 levels in the blood. IV (below) by x-ray crystallography, achieved between These mutations result in greater production of ROS 1995 and 1998, were landmarks in the study of mito- and perhaps greater tissue damage during succinate chondrial electron transfer, providing the structural oxidation.■ framework to integrate the many biochemical observa- Other substrates for mitochondrial dehydrogenases tions on the functions of the respiratory complexes pass electrons into the respiratory chain at the level of Based on the structure of Complex II and detailed ubiquinone, but not through Complex II. The first step biochemical studies of the redox reactions, a reasonable in the B oxidation of fatty acyl-CoA, catalyzed by the model has been proposed for the passage of electrons
flavoprotein acyl-CoA dehydrogenase (see Fig. 17–8), involves transfer of electrons from the substrate to the FAD of the dehydrogenase, then to electron-transferring flavoprotein (ETF), which in turn passes its electrons to ETF : ubiquinone oxidoreductase (Fig. 19–8). This enzyme transfers electrons into the respiratory chain by reducing ubiquinone. Glycerol 3-phosphate, formed either from glycerol released by triacylglycerol breakdown or by the reduction of dihydroxyacetone phosphate from glycolysis, is oxidized by glycerol 3-phosphate dehydrogenase (see Fig. 17–4). This enzyme is a flavoprotein located on the outer face of the inner mitochondrial membrane, and like succinate dehydrogenase and acyl-CoA dehydrogenase it channels electrons into the respiratory chain by reducing ubiquinone (Fig. 19–8). The important role of glycerol 3-phosphate dehydrogenase in shuttling reducing equivalents from cytosolic NADH into the mitochondrial matrix is described in Section 19.2 (see Fig. 19–28). The effect of each of these electron-transferring enzymes is to contribute to the pool of reduced ubiquinone. QH2 from all these reactions is reoxidized by Complex III. Complex III: Ubiquinone to Cytochrome c The next respiratory complex, Complex III, also called cytochrome bc1 complex or ubiquinone:cytochrome c oxidoreductase, couples the transfer of electrons from ubiquinol (QH2) to cytochrome c with the vectorial transport of protons from the matrix to the intermembrane space. The determination of the complete structure of this huge complex (Fig. 19–11) and of Complex IV (below) by x-ray crystallography, achieved between 1995 and 1998, were landmarks in the study of mitochondrial electron transfer, providing the structural framework to integrate the many biochemical observations on the functions of the respiratory complexes. Based on the structure of Complex III and detailed biochemical studies of the redox reactions, a reasonable model has been proposed for the passage of electrons 19.1 Electron-Transfer Reactions in Mitochondria 699 The heme b of Complex II is apparently not in the direct path of electron transfer; it may serve instead to reduce the frequency with which electrons “leak” out of the system, moving from succinate to molecular oxygen to produce the reactive oxygen species (ROS) hydrogen peroxide (H2O2) and the superoxide radical (O2 ) described in Section 19.5. Humans with point mutations in Complex II subunits near heme b or the quinone-binding site suffer from hereditary paraganglioma. This inherited condition is characterized by benign tumors of the head and neck, commonly in the carotid body, an organ that senses O2 levels in the blood. These mutations result in greater production of ROS and perhaps greater tissue damage during succinate oxidation. ■ Other substrates for mitochondrial dehydrogenases pass electrons into the respiratory chain at the level of ubiquinone, but not through Complex II. The first step in the oxidation of fatty acyl–CoA, catalyzed by the FIGURE 19–10 Structure of Complex II (succinate dehydrogenase) of E. coli (PDB ID 1NEK). The enzyme has two transmembrane subunits, C (green) and D (blue); the cytoplasmic extensions contain subunits B (orange) and A (purple). Just behind the FAD in subunit A (gold) is the binding site for succinate (occupied in this crystal structure by the inhibitor oxaloacetate, green). Subunit B has three sets of Fe-S centers (yellow and red); ubiquinone (yellow) is bound to subunit C; and heme b (purple) is sandwiched between subunits C and D. A cardiolipin molecule is so tightly bound to subunit C that it shows up in the crystal structure (gray spacefilling). Electrons move (blue arrows) from succinate to FAD, then through the three Fe-S centers to ubiquinone. The heme b is not on the main path of electron transfer but protects against the formation of reactive oxygen species (ROS) by electrons that go astray. Substrate binding site Cytoplasm (N side) C B D A FAD Fe-S centers Periplasm (P side) Cardiolipin Ubiquinone QH2 Heme b 8885d_c19_690-750 3/1/04 11:32 AM Page 699 mac76 mac76:385_reb:
