20.2 What Are Reduction Potentials, and How Are They Used to Account for Free Energy Changes in Redox Reactions? 593 The smooth outer membrane is about 30% to 40% lipid and 60% to 70% protein and has a relatively high concentration of phosphatidylinositol. The outer mem- brane contains significant amounts of porin—a transmembrane protein, rich in -sheets, that forms large channels across the membrane, permitting free diffusion of molecules with molecular weights of about 10,000 or less. The outer membrane plays a prominent role in maintaining the shape of the mitochondrion. The inner membrane is richly packed with proteins, which account for nearly 80% of its weight; thus, its density is higher than that of the outer membrane. The fatty acids of inner membrane lipids are highly unsaturated. Cardiolipin and diphosphatidyl- glycerol (see Chapter 8) are abundant. The inner membrane lacks cholesterol and is quite impermeable to molecules and ions. Species that must cross the mitochon- drial inner membrane—ions, substrates, fatty acids for oxidation, and so on—are carried by specific transport proteins in the membrane. Notably, the inner mem- brane is extensively folded (Figure 20.1). The folds, known as cristae, provide the inner membrane with a large surface area in a small volume. During periods of ac- tive respiration, the inner membrane appears to shrink significantly, leaving a com- paratively large intermembrane space. The Mitochondrial Matrix Contains the Enzymes of the TCA Cycle The space inside the inner mitochondrial membrane is called the matrix, and it contains most of the enzymes of the TCA cycle and fatty acid oxidation. (An im- portant exception, succinate dehydrogenase of the TCA cycle, is located in the in- ner membrane itself.) In addition, mitochondria contain circular DNA molecules, along with ribosomes and the enzymes required to synthesize proteins coded within the mitochondrial genome. Although some of the mitochondrial proteins are made this way, most are encoded by nuclear DNA and synthesized by cytosolic ribosomes. 20.2 What Are Reduction Potentials, and How Are They Used to Account for Free Energy Changes in Redox Reactions? On numerous occasions in earlier chapters, we have stressed that NADH and reduced flavoproteins ([FADH 2 ]) are forms of metabolic energy. These reduced coenzymes have a strong tendency to be oxidized—that is, to transfer electrons to other species. (a) (b) Matrix Cristae Intermembrane space Inner membrane Outer membrane FIGURE 20.1 (a) A drawing of a mitochondrion with components labeled. (b) Tomography of a rat liver mito- chondrion.The tubular structures in red, yellow, green,purple, and aqua represent individual cristae formed from the inner mitochondrial membrane. (b, Frey,T. G., and Mannella, C. A.,2000.The internal structure of mitochondria. Trends in Biochemical Sciences 25:319–324.) 594 Chapter 20 Electron Transport and Oxidative Phosphorylation Oxidative phosphorylation converts the energy of electron transfer into the energy of phosphoryl transfer stored in the phosphoric anhydride bonds of ATP. Just as the group transfer potential was used in Chapter 3 to quantitate the energy of phospho- ryl transfer, the standard reduction potential, denoted by Ᏹ o Ј, quantitates the ten- dency of chemical species to be reduced or oxidized. The standard reduction poten- tial difference describing electron transfer between two species, is related to the free energy change for the process by ⌬G°ЈϭϪnᏲ⌬Ᏹ o Ј (20.2) where n represents the number of electrons transferred; Ᏺ is Faraday’s constant, 96,485 J/V и mol; and ⌬Ᏹ o Ј is the difference in reduction potentials between the donor and acceptor. This relationship is straightforward, but it depends on a stan- dard of reference by which reduction potentials are defined. Standard Reduction Potentials Are Measured in Reaction Half-Cells Standard reduction potentials are determined by measuring the voltages generated in reaction half-cells (Figure 20.2). A half-cell consists of a solution containing 1 M concentrations of both the oxidized and reduced forms of the substance whose re- duction potential is being measured and a simple electrode. (Together, the oxidized and reduced forms of the substance are referred to as a redox couple.) Such a sample half-cell is connected to a reference half-cell and electrode via a conductive bridge (usually a salt-containing agar gel). A sensitive potentiometer (voltmeter) con- nects the two electrodes so that the electrical potential (voltage) between them can be measured. The reference half-cell normally contains 1 M H ϩ in equilibrium with H 2 gas at a pressure of 1 atm. The H ϩ /H 2 reference half-cell is arbitrarily assigned a standard reduction potential of 0.0 V. The standard reduction potentials of all other redox couples are defined relative to the H ϩ /H 2 reference half-cell on the basis of the sign and magnitude of the voltage (electromotive force, emf) registered on the po- tentiometer (Figure 20.2). If electron flow between the electrodes is toward the sample half-cell, reduction occurs spontaneously in the sample half-cell and the reduction potential is said to be positive. If electron flow between the electrodes is away from the sample half-cell and toward the reference cell, the reduction potential is said to be negative because electron loss (oxidation) is occurring in the sample half-cell. Strictly speaking, the standard reduction potential, Ᏹ o Ј, is the electromotive force generated at 25°C and pH 7.0 by a sample half-cell (containing 1 M concentrations of the oxidized and re- duced species) with respect to a reference half-cell. (Note that the reduction po- tential of the hydrogen half-cell is pH-dependent. The standard reduction poten- tial, 0.0 V, assumes 1 M H ϩ . The hydrogen half-cell measured at pH 7.0 has an Ᏹ o Ј of Ϫ0.421 V.) Two Examples Figure 20.2a shows a sample/reference half-cell pair for measure- ment of the standard reduction potential of the acetaldehyde/ethanol couple. Be- cause electrons flow toward the reference half-cell and away from the sample half-cell, the standard reduction potential is negative, specifically Ϫ0.197 V. In contrast, the fumarate/succinate couple (Figure 20.2b) causes electrons to flow from the reference half-cell to the sample half-cell; that is, reduction occurs, and the reduction potential is thus positive. For each half-cell, a half-cell reaction describes the reaction taking place. For the fumarate/succinate half-cell coupled to a H ϩ /H 2 reference half-cell, the reaction occurring is indeed a reduction of fumarate: Fumarate ϩ 2 H ϩ ϩ 2 e Ϫ ⎯⎯→succinate Ᏹ o Ј ϭ ϩ0.031 V (20.3) Reduced donor Oxidized donor Oxidized acceptor Reduced acceptor ne Ϫ Agar bridge Electron flow Electron flow Fumarate Succinate Agar bridge (a) Ethanol acetaldehyde Electron flow Electron flow Potentiometer –0.197 V Ethanol acetaldehyde H + Reference /1 atm H 2 Sample: acetaldehyde/ ethanol H + H 2 H + Reference /1 atm H 2 Sample: fumarate/ succinate 2 H + (b) Fumarate succinate H 2 2 +0.031 V ACTIVE FIGURE 20.2 Experimental apparatus used to measure the standard reduction potential of the indicated redox couples: (a) the acetaldehyde/ethanol couple, (b) the fumarate/ succinate couple. Test yourself on the concepts in this figure at www.cengage.com/login. 20.2 What Are Reduction Potentials, and How Are They Used to Account for Free Energy Changes in Redox Reactions? 595 However, the reaction occurring in the acetaldehyde/ethanol half-cell is the oxida- tion of ethanol: Ethanol⎯⎯→acetaldehyde ϩ 2 H ϩ ϩ 2 e Ϫ Ᏹ o ЈϭϪ0.197 V (20.4) Ᏹ o Values Can Be Used to Predict the Direction of Redox Reactions Some typical half-cell reactions and their respective standard reduction potentials are listed in Table 20.1. Whenever reactions of this type are tabulated, they are uniformly written as reduction reactions, regardless of what occurs in the given half-cell. The sign of the standard reduction potential indicates which reaction really occurs when the given half-cell is combined with the reference hydrogen half-cell. Redox couples that Reduction Half-Reaction Ᏹ o (V) ᎏ 1 2 ᎏ O 2 ϩ 2 H ϩ ϩ 2 e Ϫ 88n H 2 O 0.816 Fe 3ϩ ϩ e Ϫ 88n Fe 2ϩ 0.771 Photosystem P700 0.430 NO 3 Ϫ ϩ 2 H ϩ ϩ 2 e Ϫ 88n NO 2 Ϫ ϩ H 2 O 0.421 Cytochrome f (Fe 3ϩ ) ϩ e Ϫ 88n cytochrome f (Fe 2ϩ ) 0.365 Cytochrome a 3 (Fe 3ϩ ) ϩ e Ϫ 88n cytochrome a 3 (Fe 2ϩ ) 0.350 Cytochrome a(Fe 3ϩ ) ϩ e Ϫ 88n cytochrome a(Fe 2ϩ ) 0.290 Rieske Fe-S(Fe 3ϩ ) ϩ e Ϫ 88n Rieske Fe-S(Fe 2ϩ ) 0.280 Cytochrome c (Fe 3ϩ ) ϩ e Ϫ 88n cytochrome c (Fe 2ϩ ) 0.254 Cytochrome c 1 (Fe 3ϩ ) ϩ e Ϫ 88n cytochrome c 1 (Fe 2ϩ ) 0.220 UQH иϩH ϩ ϩ e Ϫ 88n UQH 2 (UQ ϭ coenzyme Q) 0.190 UQ ϩ 2 H ϩ ϩ 2 e Ϫ 88n UQH 2 0.060 Cytochrome b H (Fe 3ϩ ) ϩ e Ϫ 88n cytochrome b H (Fe 2ϩ ) 0.050 Fumarate ϩ 2 H ϩ ϩ 2 e Ϫ 88n succinate 0.031 UQ ϩ H ϩ ϩ e Ϫ 88n UQH и 0.030 Cytochrome b 5 (Fe 3ϩ ) ϩ e Ϫ 88n cytochrome b 5 (Fe 2ϩ ) 0.020 [FAD] ϩ 2 H ϩ ϩ 2 e Ϫ 88n [FADH 2 ] 0.003–0.091* Cytochrome b L (Fe 3ϩ ) ϩ e Ϫ 88n cytochrome b L (Fe 2ϩ ) Ϫ0.100 Oxaloacetate ϩ 2 H ϩ ϩ 2 e Ϫ 88n malate Ϫ0.166 Pyruvate ϩ 2 H ϩ ϩ 2 e Ϫ 88n lactate Ϫ0.185 Acetaldehyde ϩ 2 H ϩ ϩ 2 e Ϫ 88n ethanol Ϫ0.197 FMN ϩ 2 H ϩ ϩ 2 e Ϫ 88n FMNH 2 Ϫ0.219 FAD ϩ 2 H ϩ ϩ 2 e Ϫ 88n FADH 2 Ϫ0.219 Glutathione (oxidized) ϩ 2 H ϩ ϩ 2 e Ϫ 88n 2 glutathione (reduced) Ϫ0.230 Lipoic acid ϩ 2 H ϩ ϩ 2 e Ϫ 88n dihydrolipoic acid Ϫ0.290 1,3-Bisphosphoglycerate ϩ 2 H ϩ ϩ 2 e Ϫ 88n Ϫ0.290 glyceraldehyde-3-phosphate ϩ P i NAD ϩ ϩ 2 H ϩ ϩ 2 e Ϫ 88n NADH ϩ H ϩ Ϫ0.320 NADP ϩ ϩ 2 H ϩ ϩ 2 e Ϫ 88n NADPH ϩ H ϩ Ϫ0.320 Lipoyl dehydrogenase [FAD] ϩ 2 H ϩ ϩ 2 e Ϫ 88n Ϫ0.340 lipoyl dehydrogenase [FADH 2 ] ␣-Ketoglutarate ϩ CO 2 ϩ 2 H ϩ ϩ 2 e Ϫ 88n isocitrate Ϫ0.380 2 H ϩ ϩ 2 e Ϫ 88n H 2 Ϫ0.421 Ferredoxin (spinach) (Fe 3ϩ ) ϩ e Ϫ 88n ferredoxin (spinach) (Fe 2ϩ ) Ϫ0.430 Succinate ϩ CO 2 ϩ 2 H ϩ ϩ 2 e Ϫ 88n ␣-ketoglutarate ϩ H 2 O Ϫ0.670 *Typical values for reduction of bound FAD in flavoproteins such as succinate dehydrogenase (see Bonomi, F., Pagani, S., Cerletti, P., and Giori, C.,1983. Modification of the thermodynamic properties of the electron-transferring groups in mito- chondrial succinate dehydrogenase upon binding of succinate. European Journal of Biochemistry 134:439–445). TABLE 20.1 Standard Reduction Potentials for Several Biological Reduction Half-Reactions 596 Chapter 20 Electron Transport and Oxidative Phosphorylation have large positive reduction potentials have a strong tendency to accept electrons, and the oxidized form of such a couple (O 2 , for example) is a strong oxidizing agent. Re- dox couples with large negative reduction potentials have a strong tendency to un- dergo oxidation (that is, donate electrons), and the reduced form of such a couple (NADPH, for example) is a strong reducing agent. Ᏹ o Values Can Be Used to Analyze Energy Changes in Redox Reactions The half-reactions and reduction potentials in Table 20.1 can be used to analyze en- ergy changes in redox reactions. The oxidation of NADH to NAD ϩ can be coupled with the reduction of ␣-ketoglutarate to isocitrate: NAD ϩ ϩ isocitrate ⎯⎯→NADH ϩ H ϩ ϩ ␣-ketoglutarate ϩ CO 2 (20.5) This is the isocitrate dehydrogenase reaction of the TCA cycle. Writing the two half- cell reactions, we have NAD ϩ ϩ 2 H ϩ ϩ 2 e Ϫ ⎯⎯→NADH ϩ H ϩ Ᏹ o ЈϭϪ0.32 V (20.6) ␣-Ketoglutarate ϩ CO 2 ϩ 2 H ϩ ϩ 2 e Ϫ ⎯⎯→isocitrate Ᏹ o ЈϭϪ0.38 V (20.7) In a spontaneous reaction, electrons are donated by (flow away from) the half- reaction with the more negative reduction potential and are accepted by (flow to- ward) the half-reaction with the more positive reduction potential. Thus, in the pre- sent case, isocitrate donates electrons and NAD ϩ accepts electrons. The convention defines ⌬Ᏹ o Ј as ⌬Ᏹ o ЈϭᏱ o Ј (acceptor) Ϫ Ᏹ o Ј (donor) (20.8) In the present case, isocitrate is the donor and NAD ϩ the acceptor, so we write ⌬Ᏹ o ЈϭϪ0.32 V Ϫ (Ϫ0.38 V) ϭϩ0.06 V (20.9) From Equation 20.2, we can now calculate ⌬G°Ј as ⌬G°ЈϭϪ(2)(96.485 kJ/V и mol)(0.06 V) (20.10) ⌬G°ЈϭϪ11.58 kJ/mol Note that a reaction with a net positive ⌬Ᏹ o Ј yields a negative ⌬G°Ј, indicating a spontaneous reaction. The Reduction Potential Depends on Concentration We have already noted that the standard free energy change for a reaction, ⌬G°Ј, does not reflect the actual conditions in a cell, where reactants and products are not at standard-state concentrations (1 M). Equation 3.13 was introduced to permit calcu- lations of actual free energy changes under non–standard-state conditions. Similarly, standard reduction potentials for redox couples must be modified to account for the actual concentrations of the oxidized and reduced species. For any redox couple, ox ϩ ne Ϫ 34 red (20.11) the actual reduction potential is given by Ᏹ ϭ Ᏹ o Јϩ(RT/nᏲ) ln (20.12) Reduction potentials can also be quite sensitive to molecular environment. The in- fluence of environment is especially important for flavins, such as FAD/FADH 2 and FMN/FMNH 2 . These species are normally bound to their respective flavoproteins; the reduction potential of bound FAD, for example, can be very different from the value shown in Table 20.1 for the free FAD/FADH 2 couple of Ϫ0.219 V. Problem 7 at the end of the chapter addresses this case. [ox] ᎏ [red] 20.3 How Is the Electron-Transport Chain Organized? 597 20.3 How Is the Electron-Transport Chain Organized? As we have seen, the metabolic energy from oxidation of food materials—sugars, fats, and amino acids—is funneled into formation of reduced coenzymes (NADH) and re- duced flavoproteins ([FADH 2 ]). The electron-transport chain reoxidizes the coen- zymes and channels the free energy obtained from these reactions into the creation of a proton gradient. This reoxidation process involves the removal of both protons and electrons from the coenzymes. Electrons move from NADH and [FADH 2 ] to mo- lecular oxygen, O 2 , which is the terminal acceptor of electrons in the chain. The re- oxidation of NADH, NADH (reductant) ϩ H ϩ ϩ O 2 (oxidant)⎯⎯→NAD ϩ ϩ H 2 O (20.13) involves the following half-reactions: NAD ϩ ϩ 2 H ϩ ϩ 2 e Ϫ ⎯⎯→NADH ϩ H ϩ Ᏹ o ЈϭϪ0.32 V (20.14) ᎏ 1 2 ᎏ O 2 ϩ 2 H ϩ ϩ 2 e Ϫ ⎯⎯→H 2 O Ᏹ o Јϭϩ0.816 V (20.15) Here, half-reaction 20.15 is the electron acceptor and half-reaction 20.14 is the elec- tron donor. Then ⌬Ᏹ o Јϭ0.816 Ϫ (Ϫ0.32) ϭ 1.136 V (20.16) and, according to Equation 20.2, the standard-state free energy change, ⌬G°Ј, is Ϫ219 kJ/mol. Molecules along the electron-transport chain have reduction poten- tials between the values for the NAD ϩ /NADH couple and the oxygen/H 2 O couple, so electrons move down the energy scale toward progressively more positive reduc- tion potentials (Figure 20.3). Although electrons move from more negative to more positive reduction po- tentials in the electron-transport chain, it should be emphasized that the electron 0 +200 +400 +600 –200 –400 Complex I Complex II Complex III Complex IV NAD + /NADH FMN (Fe/S)N1 (Fe/S)N4 (Fe/S)N3 (Fe/S)N2 Rieske Fe/S (Fe/S)S1 (Fe/S)S3 FAD Fum/Succ UQ 10 b L b H Cu A c 1 a 3 c a (mV) FIGURE 20.3 Ᏹ o Ј and Ᏹ values for the components of the mitochondrial electron-transport chain.Values indi- cated are consensus values for animal mitochondria. Black bars represent Ᏹ o Ј; red bars, Ᏹ. 598 Chapter 20 Electron Transport and Oxidative Phosphorylation carriers do not operate in a simple linear sequence. This will become evident when the individual components of the electron-transport chain are discussed in the following paragraphs. The Electron-Transport Chain Can Be Isolated in Four Complexes The electron-transport chain involves several different molecular species, including: 1. Flavoproteins, which contain tightly bound FMN or FAD as prosthetic groups and which may participate in one- or two-electron transfer events. 2. Coenzyme Q, also called ubiquinone (and abbreviated CoQ or UQ) (see Figure 20.5), which can function in either one- or two-electron transfer reactions. 3. Several cytochromes (proteins containing heme prosthetic groups [see Chapter 5], which function by carrying or transferring electrons), including cytochromes b, c, c 1 , a, and a 3 . Cytochromes are one-electron transfer agents in which the heme iron is converted from Fe 2ϩ to Fe 3ϩ and back. 4. A number of iron–sulfur proteins, which participate in one-electron transfers in- volving the Fe 2ϩ and Fe 3ϩ states. 5. Protein-bound copper, a one-electron transfer site that converts between Cu ϩ and Cu 2ϩ . All these intermediates except for cytochrome c are membrane associated (either in the mitochondrial inner membrane of eukaryotes or in the plasma membrane of prokaryotes). Three types of proteins involved in this chain—flavoproteins, cytochromes, and iron–sulfur proteins—possess electron-transferring prosthetic groups. The components of the electron-transport chain can be purified from the mito- chondrial inner membrane. Solubilization of the membranes containing the electron- transport chain results in the isolation of four distinct protein complexes, and the com- plete chain can thus be considered to be composed of four parts: (I) NADH–coenzyme Q reductase, (II) succinate–coenzyme Q reductase, (III) coenzyme Q–cytochrome c reductase, and (IV) cytochrome c oxidase (Figure 20.4). Complex I accepts electrons from NADH, serving as a link between glycolysis, the TCA cycle, fatty acid oxidation, NADH–coenzyme Q oxidoreductase Succinate–coenzyme Q oxidoreductase Coenzyme Q–cytochrome c oxidoreductase Cytochrome c oxidase Fatty acyl-CoA dehydrogenase Electron-transferring flavoprotein, FAD, Fe-S centers NADH dehydrogenase, FMN, Fe-S centers Succinate dehydrogenase, FAD (covalent), Fe-S centers, b-type heme UQ/UQH 2 pool Flavoprotein 3Flavoprotein 1 Flavoprotein 2 Sn-glycerophosphate dehydrogenase FAD, Fe-S centers Cytochrome bc 1 complex, 2 b-type hemes, Rieske Fe-S center, C-type heme (cyt c 1 ) Flavoprotein 4 Complex II O 2 H 2 O Cytochrome c 1 2 Complex I Complex III Cytochrome aa 3 complex, 2 a-type hemes, Cu ions Complex IV FIGURE 20.4 An overview of the complexes and pathways in the mitochondrial electron-transport chain. (Adapted from Nicholls, D. G., and Ferguson, S. J., 2002. Bioener- getics 3. London: Academic Press.) 20.3 How Is the Electron-Transport Chain Organized? 599 and the electron-transport chain. Complex II includes succinate dehydrogenase and thus forms a direct link between the TCA cycle and electron transport. Complexes I and II produce a common product, reduced coenzyme Q (UQH 2 ), which is the sub- strate for coenzyme Q–cytochrome c reductase (Complex III). As shown in Figure 20.4, there are two other ways to feed electrons to UQ: the electron-transferring flavoprotein, which transfers electrons from the flavoprotein-linked step of fatty acyl- CoA dehydrogenase, and sn-glycerophosphate dehydrogenase. Complex III oxidizes UQH 2 while reducing cytochrome c, which in turn is the substrate for Complex IV, cytochrome c oxidase. Complex IV is responsible for reducing molecular oxygen. Each of the complexes shown in Figure 20.4 is a large multisubunit complex embedded within the inner mitochondrial membrane. Complex I Oxidizes NADH and Reduces Coenzyme Q As its name implies, this complex transfers a pair of electrons from NADH to coen- zyme Q, a small, hydrophobic, yellow compound. Another common name for this en- zyme complex is NADH dehydrogenase. The complex (with an estimated mass of 980 kD) involves at least 45 polypeptide chains, one molecule of flavin mononu- cleotide (FMN), and eight or nine Fe-S clusters, together containing a total of 20 to 26 iron atoms (Table 20.2). By virtue of its dependence on FMN, NADH–UQ reduc- tase is a flavoprotein. Although the precise mechanism of the NADH–UQ reductase is unknown, the first step involves binding of NADH to the enzyme on the matrix side of the inner mitochondrial membrane and transfer of electrons from NADH to tightly bound FMN: NADH ϩ [FMN] ϩ H ϩ ⎯⎯→[FMNH 2 ] ϩ NAD ϩ (20.17) The second step involves the transfer of electrons from the reduced [FMNH 2 ] to a series of Fe-S proteins, including both 2Fe-2S and 4Fe-4S clusters (see page 577). The versatile redox properties of the flavin group of FMN are important here. NADH is a two-electron donor, whereas the Fe-S proteins are one-electron transfer agents. The flavin of FMN has three redox states—the oxidized, semi- quinone, and reduced states. It can act as either a one-electron or a two-electron transfer agent and may serve as a critical link between NADH and the Fe-S proteins. The final step of the reaction involves the transfer of two electrons from iron– sulfur clusters to coenzyme Q. Coenzyme Q is a mobile electron carrier. Its isoprenoid Mass Prosthetic Complex (kD) Subunits Group Binding Site for: NADH–UQ reductase 980 Ն45 FMN NADH (matrix side) Fe-S UQ (lipid core) Succinate–UQ reductase 140 4 FAD Succinate (matrix side) Fe-S UQ (lipid core) UQ–Cyt c reductase 250 9–10 Heme b L Cyt c (intermembrane Heme b H space side) Heme c 1 Fe-S Cytochrome c 13 1 Heme c Cyt c 1 Cyt a Cytochrome c oxidase 162 13 Heme a Cyt c (intermembrane Heme a 3 space side) Cu A Cu B TABLE 20.2 Protein Complexes of the Mitochondrial Electron-Transport Chain 600 Chapter 20 Electron Transport and Oxidative Phosphorylation tail makes it highly hydrophobic, and it diffuses freely in the hydrophobic core of the inner mitochondrial membrane. As a result, it shuttles electrons from Complexes I and II to Complex III. The redox cycle of UQ is shown in Figure 20.5. The structural and functional organization of Complex I is shown in Figure 20.6. Complex I Transports Protons from the Matrix to the Cytosol The oxidation of one NADH and the reduction of one UQ by NADH–UQ reductase results in the net O• OH CH 3 R + e – OH H 3 CO H 3 CO CH 3 R OH Coenzyme Q, oxidized form + H + H + e – O O H 3 CO H 3 CO CH 3 (CH 2 CH C CH 2 ) 10 H CH 3 O CH 3 O CH 3 (a) Semiquinone intermediate (QH •) Coenzyme Q, reduced form (QH 2 , ubiquinol) (Q, ubiquinone) (b) FIGURE 20.5 (a) The three oxidation states of coenzyme Q. (b) A space-filling model of coenzyme Q. HUMAN BIOCHEMISTRY Solving a Medical Mystery Revolutionized Our Treatment of Parkinson’s Disease A tragedy among illegal drug users was the impetus for a revolu- tionary treatment of Parkinson’s disease. In 1982, several mysteri- ous cases of paralysis came to light in southern California. The vic- tims, some of them teenagers, were frozen like living statues, unable to talk or move. The case was baffling at first, but it was soon traced to a batch of synthetic heroin that contained MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) as a contaminant. MPTP is rapidly converted in the brain to MPP ϩ (1-methyl-4- phenylpyridine) by the enzyme monoamine oxidase B. MPP ϩ is a potent inhibitor of mitochondrial Complex I (NADH–UQ reduc- tase), and it acts preferentially in the substantia nigra, an area of the brain that is essential to movement and also the region of the brain that deteriorates slowly in Parkinson’s disease. Parkinson’s disease results from the inability of the brain to produce sufficient quantities of dopamine, a neurotransmitter. Neurologist J. William Langston, asked to consult on the treat- ment of some of these patients, recognized that the symptoms of this drug-induced disorder were in fact similar to those of parkin- sonism. He began treatment of the patients with L-dopa, which is decarboxylated in the brain to produce dopamine. The treated pa- tients immediately regained movement. Langston then took a bold step. He implanted fetal brain tissue into the brains of several of the affected patients, prompting substantial recovery from the Parkinson-like symptoms. Langston’s innovation sparked a revolu- tion in the use of tissue implantation for the treatment of neu- rodegenerative diseases. Other toxins may cause similar effects in neural tissue. Timothy Greenmyre at Emory University has shown that rats exposed to the pesticide rotenone (see Figure 20.27) over a period of weeks expe- rience a gradual loss of function in dopaminergic neurons and then develop symptoms of parkinsonism, including limb tremors and rigidity. This finding supports earlier research that links long-term pesticide exposure to Parkinson’s disease. MPTP MPP + Cell death in substantia nigr a Monoamine oxidase B N H H H HCH 3 H H H + N CH 3 + 20.3 How Is the Electron-Transport Chain Organized? 601 transport of protons from the matrix side to the cytosolic side of the inner mem- brane. The cytosolic side, where H ϩ accumulates, is referred to as the P (for posi- tive) face; similarly, the matrix side is the N (for negative) face. Some of the energy liberated by the flow of electrons through this complex is used in a coupled process to drive the transport of protons across the membrane. (This is an example of ac- tive transport, a phenomenon examined in detail in Chapter 9.) Available experi- mental evidence suggests a stoichiometry of four H ϩ transported per two electrons passed from NADH to UQ. Complex II Oxidizes Succinate and Reduces Coenzyme Q Complex II is perhaps better known by its other name—succinate dehydrogenase, the only TCA cycle enzyme that is an integral membrane protein in the inner mitochondrial membrane. This complex (Figure 20.7) has a mass of 124 kD and is composed of two hydrophilic subunits, a flavoprotein (Fp, 68 kD) and an iron–sulfur protein (Ip, 29 kD), and two hydrophobic, membrane-anchored sub- units (15 kD and 11 kD), which contain one heme b and provide the binding site for UQ. Fp contains an FAD covalently bound to a His residue (see Figure 19.12), and Ip contains three Fe-S centers: a 4Fe-4S cluster, a 3Fe-4S cluster, and a 2Fe-2S cluster. When succinate is converted to fumarate in the TCA cycle, concomitant re- duction of bound FAD to FADH 2 occurs in succinate dehydrogenase. This FADH 2 NADH + + H + 2 H + 2 H + 2 H + (a) 2 H + 2 H + FMN FMNH 2 2 Fe-S centers 2 Fe-S centers UQ UQH 2 NAD + e – 2 e – 2 (b) 12 13 7/11/14 8/10 (c) N1a N6a N3 FMN N1b N6b N4 N5 N7 N2 ACTIVE FIGURE 20.6 (a) Structural organization of mammalian Complex I, based on electron microscopy, showing functional relationships within the L-shaped complex. Electron flow from NADH to UQH 2 in the membrane pool is indicated. (b) Structure of the hydrophilic domain of Complex I from Thermus thermophilus is shown on a model of the membrane-associated complex (pdb id ϭ 2FUG).The locations of individual subunits are indicated. (c) Arrangement of the redox centers in Complex I.The various iron–sulfur centers of Complex I are designated by capital N. (Part a adapted from Janssen, R. J., Nijtmans, L. G., van den Heuvel, L. P., and Smeitink, J.A., 2006. Mitochondrial complex I: Structure, function, and pathology. Journal of Inherited Metabolic Diseases 29:499–515; and parts b and c adapted from Figure 1 of Sazanov, L., and Hinchliffe, P., 2006.Structure of the hydrophilic domain of respiratory Complex I from Thermus thermophilus. Science 311:1430–1436.) Test yourself on the concepts in this figure at www.cengage.com/login. 602 Chapter 20 Electron Transport and Oxidative Phosphorylation transfers its electrons immediately to Fe-S centers, which pass them on to UQ. Elec- tron flow from succinate to UQ, Succinate ⎯⎯→ fumarate ϩ 2 H ϩ ϩ 2 e Ϫ (20.18) UQ ϩ 2 H ϩ ϩ 2 e Ϫ ⎯⎯→ UQH 2 (20.19) Net rxn: Succinate ϩ UQ ⎯⎯→ fumarate ϩ UQH 2 ⌬Ᏹ o Јϭ0.029 V (20.20) yields a net reduction potential of 0.029 V. (Note that the first half-reaction is writ- ten in the direction of the e Ϫ flow. As always, ⌬Ᏹ o Ј is calculated according to Equa- tion 20.8.) The small free energy change of this reaction does not contribute to the transport of protons across the inner mitochondrial membrane. This is a crucial point because (as we will see) proton transport is coupled with ATP synthesis. Oxidation of one FADH 2 in the electron-transport chain results in synthesis of approximately two molecules of ATP, compared with the approximately three ATPs produced by the oxidation of one NADH. Other flavoproteins can also supply (a) UQ UQH 2 Intermembrane space Matrix Complex III Complex II 2Fe 2+ 2Fe 3+ FAD Succinate Fumarate FADH 2 2 H + 3Fe4S 4Fe4S 2Fe2S Heme b (b) (c) Heme b Fe-S centers FAD ACTIVE FIGURE 20.7 (a) A scheme for electron flow in Complex II. Oxidation of succinate occurs with reduction of [FAD]. Electrons are then passed to Fe-S centers and then to coenzyme Q (UQ). Proton transport does not occur in this complex. (b) The structure of Complex II from pig heart (pdb id ϭ 1ZOY). (c) The arrangement of redox centers. Electron flow is from bottom to top. Test yourself on the concepts in this figure at www.cengage.com/login.