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: