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: