Chapter 19

Oxidative Phosphorylation

Textbook pages 2363–2493 (Lehninger, 8e) · 25 MCQs below · Source: printed chapter text extracted from the PDF

CHAPTER 19 OXIDATIVE PHOSPHORYLATION mitochondria challenged and fascinated biochemists for much of the twentieth century. Our current exploration of the topic is guided by ﬁve principles: Mitochondria play a central role in eukaryotic aerobic metabolism. ATP production is not the only important mitochondrial function. Mitochondria play host to the citric acid cycle, the fatty acid β -oxidation pathway, and the pathways of amino acid oxidation. The mitochondria also act in thermogenesis, steroid synthesis, and apoptosis (programmed cell death). The discovery of these diverse and important roles of mitochondria has stimulated much current research on the biochemistry of this organelle. Mitochondria trace their evolutionary origin to bacteria. Over 1.45 billion years ago, an endosymbiotic relationship arose between bacteria and a primitive eukaryote or eukaryotic progenitor. Mitochondria are ubiquitous in modern eukaryotes, and their bacterial origin is evident in almost every aspect of their structure and function. Electrons ﬂow from electron donors (oxidizable substrates) through a chain of membrane-bound carriers to a ﬁnal electron acceptor with a large reduction potential. The ﬁnal acceptor is molecular oxygen, O2. The appearance of oxygen in the atmosphere some 2.3 billion years ago, and its harnessing in living systems via the evolution of oxidative phosphorylation, made more complex life forms possible. The free energy made available by “downhill” (exergonic) electron ﬂow is coupled to the “uphill” transport of protons across a proton-impermeable membrane. The free energy of fuel oxidation is thus conserved as a transmembrane electrochemical potential. The transmembrane ﬂow of protons back down their electrochemical gradient through speciﬁc protein channels provides the free energy for synthesis of ATP. This process is catalyzed by a membrane protein complex (ATP synthase) that couples proton ﬂow to phosphorylation of ADP. Principles 3 through 5, illustrated in Figure 19-1, encapsulate the theory, 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 ideas of twentieth-century biology. It provides insight into the processes of oxidative phosphorylation and photophosphorylation in plants, and into such apparently disparate energy transductions as active transport across membranes and the motion of bacterial ﬂagella. FIGURE 19-1 The chemiosmotic mechanism for ATP synthesis in mitochondria. Electrons move spontaneously through a chain of membrane-bound carriers, the respiratory chain, driven by the high reduction potential of oxygen and the relatively low reduction potentials of the various reduced substrates (fuels) that undergo oxidation in the mitochondrion. Electron flow creates an electrochemical potential by the transmembrane movement of protons and positive charge. This electrochemical potential drives ATP synthesis by a membrane-bound enzyme (ATP synthase) that is fundamentally similar in mitochondria and chloroplasts, and in bacteria and archaea as well. This chapter begins with a description of the components of the mitochondrial electron-transfer chain — the respiratory chain — and their organization into large functional complexes in the inner mitochondrial membrane, the path of electron ﬂow through these complexes, and the proton movements that accompany this ﬂow. We then consider the remarkable enzyme complex that captures, by “rotational catalysis,” 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. The metabolic role of mitochondria is so critical to cellular and organismal function that defects in mitochondrial function have serious medical consequences. Mitochondria are central to neuronal and muscular function, and to the regulation of whole- body energy metabolism and body weight. Human neurodegenerative diseases, as well as cancer, diabetes, and obesity, are recognized as possible results of compromised mitochondrial function, and one theory of aging is based on gradual loss of mitochondrial integrity. We consider the diverse functions of mitochondria, and the consequences of defective mitochondrial function in humans.

19.1 The Mitochondrial RespiratoryChain Albert L. Lehninger, 1917–1986 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 enzymological studies of biological energy transductions. Mitochondria, like gram-negative bacteria, have two membranes (Fig. 19-2a). 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 ATP synthase. FIGURE 19-2 Biochemical anatomy of a mitochondrion. (a) The outer membrane has pores that make it permeable to small molecules and ions, but not to proteins. The cristae 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. (b) A typical animal cell has hundreds or thousands of mitochondria. This endothelial cell from bovine pulmonary artery was stained with fluorescent probes for actin (blue), for DNA (red), and for mitochondria (yellow). Notice the variability in length of the mitochondria. (c) The mitochondria of heart muscle (blue in this colorized electron micrograph) have more profuse cristae and thus a much larger area of inner membrane, with more than three times as many sets of respiratory chains as (d) liver mitochondria. Muscle and liver mitochondria are about the size of a bacterium — 1to10μm long. 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. The mitochondrial matrix, enclosed by the inner membrane, contains the pyruvate dehydrogenase complex and the enzymes of the citric acid cycle, the fatty acid β -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 mitochondrial 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 α -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. Mammalian mitochondria have about 1,200 proteins, according to current best estimates. The functions of up to 25% of these remain partly or entirely enigmatic. The bean-shaped representation of a mitochondrion in Figure 19-2a is an oversimpliﬁcation, derived in part from early studies in which thin sections of cells were observed in the electron microscope. Three-dimensional images obtained either by reconstruction from serial sections or by confocal microscopy reveal great variation in mitochondrial size and shape. In living cells stained with mitochondrion-speciﬁc ﬂuorescent dyes, large numbers of variously shaped mitochondria are seen, clustered about the nucleus (Fig. 19- 2b). Tissues with a high demand for aerobic metabolism (brain, skeletal and heart muscle, liver, and eye, for example) contain many hundreds or thousands of mitochondria per cell, and in general, mitochondria of cells with high metabolic activity have more, and more densely packed, convolutions, or cristae (Fig. 19-2c, d). During cell growth and division, mitochondria, like bacteria, divide by ﬁssion, and under some circumstances individual mitochondria fuse to form larger, more-extended structures. Stressful conditions, such as the presence of electron-transfer inhibitors or mutations in an electron carrier, trigger mitochondrial ﬁssion and sometimes mitophagy — the breakdown of mitochondria and recycling of the amino acids, nucleotides, and lipids released. As stress is relieved, small mitochondria fuse to form long, thin, tubular organelles. Electrons Are Funneled to UniversalElectron Acceptors Oxidative phosphorylation begins with the entry of electrons into the series of electron carriers called 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: Reducedsubstrate+ NAD+ ⇌ oxidized substrate+ NADH + H+ Reducedsubstrate + NADP+ ⇌ oxidized substrate+ NADPH + H+ Most dehydrogenases that act in catabolism are speciﬁc for NAD+ as electron acceptor (Table 19-1). Some are in the cytosol, many are in mitochondria, and still others have mitochondrial and cytosolic isozymes. TABLE 19-1 Some Important Reactions Catalyzed by NAD(P)+-Linked Dehydrogenases Reaction Location NAD+-linked α-Ketoglutarate+ CoA+ NAD+ ⇌ succinyl-CoA+ CO2+ NADH + H+ M a b L-M alate+ NAD+ ⇌ oxaloacetate+ NADH + H+ M and C Pyruvate+ CoA+ NAD+ ⇌ acetyl-CoA+ CO2+ NADH + H+ M G lyceraldehyde3-phosphate+ Pi+ NAD+ ⇌ 1,3-bisphosphoglycerate+ NADH + H+ C Lactate+ NAD+ ⇌ pyruvate+ NADH + H+ C β-Hydroxyacyl-CoA+ NAD+ ⇌ β-ketoacyl-CoA+ NADH + H+ M NADP+-linked G lucose6-phosphate+ NADP+ ⇌ 6-phosphogluconate+ NADPH + H+ C L-M alate+ NADP+ ⇌ pyruvate+ CO2+ NADPH + H+ C NAD+- or NADP+-linked L-G lutamate+ H2O+ NAD(P)+ ⇌ α-ketoglutarate+ NH+4 + NAD(P)H M Isocitrate+ NAD(P)+ ⇌ α-ketoglutarate+ CO2+ NAD(P)H + H+ M and C These reactions and their enzymes are discussed in Chapters 14, 16, 17, and 18. M designates mitochondria; C designates cytosol. NAD+-linked dehydrogenases remove two hydrogen atoms from their substrates. One of these is transferred as a hydride ion (:H−) to NAD+, and the other is released as H+ in the medium (see Fig. 13-24). NADH and NADPH are water-soluble electron carriers that associate reversibly with dehydrogenases. About 70% of the cellular NAD pool is in the mitochondria. NADH carries electrons from catabolic reactions to their point of entry into the respiratory chain, the NADH dehydrogenase complex described below. NADPH is primarily involved in biosynthetic (anabolic) reactions, and much of it is concentrated in the cytosol. Cells maintain separate pools of NADPH and NADH, with different redox potentials. This is accomplished by holding the ratio [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. a b Flavoproteins contain a very tightly, sometimes covalently, bound ﬂavin nucleotide, either FMN or FAD (see Fig. 13-27). The oxidized ﬂavin nucleotide can accept either one electron (yielding the semiquinone form) or two (yielding FADH2 or FM NH2). Electron transfer occurs because the ﬂavoprotein has a higher reduction potential than the compound oxidized. Recall that reduction potential is a quantitative measure of the relative tendency of a given chemical species to accept electrons in an oxidation- reduction reaction (p. 490). 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 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 electron-transfer 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 ofMembrane-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 called 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. 19-3). Ubiquinone can accept one electron to become the semiquinone radical (∙QH or ubisemiquinone) or two electrons to form ubiquinol (QH2) and, like ﬂavoprotein carriers, it can act at the junction between a two- electron donor and a one-electron acceptor. Small and hydrophobic, ubiquinone is not bound to proteins but instead 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.

FIGURE 19-3 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. The cytochromes are proteins with characteristic strong absorption of visible light, due to their iron-containing heme prosthetic groups (Fig. 19-4a). 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. 19-4b). The longest-wavelength 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. FIGURE 19-4 Prosthetic groups of cytochromes. (a) 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. 5-1b). Heme c is covalently bound to the protein of cytochrome c through thioether bonds to two Cys residues. Heme a, found in a-type cytochromes, has a long isoprenoid tail attached to one of the five-membered rings. The conjugated double-bond system (shaded light red) of the porphyrin ring has delocalized π electrons that are relatively easily excited by photons with the wavelengths of visible light, which accounts for the strong absorption by hemes (and related compounds) in the visible region of the spectrum. (b) Absorption spectra of cytochrome c (cyt c) in its oxidized (blue) and reduced (red) forms. The characteristic α , β , and γ bands of the reduced form are labeled. The hemes 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. 19-4). 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 soluble cytochrome c that associates through electrostatic interactions with the outer surface of the inner membrane. In iron-sulfur proteins, 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) 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 aer 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 Fe-S 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 −0.65V to +0.45V, depending on the microenvironment of the iron within the protein. FIGURE 19-5 Iron-sulfur centers. The Fe-S centers of iron-sulfur proteins may be as simple as shown in (a), with a single Fe ion surrounded by the S atoms of four Cys residues; inorganic S is yellow and the S of Cys is orange. 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. (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. [(d) Data from PDB ID 1FRD, B. L. Jacobson et al., Biochemistry 32:6788, 1993.] 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 (see Fig. 20-12). First, the standard reduction potentials of the individual electron carriers have been determined experimentally (Table 19-2). 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 → cytochromeb → cytochrome c1 → cytochromec →cytochromea → cytochromea3 → 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 concentrations of reduced and oxidized forms (see Eqn 13-5, p. 491). A second method for determining the sequence 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. TABLE 19-2 Standard Reduction Potentials of Respiratory Chain and Related Electron Carriers Redox reaction (half-reaction) E′° (V) 2H+ + 2e− → H2 −0.414 NAD+ + H+ + 2e− → NADH −0.320 NADP+ + H+ + 2e− → NADPH −0.324 NADHdehydrogenase(FM N)+ 2H+ + 2e− → NADHdehydrogenase(FM NH2) −0.30 Ubiquinone+ 2H+ + 2e− → ubiquinol 0.045 Cytochromeb(Fe3+)+ e− → cytochromeb(Fe2+) 0.077 Cytochromec1(Fe3+)+ e− → cytochromec1(Fe2+) 0.22 Cytochromec(Fe3+)+ e− → cytochromec(Fe2+) 0.254 Cytochromea(Fe3+)+ e− → cytochromea(Fe2+) 0.29 Cytochromea3(Fe3+)+ e− → cytochromea3(Fe2+) 0.35 ½O2+ 2H+ + 2e− → H2O 0.817 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 aer 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. 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 aer the block become oxidized (light red). Electron Carriers Function in MultienzymeComplexes 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 electron-carrier complexes, each capable of catalyzing electron transfer through a portion of the chain (Fig. 19-7; Table 19-3). 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. 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 membrane, and the fragments are gently dissolved in a second detergent. The resulting mixture of inner membrane proteins is resolved by ion-exchange chromatography into several 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 electron 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. TABLE 19-3 The Protein Components of the Mitochondrial Respiratory Chain Enzyme complex/protein Mass (kDa) Number of subunits Prosthetic group(s) I NADH dehydrogenase 850 45 (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 204 13 (3–4) Hemes; CuA,CuB Number of subunits in bacterial complexes is shown in parentheses. Mass and subunit data are for the monomeric form. Cytochrome c is not part of an enzyme complex; it moves between Complexes III and IV as a freely soluble protein. We now look in more detail at the structure and function of each complex of the mitochondrial respiratory chain. Complex I: NADH to Ubiquinone In mammals, Complex I, also called NADH:ubiquinone oxidoreductase or NADH dehydrogenase, is a large enzyme composed of 45 different polypeptide chains, including an FMN-containing ﬂavoprotein and at least 8 iron-sulfur centers. Complex I is L-shaped, with one arm embedded in the inner membrane and the other extending into the matrix. Comparative studies of Complex I in bacteria and other organisms show that 7 a b c b a b c polypeptides in the membrane arm and 7 in the matrix arm are conserved and essential (Fig. 19-8). FIGURE 19-8 Structure of Complex I (NADH:ubiquinone oxidoreductase). Complex I catalyzes the transfer of a hydride ion from NADH to FMN. From the FMN, two electrons pass through a series of Fe-S centers to the Fe-S center 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. The protons travel a path dictated by subunit conformation changes triggered by the electron flow. Proton flux produces an electrochemical potential across the inner mitochondrial membrane ( side negative, side positive). Three of the membrane subunits (subunits Nqo12, Nqo13, and Nqo14) are structurally related to a known Na+-H+ antiporter, and the path of proton movement may be similar in both cases. The fourth putative proton pathway is through an integral subunit closest to the Q-binding site. A long helix (not visible in this view) lying along the surface of the membrane arm may coordinate the action of all four proton pumps when Q is reduced. [Data from PDB ID 4HEA, R. Baradaran et al., Nature 494:443, 2013.] Complex I catalyzes two simultaneous and obligately coupled processes: (1) the exergonic transfer to ubiquinone of a hydride ion from NADH and a proton from the matrix, expressed by NADH + H+ + Q → NAD+ + QH2 (19-1) and (2) the endergonic transfer of four protons from the matrix to the intermembrane space. Protons are moved against a transmembrane proton gradient in this process. 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 oen 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 → NAD+ + QH2+ 4H+P (19-2) 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. TABLE 19-4 Agents That Interfere with Oxidative Phosphorylation Type of interference Compound Target/mode of action Inhibition of electron transfer Cyanide Carbon monoxide Inhibit cytochrome oxidase a Antimycin A Blocks electron transfer from cytochrome b to cytochrome c1 Myxothiazol Rotenone Amytal Piericidin A Prevent electron transfer from Fe-S center to ubiquinone Inhibition of ATP synthase Aurovertin Inhibit F1 Oligomycin Venturicidin Inhibit Fo DCCD Blocks proton flow through Fo Uncoupling of phosphorylation from electron transfer FCCP DNP Hydrophobic proton carriers Valinomycin K+ ionophore Uncoupling protein 1 In brown adipose tissue, forms proton- conducting pores in inner mitochondrial membrane Inhibition of ATP-ADP exchange Atractyloside Inhibits adenine nucleotide translocase DCCD, dicyclohexylcarbodiimide; FCCP, cyanide-p-trifluoromethoxyphenylhydrazone; DNP, 2,4- dinitrophenol. Three of the seven integral protein subunits of the membrane arm are related to a Na+-H+ antiporter and are believed to be responsible for pumping three protons; a fourth subunit in the membrane arm, that nearest the Q-binding site, is probably responsible for pumping the fourth proton (Fig. 19-8). How is the reduction of ubiquinone coupled to proton pumping? Reduction of Q occurs far away from the membrane arm of the protein, where proton a pumping occurs, so the coupling is clearly indirect. The high-resolution view of Complex I from crystallographic and cryo-EM studies suggests that reduction of Q is coupled to a long-range conformational change conducted to all subunits along the hydrophilic core of the transmembrane arm. It seems likely that all four protons are pumped simultaneously, so that the energy from a strongly exergonic reaction (Q reduction) is broken into smaller packets, a common strategy employed by living organisms. 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. 586). Complex II couples the oxidation of succinate at one site with the reduction of ubiquinone at another site about 40 Å away. Although smaller and simpler than Complex I, Complex II contains ﬁve prosthetic groups of two types and four different protein subunits (Fig. 19-9). 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 Q, the ﬁnal electron acceptor in the reaction catalyzed by Complex II. Subunits A and B extend into the matrix; they contain three 2Fe-2S centers, bound FAD, and a binding site for the substrate, succinate. Although the overall path of electron transfer is long (from the succinate-binding site to FAD, then through the Fe-S centers to the Q-binding site), none of the individual electron-transfer distances exceeds about 11 Å — a reasonable distance for rapid electron transfer (Fig. 19-9). Electron transfer through Complex II is not accompanied by proton pumping across the inner membrane, although the QH2 produced by succinate oxidation will be used by Complex III to drive proton transfer. Because Complex II functions in the citric acid cycle, factors that affect its activity (such as the availability of oxidized Q) probably serve to coordinate that cycle with mitochondrial electron transfer. FIGURE 19-9 Structure of Complex II (succinate dehydrogenase). This complex (porcine) has two transmembrane subunits, C and D; subunits A and B extend into the matrix. Just behind the FAD in subunit A is the binding site for succinate. Subunit B has three Fe-S centers, ubiquinone is bound to subunit B, and heme b is sandwiched between subunits C and D. Two phosphatidylethanolamine molecules are so tightly bound to subunit D that they show up in the crystal structure. 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. [Data from PDB ID 1ZOY, F. Sun et al., Cell 121:1043, 2005.] The heme b of Complex II is apparently not in the direct path of electron transfer; it serves 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 (∙O−2), as described below. Some individuals with point mutations in Complex II subunits near heme b or the ubiquinone-binding site suffer from hereditary paraganglioma, 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, which cause DNA damage and genome instability that can lead to cancer. Mutations that affect the succinate-binding region in Complex II may lead to degenerative changes in the central nervous system, and some mutations are associated with tumors of the adrenal medulla. Complex III: Ubiquinone to Cytochrome c Electrons from reduced ubiquinone (ubiquinol, QH2) pass through two more large protein complexes in the inner mitochondrial membrane before reaching the ultimate electron acceptor, O2.

Complex III (also called cytochrome bc1 complex or ubiquinone:cytochrome c oxidoreductase) couples the transfer of electrons from ubiquinol to cytochrome c with the vectorial transport of protons from the matrix to the intermembrane space. The functional unit of Complex III (Fig. 19-10) is a dimer. Each monomer consists of three proteins central to the action of the complex: cytochrome b, cytochrome c1, and the Rieske iron-sulfur protein. (Several other proteins associated with Complex III in vertebrates are not conserved across the phyla and presumably play subsidiary roles.) The two cytochrome b monomers surround a cavern in the middle of the membrane, in which ubiquinone is free to move from the matrix side of the membrane (site QN on one monomer) to the intermembrane space (site QP on the other monomer) as it shuttles electrons and protons across the inner mitochondrial membrane. FIGURE 19-10 Structure of Complex III (cytochrome bc1 complex). The complex (bovine) is a dimer of identical monomers, each with 11 diﬀerent subunits. The functional core of each monomer consists of three subunits: cytochrome b (green), with its two hemes (bH andbL); the Rieske iron-sulfur protein (purple), with its 2Fe-2S centers; and cytochrome c1 (blue), with its heme. Cytochrome c1 and the Rieske iron-sulfur protein project from the surface and can interact with cytochrome c (not part of the functional complex) in the intermembrane space. The complex has two distinct binding sites for ubiquinone, QN and QP, which correspond to the sites of inhibition by two drugs that block oxidative phosphorylation. Antimycin A, which blocks electron flow from cytochrome b to cytochrome c1, specifically from heme bH to Q, binds at QN, close to heme bH on the (matrix) side of the membrane. Myxothiazol, which prevents electron flow from QH2 to the Rieske iron-sulfur protein, binds at QP, near the 2Fe-2S center and heme bL on the side. The dimeric structure is essential to the function of Complex III. The interface between monomers forms two caverns, each containing a QP site from one monomer and a QN site from the other. The ubiquinone intermediates move within these sheltered caverns. [Data from PDB ID 1BGY, S. Iwata et al., Science 281:64, 1998.] To account for the role of Q in energy conservation, Mitchell proposed the Q cycle (Fig. 19-11). As electrons move from QH2 through Complex III, QH2 is oxidized with the release of protons on one side of the membrane (at QP), while at the other site (QN), Q is reduced and protons are taken up. FIGURE 19-11 The Q cycle, shown in two stages. The path of electrons through Complex III is shown with blue arrows; the movement of various forms of ubiquinone, with black arrows. (a) In the first stage, Q on the side is reduced to the semiquinone radical, which moves back into position (dotted line) to accept another electron. (b) In the second stage, the semiquinone radical is converted to QH2. Meanwhile, on the side of the membrane, two molecules of QH2 are oxidized to Q, releasing two protons per Q molecule (four protons in all) into the intermembrane space. Each QH2 donates one electron (via the Rieske Fe-S center) to cytochrome c1, and one electron (via cytochrome b) to a molecule of Q near the side, reducing it in two steps to QH2. This reduction also consumes two protons per Q, which are taken up from the matrix ( side). Reduced cytochrome c1 passes electrons one at a time to cytochrome c, which dissociates and carries electrons to Complex IV. In each cycle, one reduction of Q at the QN site is coupled with two oxidations of QH2 at the QP site by consuming two protons from the matrix and releasing four protons into the intermembrane space. The Q cycle is most easily understood as occurring in two stages, with two active sites where ubiquinone is either oxidized or reduced. In both stages, one QH2 is oxidized at active site 1, shedding two H+ and two electrons. The protons are released into the intermembrane space. The two electrons take different paths, with one reducing cytochrome c and the other reducing a molecule of Q at active site 2. Two electrons are required at active site 2 to fully reduce the Q to QH2, one in each stage. Reducing one Q at one site while oxidizing two QH2 at another may seem counterproductive at ﬁrst glance. However, the two processes have complementary functions. The oxidation of two QH2 is moving four protons to the intermembrane space and two electrons to cytochrome c. At the same time, the reduction of Q at the other site (using the other two electrons from the oxidation of QH2 at site 1) is pulling protons from the matrix, creating a net movement of protons from the matrix to the intermembrane space. The QH2 produced at active site 2 becomes a substrate for oxidation at active site 1 in subsequent turns of the cycle, and vice versa. The net equation for the redox reactions of the Q cycle is QH2+ 2 cyt c (oxidized)+ 2H+N → Q+ 2 cyt c (reduced)+ 4H+P (19-3) The Q cycle accommodates the switch between the two-electron carrier ubiquinol (the reduced form of ubiquinone) and the one-electron carriers — hemes bL and bH of cytochrome b, and cytochromes c1 and c — and results in the uptake of two protons on the N side and the release of four protons on the P side, per pair of electrons passing through Complex III to cytochrome c. Two of the protons released on the P side are electrogenic; the other two are electroneutral, balanced by the two charges (electrons) passed to cytochrome c on the P side. Although the path of electrons through this segment of the respiratory chain is complicated, the net effect of the transfer is simple: QH2 is oxidized to Q, two molecules of cytochrome c are reduced, and two protons are moved from the N side to the P side of the inner mitochondrial membrane. Cytochrome c is a soluble protein of the intermembrane space, which associates reversibly with the P side of the inner membrane. Aer its single heme accepts an electron from Complex III, cytochrome c moves in the intermembrane space to Complex IV to donate the electron to a binuclear copper center. Complex IV: Cytochrome c to O2 In the ﬁnal step of the respiratory chain, Complex IV, also called cytochrome oxidase, carries electrons from cytochrome c to molecular oxygen, reducing it to H2O.

Complex IV is a large, dimeric enzyme of the inner mitochondrial membrane, each monomer having 13 subunits and Mr of 204,000. Bacteria contain a form that is much simpler, with only 3 or 4 subunits per monomer, but still capable of catalyzing both electron transfer and proton pumping. Comparison of the mitochondrial and bacterial complexes suggests that these 3 subunits have been conserved in evolution; in multicellular organisms, the other 10 subunits contribute to the assembly or stability of Complex IV (Fig. 19-12). FIGURE 19-12 Structure of Complex IV (cytochrome oxidase). (a) This complex (bovine) has 13 subunits in each identical monomer of its dimeric structure. Subunit I has two heme groups, a and a3, near a single copper ion, CuB (not visible here). Heme a3 and CuB form a binuclear Fe-Cu center. Subunit II contains two Cu ions complexed with the —SH groups of two Cys residues in a binuclear center, CuA, that resembles the 2Fe-2S centers of iron-sulfur proteins. This binuclear center and the cytochrome c–binding site are located in a domain of subunit II that protrudes from the side of the inner membrane (into the intermembrane space). Subunit III is essential for rapid proton movement through subunit II. The roles of the other 10 subunits in mammalian Complex IV are not fully understood, although some function in assembly or stabilization of the complex. (b) The binuclear center of CuA. The Cu ions (blue spheres) share electrons equally. When the center is reduced, the ions have the formal charges Cu1+Cu1+; when oxidized, Cu1.5+Cu1.5+. Six amino acid residues are ligands around the Cu ions: Glu, Met, two His, and two Cys. [Data from PDB ID 1OCC, T. Tsukihara et al., Science 272:1136, 1996.] Subunit II of Complex IV contains two Cu ions complexed with the —SH groups of two Cys residues in a binuclear center (CuA; Fig. 19-12b) that resembles the 2Fe-2S centers of iron-sulfur proteins. Subunit I contains two heme groups, designated a and a3, and another copper ion (CuB). Heme a3 and CuB form a second binuclear center that accepts electrons from heme a and transfers them to O2 bound to heme a3. The detailed role of subunit III is not clear, but its presence is essential to Complex IV function. Electron transfer through Complex IV is from cytochrome c to the CuA center, to heme a, to the heme a3–CuB center, and ﬁnally to O2 (Fig. 19-13a). For every four electrons passing through this complex, the enzyme consumes four “substrate” H+ from the matrix (N side) in converting O2 to two H2O. It also uses the energy of this redox reaction to pump four protons outward into the intermembrane space (P side) for each four electrons that pass through, adding to the electrochemical potential produced by redox-driven proton transport through Complexes I and III. The overall reaction catalyzed by Complex IV is 4 cyt c(reduced)+ 8H+N+ O2 → 4 cyt c (oxidized)+ 4H+P + 2H2O (19-4) FIGURE 19-13 Path of electrons through Complex IV. (a) For simplicity, only one monomer of the dimeric bovine Complex IV is shown. The three proteins critical to electron flow are subunits I, II, and III. The larger green structure includes the other 10 proteins in each monomer of the dimeric complex. Electron transfer through Complex IV begins with cytochrome c (top). Two molecules of reduced cytochrome c each donate an electron to the binuclear center CuA. From here, electrons pass through heme a to the Fe-Cu center (heme a3 and CuB). Oxygen now binds to heme a3 and is reduced to its peroxy derivative (O2−2; not shown here) by two electrons from the Fe-Cu center. Delivery of two more electrons from cytochrome c (top), for a total of four electrons, converts the O2−2 to two molecules of water, with consumption of four “substrate” protons from the matrix. At the same time, four protons are pumped from the matrix for every four electrons passing through Complex IV. A simplified reaction sequence is presented in (b). Intermediate complexes O, R, A, PR, F, and H represent only a prominent subset of the species for which there is experimental evidence, with some steps and intermediate structures still being debated. The four electrons are introduced in separate steps, and H2O is released in two separate steps. Note that the O2 in this reaction is the ﬁnal acceptor for electrons originating from the many sources already described, and the stoichiometries between electron sources and O2 molecules consumed help to deﬁne the energetics of the systems. In this chapter, stoichiometries are sometimes presented, as here, in terms of one molecule of O2. For calculation simplicity, the stoichiometries will be presented in terms of ½O2 in some examples to come. At Complex IV, O2 is reduced at redox centers that carry only one electron at a time. A reaction scheme is presented in Figure 19-13b. Normally the incompletely reduced oxygen intermediates remain tightly bound to the complex until completely converted to water. However, a small fraction of oxygen intermediates escape. These intermediates are reactive oxygen species that can damage cellular components unless eliminated by defense mechanisms described below. Mitochondrial Complexes Associate inRespirasomes Although the four electron-transferring complexes can be separated in the laboratory, in the intact mitochondrion, three of the four respiratory complexes associate with each other in the inner membrane. Combinations of Complexes I and III, III and IV, and I, III, and IV are formed in organisms ranging from yeast to plants to mammals. The supercomplex containing Complexes I, III, and IV has been called the respirasome. Unlike the other three complexes, Complex II is generally found free-ﬂoating within the membrane. Structural characterization of the various supercomplexes has been advanced by cryo-EM (Fig. 19-14). The functional signiﬁcance of supercomplexes has not been determined. Researchers have suggested that they may facilitate electron transfers or limit the production of reactive oxygen species. Local pools of the electron carriers cytochrome c and ubiquinone are not constrained within supercomplexes, but instead readily diffuse between them. FIGURE 19-14 A respirasome composed of Complexes I, III, and IV. (a) Purified supercomplexes containing Complexes III and IV (from yeast), as determined by cryo-EM. (b) The structure of a respirasome composed of mammalian (porcine and bovine) Complexes I, III, and IV. Two views are shown. [Data from (a) PDB ID 6GIQ, S. Rathore et al., Nat. Struct. Mol. Biol. 26:50, 2019; (b) PDB ID 5GPN, J. Gu et al., Nature 537:639, 2016.] Other Pathways Donate Electrons to theRespiratory Chain via Ubiquinone Several other electron-transfer reactions can reduce ubiquinone in the inner mitochondrial membrane (Fig. 19-15). In the ﬁrst step of the β oxidation of fatty acyl–CoA, catalyzed by the ﬂavoprotein acyl-CoA dehydrogenase (see Fig. 17-8), electrons pass from the substrate to the FAD of the dehydrogenase, then to electron-transferring ﬂavoprotein (ETF). ETF passes its electrons to ETF: ubiquinone oxidoreductase, which reduces Q in the inner mitochondrial membrane to QH2. Glycerol 3-phosphate, formed either from glycerol released by triacylglycerol breakdown or from the reduction of dihydroxyacetone phosphate from glycolysis, is oxidized by glycerol 3- phosphate dehydrogenase (see Fig. 17-4), a ﬂavoprotein located on the outer face of the inner mitochondrial membrane. The electron acceptor in this reaction is Q; the QH2 produced enters the pool of QH2 in the membrane. 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. 19-32). Dihydroorotate dehydrogenase, which acts in the synthesis of pyrimidines (see Fig. 22-38), is also on the outside of the inner mitochondrial membrane and donates electrons to Q in the respiratory chain. The reduced QH2 passes its electrons through Complex III and ultimately to O2. FIGURE 19-15 Paths of electron transfer to ubiquinone in the respiratory chain. Electrons from NADH in the matrix pass through the FMN of a flavoprotein (NADH dehydrogenase) to a series of Fe-S centers (in Complex I) and then to Q. Electrons from succinate oxidation in the citric acid cycle pass through a flavoprotein with several Fe-S centers (Complex II) on the way to Q. Acyl-CoA dehydrogenase, the first enzyme of fatty acid β oxidation, transfers electrons to electron-transferring flavoprotein (ETF), from which they pass to Q via ETF:ubiquinone oxidoreductase. Dihydroorotate, an intermediate in the biosynthetic pathway to pyrimidine nucleotides, donates two electrons to Q through a flavoprotein (dihydroorotate dehydrogenase). And glycerol 3-phosphate, an intermediate of glycolysis in the cytosol, donates electrons to a flavoprotein (glycerol 3-phosphate dehydrogenase) on the outer face of the inner mitochondrial membrane, from which they pass to Q.QH2 freely diﬀuses through the membrane (black dashed arrow), and can interact with several additional complexes. The Energy of Electron Transfer Is EﬀicientlyConserved in a Proton Gradient The transfer of two electrons from NADH through the respiratory chain to molecular oxygen can be summarized as 2 NADH + 2H+ + O2 → 2 NAD+ + 2H2O (19-5) This net reaction is highly exergonic. For the redox pair NAD+/NADH, E′° is −0.320V, and for the pair O2/H2O, E′° is 0.816V. The ΔE′° for this reaction is therefore 1.14 V, and the standard free-energy change (see Eqn 13-7, p. 492) is ΔG′°=−nFΔE′° =−2(96.5kJ/V∙mol)(1.14V) =−220kJ/mol(ofNADH) (19-6) This standard free-energy change is based on the assumption of equal concentrations (1 M) of NADH and NAD+. In actively respiring mitochondria, the actions of many dehydrogenases keep the actual [NADH]/[NAD+] ratio well above unity, and the real free-energy change for the reaction shown in Equation 19-5 is therefore substantially greater (more negative) than −220kJ/mol. A similar calculation for the oxidation of succinate shows that electron transfer from succinate (E′°forfumarate/succinate= 0.031V) to O2 has a smaller, but still negative, standard free-energy change of about −150kJ/mol. Much of this energy is used to pump protons out of the matrix. For each pair of electrons transferred to O2, four protons are pumped out by Complex I, four by Complex III, and two by Complex IV (Fig. 19-16). The vectorial equation for the process is therefore 2NADH + 22H+N+ O2 → 2NAD+ + 20H+P + 2H2O (19-7) FIGURE 19-16 Summary of the flow of electrons and protons through the four complexes of the respiratory chain. Electrons reach Q through Complexes I and II (as well as through several other paths shown in Fig. 19-15). Reduced Q (QH2) serves as a mobile carrier of electrons and protons. It passes electrons to Complex III, which passes them to another mobile connecting link, cytochrome c. Complex IV then transfers electrons from reduced cytochrome c to O2. Electron flow through Complexes I, III, and IV is accompanied by proton eﬀlux from the matrix into the intermembrane space. In bovine heart, the approximate ratios of Complexes I:II:III:IV are 1.1:1.3:3.0:6.7. Broken lines indicate the diﬀusion of Q in the plane of the inner membrane, and of cytochrome c through the intermembrane space. [Data from Complex I: PDB ID 4HEA, R. Baradaran et al., Nature 494:443, 2013; Complex II: PDB ID 1ZOY, F. Sun et al., Cell 121:1043, 2005; Complex III: PDB ID 1BGY, S. Iwata et al., Science 281:64, 1998; cytochrome c: PDB ID 1HRC, G. W. Bushnell et al., J. Mol. Biol. 214:585, 1990; Complex IV: PDB ID 1OCC, T. Tsukihara et al., Science 272:1136, 1996.] The electrochemical energy inherent in this difference in proton concentration and separation of charge represents a temporary conservation of much of the energy of electron transfer. The energy stored in such a gradient, termed the proton-motive force, has two components: (1) the chemical potential energy due to the difference in concentration of a chemical species (H+) in the two regions separated by the membrane, and (2) the electrical potential energy that results from the separation of charge when a proton moves across the membrane without a counterion (Fig. 19-17). FIGURE 19-17 Proton-motive force. The inner mitochondrial membrane separates two compartments of diﬀerent [H+], resulting in diﬀerences in chemical concentration (ΔpH) and charge distribution (Δψ) across the membrane. The net eﬀect is the proton-motive force (ΔG), which can be calculated as shown here. As we saw in Chapter 11, the free-energy change for the creation of an electrochemical gradient by an ion pump is ΔG= RT ln(C2/C1)+ ZFΔψ (19-8) where C2 and C1 are the concentrations of an ion in two regions, and C2> C1; Z is the absolute value of its electrical charge (1 for a proton); and Δψ is the transmembrane difference in electrical potential, measured in volts. For protons, ln(C2/C1)= 2.3(log[H+]P −log[H+]N) = 2.3(pHN−pHP)= 2.3 ΔpH and Equation 19-8 reduces to ΔG= 2.3RTΔpH + FΔψ (19-9) In actively respiring mitochondria, the measured Δψ is 0.15 to 0.20 V, and the pH of the matrix is about 0.75 units more alkaline than that of the intermembrane space. WORKED EXAMPLE 19-1 Energetics ofElectron Transfer Calculate the amount of energy conserved in the proton gradient across the inner mitochondrial membrane per pair of electrons transferred through the respiratory chain from NADH to oxygen. Assume Δψ is 0.15 V and the pH difference is 0.75 unit at body temperature of 37 °C. SOLUTION: Equation 19-9 gives the free-energy change when one mole of protons moves across the inner membrane. Substituting the values of the constants R and F, 310 K for T, and the measured values for ΔpH (0.75 unit) and Δψ (0.15 V) in this equation gives ΔG= 19kJ/mol (of protons). Because the transfer of two electrons from NADH to O2 is accompanied by the outward pumping of 10 protons (Eqn 19-7), roughly 190 kJ (of the 220 kJ released by oxidation of 1 mol of NADH) is conserved in the proton gradient. When protons ﬂow spontaneously down their electrochemical gradient, energy is made available to do work. In mitochondria, chloroplasts, and aerobic bacteria, the electrochemical energy in the proton gradient drives the synthesis of ATP from ADP and Pi. We return to the energetics and stoichiometry of ATP synthesis driven by the electrochemical potential of the proton gradient in Section 19.2. Reactive Oxygen Species Are Generatedduring Oxidative Phosphorylation Several steps in the path of oxygen reduction in mitochondria have the potential to produce reactive oxygen species (superoxide, hydrogen peroxide, and hydroxyl radicals) that can damage cells. Some intermediates in the electron-transfer system, such as the partially reduced ubisemiquinone, can react directly with oxygen to form the superoxide radical (∙Q−) as an intermediate. The ∙Q− radical is formed when a single electron is passed to O2 in the reaction O2+ e− → ∙O−2 Successive reduction of the superoxide radical with additional electrons produces H2O2, hydroxyl radicals (∙OH), and ﬁnally H2O. The very reactive hydroxyl radical can be especially damaging (Fig. 19-18). FIGURE 19-18 ROS formation in mitochondria and mitochondrial defenses. When the rate of electron entry into the respiratory chain and the rate of electron transfer through the chain are mismatched, superoxide radical (∙O−2) production increases at Complexes I and III as the partially reduced ubiquinone radical (∙Q−) donates an electron to O2. Superoxide acts on aconitase, a 4Fe-4S protein (not shown), to release Fe2+. Fe2+ in turn will react with hydrogen peroxide (in a process called the Fenton reaction) to produce the highly reactive hydroxyl free radical (∙OH). The reactions shown in blue defend the cell against the damaging eﬀects of superoxide. Reduced glutathione (GSH; see Fig. 22-29) donates electrons for the reduction of H2O2 and of the oxidized Cys residues (—S—S—) of enzymes and other proteins; GSH is regenerated from the oxidized form (GSSG) by reduction with NADPH. Reactive oxygen species (ROS) can wreak havoc, reacting with and damaging enzymes, membrane lipids, and nucleic acids. In actively respiring mitochondria, 0.2% to as much as 2% of the O2 used in respiration forms ∙O−2 — more than enough to have lethal effects unless the free radical is quickly disposed of. Factors that slow the ﬂow of electrons through the respiratory chain increase the formation of superoxide, perhaps by prolonging the lifetime of ∙O−2 generated in the Q cycle. The formation of ROS is favored when two conditions are met: (1) mitochondria are not making ATP (for lack of ADP or O2) and therefore have a large proton-motive force and a high QH2/Q ratio, and (2) there is a high NADH/NAD+ ratio in the matrix. In these situations, the mitochondrion is under oxidative stress — more electrons are available to enter the respiratory chain than can be immediately passed through to oxygen. When the supply of electron donors (NADH) is matched with that of electron acceptors, there is less oxidative stress, and ROS production is much reduced. Although overproduction of ROS is clearly detrimental, low levels of ROS are used by the cell as a signal reﬂecting the insufﬁcient supply of oxygen (hypoxia), triggering metabolic adjustments (see Fig. 19-34). To prevent oxidative damage by ∙O−2, cells have the enzyme superoxide dismutase, which catalyzes the reaction 2∙O−2+ 2H+ → H2O2+ O2 The hydrogen peroxide (H2O2) thus generated is rendered harmless by glutathione peroxidase (Fig. 19-18). Glutathione reductase recycles the oxidized glutathione to its reduced form, using electrons from the NADPH generated by nicotinamide nucleotide transhydrogenase (in the mitochondrion) or by the pentose phosphate pathway (in the cytosol; see Fig. 14-30). Reduced glutathione also serves to keep protein sulfhydryl groups in their reduced state, preventing some of the deleterious effects of oxidative stress. SUMMARY 19.1 The MitochondrialRespiratory Chain Chemiosmotic theory provides the intellectual framework for understanding many biological energy transductions, including oxidative phosphorylation and photophosphorylation. The energy of electron ﬂow is conserved by the concomitant pumping of protons across the membrane, producing an electrochemical gradient, the proton-motive force. In mitochondria, hydride ions removed from substrates (such as α - ketoglutarate and malate) by NAD-linked dehydrogenases donate electrons to the respiratory chain, which transfers the electrons to molecular O2, reducing it to H2O. Reducing equivalents from NADH are passed through a series of carrier types that include ubiquinone, Fe-S centers, and cytochromes. Electron carriers are arranged in multiprotein complexes, which couple electron transfer to the generation of proton gradients. Complex I transfers electrons from NADH to ubiquinone. Complex II harvests electrons from the oxidation of succinate, also transferring them to ubiquinone. Ubiquinone diffuses through the inner mitochondrial membrane, and transfers the electrons to cytochrome b, the ﬁrst carrier in Complex III. In this complex, electrons move ﬁrst to an Fe-S center. The Fe-S center passes electrons, one at a time, through cytochrome c and into Complex IV, cytochrome oxidase. This copper-containing enzyme, which also contains cytochromes a and a3, accumulates electrons, then passes them to O2, reducing it to H2O. Complexes I, III, and IV form supercomplexes that facilitate electron ﬂow between them. Some electrons enter this chain of carriers through alternative paths. Electrons derived from the oxidation of fatty acids pass to ubiquinone via the electron-transferring ﬂavoprotein. The oxidation of glycerol phosphate and of dihydroorotate also sends electrons into the respiratory chain at the level of QH2. Much of the free energy generated by electron transfer and the reduction of oxygen to form water is recovered and stored in the form of an electrochemical proton gradient across the mitochondrial inner membrane. Potentially harmful reactive oxygen species produced in mitochondria are inactivated by a set of protective enzymes, including superoxide dismutase and glutathione peroxidase. Low levels of ROS serve as signals coordinating mitochondrial oxidative phosphorylation with other metabolic pathways. 19.2 ATP Synthesis How is a concentration gradient of protons transformed into ATP? We have seen that electron transfer releases, and the proton- motive force conserves, more than enough free energy (about 190 kJ) per “mole” of electron pairs to drive the formation of a mole of ATP, which requires about 50 kJ (p. 480). Mitochondrial oxidative phosphorylation therefore poses no thermodynamic problem. But what is the chemical mechanism that couples proton ﬂux with phosphorylation? In the Chemiosmotic Model, Oxidation and Phosphorylation Are Obligately Coupled The chemiosmotic model, proposed by Peter Mitchell, is the paradigm for energy coupling. According to the model (Fig. 19- 19), the electrochemical energy inherent in the difference in proton concentration and the separation of charge across the inner mitochondrial membrane — the proton-motive force — drives the synthesis of ATP as protons ﬂow passively back into the matrix through a proton pore in ATP synthase. To emphasize this crucial role of the proton-motive force, the equation for ATP synthesis is sometimes written AD P + Pi+ nH+P → AT P + H2O + nH+N (19-10) FIGURE 19-19 Chemiosmotic model. In this simple representation of the chemiosmotic theory applied to mitochondria, electrons from NADH and other oxidizable substrates pass through a chain of carriers arranged asymmetrically in the inner membrane. Electron flow is accompanied by proton transfer across the membrane, producing both a chemical gradient (ΔpH) and an electrical gradient (Δψ), which, combined, create the proton-motive force. The inner mitochondrial membrane is impermeable to protons; protons can reenter the matrix only through proton-specific channels (Fo). The proton- motive force that drives protons back into the matrix provides the energy for ATP synthesis, catalyzed by the F1 complex associated with Fo.

Peter Mitchell, 1920–1992 Mitchell used the term “chemiosmotic” to describe enzymatic reactions that involve, simultaneously, a chemical reaction and a transport process, and the overall process is sometimes referred to as “chemiosmotic coupling.” Here, “coupling” refers to the obligate connection between mitochondrial ATP synthesis and electron ﬂow through the respiratory chain; neither of the two processes can proceed without the other. The operational deﬁnition of coupling is shown in Figure 19-20. When isolated mitochondria are suspended in a buffer containing ADP, Pi, and an oxidizable substrate such as succinate, three easily measured processes occur: (1) the substrate is oxidized (succinate yields fumarate), (2) O2 is consumed, and (3) ATP is synthesized. Oxygen consumption and ATP synthesis depend on the presence of an oxidizable substrate (succinate in this case) as well as ADP and Pi. FIGURE 19-20 Coupling of electron transfer and ATP synthesis in mitochondria. In experiments to demonstrate coupling, mitochondria are suspended in a buﬀered medium, and an O2 electrode monitors O2 consumption. At intervals, samples are removed and assayed for the presence of ATP. (a) Addition of ADP and Pi alone results in little or no increase in either respiration (O2 consumption; black) or ATP synthesis (red). When succinate is added, respiration begins immediately, and ATP is synthesized. Addition of cyanide (CN−), which blocks electron transfer between cytochrome oxidase (Complex IV) and O2, inhibits both respiration and ATP synthesis. (b) Mitochondria provided with succinate respire and synthesize ATP only when ADP and Pi are added. Subsequent addition of venturicidin or oligomycin, inhibitors of ATP synthase, blocks both ATP synthesis and respiration. Dinitrophenol (DNP) is an uncoupler, allowing respiration to continue without ATP synthesis. Because substrate oxidation drives ATP synthesis, inhibitors of electron transfer block ATP synthesis (Fig. 19-20a). The converse is also true: inhibition of ATP synthesis blocks electron transfer in intact mitochondria. When isolated mitochondria are given O2 and oxidizable substrates, but not ADP (Fig. 19-20b), no ATP synthesis can occur and electron transfer to O2 does not proceed. Henry Lardy, who pioneered the use of antibiotics to explore mitochondrial function, showed coupling of oxidation and phosphorylation by using oligomycin and venturicidin. These toxic antibiotics bind to the ATP synthase in mitochondria, inhibiting both ATP synthesis and the transfer of electrons through the chain of carriers to O2 (Fig. 19-20b). As oligomycin does not interact with the electron carriers, it follows that electron transfer and ATP synthesis are obligately coupled: neither reaction occurs without the other. Henry Lardy, 1917–2010 Chemiosmotic theory readily explains the dependence of electron transfer on ATP synthesis in mitochondria. When the ﬂow of protons into the matrix through the proton channel of ATP synthase is blocked (with oligomycin, for example), no path exists for the return of protons to the matrix, and the continued extrusion of protons driven by the activity of the respiratory chain generates a large proton gradient. The proton-motive force builds up until the cost (free energy) of pumping protons out of the matrix against this gradient equals or exceeds the energy released by the transfer of electrons from NADH to O2. At this point electron ﬂow must stop; the free energy for the overall process of electron ﬂow coupled to proton pumping becomes zero, and the system is at equilibrium. Certain conditions and reagents, however, can uncouple oxidation from phosphorylation. When intact mitochondria are disrupted by treatment with detergent or by physical shear, the resulting membrane fragments can still catalyze electron transfer from succinate or NADH to O2, but no ATP synthesis is coupled to this respiration. Certain chemical compounds cause uncoupling without physically disrupting mitochondrial structure. Chemical uncouplers include 2,4-dinitrophenol (DNP) and carbonylcyanide- p-triﬂuoromethoxyphenylhydrazone (FCCP) (Table 19-4; Fig. 19- 21), weak acids with hydrophobic properties that permit them to diffuse readily across mitochondrial membranes. Aer entering the matrix in the protonated form, they can release a proton, thus dissipating the proton gradient. Resonance stabilization delocalizes the charge on the anionic forms, making them sufﬁciently hydrophobic to diffuse back across the membrane, where they can pick up a proton and repeat the process. Ionophores such as valinomycin (see Fig. 11-43) allow inorganic ions to pass easily through membranes. Ionophores uncouple electron transfer from oxidative phosphorylation by dissipating the electrical contribution to the electrochemical gradient across the mitochondrial membrane. FIGURE 19-21 Two chemical uncouplers of oxidative phosphorylation. Both DNP and FCCP have a dissociable proton (red) and are very hydrophobic. They carry protons across the inner mitochondrial membrane, dissipating the proton gradient. Both also uncouple photophosphorylation. A prediction of the chemiosmotic theory is that, because the role of electron transfer in mitochondrial ATP synthesis is simply to pump protons to create the electrochemical potential of the proton-motive force, an artiﬁcially created proton gradient should be able to replace electron transfer in driving ATP synthesis. This has been experimentally conﬁrmed (Fig. 19-22). In the absence of an oxidizable substrate, the proton-motive force alone sufﬁces to drive ATP synthesis. FIGURE 19-22 Evidence for the role of a proton gradient in ATP synthesis. An artificially imposed electrochemical gradient can drive ATP synthesis in the absence of an oxidizable substrate as electron donor. In this two-step experiment, (a) isolated mitochondria are first incubated in a pH 9 buﬀer containing 0.1 KCl. Slow leakage of buﬀer and KCl into the mitochondria eventually brings the matrix into equilibrium with the surrounding medium. No oxidizable substrates are present. (b) Mitochondria are now removed from the pH 9 buﬀer and resuspended in pH 7 buﬀer containing valinomycin but no KCl. The change in buﬀer creates a diﬀerence of two pH units across the inner mitochondrial membrane. The outward flow of K+, carried by valinomycin down the K+ ion concentration gradient without a counterion, creates a charge imbalance across the membrane (matrix negative). The sum of the chemical potential provided by the pH diﬀerence and the electrical potential provided by the separation of charges is a proton-motive force large enough to support ATP synthesis in the absence of an oxidizable substrate. ATP Synthase Has Two Functional Domains, F0 and F1 Mitochondrial ATP synthase is an F-type ATPase (see Fig. 11-40b) similar in structure and mechanism to the ATP synthases of bacteria and (as we will see in Chapter 20) chloroplasts. This large enzyme complex of the inner mitochondrial membrane catalyzes the formation of ATP from ADP and Pi, driven by the ﬂow of protons from the P to the N side of the membrane (Eqn 19- 10). ATP synthase, also called Complex V to relate it to the electron-transfer complexes described in the last section, has two distinct components. These are F1, a peripheral membrane protein, and Fo (o denoting oligomycin-sensitive), which is integral to the membrane. F1, the ﬁrst factor recognized as essential for oxidative phosphorylation, was identiﬁed and puriﬁed by Efraim Racker and his colleagues in the early 1960s. In the laboratory, small membrane vesicles formed from inner mitochondrial membranes carry out ATP synthesis coupled to electron transfer. When F1 is gently extracted, the “stripped” vesicles still contain intact respiratory chains and the Fo portion of ATP synthase. The vesicles can catalyze electron transfer from NADH to O2 but cannot produce a proton gradient: Fo has a proton pore through which protons leak as fast as they are pumped by electron transfer, and without a proton gradient the F1-depleted vesicles cannot make ATP. Isolated F1 catalyzes ATP hydrolysis (the reversal of synthesis) and was therefore originally called F1 ATPase. When puriﬁed F1 is added back to the depleted vesicles, it reassociates with Fo, plugging its proton pore and restoring the membrane’s capacity to couple electron transfer and ATP synthesis. ATP Is Stabilized Relative to ADP on the Surface of F1 Isotope exchange experiments using puriﬁed F1 reveal an extraordinary fact about the enzyme’s catalytic mechanism: on the enzyme surface, the reaction AD P + Pi ⇌ AT P + H2O is readily reversible — the free-energy change for ATP synthesis is close to zero. When ATP is hydrolyzed by F1 in the presence of 18O -labeled water, the Pi released contains an 18O atom. Careful measurement of the 18O content of Pi formed in vitro by F1- catalyzed hydrolysis of ATP reveals that the Pi has not one but three or four 18O atoms (Fig. 19-23). This indicates that the terminal pyrophosphate bond in ATP is cleaved and re-formed repeatedly before Pi leaves the enzyme surface. This exchange reaction occurs in unenergized FoF1 complexes (with no proton gradient) and with isolated F1 — the exchange does not require the input of energy.

FIGURE 19-23 Catalytic mechanism of F1. (a) An 18O-exchange experiment. F1 solubilized from mitochondrial membranes is incubated with ATP in the presence of 18O-labeled water. At intervals, a sample of the solution is withdrawn and analyzed for the incorporation of 18O into the Pi produced from ATP hydrolysis. In minutes, the Pi contains three or four 18O atoms, indicating that both ATP hydrolysis and ATP synthesis have occurred several times during the incubation. (b) The likely transition state complex for ATP hydrolysis and synthesis by ATP synthase. The α subunit is shown in gray, β in purple. The positively charged residues β-Arg182 and α-Arg376 coordinate two oxygens of the pentavalent phosphate intermediate; β-Lys155 interacts with a third oxygen, and the M g2+ ion further stabilizes the intermediate. The blue sphere represents the leaving group (H2O). These interactions result in ready equilibration of ATP and AD P + Pi in the active site. [(b) Data from PDB ID 1BMF, J. P. Abrahams et al., Nature 370:621, 1994.] Kinetic studies of the initial rates of ATP synthesis and hydrolysis conﬁrm the conclusion that ΔG ′° for ATP synthesis on the enzyme is near zero. From the measured rates of hydrolysis (k1= 10s−1) and synthesis (k−1= 24s−1), the calculated equilibrium constant for the reaction Enz-AT P ⇌ Enz-(AD P + Pi) is K ′eq = = = 2.4k−1 k1 24 s−1 10 s−1 From this K ′eq, the calculated apparent ΔG ′° is close to zero. This is much different from the K ′eq of about 105(ΔG ′°= −30.5kJ /mol) for the hydrolysis of ATP free in solution (i.e., not on the enzyme surface). What accounts for the huge difference? ATP synthase stabilizes ATP relative to AD P + Pi by binding ATP more tightly, releasing enough energy to counterbalance the cost of making ATP. Careful measurements of the binding constants show that FoF1 binds ATP with very high afﬁnity (Kd ≤ 10−12M ) and ADP with much lower afﬁnity (Kd ≈ 10−5M ). The difference in Kd corresponds to a difference of about 40 kJ/mol in binding energy, and this binding energy drives the equilibrium toward formation of the product ATP. The Proton Gradient Drives the Release of ATP from the Enzyme Surface Although ATP synthase equilibrates ATP with AD P + Pi, in the absence of a proton gradient the newly synthesized ATP does not leave the surface of the enzyme. Effectively, the enzyme cannot turn over and synthesize a second molecule of ATP. It is the proton gradient that causes the enzyme to release the ATP formed on its surface. The reaction coordinate diagram of the process (Fig. 19-24) illustrates the difference between the mechanism of ATP synthase and that of many other enzymes that catalyze endergonic reactions. FIGURE 19-24 Reaction coordinate diagrams for ATP synthase and for a more typical enzyme. In a typical enzyme-catalyzed reaction (le ), reaching the transition state (‡) between substrate and product is the major energy barrier to overcome. In the reaction catalyzed by ATP synthase (right), release of ATP from the enzyme, not formation of ATP, is the major energy barrier. The free-energy change for the formation of ATP from ADP and Pi in aqueous solution is large and positive, but on the enzyme surface, the very tight binding of ATP provides suﬀicient binding energy to bring the free energy of the enzyme-bound ATP close to that of AD P + Pi, so the reaction is readily reversible. The equilibrium constant is near 1. The free energy required for the release of ATP is provided by the proton-motive force. For the continued synthesis of ATP, the enzyme must cycle between a form that binds ATP very tightly and a form that releases ATP. Chemical and crystallographic studies of the ATP synthase have revealed the structural basis for this alternation in function. Each β Subunit of ATP Synthase Can Assume Three Diﬀerent Conformations Mitochondrial F1 has nine subunits of ﬁve different types, with the composition α3β3γδε. Each of the three β subunits has one catalytic site for ATP synthesis. The crystallographic determination of the F1 structure by John E. Walker and colleagues revealed structural details that help explain the catalytic mechanism of the enzyme. The knoblike portion of F1 is a ﬂattened sphere, 8 nm by 10 nm, consisting of alternating α and β subunits arranged like the sections of an orange (Fig. 19- 25a–d). Although the amino acid sequences of the three β subunits are identical, their conformations diﬀer. The conformational differences extend to differences in their ATP/ADP-binding sites. When the protein is crystallized in the presence of ADP and App(NH)p, a close structural analog of ATP that cannot be hydrolyzed by the ATPase activity of F1, the binding site of one of the three β subunits is ﬁlled with App(NH)p, the second is ﬁlled with ADP, and the third is empty. The corresponding β subunit conformations are designated β - ATP, β -ADP, and β -empty (Fig. 19-25b). This difference in nucleotide binding among the three subunits is critical to the mechanism of the complex. The polypeptides that make up the stalk in the F1 crystal structure are asymmetrically arranged. One domain of the single γ subunit makes up a central sha that passes through F1. Another globular domain of γ helps to stabilize the β -empty conformation in a β subunit it is transiently associated with (Fig. 19-25c).

FIGURE 19-25 Mitochondrial ATP synthase complex. (a) A cartoon representation of the FoF1 complex. The dimeric form is found in eukaryotic mitochondria. The monomeric form is observed in bacteria. (b) F1 viewed from above (that is, from the side of the membrane), showing the three β (shades of purple) and three α (shades of gray) subunits and the central sha (γ subunit, green). Each β subunit, near its interface with the neighboring α subunit, has a nucleotide-binding site critical to the catalytic activity. The single γ subunit associates primarily with one of the three αβ pairs, forcing each of the three β subunits into slightly diﬀerent conformations, with diﬀerent nucleotide-binding sites. In the crystalline enzyme, one subunit, β -ADP, has ADP (yellow) in its binding site; the next, β -ATP, has ATP (red); and the third, β -empty, has no bound nucleotide. (c) The entire enzyme viewed from the side (in the plane of the membrane). The F1 portion has three α subunits and three β subunits arranged like the segments of an orange around a central sha , the γ subunit (green). (Two α subunits and one β subunit have been omitted to reveal the γ subunit and the binding sites for ATP and ADP on the β subunits.) The δ subunit confers oligomycin sensitivity on the ATP synthase, and the ε subunit may serve to inhibit the enzyme’s ATPase activity under some circumstances. The Fo subunit consists of one a subunit and two b subunits, which anchor the FoF1 complex in the membrane and act as a stator (the stationary part of a rotary system), holding the α and β subunits in place. Fo also includes the c ring, made up of a number (8 to 17, depending on the species) of identical c subunits, small, hydrophobic proteins. The c ring and the a subunit interact to provide a transmembrane path for protons. Each of the c subunits in Fo has a critical Asp residue near the middle of the membrane, which undergoes protonation/deprotonation during the catalytic cycle of the ATP synthase. Shown here is the homologous c11 ring of the Na+-ATP ase of Ilyobacter tartaricus, for which the structure is well established. The Na+-binding sites, which correspond to the proton-binding sites of the FoF1 complex, are shown with their bound Na+ ions (red spheres). (d) A view of Fo perpendicular to the membrane. As in (c), red spheres represent the Na+- or proton-binding sites in Asp residues. [(a) Information from W. Kühlbrandt and K. M. Davies, Trends Biochem. Sci. 41:106, 2016. (b, c, d) Data from F1: PDB ID 1BMF, J. P. Abrahams et al., Nature 370:621, 1994; PDB ID 1JNV, A. C. Hausrath et al., J. Biol. Chem. 276:47,227, 2001; PDB ID 2A7U, S. Wilkens et al., Biochemistry 44:11,786, 2005; PDB ID 2CLY, V. Kane Dickson et al., EMBO J. 25:2911, 2006; Fo: PDB ID 1B9U, O. Dmitriev et al., J. Biol. Chem. 274:15,598, 1999; c ring: PDB ID 1YCE, T. Meier et al., Science 308:659, 2005.] The Fo complex, with its proton pore, is composed of three subunits, a, b, and c, in the proportion ab2cn, where n ranges from 8 to 17, depending on the species. Subunit c is a small (M r 8,000), very hydrophobic polypeptide, consisting almost entirely of two transmembrane helices, with a small loop extending from the matrix side of the membrane. The crystal structure of the yeast FoF1 shows 10 c subunits, each with two transmembrane helices roughly perpendicular to the plane of the membrane and arranged in two concentric circles to create the c ring. The inner circle is made up of the amino-terminal helices of each c subunit; the outer circle, about 55 Å in diameter, is made up of the carboxyl-terminal helices. The c subunits in the c ring rotate together as a unit around an axis perpendicular to the membrane. The ε and γ subunits of F1 form a leg-and-foot that projects from the bottom (membrane) side of F1 and stands ﬁrmly on the ring of c subunits. The a subunit consists of several hydrophobic helices that span the membrane in close association with one of the c subunits in the c ring. Rotational Catalysis Is Key to the Binding-Change Mechanism for ATP Synthesis On the basis of detailed kinetic and binding studies of the reactions catalyzed by FoF1, Paul Boyer proposed a rotational catalysis mechanism in which the three active sites of F1 take turns catalyzing ATP synthesis (Fig. 19-26). A given β subunit starts in the β -ADP conformation, which binds ADP and Pi from the surrounding medium. The subunit now changes conformation, assuming the β -ATP form that tightly binds and stabilizes ATP, bringing about the ready equilibration of AD P + Pi with ATP on the enzyme surface. Finally, the subunit changes to the β -empty conformation, which has very low afﬁnity for ATP, and the newly synthesized ATP leaves the enzyme surface. Another round of catalysis begins when this subunit again assumes the β -ADP form and binds ADP and Pi. FIGURE 19-26 Binding-change model for ATP synthase. The F1 complex has three nonequivalent adenine nucleotide–binding sites, one for each pair of α and β subunits. At any given moment, one of these sites is in the β -ATP conformation (which binds ATP tightly), a second is in the β -ADP (loose-binding) conformation, and a third is in the β -empty (very-loose- binding) conformation. In this view from the side, the proton-motive force causes rotation of the central sha — the γ subunit, shown as a green arrowhead — which comes into contact with each αβ subunit pair in succession. This produces a cooperative conformational change in which the β -ATP site is converted to the β -empty conformation, and ATP dissociates; the β -ADP site is converted to the β -ATP conformation, which promotes condensation of bound AD P + Pi to form ATP; and the β - empty site becomes a β -ADP site, which loosely binds AD P + Pi entering from the solvent. Note that the direction of rotation reverses when the ATP synthase is acting as an ATPase, as in the experiment depicted in Figure 19- 27. The conformational changes central to this mechanism are driven by the passage of protons through the Fo portion of ATP synthase. The streaming of protons through the Fo pore causes the c ring and the attached γ subunit to rotate about the long axis of γ , which is perpendicular to the plane of the membrane. The γ subunit passes through the center of the α3β3 spheroid, which is held stationary relative to the membrane surface by the b2 and δ subunits (Fig. 19-25a). With each rotation of 120°, γ comes into contact with a different β subunit, and the contact forces that β subunit into the β -empty conformation. The three β subunits interact in such a way that when one assumes the β -empty conformation, its neighbor to one side must assume the β -ADP form, and the other neighbor the β -ATP form. Thus, one complete rotation of the γ subunit causes each β subunit to cycle through all three of its possible conformations, and for each rotation, three ATP are synthesized and released from the enzyme surface. One strong prediction of this binding-change model is that the γ subunit should rotate in one direction when FoF1 is synthesizing ATP and in the opposite direction when the enzyme is hydrolyzing ATP. This prediction of rotation with ATP hydrolysis was conﬁrmed in elegant experiments in the laboratories of Masasuke Yoshida and Kazuhiko Kinosita, Jr. The rotation of γ in a single F1 molecule was observed microscopically by attaching a long, thin, ﬂuorescent actin polymer to γ and watching it move relative to α3β3 immobilized on a microscope slide as ATP was hydrolyzed. (The expected reversal of the rotation when ATP is being synthesized could not be tested in this experiment; there is no proton gradient to drive ATP synthesis.) When the entire FoF1 complex (not just F1) was used in a similar experiment, the entire ring of c subunits rotated with γ (Fig. 19-27). The “sha” rotated in the predicted direction through 360°. The rotation was not smooth but occurred in three discrete steps of 120°. As calculated from the known rate of ATP hydrolysis by one F1 molecule and from the frictional drag on the long actin polymer, the efﬁciency of this mechanism in converting chemical energy into motion is close to 100%. It is, in Boyer’s words, “a splendid molecular machine!” FIGURE 19-27 Experimental demonstration of rotation of Fo and γ. This fundamental property of the ATP synthase reaction was demonstrated in several creative ways. (a) In one experiment, illustrated in this cartoon, F1, genetically engineered to contain a run of His residues, was tightly adhered to a microscope slide coated with a Ni complex; biotin was covalently attached to a c subunit of Fo. The protein avidin, which binds biotin very tightly, was covalently attached to long filaments of actin labeled with a fluorescent probe. Biotin-avidin binding then attached the actin filaments to the c subunit. When ATP was provided as substrate for the ATPase activity of F1, the labeled filament rotated in one direction, proving that the Fo cylinder of c subunits rotates. (b) In another experiment, a fluorescent actin filament was attached directly to the γ subunit. The series of fluorescence micrographs (read le to right) shows the position of the actin filament at intervals of 133 ms. Note that as the filament rotated, it made discrete jumps rather than smooth rotation about the circle. The cylinder and sha move as one unit. [(a) Information from Y. Sambongi et al., Science 286:1722, 1999.] A model that illustrates how proton ﬂow and rotary motion are coupled in the Fo complex is shown in Figure 19-28. The a subunit is stationary, while the c ring rotates. Critical interactions occur between conserved amino acids in the a and c subunits. The individual subunits in the c ring are arranged in a circle with only a few in contact with the a subunit at any moment. Protons diffuse across the membrane through a path made up of both a and c subunits. Transient protonation of a key Glu residue in each c subunit elicits conformation changes that drive rotation and transmit protons between hydrophilic half channels positioned on each side of the membrane. The rotary movement of the c ring is made unidirectional by the large difference in proton concentration across the membrane. The number of protons that must be transferred to produce one complete rotation of the c ring is equal to the number of c subunits in the ring. Structural studies of the c ring have shown that the number of c subunits differs in different organisms (Fig. 19-29). In bovine mitochondria the number is 8, in yeast mitochondria and in Escherichia coli it is 10, and the number of c subunits can range as high as 17, as is seen in the soil bacterium Burkholderia pseudomallei. The rate of rotation in intact mitochondria has been estimated at about 6,000 rpm — 100 rotations per second. FIGURE 19-28 A model for proton-driven rotation of the c ring. The a subunit of the Fo complex of the ATP synthase (see Fig. 19-25a) has two hydrophilic half-channels for protons, one leading from the side to the middle of the membrane, the other leading from the middle of the membrane to the side (matrix). The function of the stationary a subunit is to conduct protons to and from the c ring subunits to drive c ring rotational motion. Individual c subunits in Fo (the total number varies from 8 to 17 in diﬀerent species) are arranged in a circle about a central core. Each c subunit has a critical Glu residue (an Asp in some species) about midway across the membrane, with a perturbed pKa allowing it to donate or accept a proton (red H+) at pH near neutrality. The cycle that c subunits go through is illustrated. One c subunit is initially positioned so that a proton that enters the half-channel on the side (where the proton concentration is relatively high) encounters and protonates a conserved His residue in the a subunit, transferring it to the c subunit Glu residue. This triggers a rotation-facilitating conformational change in the protonated c subunit as the Glu loses its negative charge. The now neutral Glu residue is sequestered in the hydrophobic membrane layer as it rotates as part of the c ring . As the c ring rotates, the c subunit we are following eventually makes contact with the channel to the side of the membrane, where the environment is relatively alkaline, and the proton is released . As the Glu reacquires its negative charge, another rotation-facilitating conformational change occurs such that the Glu interacts transiently with a conserved Arg residue in the immobile a subunit . The interaction with the Arg is disrupted as the Glu is again protonated by the His residue, in motions that again facilitate rotation. The c subunits positioned near the half channels are providing the rotational driving force at any given moment, as they are the ones undergoing conformation changes associated with protonation and deprotonation. The orientation of the proton gradient dictates the direction of proton flow and makes rotation of the c ring essentially unidirectional. [Information from W. Kühlbrandt and K. M. Davies, Trends Biochem. Sci. 41:106, 2016.] FIGURE 19-29 Species diﬀerences in number of c subunits in the c ring of the Fo complex. The structures of the c rings from several species have been determined by x-ray crystallography. Each helix in the inner ring is half of a hairpin-shaped c subunit; the outer ring of helices forms the other half of the hairpin structure. The essential Glu residue (Asp in some species) is shown as a red dot. Views of the c ring perpendicular to the membrane show the number of c subunits for (a) bovine mitochondria (8 subunits) and (b) yeast mitochondria (10). Atomic force microscopy has been used to visualize the c rings of (c) a thermophilic bacterium, Bacillus species TA2.A1 (13 subunits), and (d) spinach (14). According to the model in Figure 19-28, diﬀerent numbers of c subunits in the c ring should result in diﬀerent ratios of ATP formed per pair of electrons passing through the respiratory chain (i.e., diﬀerent P/O ratios). [(a) Data from PDB ID 1OHH, E. Cabezon et al., Nat. Struct. Biol. 10:744, 2003. (c) D. Matthies et al., J. Mol. Biol. 388:611, 2009. (d) H. Seelert et al., Nature 405:418, 2000. Republished with permission of Elsevier from J. Mol. Biol., Matthies et al., Vol. 388(3), ©2009; permission conveyed through Copyright Clearance Center, Inc.] Chemiosmotic Coupling Allows Nonintegral Stoichiometries of O2 Consumption and ATP Synthesis The overall reaction equation for ATP synthesis has the following form: xAD P + xPi+ ½O2+ H+ + NAD H → xAT P + H2O + NAD + (19-11) The value of x is sometimes called the P/O ratio or the P/2e− ratio. When a proton gradient is coupled to ATP synthesis as described above, there is no theoretical requirement for P/O to be integral. The relevant questions about stoichiometry become these: How many protons are pumped outward by electron transfer from one NADH to O2? and How many protons must ﬂow inward through the FoF1 complex to drive the synthesis of one ATP? The measurement of proton ﬂuxes is technically complicated; the investigator must take into account the buffering capacity of mitochondria, nonproductive leakage of protons across the inner membrane, and use of the proton gradient for functions other than ATP synthesis, such as driving the transport of substrates across the inner mitochondrial membrane (described below). When NADH or succinate (which sends electrons into the respiratory chain at the level of ubiquinone) is the oxidizable substrate, the consensus experimental values for number of protons pumped out per pair of electrons are 10 and 6, respectively. The most widely accepted experimental value for number of protons required to drive the synthesis of an ATP molecule is 4, of which 1 is used in transporting Pi, ATP, and ADP across the mitochondrial membrane (see below). If 10 protons are pumped out per NADH and 4 must ﬂow in to produce 1 ATP, the proton-based P/O ratio is 2.5 for NADH as the electron donor and 1.5 (6/4) for succinate. However, as we will see in Worked Example 19-2, the proton stoichiometry of ATP synthesis by ATP synthase depends upon the number of c units in Fo, which ranges from 8 to 17, depending on the species. WORKED EXAMPLE 19-2 Stoichiometry of ATP Production: Eﬀect of c Ring Size (a) If the ATP synthase of bovine mitochondria has 8 c subunits per c ring, what is the predicted ratio of ATP formed per NADH oxidized? (b) What is the predicted value for yeast mitochondria, with 10 c subunits per ATP synthase? (c) What are the comparable values for electrons entering the respiratory chain from FAD H2? SOLUTION: (a) Here we are asked to determine how many ATP molecules are produced per NADH. This is another way of asking us to calculate the P/O ratio, or x, in Equation 19-11. If the c ring has 8 c subunits, then one full rotation will transfer 8 protons to the matrix and produce 3 ATP molecules. But this synthesis also requires the transport of 3 Pi into the matrix, at a cost of 1 proton each, adding 3 more protons to the total number required. This brings the total cost to (11protons)/(3AT P)= 3.7 protons/AT P. The generally agreed value for the number of protons pumped out per pair of electrons transferred from NADH is 10 (Eqn 19-7). So, oxidizing 1 NADH produces (10 protons)/(3.7protons/AT P)= 2.7AT P. (b) If the c ring has 10 c subunits, then one full rotation will transfer 10 protons to the matrix and produce 3 ATP molecules. Adding in the 3 protons to transport the 3 Pi into the matrix brings the total cost to (13protons)/(3AT P)= 4.3protons/AT P. Oxidizing 1 NADH produces (10protons)/(4.3protons/AT P)= 2.3AT P. (c) When electrons enter the respiratory chain from FAD H2 (at ubiquinone), only 6 protons are available to drive ATP synthesis. This changes the calculation for bovine mitochondria to (6protons)/(3.7protons/AT P)= 1.6AT P per pair of electrons from FAD H2. For yeast mitochondria, the calculation is (6protons)/(4.3protons/AT P)= 1.4AT P per pair of electrons from FAD H2. These calculated values of x, or the P/O ratio, deﬁne a range that includes the experimental values of 2.5 AT P/NAD H and 1.5AT P/FAD H2, and we therefore use these values throughout this book. The Proton-Motive Force Energizes Active Transport Although the primary role of the proton gradient in mitochondria is to furnish energy for the synthesis of ATP, the proton-motive force also drives several transport processes essential to oxidative phosphorylation. The inner mitochondrial membrane is generally impermeable to charged species, but two speciﬁc systems transport ADP and Pi into the matrix and ATP out to the cytosol (Fig. 19-30). FIGURE 19-30 Adenine nucleotide and phosphate translocases. Transport systems of the inner mitochondrial membrane carry ADP and Pi into the matrix and newly synthesized ATP into the cytosol. The adenine nucleotide translocase is an antiporter; the same protein moves ADP into the matrix and ATP out. The eﬀect of replacing ATP 4− with AD P3− in the matrix is the net eﬀlux of one negative charge, which is favored by the charge diﬀerence across the inner membrane (outside positive). At pH 7, Pi is present as both HPO2− 4 and H2PO− 4; the phosphate translocase is specific for H2PO− 4. There is no net flow of charge during symport of H2PO− 4 and H+, but the relatively low proton concentration in the matrix favors the inward movement of H+. Thus the proton-motive force is responsible both for providing the energy for ATP synthesis and for transporting substrates (ADP and Pi) into and product (ATP) out of the mitochondrial matrix. All three of these transport systems can be isolated as a single membrane- bound complex (ATP synthasome). The adenine nucleotide translocase, integral to the inner membrane, binds AD P3− in the intermembrane space and transports it into the matrix in exchange for an AT P4− molecule simultaneously transported outward (see Fig. 13-11 for the ionic forms of ATP and ADP). Because this antiporter moves four negative charges out for every three moved in, its activity is favored by the transmembrane electrochemical gradient, which gives the matrix a net negative charge; the proton-motive force drives ATP-ADP exchange. Adenine nucleotide translocase is speciﬁcally inhibited by atractyloside, a toxic glycoside produced by a species of thistle. If the transport of ADP into and ATP out of mitochondria is inhibited, cytosolic ATP cannot be regenerated from ADP, explaining the toxicity of atractyloside. A second membrane transport system essential to oxidative phosphorylation is the phosphate translocase, which promotes symport of one H2PO−4 and one H+ into the matrix. This transport process, too, is favored by the transmembrane proton gradient (Fig. 19-30). Notice that the process requires movement of one proton from the P side to the N side of the inner membrane, consuming some of the energy of electron transfer. A complex of the ATP synthase and both translocases, the ATP synthasome, can be isolated from mitochondria by gentle dissection with detergents, suggesting that the functions of these three proteins are very tightly integrated. ATP and ADP cross the outer mitochondrial membrane via the voltage-dependent anion channel (VDAC), a 19-stranded β barrel with an opening about 27 Å wide, connecting the cytosol and the intermembrane space. Each VDAC, when open, can move 105 ATP molecules per second. The opening is gated by voltage, as its name indicates, and under some conditions VDAC is closed to ATP. Shuttle Systems Indirectly Convey Cytosolic NADH into Mitochondria for Oxidation The NADH dehydrogenase of the inner mitochondrial membrane of animal cells can accept electrons only from NADH in the matrix. Given that the inner membrane is not permeable to NADH, how can the NADH generated by glycolysis in the cytosol be reoxidized to NAD + by O2 via the respiratory chain? Special shuttle systems carry reducing equivalents from cytosolic NADH into mitochondria by an indirect route. The most active NADH shuttle, which functions in liver, kidney, and heart mitochondria, is the malate-aspartate shuttle (Fig. 19-31). The reducing equivalents of cytosolic NADH are ﬁrst transferred to cytosolic oxaloacetate to yield malate, catalyzed by cytosolic malate dehydrogenase. The malate thus formed passes through the inner membrane via the malate–α -ketoglutarate transporter. Within the matrix, the reducing equivalents are passed to NAD + by the action of matrix malate dehydrogenase, forming NADH; this NADH can pass electrons directly to the respiratory chain. About 2.5 molecules of ATP are generated as this pair of electrons passes to O2. Cytosolic oxaloacetate must be regenerated by transamination reactions and the activity of membrane transporters to start another cycle of the shuttle. FIGURE 19-31 Malate-aspartate shuttle. This shuttle for transporting reducing equivalents from cytosolic NADH into the mitochondrial matrix is used in liver, kidney, and heart. NADH in the cytosol enters the intermembrane space through openings in the outer membrane (porins), then passes two reducing equivalents to oxaloacetate, producing malate. Malate crosses the inner membrane via the malate–α - ketoglutarate transporter. In the matrix, malate passes two reducing equivalents to NAD +, and the resulting NADH is oxidized by the respiratory chain; the oxaloacetate formed from malate cannot pass directly into the cytosol. Oxaloacetate is first transaminated to aspartate, and aspartate can leave via the glutamate-aspartate transporter. Oxaloacetate is regenerated in the cytosol, completing the cycle, and glutamate produced in the same reaction enters the matrix via the glutamate-aspartate transporter. Skeletal muscle and brain use a different NADH shuttle, the glycerol 3-phosphate shuttle (Fig. 19-32). It differs from the malate-aspartate shuttle in that it delivers the reducing equivalents from NADH through FAD in glycerol 3-phosphate dehydrogenase to ubiquinone and thus into Complex III, not Complex I (Fig. 19-15), providing enough energy to synthesize only 1.5 ATP molecules per pair of electrons. FIGURE 19-32 Glycerol 3-phosphate shuttle. This alternative means of moving reducing equivalents from the cytosol to the respiratory chain operates in skeletal muscle and the brain. In the cytosol, dihydroxyacetone phosphate accepts two reducing equivalents from NADH in a reaction catalyzed by cytosolic glycerol 3-phosphate dehydrogenase. An isozyme of glycerol 3-phosphate dehydrogenase bound to the outer face of the inner membrane then transfers two reducing equivalents from glycerol 3- phosphate in the intermembrane space to ubiquinone. Note that this shuttle does not involve membrane transport systems. The mitochondria of plants have an externally oriented NADH dehydrogenase that can transfer electrons directly from cytosolic NADH into the respiratory chain at the level of ubiquinone. Because this pathway bypasses the NADH dehydrogenase of Complex I and the associated proton movement, the yield of ATP from cytosolic NADH is less than that from NADH generated in the matrix (Box 19-1). BOX 19-1 Hot, Stinking Plants and Alternative Respiratory Pathways Many flowering plants attract insect pollinators by releasing odorant molecules that mimic an insect’s natural food sources or potential egg-laying sites. Plants pollinated by flies or beetles that normally feed on or lay their eggs in dung or carrion sometimes use foul-smelling compounds to attract these insects. One family of stinking plants is the Araceae, which includes philodendrons, arum lilies, and skunk cabbages. These plants have tiny flowers densely packed on an erect structure, the spadix, surrounded by a modified leaf, the spathe. The spadix releases odors of rotting flesh or dung. Before pollination the spadix also heats up, in some species to as much as 20 to 40 °C above ambient temperatures. Heat production (thermogenesis) helps evaporate odorant molecules for better dispersal, and because rotting flesh and dung are usually warm from the hyperactive metabolism of scavenging microbes, the heat itself might also attract insects. In the case of the eastern skunk cabbage (Fig. 1), which flowers in late winter or early spring when snow still covers the ground, thermogenesis allows the spadix to grow up through the snow. FIGURE 1 Eastern skunk cabbage. How does a skunk cabbage heat its spadix? The mitochondria of plants, fungi, and unicellular eukaryotes have respiratory chains that are essentially the same as those in animals, but they also have an alternative respiratory pathway. A QH2 oxidase transfers electrons from the ubiquinone pool directly to oxygen, bypassing the two proton-translocating steps of Complexes III and IV (Fig. 2). Energy that might have been conserved as ATP is instead released as heat. Plant mitochondria also have an alternative NADH dehydrogenase, insensitive to the Complex I inhibitor rotenone (see Table 19-4), that transfers electrons from NADH in the matrix directly to ubiquinone, bypassing Complex I and its associated proton pumping. And plant mitochondria have yet another NADH dehydrogenase, on the external face of the inner membrane, that transfers electrons from NADPH or NADH in the intermembrane space to ubiquinone, again bypassing Complex I. Thus when electrons enter the alternative respiratory pathway through the rotenone-insensitive NADH dehydrogenase, the external NADH dehydrogenase, or succinate dehydrogenase (Complex II), and pass to O2 via the cyanide-resistant alternative oxidase, energy is not conserved as ATP but is released as heat. A skunk cabbage can use the heat to melt snow, produce a foul stench, or attract beetles or flies. FIGURE 2 Electron carriers of the inner membrane of plant mitochondria. Electrons can flow through Complexes I, III, and IV, as in animal mitochondria, or through plant-specific alternative carriers by the paths shown with blue arrows. SUMMARY 19.2 ATP Synthesis The chemiosmotic theory describes the coupling of ATP synthesis to an electrochemical proton gradient. The ﬂow of electrons through Complexes I, III, and IV results in pumping of protons across the inner mitochondrial membrane, making the matrix alkaline relative to the intermembrane space. This proton gradient provides the energy, in the form of the proton-motive force, for ATP synthesis from ADP and Pi. ATP synthase has two major components, called Fo and F1. Both components have multiple subunits. The overall complex spans the inner mitochondrial membrane. ATP synthesis is reversible within the active site on the β subunits of the F1 complex. Very tight binding to ATP offsets the negative ΔG for ATP hydrolysis in solution. Release of ATP from ATP synthase is promoted by the transmembrane proton gradient. The subunits of the F1 complex cycle from (AD P + Pi)–bound to ATP-bound to empty conformations. ATP synthase carries out “rotational catalysis,” in which the ﬂow of protons through Fo causes the c ring to rotate and in turn trigger the subunit conformational changes in F1. The ratio of ATP synthesized per ½O2 reduced to H2O (the P/O ratio) is about 2.5 when electrons enter the respiratory chain at Complex I, and 1.5 when electrons enter at ubiquinone. This ratio varies among species, depending on the number of c subunits in the Fo complex. Energy conserved in a proton gradient can drive solute transport uphill across a membrane. The inner mitochondrial membrane is impermeable to NADH and NAD +, but NADH equivalents are moved from the cytosol to the matrix by either of two shuttles. NADH equivalents moved in by the malate-aspartate shuttle enter the respiratory chain at Complex I and yield a P/O ratio of 2.5; those moved in by the glycerol 3-phosphate shuttle enter at ubiquinone and give a P/O ratio of 1.5. 19.3 Regulation of Oxidative Phosphorylation Oxidative phosphorylation produces most of the ATP made in aerobic cells. Complete oxidation of a molecule of glucose to CO2 yields 30 or 32 ATP (Table 19-5). By comparison, glycolysis under anaerobic conditions (lactate fermentation) yields only 2 ATP per glucose. Clearly, the evolution of oxidative phosphorylation provided a tremendous increase in the energy efﬁciency of catabolism. Complete oxidation to CO2 of the coenzyme A derivative of palmitate (16:0), which also occurs in the mitochondrial matrix, yields 108 ATP per palmitoyl-CoA (see Table 17-1). A similar calculation can be made for the ATP yield from oxidation of each of the amino acids (Chapter 18). Aerobic oxidative pathways that result in electron transfer to O2 accompanied by oxidative phosphorylation therefore account for the vast majority of the ATP produced in catabolism, so the regulation of ATP production by oxidative phosphorylation to match the cell’s ﬂuctuating needs for ATP is absolutely essential. TABLE 19-5 ATP Yield from Complete Oxidation of Glucose Process Direct product Final ATP Glycolysis 2 NADH (cytosolic) 2 ATP 3 or 5 2 Pyruvate oxidation (two per glucose) 2 NADH (mitochondrial matrix) 5 a Acetyl-CoA oxidation in citric acid cycle (two per glucose) 6 NADH (mitochondrial matrix) 2FADH2 2 ATP or 2 GTP 15 3 2 Total yield per glucose 30 or 32 If the malate/aspartate shuttle is used to transfer reducing equivalents into the mitochondrion, the yield is 5 ATP. If the glycerol 3-phosphate shuttle is used, the yield is 3 ATP. Oxidative Phosphorylation Is Regulated by Cellular Energy Needs The rate of respiration (O2 consumption) in mitochondria is generally limited by the availability of ADP as a substrate for phosphorylation. Dependence of the rate of O2 consumption on the availability of the Pi acceptor, ADP (Fig. 19-20b), the acceptor control of respiration, can be remarkable. In some animal tissues, the acceptor control ratio, the ratio of the maximal rate of ADP-induced O2 consumption to the basal rate in the absence of ADP, is at least 10. The intracellular concentration of ADP is one measure of the energy status of cells. Another, related measure is the mass- action ratio of the ATP-ADP system, [ATP]/([ADP][Pi]). Usually this ratio is very high, so the ATP-ADP system is almost fully a phosphorylated. When the rate of some energy-requiring process (protein synthesis, for example) increases, the rate of breakdown of ATP to ADP and Pi increases, lowering the mass-action ratio. With more ADP available for oxidative phosphorylation, the rate of respiration increases, causing regeneration of ATP. This continues until the mass-action ratio returns to its normal high level, at which point respiration slows again. The rate of oxidation of cellular fuels is regulated with such sensitivity and precision that the [ATP]/([ADP][Pi]) ratio ﬂuctuates only slightly in most tissues, even during extreme variations in energy demand. In short, ATP is formed only as fast as it is used in energy-requiring cellular activities. An Inhibitory Protein Prevents ATP Hydrolysis during Hypoxia We have already encountered ATP synthase as an ATP-driven proton pump (see Fig. 11-40), catalyzing the reverse of ATP synthesis under some experimental conditions. When a cell is hypoxic (deprived of oxygen), as in a heart attack or a stroke, electron transfer to oxygen slows, and so does the pumping of protons. The proton-motive force soon collapses. Under these conditions, the ATP synthase could operate in reverse, hydrolyzing ATP made by glycolysis to pump protons outward and causing a disastrous drop in ATP levels. This is prevented by a small (84 amino acids) protein inhibitor, IF1. IF1 simultaneously binds to two ATP synthase molecules, inhibiting the enzyme’s activity in both directions (Fig. 19-33). IF1 is inhibitory only in its dimeric form, which is favored at pH lower than 6.5. In a cell starved for oxygen, the main source of ATP becomes glycolysis, and the pyruvic or lactic acid thus formed lowers the pH in the cytosol and the mitochondrial matrix. This favors IF1 dimerization, leading to inhibition of ATP synthase and thereby preventing any wasteful hydrolysis of ATP. When aerobic metabolism resumes, production of pyruvic acid slows, the pH of the cytosol rises, the IF1 dimer is destabilized, and the inhibition of ATP synthase is lied. IF1 is an intrinsically disordered protein (p. 117); it acquires a favored conformation only on interaction with ATP synthase. In many tumors and cancer cell lines, which rely more heavily on glycolysis for ATP generation, IF1 is expressed at unusually high levels. FIGURE 19-33 Structure of bovine F1-ATPase in a complex with its regulatory protein IF1. Two F1 molecules are viewed here from the side, as in Figure 19-25b. The inhibitor IF1 (red) binds to the αβ interface of the subunits in the diphosphate (ADP) conformation (α -ADP and β -ADP), freezing the two F1 complexes and thereby blocking ATP hydrolysis (and synthesis). (Parts of the IF1 α-helices that link the two F1 molecules failed to resolve in the crystals of F1 and are modeled based on the crystal structure of isolated IF1.) [Data from PDB ID 1OHH, E. Cabezon et al., Nat. Struct. Biol. 10:744, 2003.] Hypoxia Leads to ROS Production and Several Adaptive Responses In hypoxic cells there is an imbalance between the input of electrons from fuel oxidation in the mitochondrial matrix and transfer of electrons to molecular oxygen, leading to increased formation of reactive oxygen species. In addition to the glutathione peroxidase system (Fig. 19-18), cells have two other lines of defense against ROS (Fig. 19-34). One is regulation of pyruvate dehydrogenase (PDH), the enzyme that delivers acetyl- CoA to the citric acid cycle (Chapter 16). Under hypoxic conditions, PDH kinase phosphorylates mitochondrial PDH, inactivating it and slowing the delivery of FADH2 and NADH from the citric acid cycle to the respiratory chain. A second means of preventing ROS formation is the replacement of one subunit of Complex IV, known as COX4-1, with another subunit, COX4-2, that is better suited to hypoxic conditions. With COX4-1, the catalytic properties of Complex IV are optimal for respiration at normal oxygen concentrations; with COX4-2, Complex IV is optimized for operation under hypoxic conditions. FIGURE 19-34 Regulation of gene expression by hypoxia-inducible factor (HIF-1) to reduce ROS formation. Under conditions of low oxygen (hypoxia), HIF-1 is synthesized in greater amounts and acts as a transcription factor, increasing synthesis of the glucose transporter, glycolytic enzymes, pyruvate dehydrogenase kinase (PDH kinase), lactate dehydrogenase, a protease that degrades the cytochrome oxidase subunit COX4-1, and cytochrome oxidase subunit COX4-2. These changes counter the formation of ROS by decreasing the supply of NADH and FADH2 and making cytochrome oxidase of Complex IV more eﬀective. [Information from D. A. Harris, Bioenergetics at a Glance, p. 36, Blackwell Science, 1995.] The changes in PDH activity and the COX4-2 content of Complex IV are both mediated by HIF-1, the hypoxia-inducible factor. HIF- 1 (another intrinsically disordered protein) accumulates in hypoxic cells and, acting as a transcription factor, triggers increased synthesis of PDH kinase, COX4-2, and a protease that degrades COX4-1. HIF-1 is a master regulator of O2 homeostasis. Recall that it also mediates the changes in glucose transport and glycolytic enzymes that produce the Warburg effect, the dependence on glycolysis (not mitochondrial respiration) for ATP production, even in the presence of sufﬁcient oxygen (see Box 14- 1). When these mechanisms for dealing with ROS are insufﬁcient, either due to genetic mutation affecting one of the protective proteins or under conditions of very high rates of ROS production, mitochondrial function is compromised. Mitochondrial damage is thought to be involved in aging, heart failure, certain rare cases of diabetes (described below), and several maternally inherited genetic diseases that affect the nervous system. ATP-Producing Pathways Are Coordinately Regulated The major catabolic pathways have overlapping and concerted regulatory mechanisms that allow them to function together in an economical and self-regulating manner to produce ATP and biosynthetic precursors. The relative concentrations of ATP and ADP control not only the rates of electron transfer and oxidative phosphorylation but also the rates of the citric acid cycle, pyruvate oxidation, and glycolysis (Fig. 19-35). Whenever ATP consumption increases, the rate of electron transfer and oxidative phosphorylation increases. Simultaneously, the rate of pyruvate oxidation via the citric acid cycle increases, increasing the ﬂow of electrons into the respiratory chain. These events, in turn, can evoke an increased rate of glycolysis, increasing the rate of pyruvate formation. When conversion of ADP to ATP lowers the ADP concentration, acceptor control slows electron transfer and thus oxidative phosphorylation. Glycolysis and the citric acid cycle are also slowed, because ATP is an allosteric inhibitor of the glycolytic enzyme phosphofructokinase-1 (see Fig. 14-23) and of pyruvate dehydrogenase (see Fig. 16-18).

FIGURE 19-35 Regulation of ATP-producing pathways. This diagram shows the coordinated regulation of glycolysis, pyruvate oxidation, the citric acid cycle, and oxidative phosphorylation by the relative concentrations of ATP, ADP, and AMP, and by NADH. High [ATP] (or low [ADP] and [AMP]) produces low rates of glycolysis, pyruvate oxidation, acetate oxidation via the citric acid cycle, and oxidative phosphorylation. All four pathways are accelerated when the use of ATP and the formation of ADP, AMP, and Pi increase. The ability of citrate to inhibit both glycolysis and the citric acid cycle reinforces the action of the adenine nucleotide system. In addition, increased [NADH] and [acetyl-CoA] also inhibit the oxidation of pyruvate to acetyl-CoA, and a high [NADH]/[NAD+] ratio inhibits the dehydrogenase reactions of the citric acid cycle (see Fig. 16-18). Phosphofructokinase-1 is also inhibited by citrate, the ﬁrst intermediate of the citric acid cycle. When the cycle is “idling,” citrate accumulates within mitochondria, then is transported into the cytosol. When the cytosolic concentrations of both ATP and citrate rise, they produce a concerted allosteric inhibition of phosphofructokinase-1 that is greater than the sum of their individual effects, slowing glycolysis. SUMMARY 19.3 Regulation of Oxidative Phosphorylation Oxidative phosphorylation is regulated by cellular energy demands. Intracellular [ADP] and the mass-action ratio [ATP]/([ADP][Pi]) are measures of a cell’s energy status. In hypoxic (oxygen-deprived) cells, a protein inhibitor blocks ATP hydrolysis by the reverse activity of ATP synthase, preventing a drastic drop in [ATP]. The adaptive responses to hypoxia, mediated by HIF-1, slow electron transfer into the respiratory chain and modify Complex IV to act more efﬁciently under low-oxygen conditions. ATP and ADP concentrations set the rate of electron transfer through the respiratory chain via a series of coordinated controls on respiration, glycolysis, and the citric acid cycle. 19.4 Mitochondria in Thermogenesis, Steroid Synthesis, and Apoptosis Although ATP production is a central role for the mitochondrion, this organelle has other functions that, in speciﬁc tissues or under speciﬁc circumstances, are also crucial. In adipose tissue, mitochondria generate heat to protect vital organs from low ambient temperature; in the adrenal glands and the gonads, mitochondria are the sites of steroid hormone synthesis; and in most or all tissues, they are key participants in apoptosis (programmed cell death). Uncoupled Mitochondria in Brown Adipose Tissue Produce Heat We noted above that respiration slows when the cell is adequately supplied with ATP. There is a remarkable and instructive exception to this general rule. Most newborn mammals, including humans, have a type of adipose tissue called brown adipose tissue (BAT; p. 852), in which fuel oxidation serves not to produce ATP but to generate heat to keep the newborn warm. This specialized adipose tissue is brown because of the presence of large numbers of mitochondria and thus high concentrations of cytochromes, with heme groups that are strong absorbers of visible light. There are at least two mechanisms of thermogenesis. The mitochondria of brown adipocytes are much like those of other mammalian cells, except in having a unique protein in their inner membrane. Uncoupling protein 1 (UCP1), a long-chain fatty acid/H+ symporter, provides a path for protons to return to the matrix without passing through the FoF1 complex (Fig. 19-36). As a result of this short-circuiting of protons, the energy of oxidation is not conserved by ATP formation but is dissipated as heat, which contributes to maintaining body temperature. However, UCP1 is only part of the story, and heat generation occurs in mammals even when it is absent. The thermogenic action of UCP1 is supplemented by a futile cycle involving creatine and phosphocreatine (Fig. 19-36). ATP synthesized in the mitochondrial matrix is exported to the intermembrane space via the adenine nucleotide translocator, an ADP/ATP antiporter. There, the ATP is used to phosphorylate creatine to create phosphocreatine and ADP. The ADP is transported back into the matrix. Hydrolysis of phosphocreatine completes a futile cycle that liberates heat.

FIGURE 19-36 Two mechanisms of thermogenesis in mitochondria. UCP1, an uncoupling protein in the mitochondria of brown adipose tissue, causes the energy conserved by proton pumping to be dissipated as heat by providing an alternative route for protons to reenter the mitochondrial matrix. A futile cycle in which creatine is phosphorylated by creatine kinase (CK), using ATP and producing ADP transported by the ATP/ADP carrier (AAC), also generates heat. Hibernating animals also depend on the activity of uncoupled BAT mitochondria to generate heat during their long dormancy (see Box 17-1). We will return to the role of UCP1 when we discuss the regulation of body mass in Chapter 23 (pp. 867–869). Mitochondrial P-450 Monooxygenases Catalyze Steroid Hydroxylations Mitochondria are the site of biosynthetic reactions that produce steroid hormones, including the sex hormones, glucocorticoids, mineralocorticoids, and vitamin D hormone. These compounds are synthesized from cholesterol or a related sterol in a series of hydroxylations catalyzed by enzymes of the cytochrome P-450 family (see Box 21-1), all of which have a critical heme group (its absorption at 450 nm gives this family its name). In the hydroxylation reactions, one atom of molecular oxygen is incorporated into the substrate and the second is reduced to H2O, making cytochrome P-450 enzymes monooxygenases: R—H + O2+ NAD PH + H+ → R—OH + H2O + NAD P+ In this reaction, two species are oxidized: NADPH and R—H. There are dozens of P-450 enzymes, all situated in the inner mitochondrial membrane with their catalytic site exposed to the matrix. Steroidogenic cells are packed with mitochondria specialized for steroid synthesis; the mitochondria are generally larger than those in other tissues and have more extensive and highly convoluted inner membranes (Fig. 19-37). FIGURE 19-37 Mitochondria of adrenal gland, specialized for steroid synthesis. As seen in this electron micrograph of a thin section of adrenal gland, mitochondria are profuse and have extensive cristae, providing a large surface for the P-450 enzymes of the inner membrane. The path of electron ﬂow in the mitochondrial P-450 system is complex, involving a ﬂavoprotein and an iron-sulfur protein that carry electrons from NADPH to the P-450 heme (Fig. 19-38). All P- 450 enzymes have a heme that interacts with O2 and a substrate- binding site that confers speciﬁcity. FIGURE 19-38 Path of electron flow in mitochondrial cytochrome P-450 reactions in adrenal gland. Two electrons are transferred from NADPH to the FAD-containing flavoprotein adrenodoxin reductase, which passes the electrons, one at a time, to adrenodoxin, a small, soluble 2Fe-2S protein. Adrenodoxin passes single electrons to the cytochrome P-450 hydroxylase, which interacts directly with O2 and the substrate (R—H) to form the products, H2O and R—OH. Another large family of P-450 enzymes is found in the endoplasmic reticulum of hepatocytes. These enzymes catalyze reactions similar to the mitochondrial P-450 reactions, but their substrates include a wide variety of hydrophobic compounds, many of which are xenobiotics — compounds not found in nature but synthesized industrially. The P-450 enzymes of the ER have very broad and overlapping substrate speciﬁcities. Hydroxylation of the hydrophobic compounds makes them more water-soluble, and they can then be cleared by the kidneys and excreted in urine. Among the substrates for these P-450 oxygenases are many commonly used prescription drugs. Metabolism by P-450 enzymes limits a drug’s lifetime in the bloodstream and thus its therapeutic effects. Humans differ in their genetic complement of P-450 enzymes in the ER, as well as in the extent to which certain P-450 enzymes have been induced, such as by a history of ethanol ingestion. In principle, therefore, an individual’s genetics and personal history should be considered in determining therapeutic drug doses. In practice, this precise tailoring of dosage is not yet economically feasible, but it may become so. Mitochondria Are Central to the Initiation of Apoptosis Apoptosis, also called programmed cell death, is a process in which individual cells die for the good of the organism, such as in the course of normal embryonic development, and the organism conserves the cells’ molecular components (amino acids, nucleotides, and so forth). Apoptosis may be triggered by an external signal, acting at a plasma membrane receptor, or by internal events such as DNA damage, viral infection, oxidative stress from the accumulation of ROS, or other stress such as a heat shock. Mitochondria play a critical role in triggering apoptosis. When a stressor gives the signal for cell death, one early consequence is an increase in the permeability of the outer mitochondrial membrane, allowing cytochrome c to escape from the intermembrane space into the cytosol (Fig. 19-39). The increased permeability is due to the opening of the permeability transition pore complex (PTPC), a multisubunit protein in the outer membrane; its opening and closing are affected by several proteins that stimulate or suppress apoptosis. When released into the cytosol, cytochrome c interacts with monomers of the protein Apaf-1 (apoptosis protease activating factor-1), causing the formation of an apoptosome composed of seven Apaf-1 and seven cytochrome c molecules. The apoptosome provides the platform on which the proenzyme procaspase-9 is activated to caspase-9, a member of a family of highly speciﬁc proteases, called the caspases, involved in apoptosis. These cysteine proteases cleave proteins only on the carboxyl-terminal side of Asp residues, thus the name “caspases.” Caspase-9 initiates a cascade of proteolytic activations, with one caspase activating a second, and this in turn activating a third, and so forth (see Fig. 12-42). Note that this role of cytochrome c in apoptosis is a clear case of “moonlighting,” in that one protein plays two very different roles in the cell (see Box 16-1).

FIGURE 19-39 Role of cytochrome c in apoptosis. Cytochrome c is a small, soluble, mitochondrial protein, located in the intermembrane space, that carries electrons between Complex III and Complex IV during respiration. In a completely separate role, as outlined here, it acts as a trigger for apoptosis by stimulating the activation of a family of proteases called caspases. [Information from S. J. Riedl and G. S. Salvesen, Nat. Rev. Mol. Cell Biol. 8:409, 2007, Fig. 3.] SUMMARY 19.4 Mitochondria in Thermogenesis, Steroid Synthesis, and Apoptosis In the brown adipose tissue of newborns, electron transfer is uncoupled from ATP synthesis, and the energy of fuel oxidation is dissipated as heat. Hibernating animals use this strategy to avoid freezing. Hydroxylation reaction steps in the synthesis of steroid hormones in steroidogenic tissues (adrenal gland, gonads, liver, and kidney) take place in specialized mitochondria. Key reactions are catalyzed by a family of P-450 monooxygenases. Mitochondria play a central role in apoptosis. Mitochondrial cytochrome c, released into the cytosol, participates in activation of caspase-9, one of the proteases involved in apoptosis. 19.5 Mitochondrial Genes: Their Origin and the Eﬀects of Mutations Mitochondria contain their own genome, a circular, double- stranded DNA (mtDNA) molecule. Each of the hundreds or thousands of mitochondria in a typical cell has about ﬁve copies of this genome. The human mitochondrial chromosome (Fig. 19- 40) contains 37 genes (16,569 bp), including 13 that encode subunits of proteins of the respiratory chain (Table 19-6); the remaining genes code for rRNA and tRNA molecules essential to the protein-synthesizing machinery of mitochondria. To synthesize these 13 protein subunits, mitochondria have their own ribosomes, distinctly different from those in the cytoplasm. The great majority of mitochondrial proteins — about 1,200 different types — are encoded by nuclear genes, synthesized on cytoplasmic ribosomes, then imported into and assembled in the mitochondria (Chapter 27). FIGURE 19-40 Mitochondrial genes and mutations. A map of human mitochondrial DNA, showing the genes that encode proteins of Complex I, the NADH dehydrogenase (ND1 to ND6); the cytochrome b of Complex III (Cyt b); the subunits of cytochrome oxidase, Complex IV (COI to COIII); and two subunits of ATP synthase (ATPase6 and ATPase8). The colors of the genes correspond to those of the complexes shown in Figure 19-7. Also included here are the genes for ribosomal RNAs (rRNA) and for some mitochondrion-specific transfer RNAs; tRNA specificity is indicated by the one-letter codes for amino acids. Arrows indicate the positions of mutations that cause Leber hereditary optic neuropathy (LHON) and myoclonic epilepsy with ragged-red fibers (MERRF) syndrome. Numbers in parentheses indicate the position of the altered nucleotides (nucleotide 1 is at the top of the circle, and numbering proceeds counterclockwise). [Information from M. A. Morris, J. Clin. Neuroophthalmol. 10:159, 1990.] TABLE 19-6 Respiratory Proteins Encoded by Mitochondrial Genes in Humans Complex Number of subunits Number of subunits encoded by mtDNA I NADH dehydrogenase 45 7 II Succinate dehydrogenase 4 0 III Ubiquinone:cytochrome c oxidoreductase 11 1 IV Cytochrome oxidase 13 3 V ATP synthase 8 2 Mitochondria Evolved from Endosymbiotic Bacteria The existence of mitochondrial DNA, ribosomes, and tRNAs supports the theory of the endosymbiotic origin of mitochondria (see Fig. 1-37), which holds that the ﬁrst organisms capable of aerobic metabolism, including respiration-linked ATP production, were bacteria. Primitive eukaryotes that lived anaerobically (by fermentation) acquired the ability to carry out oxidative phosphorylation when they established a symbiotic relationship with bacteria living in their cytosol. Aer a long period of evolution and the movement of many bacterial genes into the nucleus of the “host” eukaryote, the endosymbiotic bacteria eventually became mitochondria. This hypothesis presumes that early free-living bacteria had the enzymatic machinery for oxidative phosphorylation. And it predicts that their modern bacterial descendants must have respiratory chains closely similar to those of modern eukaryotes. They do. Aerobic bacteria carry out NAD-linked electron transfer from substrates to O2, coupled to the phosphorylation of cytosolic ADP. The dehydrogenases are located in the bacterial cytosol, and the respiratory chain in the plasma membrane. The electron carriers translocate protons outward across the plasma membrane as electrons are transferred to O2. Bacteria such as E. coli have FoF1 complexes in their plasma membranes; the F1 portion protrudes into the cytosol and catalyzes ATP synthesis from ADP and Pi as protons ﬂow back into the cell through the proton channel of Fo. The respiration-linked extrusion of protons across the bacterial plasma membrane also provides the driving force for other processes. Certain bacterial transport systems bring about uptake of extracellular nutrients (lactose, for example) against a concentration gradient, in symport with protons. And the rotary motion of bacterial ﬂagella is provided by “proton turbines,” molecular rotary motors driven not by ATP but directly by the transmembrane electrochemical potential generated by respiration-linked proton pumping (Fig. 19-41). It seems likely that the chemiosmotic mechanism evolved early, before the emergence of eukaryotes. FIGURE 19-41 Rotation of bacterial flagella by proton-motive force. The sha and rings at the base of the flagellum make up a rotary motor that has been called a “proton turbine.” Protons ejected by electron transfer flow back into the cell through the turbine, causing rotation of the sha of the flagellum. This motion diﬀers fundamentally from the motion of muscle and of eukaryotic flagella and cilia, for which ATP hydrolysis is the energy source. Mutations in Mitochondrial DNA Accumulate throughout the Life of the Organism The respiratory chain is the major producer of reactive oxygen species in cells, so mitochondrial contents, including the mitochondrial genome, suffer the greatest exposure to, and damage by, ROS. Moreover, the mitochondrial DNA replication system is less effective than the nuclear system at correcting mistakes made during replication and at repairing DNA damage. As a consequence, defects in mtDNA accumulate over time. One theory of aging is that this gradual accumulation of defects is the primary cause of many of the “symptoms” of aging, which include, for example, progressive weakening of skeletal and heart muscle. A unique feature of mitochondrial inheritance is the variation among individual cells, and between one individual organism and another, in the effects of a mtDNA mutation. A typical cell has hundreds or thousands of mitochondria, each with multiple copies of its own genome (Fig. 19-2b). Animals inherit essentially all of their mitochondria from the female parent. Eggs are large and contain 105 or 106 mitochondria; sperm are much smaller and contain far fewer mitochondria — perhaps 100 to 1,000. Furthermore, there is an active mechanism for targeting sperm- derived mitochondria for degradation in the fertilized egg. Just aer fertilization, maternal phagosomes migrate to the site of sperm entry, engulf sperm mitochondria, and degrade them. Suppose that, in a female organism, damage to one mitochondrial genome occurs in a germ cell from which oocytes develop, such that the germ cell contains mainly mitochondria with wild-type genes but one mitochondrion with a mutant gene. During the course of oocyte maturation, as this germ cell and its descendants repeatedly divide, the defective mitochondrion replicates and its progeny, all defective, are randomly distributed to daughter cells. Eventually, the mature egg cells contain different proportions of the defective mitochondria. When an egg cell is fertilized and undergoes the many divisions of embryonic development, the resulting somatic cells differ in their proportion of mutant mitochondria (Fig. 19-42a). This heteroplasmy (in contrast to homoplasmy, in which every mitochondrial genome in every cell is the same) results in mutant phenotypes of varying degrees of severity. Cells (and tissues) containing mostly wild-type mitochondria have the wild-type phenotype; they are essentially normal. Other heteroplasmic cells have intermediate phenotypes, some almost normal, others (with a high proportion of mutant mitochondria) abnormal (Fig. 19-42b). If the abnormal phenotype is associated with a disease, individuals with the same mtDNA mutation may have disease symptoms of differing severity — depending on the number and distribution of affected mitochondria.

FIGURE 19-42 Heteroplasmy in mitochondrial genomes. (a) When a mature egg cell is fertilized, all of the mitochondria in the resulting diploid cell (zygote) are maternal; none come from the sperm. If some fraction of the maternal mitochondria have a mutant gene, the random distribution of mitochondria during subsequent cell divisions yields some daughter cells with mostly mutant mitochondria, some with mostly wild-type mitochondria, and some in between. Thus daughter cells show a varying degree of heteroplasmy. (b) Diﬀerent degrees of heteroplasmy produce diﬀerent cellular phenotypes. This section of human muscle tissue is from an individual with defective cytochrome oxidase. The cells were stained so that wild-type cells are blue and cells with mutant cytochrome oxidase are brown. As the micrograph shows, diﬀerent cells in the same tissue are aﬀected to diﬀerent degrees by the mitochondrial mutation. [(b) Courtesy of Rob Taylor. Reprinted with permission from R. W. Taylor and D. M. Turnbull, Nat. Rev. Genet. 6:389, 2005, Fig. 2a.] Some Mutations in Mitochondrial Genomes Cause Disease About 1 in 5,000 people have a disease-causing mutation in a mitochondrial protein that reduces the cell’s capacity to produce ATP. A growing number of these diseases have been attributed to mutations in mitochondrial genes. Some tissues and cell types — neurons, myocytes of both skeletal and cardiac muscle, and β cells of the pancreas — are less able than others to tolerate lowered ATP production and are therefore more affected by mutations in mitochondrial proteins. A group of genetic diseases known as the mitochondrial encephalomyopathies affect primarily the brain and skeletal muscle. These diseases are invariably inherited from the mother, because, as noted above, a developing embryo derives all its mitochondria from the egg. The rare disease Leber hereditary optic neuropathy (LHON) affects the central nervous system, including the optic nerves, causing bilateral loss of vision in early adulthood. A single base change in the mitochondrial gene ND4 (Fig. 19-40) changes an Arg residue to a His residue in a polypeptide of Complex I, and the result is mitochondria partially defective in electron transfer from NADH to ubiquinone. Although these mitochondria can produce some ATP by electron transfer from succinate, they apparently cannot supply sufﬁcient ATP to support the very active metabolism of neurons, including the optic nerve. A single base change in the mitochondrial gene for cytochrome b, a component of Complex III, also produces LHON, demonstrating that the pathology results from a general reduction of mitochondrial function, not speciﬁcally from a defect in electron transfer through Complex I. A mutation in the mitochondrial gene ATP6 affects the proton pore in ATP synthase, leading to low rates of ATP synthesis while leaving the respiratory chain intact. Oxidative stress due to the continued supply of electrons from NADH increases the production of ROS, and the damage to mitochondria caused by ROS sets up a vicious cycle. Half of individuals with this mutant gene die within days or months of birth. Myoclonic epilepsy with ragged-red ﬁbers (MERRF) syndrome is caused by a mutation in the mitochondrial gene that encodes a tRNA speciﬁc for lysine (tRNALys). This disease, characterized by uncontrollable muscular jerking, results from defective production of several of the proteins that require mitochondrial tRNAs for their synthesis. Skeletal muscle ﬁbers of individuals with MERRF syndrome have abnormally shaped mitochondria that sometimes contain paracrystalline structures (Fig. 19-43). Other mutations in mitochondrial genes are believed to be responsible for the progressive muscular weakness that characterizes mitochondrial myopathy and for enlargement and deterioration of the heart muscle in hypertrophic cardiomyopathy. FIGURE 19-43 Paracrystalline inclusions in MERRF syndrome mitochondrion. Electron micrograph of an abnormal mitochondrion from the muscle of an individual with MERRF syndrome, showing the paracrystalline protein inclusions sometimes present in the mutant mitochondria. [From Regionalized Pathology Correlates with Augmentation of mtDNA Copy Numbers in a Patient with Myoclonic Epilepsy with Ragged- Red Fibers (MERRF-Syndrome). PLOS ONE, Anja Brinckmann et al., October 20, 2010. https://doi.org/10.1371/journal.pone.0013513] If a prospective mother is known to carry a pathogenic mitochondrial gene, the technique of mitochondrial donation can circumvent the passage of that mutant gene to her offspring. A prospective mother’s nuclear genes are microscopically transplanted into an enucleated ovum from a donor with healthy mitochondria, then the ovum is fertilized in vitro and the resulting embryo is transplanted into the mother’s uterus. This and similar “three-parent baby” procedures, which were approved in the United Kingdom in 2015, raise ethical issues that are being vigorously debated. Mitochondrial disease can also result from mutations in any of the ~1,200 nuclear genes that encode mitochondrial proteins. For example, a mutation in one of the nuclear-encoded proteins of Complex IV, COX6B1, results in severe defects in brain development and thickened walls of the heart muscle. Other nuclear genes encode proteins essential for the assembly of mitochondrial complexes. Mutations in these genes can also lead to serious mitochondrial disease. A Rare Form of Diabetes Results from Defects in the Mitochondria of Pancreatic β Cells The insulin so important to glucose homeostasis in all humans is produced and exported from pancreatic β cells. Insulin export hinges on the ATP concentration in those cells. When blood glucose is high, β cells take up glucose and oxidize it by glycolysis and the citric acid cycle, raising [ATP] above a threshold level (Fig. 19-44). When [ATP] exceeds this threshold, an ATP-gated K+ channel in the plasma membrane closes, depolarizing the membrane and triggering insulin release (see Fig. 23-24).

FIGURE 19-44 A mitochondrial defect prevents insulin secretion. In the normal situation, as depicted here, when the blood glucose level rises, production of ATP in β cells increases. ATP, by blocking K+ channels, depolarizes the plasma membrane and thus opens voltage-gated Ca2+ channels. The resulting influx of Ca2+ triggers exocytosis of insulin- containing secretory vesicles, releasing insulin. When oxidative phosphorylation in β cells is defective, [ATP] is never suﬀicient to trigger this process, and insulin is not released. Normal insulin release can be compromised in several ways. Pancreatic β cells with defects in any aspect of oxidative phosphorylation may not be able to increase [ATP] above this threshold, and the resulting failure of insulin release effectively produces diabetes. For example, defects in the gene for glucokinase, the hexokinase IV isozyme present in β cells, lead to a rare form of diabetes called MODY2 (maturity onset diabetes of the young); low glucokinase activity prevents the generation of ATP concentrations above the threshold needed to trigger insulin secretion. Mutations in the mitochondrial tRNALys or tRNALeu genes also compromise mitochondrial ATP production by limiting the expression of electron transfer components encoded in the mitochondrial DNA. Type 2 diabetes mellitus is common among individuals with these defects (although such cases make up a very small fraction of all cases of diabetes). When nicotinamide nucleotide transhydrogenase, which is part of the mitochondrial defense against ROS (Fig. 19-18), is genetically defective, the accumulation of ROS damages mitochondria, slowing ATP production and blocking insulin release by β cells (Fig. 19-44). Damage caused by ROS, including damage to mtDNA, may also underlie other human diseases; there is some evidence for its involvement in Alzheimer, Parkinson, and Huntington diseases and in heart failure, as well as in aging. SUMMARY 19.5 Mitochondrial Genes: Their Origin and the Eﬀects of Mutations A small proportion of human mitochondrial proteins, 13 in all, are encoded by the mitochondrial genome and synthesized in mitochondria. About 1,200 mitochondrial proteins are encoded by nuclear genes and imported into mitochondria aer their synthesis. Mitochondria arose from aerobic bacteria that entered into an endosymbiotic relationship with ancestral eukaryotes. Mutations in the mitochondrial genome accumulate over the life of the organism. Mutations in nuclear or mitochondrial genes that encode components of the respiratory chain, ATP synthase, and the ROS-scavenging system, and even in tRNA genes, can cause a variety of human diseases, which oen most severely affect muscle, heart, pancreatic β cells, and brain. It is possible to combine the mitochondria from one woman with the nuclear genes of another to create an ovum free of a mutation that would have led to a mitochondrial disease. Mitochondrial defects in pancreatic β cells that limit ATP production when glucose levels are high can compromise normal insulin release and give rise to a form of type 2 diabetes. Chapter Review KEY TERMS Terms in bold are deﬁned in the glossary. chemiosmotic theory cristae respiratory chain ﬂavoprotein reducing equivalent ubiquinone (coenzyme Q, Q) cytochromes iron-sulfur protein Rieske iron-sulfur protein Complex I NADH dehydrogenase vectorial Complex II succinate dehydrogenase reactive oxygen species (ROS) superoxide radical (∙O− 2) Complex III cytochrome bc1 complex Q cycle Complex IV cytochrome oxidase respirasome proton-motive force ATP synthase F1 ATPase c ring rotational catalysis binding-change model P/O ratio P/2e− ratio malate-aspartate shuttle glycerol 3-phosphate shuttle acceptor control mass-action ratio (Q) brown adipose tissue (BAT) uncoupling protein 1 (UCP1) cytochrome P-450 xenobiotics apoptosis apoptosome caspase heteroplasmy homoplasmy PROBLEMS 1. Oxidation-Reduction Reactions Complex I, the NADH dehydrogenase complex of the mitochondrial respiratory chain, promotes the following series of oxidation-reduction reactions, in which Fe3+ and Fe2+ represent the iron in iron- sulfur centers, Q is ubiquinone, QH2 is ubiquinol, and E is the enzyme: 1. NADH + H+ + E-FM N → NAD+ + E-FM NH2 2. E-FM NH2+ 2Fe3+ → E-FM N+ 2Fe2+ + 2H+ 3. 2Fe2+ + 2H+ + Q → 2Fe3+ + QH2 Sum: NADH + H+ + Q → NAD+ + QH2 For each of the three reactions catalyzed by Complex I, identify (a) the electron donor, (b) the electron acceptor, (c) the conjugate redox pair, (d) the reducing agent, and (e) the oxidizing agent. 2. All Parts of Ubiquinone Have a Function In electron transfer, only the quinone portion of ubiquinone undergoes oxidation-reduction; the isoprenoid side chain remains unchanged. What is the function of this chain? 3. Use of FAD Rather Than NAD+ in Succinate Oxidation All the dehydrogenases of glycolysis and the citric acid cycle use NAD+ (E ′° for NAD+/NADH is − 0.32V) as electron acceptor except succinate dehydrogenase, which uses covalently bound FAD (E ′° for FAD+/FADH2 in this enzyme is 0.050 V). Suggest why FAD is a more appropriate electron acceptor than NAD+ in the dehydrogenation of succinate, based on the E ′° values of fumarate/succinate (E ′°= 0.031V),NAD+/NADH, and the succinate dehydrogenase FAD/FADH2. 4. Degree of Reduction of Electron Carriers in the Respiratory Chain Mitochondrial conditions determine the degree of reduction of each carrier in the respiratory chain. For example, when NADH and O2 are abundant, the steady- state degree of reduction of the carriers decreases as electrons pass from the substrate to O2. When electron transfer is blocked, the carriers before the block become more reduced and those beyond the block become more oxidized (see Fig. 19-6). For each of the mitochondrial conditions listed, predict the state of oxidation of ubiquinone and cytochromes b, c1, c, and a+ a3. a. Abundant NADH and O2, but cyanide added b. Abundant NADH, but O2 exhausted c. Abundant O2, but NADH exhausted d. Abundant NADH and O2 5. Effect of Rotenone and Antimycin A on Electron Transfer Rotenone, a toxic natural product from plants, strongly inhibits NADH dehydrogenase of insect and ﬁsh mitochondria. Antimycin A, a toxic antibiotic, strongly inhibits the oxidation of ubiquinol. a. Explain why rotenone ingestion is lethal to some insect and ﬁsh species. b. Explain why antimycin A is a poison. c. Given that rotenone and antimycin A are equally effective in blocking their respective sites in the electron-transfer chain, which would be a more potent poison? Explain. 6. Uncouplers of Oxidative Phosphorylation In normal mitochondria, the rate of electron transfer is tightly coupled to the demand for ATP. When the rate of ATP use is relatively low, the rate of electron transfer is low; when demand for ATP increases, the electron-transfer rate increases. Under these conditions of tight coupling, the number of ATP molecules produced per atom of oxygen consumed when NADH is the electron donor — the P/O ratio — is about 2.5. a. Predict the effect of a relatively low and a relatively high concentration of uncoupling agent on the rate of electron transfer and the P/O ratio. b. Ingestion of uncouplers causes profuse sweating and an increase in body temperature. Explain this phenomenon in molecular terms. What happens to the P/O ratio in the presence of uncouplers? c. Physicians used to prescribe the uncoupler 2,4- dinitrophenol (DNP) as a weight-reducing drug. How could this agent, in principle, serve as a weight- reducing aid? Physicians no longer prescribe uncoupling agents, because some deaths occurred following their use. Why might the ingestion of uncouplers cause death? 7. Effects of Valinomycin on Oxidative Phosphorylation When investigators add the antibiotic valinomycin (see Fig. 11-43) to actively respiring mitochondria, several things happen: the yield of ATP decreases, the rate of O2 consumption increases, heat is released, and the pH gradient across the inner mitochondrial membrane increases. Does valinomycin act as an uncoupler or as an inhibitor of oxidative phosphorylation? Explain the experimental observations in terms of the antibiotic’s ability to transfer K+ ions across the inner mitochondrial membrane. 8. Cellular ADP Concentration Controls ATP Formation Although ATP synthesis requires both ADP and Pi, the rate of synthesis depends mainly on the concentration of ADP, not Pi . Why? 9. Reactive Oxygen Species Describe the role played by superoxide dismutase in ameliorating the effects of reactive oxygen species. 10. How Many Protons in a Mitochondrion? Electron transfer translocates protons from the mitochondrial matrix to the external medium, establishing a pH gradient across the inner membrane (outside more acidic than inside). The tendency of protons to diffuse back into the matrix is the driving force for ATP synthesis by ATP synthase. During oxidative phosphorylation by a suspension of mitochondria in a medium of pH 7.4, the measured pH of the matrix is 7.7. a. Calculate [H+] in the external medium and in the matrix under these conditions. b. What is the outside-to-inside ratio of [H+]? How much energy for ATP synthesis is available in this concentration difference. (Hint: See Eqn 11-4, p. 392.) c. Calculate the number of protons in a respiring liver mitochondrion, assuming its inner matrix compartment is a sphere of diameter 1.5μm. d. From these data, is the pH gradient alone sufﬁcient to generate ATP? e. If not, suggest how the necessary energy for synthesis of ATP arises. 11. Rate of ATP Turnover in Rat Heart Muscle Rat heart muscle operating aerobically ﬁlls more than 90% of its ATP needs by oxidative phosphorylation. Each gram of tissue consumes O2 at the rate of 10.0μmol/min, with glucose as the fuel source. a. Calculate the rate at which the heart muscle consumes glucose and produces ATP. b. For a steady-state ATP concentration of 5.0μmol/g of heart muscle tissue, calculate the time required (in seconds) to completely turn over the cellular pool of ATP. What does this result indicate about the need for tight regulation of ATP production? (Note: Concentrations are expressed as micromoles per gram of muscle tissue because the tissue is mostly water.) 12. Rate of ATP Breakdown in Insect Flight Muscle ATP production in the ﬂight muscle of the ﬂy Lucilia sericata results almost exclusively from oxidative phosphorylation. During ﬂight, maintaining an ATP concentration of 7.0μmol/g of ﬂight muscle requires 187 mL of O2/h∙g of body weight. Assuming that ﬂight muscle makes up 20% of the ﬂy’s weight, calculate the rate at which the ﬂight-muscle ATP pool turns over. How long would the reservoir of ATP last in the absence of oxidative phosphorylation? Assume that the glycerol 3-phosphate shuttle transfers the reducing equivalents and that O2 is at 25 °C and 101.3kPa(1atm). 13. High Blood Alanine Level Associated with Defects in Oxidative Phosphorylation Most individuals with genetic defects in oxidative phosphorylation have relatively high concentrations of alanine in their blood. Explain this in biochemical terms. 14. Compartmentalization of Citric Acid Cycle Components Isocitrate dehydrogenase is found only in mitochondria, but malate dehydrogenase is found in both the cytosol and mitochondria. What is the role of cytosolic malate dehydrogenase? 15. Transmembrane Movement of Reducing Equivalents Under aerobic conditions, extramitochondrial NADH must undergo oxidation by the mitochondrial respiratory chain. Consider a preparation of rat hepatocytes containing mitochondria and all the cytosolic enzymes. Aer the introduction of [4-3H]NADH, radioactivity soon appears in the mitochondrial matrix. Conversely, no radioactivity appears in the matrix aer the introduction of [7-14C]NADH. What do these observations reveal about the oxidation of extramitochondrial NADH by the respiratory chain? 16. NAD Pools and Dehydrogenase Activities Although both pyruvate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase use NAD+ as their electron acceptor, the two enzymes do not compete for the same cellular NAD pool. Why? 17. The Malate–α -Ketoglutarate Transport System n- Butylmalonate inhibits the transport system that conveys malate and α -ketoglutarate across the inner mitochondrial membrane (see Fig. 19-31). Suppose you add n-butylmalonate to an aerobic suspension of kidney cells using exclusively glucose as fuel. Predict the effect of this inhibitor on a. glycolysis, b. oxygen consumption, c. lactate formation, and d. ATP synthesis. 18. Time Scales of Regulatory Events in Mitochondria Compare the likely time scales for the adjustments in respiratory rate caused by a. increased [ADP] and b. reduced pO2. What accounts for the difference? 19. The Pasteur Effect When investigators add O2 to an anaerobic suspension of cells consuming glucose at a high rate, the rate of glucose consumption declines greatly as the cells consume the O2, and accumulation of lactate ceases. This effect, ﬁrst observed by Louis Pasteur in the 1860s, is characteristic of most cells capable of both aerobic and anaerobic glucose catabolism. a. Why does the accumulation of lactate cease aer the addition of O2? b. Why does the presence of O2 decrease the rate of glucose consumption? c. How does the onset of O2 consumption slow down the rate of glucose consumption? Explain in terms of speciﬁc enzymes. 20. Respiration-Deﬁcient Yeast Mutants and Ethanol Production Researchers can produce respiration-deﬁcient yeast mutants (p−; “petites”) from wild-type parents by treatment with mutagenic agents. The mutants lack cytochrome oxidase, a deﬁcit that markedly affects their metabolic behavior. One striking effect is that fermentation is not suppressed by O2 — that is, the mutants do not experience the Pasteur effect (see Problem 19). Some companies are very interested in using these mutants to ferment wood chips to ethanol for energy use. Why does the absence of cytochrome oxidase eliminate the Pasteur effect? Explain the advantages of using these mutants rather than wild-type yeast for large-scale ethanol production. 21. Mitochondrial Disease and Cancer Mutations in the genes that encode certain mitochondrial proteins are associated with a high incidence of some types of cancer. How might defective mitochondria lead to cancer? 22. Variable Severity of a Mitochondrial Disease Different individuals with a disease caused by the same speciﬁc defect in the mitochondrial genome may have symptoms ranging from mild to severe. Explain why. 23. Diabetes as a Consequence of Mitochondrial Defects Glucokinase is essential in the metabolism of glucose in pancreatic β cells. Humans with two defective copies of the glucokinase gene exhibit a severe, neonatal diabetes, whereas those with only one defective copy of the gene have a much milder form of the disease (maturity onset diabetes of the young, MODY2). Explain this difference in terms of the biology of the β cell. 24. Effects of Mutations in Mitochondrial Complex II Single nucleotide changes in the gene for succinate dehydrogenase (Complex II) are associated with midgut carcinoid tumors. Suggest a mechanism to explain this observation. DATA ANALYSIS PROBLEM 25. Membrane Fluidity and Respiration Rate The mitochondrial electron transfer complexes and the FoF1 ATP synthase are embedded in the inner mitochondrial membrane in eukaryotes and in the inner membrane of bacteria. Electrons are shuttled between complexes in part by coenzyme Q, or ubiquinone, a factor that migrates within the membrane. Jay Keasling and coworkers explored the effect of membrane ﬂuidity on rates of respiration in E. coli. E. coli naturally adjusts its membrane lipid content to maintain membrane ﬂuidity at different temperatures. Workers in the Keasling lab bioengineered an E. coli strain to allow them to control expression of the enzyme FabB, which catalyzes the limiting step in the synthesis of unsaturated fatty acids in E. coli. a. How does the content of unsaturated fatty acids affect membrane ﬂuidity? b. The researchers were able to modulate the content of unsaturated fatty acids in the membrane lipid from 15% to 80%. They did not try to completely block synthesis of unsaturated fatty acids to extend the experimental range in the membrane to 0%. Why not? c. When the cells were grown under aerobic conditions, the researchers found that bacterial growth rate increased as the concentration of unsaturated fatty acids in the membrane increased. However, when oxygen was very limited, the unsaturated fatty acid content of the membrane had no effect on growth rate. How might you explain this observation? d. The researchers measured rates of respiration, ﬁnding a strong correlation between those rates and the fraction of membrane fatty acids that was unsaturated. When the unsaturated fatty acid content of the membranes was kept low, the cells accumulated pyruvate and lactate. Explain these observations. e. Next, they measured rates of diffusion of membrane phospholipids and ubiquinone in vesicles derived from E. coli membranes. The diffusion rates increased as a function of the content of unsaturated fatty acids. These measured rates were consistent with simulations carried out to model the effects of ubiquinone diffusion on respiration. What overall conclusion can be drawn from this work? Reference Budin, I, T. de Rond, Y. Chen, L.J.G. Chan, C.J. Petzold, and J.D. Keasling. 2018. Viscous control of cellular respiration by membrane lipid composition. Science 362:1186–1189.

Stems are from the chapter Problems section; correct choices are drawn from Abbreviated Solutions to Problems (Appendix B) in the same edition.

Practice questions (from chapter Problems & Appendix B)Score: 0 / 25

1. Oxidation-Reduction Reactions Complex I, the NADH dehydrogenase complex of the mitochondrial respiratory chain, promotes the following series of oxidation-reduction reactions, in which Fe3+ and Fe2+ represent the iron in iron- sulfur centers, Q is ubiquinone, QH2 is ubiquinol, and E is the enzyme:

AReaction 1: (a), (d) NADH; (b), (e) E-FMN; (c) NAD+/NADH and E-FM N/FM NH2 Reaction 2: (a), (d) E-FM NH2; (b), (e) Fe3+; (c) E-FM N/FM NH2 and Fe3+/Fe2+ Reaction 3: (a), (d) Fe2+; (b), (e) Q; (c) Fe3+/Fe2+ and Q/QH2 Ba. Glycolysis becomes anaerobic. b. Oxygen consumption ceases. c. Lactate formation increases. d. ATP synthesis decreases to 2 ATP/glucose. Ca. All carriers reduced; CN− blocks the reduction of O2 catalyzed by cytochrome oxidase. b. All carriers reduced; in the absence of O2, the reduced carriers are not reoxidized. c. All carriers oxidized d. Early carriers more reduced; later carriers more oxidized DSuperoxide dismutase catalyzes the reduction of superoxide to hydrogen peroxide. The hydrogen peroxide can then be eliminated by glutathione peroxidase. This is a major pathway for eliminating the superoxide generated during respiration, helping to ameliorate the damaging effects of reactive oxygen species.

2. NADH + H+ + E-FM N → NAD+ + E-FM NH2

AFrom the difference in standard reduction potential (ΔE′°) for each pair of half-reactions, we can calculate ΔG′°. The oxidation of succinate by FAD is favored by the negative standard free- energy change (ΔG′°=−3.7 kJ/mol). Oxidation by NAD+ would require a large, positive, standard free-energy change (ΔG′°=68 kJ/mol). BThe citric acid cycle is stalled for lack of an acceptor of electrons from NADH. Pyruvate produced by glycolysis cannot enter the cycle as acetyl-CoA; accumulated pyruvate is transaminated to alanine and exported to the liver. Ca. 0.91 μmol/s∙g b. 5.5 s; to provide a constant level of ATP, regulation of ATP production must be tight and rapid. DReaction 1: (a), (d) NADH; (b), (e) E-FMN; (c) NAD+/NADH and E-FM N/FM NH2 Reaction 2: (a), (d) E-FM NH2; (b), (e) Fe3+; (c) E-FM N/FM NH2 and Fe3+/Fe2+ Reaction 3: (a), (d) Fe2+; (b), (e) Q; (c) Fe3+/Fe2+ and Q/QH2

3. E-FM NH2+ 2Fe3+ → E-FM N+ 2Fe2+ + 2H+

Aa. All carriers reduced; CN− blocks the reduction of O2 catalyzed by cytochrome oxidase. b. All carriers reduced; in the absence of O2, the reduced carriers are not reoxidized. c. All carriers oxidized d. Early carriers more reduced; later carriers more oxidized Ba. NADH is reoxidized via electron transfer instead of lactic acid fermentation. b. Oxidative phosphorylation is more efﬁcient. c. The high mass-action ratio of the ATP system inhibits phosphofructokinase-1. Ca. Unsaturated fatty acids increase membrane ﬂuidity. b. The cells cannot survive if their membranes contain less than 15% unsaturated fatty acids. c. Oxygen is required for respiration. If cell growth is affected by membrane ﬂuidity only in the presence of oxygen, then logically respiration rates may be affected by membrane ﬂuidity. d. The ﬁrst observation indicates that something that migrates in the membrane is l… DThe side chain makes ubiquinone soluble in lipids and allows it to diffuse in the semiﬂuid membrane.

4. 2Fe2+ + 2H+ + Q → 2Fe3+ + QH2 Sum: NADH + H+ + Q → NAD+ + QH2 For each of the three reactions catalyzed by Complex I, identify (a) the electron donor, (b) the electron acceptor, (c) the conjugate redox pair, (d) the reducing agent, and (e) the oxidizing agent.

AReactive oxygen species react with macromolecules, including DNA. If a mitochondrial defect leads to increased production of ROS, proto-oncogenes in the nuclear chromosomes can be damaged, producing oncogenes and leading to unregulated cell division and cancer (see Section 12.9). B53 μmol/s∙g. With a steady state [ATP] of 7.0 μmol/g, this is equivalent to 10 turnovers of the ATP pool per second; the reservoir would last about 0.13 s. CFrom the difference in standard reduction potential (ΔE′°) for each pair of half-reactions, we can calculate ΔG′°. The oxidation of succinate by FAD is favored by the negative standard free- energy change (ΔG′°=−3.7 kJ/mol). Oxidation by NAD+ would require a large, positive, standard free-energy change (ΔG′°=68 kJ/mol). Da. NADH is reoxidized via electron transfer instead of lactic acid fermentation. b. Oxidative phosphorylation is more efﬁcient. c. The high mass-action ratio of the ATP system inhibits phosphofructokinase-1.

5. All Parts of Ubiquinone Have a Function In electron transfer, only the quinone portion of ubiquinone undergoes oxidation-reduction; the isoprenoid side chain remains unchanged. What is the function of this chain?

Aa. NADH is reoxidized via electron transfer instead of lactic acid fermentation. b. Oxidative phosphorylation is more efﬁcient. c. The high mass-action ratio of the ATP system inhibits phosphofructokinase-1. BCytosolic malate dehydrogenase plays a key role in the transport of reducing equivalents across the inner mitochondrial membrane via the malate-aspartate shuttle. CThe side chain makes ubiquinone soluble in lipids and allows it to diffuse in the semiﬂuid membrane. DDefects in Complex II result in increased production of ROS, damage to DNA, and mutations that lead to unregulated cell division (cancer; see Section 12.9). It is not clear why the cancer tends to occur in the midgut.

6. Use of FAD Rather Than NAD+ in Succinate Oxidation All the dehydrogenases of glycolysis and the citric acid cycle use NAD+ (E ′° for NAD+/NADH is − 0.32V) as electron acceptor except succinate dehydrogenase, which uses covalently bound FAD (E ′° for FAD+/FADH2 in this enzyme is 0.050 V). Suggest why FAD is a more appropriate electron acceptor than NAD+ in the dehydrogenation of succinate, based on the E ′° values of fumarate/succinate (E ′°= 0.031V),NAD+/NADH, and the succinate dehydrogenase FAD/FADH2.

AThe citric acid cycle is stalled for lack of an acceptor of electrons from NADH. Pyruvate produced by glycolysis cannot enter the cycle as acetyl-CoA; accumulated pyruvate is transaminated to alanine and exported to the liver. BComplete lack of glucokinase (two defective alleles) makes it impossible to carry out glycolysis at a sufﬁcient rate to raise [ATP] to the threshold required for insulin secretion. CFrom the difference in standard reduction potential (ΔE′°) for each pair of half-reactions, we can calculate ΔG′°. The oxidation of succinate by FAD is favored by the negative standard free- energy change (ΔG′°=−3.7 kJ/mol). Oxidation by NAD+ would require a large, positive, standard free-energy change (ΔG′°=68 kJ/mol). Da. NADH is reoxidized via electron transfer instead of lactic acid fermentation. b. Oxidative phosphorylation is more efﬁcient. c. The high mass-action ratio of the ATP system inhibits phosphofructokinase-1.

7. Degree of Reduction of Electron Carriers in the Respiratory Chain Mitochondrial conditions determine the degree of reduction of each carrier in the respiratory chain. For example, when NADH and O2 are abundant, the steady- state degree of reduction of the carriers decreases as electrons pass from the substrate to O2. When electron transfer is blocked, the carriers before the block become more reduced and those beyond the block become more oxidized (see Fig. 19-6). For each of the mitochondrial conditions listed, predict the state of oxidation of ubiquinone and cytochromes b, c1, c, and a+ a3. a. Abundant NADH and O2, but cyanide added b. Abundant NADH, but O2 exhausted c. Abundant O2, but NADH exhausted d. Abundant NADH and O2

AThe response to (a), increased [ADP], is faster because the response to (b), reduced pO2, requires protein synthesis. BReaction 1: (a), (d) NADH; (b), (e) E-FMN; (c) NAD+/NADH and E-FM N/FM NH2 Reaction 2: (a), (d) E-FM NH2; (b), (e) Fe3+; (c) E-FM N/FM NH2 and Fe3+/Fe2+ Reaction 3: (a), (d) Fe2+; (b), (e) Q; (c) Fe3+/Fe2+ and Q/QH2 Ca. All carriers reduced; CN− blocks the reduction of O2 catalyzed by cytochrome oxidase. b. All carriers reduced; in the absence of O2, the reduced carriers are not reoxidized. c. All carriers oxidized d. Early carriers more reduced; later carriers more oxidized DThe citric acid cycle is stalled for lack of an acceptor of electrons from NADH. Pyruvate produced by glycolysis cannot enter the cycle as acetyl-CoA; accumulated pyruvate is transaminated to alanine and exported to the liver.

8. Effect of Rotenone and Antimycin A on Electron Transfer Rotenone, a toxic natural product from plants, strongly inhibits NADH dehydrogenase of insect and ﬁsh mitochondria. Antimycin A, a toxic antibiotic, strongly inhibits the oxidation of ubiquinol. a. Explain why rotenone ingestion is lethal to some insect and ﬁsh species. b. Explain why antimycin A is a poison. c. Given that rotenone and antimycin A are equally effective in blocking their respective sites in the electron-transfer chain, which would be a more potent poison? Explain.

ADefects in Complex II result in increased production of ROS, damage to DNA, and mutations that lead to unregulated cell division (cancer; see Section 12.9). It is not clear why the cancer tends to occur in the midgut. BSuperoxide dismutase catalyzes the reduction of superoxide to hydrogen peroxide. The hydrogen peroxide can then be eliminated by glutathione peroxidase. This is a major pathway for eliminating the superoxide generated during respiration, helping to ameliorate the damaging effects of reactive oxygen species. C53 μmol/s∙g. With a steady state [ATP] of 7.0 μmol/g, this is equivalent to 10 turnovers of the ATP pool per second; the reservoir would last about 0.13 s. Da. Inhibition of NADH dehydrogenase by rotenone decreases the rate of electron ﬂow through the respiratory chain, which in turn decreases the rate of ATP production. If this reduced rate is unable to meet the organism’s ATP requirements, the organism dies. b. Antimycin A strongly inhibits the oxidation of Q in the respiratory chain, reducing the rate of electron transfer and leading to the consequences described in …

9. Uncouplers of Oxidative Phosphorylation In normal mitochondria, the rate of electron transfer is tightly coupled to the demand for ATP. When the rate of ATP use is relatively low, the rate of electron transfer is low; when demand for ATP increases, the electron-transfer rate increases. Under these conditions of tight coupling, the number of ATP molecules produced per atom of oxygen consumed when NADH is the electron donor — the P/O ratio — is about 2.5. a. Predict the effect of a relatively low and a relatively high concentration of uncoupling agent on the rate of electron transfer and the P/O ratio. b. Ingestion of uncouplers causes profuse sweating and an increase in body temperature. Explain this phenomenon in molecular terms. What happens to the P/O ratio in the presence of uncouplers? c. Physicians used to prescribe the uncoupler 2,4- dinitrophenol (DNP) as a weight-reducing drug. How could this agent, in principle, serve as a weight- reducing aid? Physicians no longer prescribe uncoupling agents, because some deaths occurred following their use. Why might the ingestion of uncouplers cause death?

Aa. Glycolysis becomes anaerobic. b. Oxygen consumption ceases. c. Lactate formation increases. d. ATP synthesis decreases to 2 ATP/glucose. Ba. The rate of electron transfer necessary to meet the ATP demand increases, and thus the P/O ratio decreases. b. High concentrations of uncoupler produce P/O ratios near zero. The P/O ratio decreases and more fuel must be oxidized to generate the same amount of ATP. The extra heat released by this oxidation raises the body temperature. c. Increased activity of the respiratory chain in the presence of an uncoupler r… CReactive oxygen species react with macromolecules, including DNA. If a mitochondrial defect leads to increased production of ROS, proto-oncogenes in the nuclear chromosomes can be damaged, producing oncogenes and leading to unregulated cell division and cancer (see Section 12.9). Da. NADH is reoxidized via electron transfer instead of lactic acid fermentation. b. Oxidative phosphorylation is more efﬁcient. c. The high mass-action ratio of the ATP system inhibits phosphofructokinase-1.

10. Effects of Valinomycin on Oxidative Phosphorylation When investigators add the antibiotic valinomycin (see Fig. 11-43) to actively respiring mitochondria, several things happen: the yield of ATP decreases, the rate of O2 consumption increases, heat is released, and the pH gradient across the inner mitochondrial membrane increases. Does valinomycin act as an uncoupler or as an inhibitor of oxidative phosphorylation? Explain the experimental observations in terms of the antibiotic’s ability to transfer K+ ions across the inner mitochondrial membrane.

A53 μmol/s∙g. With a steady state [ATP] of 7.0 μmol/g, this is equivalent to 10 turnovers of the ATP pool per second; the reservoir would last about 0.13 s. BValinomycin acts as an uncoupler. It combines with K+ to form a complex that passes through the inner mitochondrial membrane, dissipating the membrane potential. ATP synthesis decreases, which causes the rate of electron transfer to increase. This results in an increase in the H+ gradient, O2 consumption, and amount of heat released. CReactive oxygen species react with macromolecules, including DNA. If a mitochondrial defect leads to increased production of ROS, proto-oncogenes in the nuclear chromosomes can be damaged, producing oncogenes and leading to unregulated cell division and cancer (see Section 12.9). DThe Pasteur effect is not observed, because the citric acid cycle and respiratory chain are inactive. Fermentation to ethanol could be accomplished in the presence of O2, which is an advantage because strict anaerobic conditions are difﬁcult to maintain.

11. Cellular ADP Concentration Controls ATP Formation Although ATP synthesis requires both ADP and Pi, the rate of synthesis depends mainly on the concentration of ADP, not Pi . Why?

ASuperoxide dismutase catalyzes the reduction of superoxide to hydrogen peroxide. The hydrogen peroxide can then be eliminated by glutathione peroxidase. This is a major pathway for eliminating the superoxide generated during respiration, helping to ameliorate the damaging effects of reactive oxygen species. BThe steady-state concentration of Pi in the cell is much higher than that of ADP. The Pi released by ATP hydrolysis changes total [Pi] very little. CThe Pasteur effect is not observed, because the citric acid cycle and respiratory chain are inactive. Fermentation to ethanol could be accomplished in the presence of O2, which is an advantage because strict anaerobic conditions are difﬁcult to maintain. Da. External medium: 4.0×10−8 M ; matrix: 2.0×10−8 M b. [H+] gradient contributes 1.7 kJ/mol toward ATP synthesis. c. 21 d. No e. From the overall transmembrane potential

12. Reactive Oxygen Species Describe the role played by superoxide dismutase in ameliorating the effects of reactive oxygen species.

A53 μmol/s∙g. With a steady state [ATP] of 7.0 μmol/g, this is equivalent to 10 turnovers of the ATP pool per second; the reservoir would last about 0.13 s. BThe response to (a), increased [ADP], is faster because the response to (b), reduced pO2, requires protein synthesis. CSuperoxide dismutase catalyzes the reduction of superoxide to hydrogen peroxide. The hydrogen peroxide can then be eliminated by glutathione peroxidase. This is a major pathway for eliminating the superoxide generated during respiration, helping to ameliorate the damaging effects of reactive oxygen species. Da. 0.91 μmol/s∙g b. 5.5 s; to provide a constant level of ATP, regulation of ATP production must be tight and rapid.

13. How Many Protons in a Mitochondrion? Electron transfer translocates protons from the mitochondrial matrix to the external medium, establishing a pH gradient across the inner membrane (outside more acidic than inside). The tendency of protons to diffuse back into the matrix is the driving force for ATP synthesis by ATP synthase. During oxidative phosphorylation by a suspension of mitochondria in a medium of pH 7.4, the measured pH of the matrix is 7.7. a. Calculate [H+] in the external medium and in the matrix under these conditions. b. What is the outside-to-inside ratio of [H+]? How much energy for ATP synthesis is available in this concentration difference. (Hint: See Eqn 11-4, p. 392.) c. Calculate the number of protons in a respiring liver mitochondrion, assuming its inner matrix compartment is a sphere of diameter 1.5μm. d. From these data, is the pH gradient alone sufﬁcient to generate ATP? e. If not, suggest how the necessary energy for synthesis of ATP arises.

Aa. External medium: 4.0×10−8 M ; matrix: 2.0×10−8 M b. [H+] gradient contributes 1.7 kJ/mol toward ATP synthesis. c. 21 d. No e. From the overall transmembrane potential Ba. All carriers reduced; CN− blocks the reduction of O2 catalyzed by cytochrome oxidase. b. All carriers reduced; in the absence of O2, the reduced carriers are not reoxidized. c. All carriers oxidized d. Early carriers more reduced; later carriers more oxidized Ca. NADH is reoxidized via electron transfer instead of lactic acid fermentation. b. Oxidative phosphorylation is more efﬁcient. c. The high mass-action ratio of the ATP system inhibits phosphofructokinase-1. DPyruvate dehydrogenase is located in mitochondria; glyceraldehyde 3-phosphate dehydrogenase, in the cytosol. The NAD pools are separated by the inner mitochondrial membrane.

14. Rate of ATP Turnover in Rat Heart Muscle Rat heart muscle operating aerobically ﬁlls more than 90% of its ATP needs by oxidative phosphorylation. Each gram of tissue consumes O2 at the rate of 10.0μmol/min, with glucose as the fuel source. a. Calculate the rate at which the heart muscle consumes glucose and produces ATP. b. For a steady-state ATP concentration of 5.0μmol/g of heart muscle tissue, calculate the time required (in seconds) to completely turn over the cellular pool of ATP. What does this result indicate about the need for tight regulation of ATP production? (Note: Concentrations are expressed as micromoles per gram of muscle tissue because the tissue is mostly water.)

Aa. The rate of electron transfer necessary to meet the ATP demand increases, and thus the P/O ratio decreases. b. High concentrations of uncoupler produce P/O ratios near zero. The P/O ratio decreases and more fuel must be oxidized to generate the same amount of ATP. The extra heat released by this oxidation raises the body temperature. c. Increased activity of the respiratory chain in the presence of an uncoupler r… Ba. NADH is reoxidized via electron transfer instead of lactic acid fermentation. b. Oxidative phosphorylation is more efﬁcient. c. The high mass-action ratio of the ATP system inhibits phosphofructokinase-1. Ca. 0.91 μmol/s∙g b. 5.5 s; to provide a constant level of ATP, regulation of ATP production must be tight and rapid. Da. Inhibition of NADH dehydrogenase by rotenone decreases the rate of electron ﬂow through the respiratory chain, which in turn decreases the rate of ATP production. If this reduced rate is unable to meet the organism’s ATP requirements, the organism dies. b. Antimycin A strongly inhibits the oxidation of Q in the respiratory chain, reducing the rate of electron transfer and leading to the consequences described in …

15. Rate of ATP Breakdown in Insect Flight Muscle ATP production in the ﬂight muscle of the ﬂy Lucilia sericata results almost exclusively from oxidative phosphorylation. During ﬂight, maintaining an ATP concentration of 7.0μmol/g of ﬂight muscle requires 187 mL of O2/h∙g of body weight. Assuming that ﬂight muscle makes up 20% of the ﬂy’s weight, calculate the rate at which the ﬂight-muscle ATP pool turns over. How long would the reservoir of ATP last in the absence of oxidative phosphorylation? Assume that the glycerol 3-phosphate shuttle transfers the reducing equivalents and that O2 is at 25 °C and 101.3kPa(1atm).

A53 μmol/s∙g. With a steady state [ATP] of 7.0 μmol/g, this is equivalent to 10 turnovers of the ATP pool per second; the reservoir would last about 0.13 s. BDifferent extents of heteroplasmy for the defective gene produce different degrees of defective mitochondrial function. CThe inner mitochondrial membrane is impermeable to NADH, but the reducing equivalents of NADH are transferred (shuttled) through the membrane indirectly: they are transferred to oxaloacetate in the cytosol, the resulting malate is transported into the matrix, and mitochondrial NAD+ is reduced to NADH. DThe citric acid cycle is stalled for lack of an acceptor of electrons from NADH. Pyruvate produced by glycolysis cannot enter the cycle as acetyl-CoA; accumulated pyruvate is transaminated to alanine and exported to the liver.

16. High Blood Alanine Level Associated with Defects in Oxidative Phosphorylation Most individuals with genetic defects in oxidative phosphorylation have relatively high concentrations of alanine in their blood. Explain this in biochemical terms.

Aa. The rate of electron transfer necessary to meet the ATP demand increases, and thus the P/O ratio decreases. b. High concentrations of uncoupler produce P/O ratios near zero. The P/O ratio decreases and more fuel must be oxidized to generate the same amount of ATP. The extra heat released by this oxidation raises the body temperature. c. Increased activity of the respiratory chain in the presence of an uncoupler r… BThe side chain makes ubiquinone soluble in lipids and allows it to diffuse in the semiﬂuid membrane. CThe citric acid cycle is stalled for lack of an acceptor of electrons from NADH. Pyruvate produced by glycolysis cannot enter the cycle as acetyl-CoA; accumulated pyruvate is transaminated to alanine and exported to the liver. Da. NADH is reoxidized via electron transfer instead of lactic acid fermentation. b. Oxidative phosphorylation is more efﬁcient. c. The high mass-action ratio of the ATP system inhibits phosphofructokinase-1.

17. Compartmentalization of Citric Acid Cycle Components Isocitrate dehydrogenase is found only in mitochondria, but malate dehydrogenase is found in both the cytosol and mitochondria. What is the role of cytosolic malate dehydrogenase?

AThe Pasteur effect is not observed, because the citric acid cycle and respiratory chain are inactive. Fermentation to ethanol could be accomplished in the presence of O2, which is an advantage because strict anaerobic conditions are difﬁcult to maintain. Ba. The rate of electron transfer necessary to meet the ATP demand increases, and thus the P/O ratio decreases. b. High concentrations of uncoupler produce P/O ratios near zero. The P/O ratio decreases and more fuel must be oxidized to generate the same amount of ATP. The extra heat released by this oxidation raises the body temperature. c. Increased activity of the respiratory chain in the presence of an uncoupler r… CCytosolic malate dehydrogenase plays a key role in the transport of reducing equivalents across the inner mitochondrial membrane via the malate-aspartate shuttle. DDefects in Complex II result in increased production of ROS, damage to DNA, and mutations that lead to unregulated cell division (cancer; see Section 12.9). It is not clear why the cancer tends to occur in the midgut.

18. Transmembrane Movement of Reducing Equivalents Under aerobic conditions, extramitochondrial NADH must undergo oxidation by the mitochondrial respiratory chain. Consider a preparation of rat hepatocytes containing mitochondria and all the cytosolic enzymes. Aer the introduction of [4-3H]NADH, radioactivity soon appears in the mitochondrial matrix. Conversely, no radioactivity appears in the matrix aer the introduction of [7-14C]NADH. What do these observations reveal about the oxidation of extramitochondrial NADH by the respiratory chain?

Aa. The rate of electron transfer necessary to meet the ATP demand increases, and thus the P/O ratio decreases. b. High concentrations of uncoupler produce P/O ratios near zero. The P/O ratio decreases and more fuel must be oxidized to generate the same amount of ATP. The extra heat released by this oxidation raises the body temperature. c. Increased activity of the respiratory chain in the presence of an uncoupler r… B53 μmol/s∙g. With a steady state [ATP] of 7.0 μmol/g, this is equivalent to 10 turnovers of the ATP pool per second; the reservoir would last about 0.13 s. CThe inner mitochondrial membrane is impermeable to NADH, but the reducing equivalents of NADH are transferred (shuttled) through the membrane indirectly: they are transferred to oxaloacetate in the cytosol, the resulting malate is transported into the matrix, and mitochondrial NAD+ is reduced to NADH. Da. Glycolysis becomes anaerobic. b. Oxygen consumption ceases. c. Lactate formation increases. d. ATP synthesis decreases to 2 ATP/glucose.

19. NAD Pools and Dehydrogenase Activities Although both pyruvate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase use NAD+ as their electron acceptor, the two enzymes do not compete for the same cellular NAD pool. Why?

ACytosolic malate dehydrogenase plays a key role in the transport of reducing equivalents across the inner mitochondrial membrane via the malate-aspartate shuttle. BPyruvate dehydrogenase is located in mitochondria; glyceraldehyde 3-phosphate dehydrogenase, in the cytosol. The NAD pools are separated by the inner mitochondrial membrane. Ca. Unsaturated fatty acids increase membrane ﬂuidity. b. The cells cannot survive if their membranes contain less than 15% unsaturated fatty acids. c. Oxygen is required for respiration. If cell growth is affected by membrane ﬂuidity only in the presence of oxygen, then logically respiration rates may be affected by membrane ﬂuidity. d. The ﬁrst observation indicates that something that migrates in the membrane is l… DComplete lack of glucokinase (two defective alleles) makes it impossible to carry out glycolysis at a sufﬁcient rate to raise [ATP] to the threshold required for insulin secretion.

20. The Malate–α -Ketoglutarate Transport System n- Butylmalonate inhibits the transport system that conveys malate and α -ketoglutarate across the inner mitochondrial membrane (see Fig. 19-31). Suppose you add n-butylmalonate to an aerobic suspension of kidney cells using exclusively glucose as fuel. Predict the effect of this inhibitor on a. glycolysis, b. oxygen consumption, c. lactate formation, and d. ATP synthesis.

Aa. Glycolysis becomes anaerobic. b. Oxygen consumption ceases. c. Lactate formation increases. d. ATP synthesis decreases to 2 ATP/glucose. BThe citric acid cycle is stalled for lack of an acceptor of electrons from NADH. Pyruvate produced by glycolysis cannot enter the cycle as acetyl-CoA; accumulated pyruvate is transaminated to alanine and exported to the liver. Ca. External medium: 4.0×10−8 M ; matrix: 2.0×10−8 M b. [H+] gradient contributes 1.7 kJ/mol toward ATP synthesis. c. 21 d. No e. From the overall transmembrane potential DComplete lack of glucokinase (two defective alleles) makes it impossible to carry out glycolysis at a sufﬁcient rate to raise [ATP] to the threshold required for insulin secretion.

21. Time Scales of Regulatory Events in Mitochondria Compare the likely time scales for the adjustments in respiratory rate caused by a. increased [ADP] and b. reduced pO2. What accounts for the difference?

Aa. All carriers reduced; CN− blocks the reduction of O2 catalyzed by cytochrome oxidase. b. All carriers reduced; in the absence of O2, the reduced carriers are not reoxidized. c. All carriers oxidized d. Early carriers more reduced; later carriers more oxidized BThe response to (a), increased [ADP], is faster because the response to (b), reduced pO2, requires protein synthesis. Ca. External medium: 4.0×10−8 M ; matrix: 2.0×10−8 M b. [H+] gradient contributes 1.7 kJ/mol toward ATP synthesis. c. 21 d. No e. From the overall transmembrane potential DValinomycin acts as an uncoupler. It combines with K+ to form a complex that passes through the inner mitochondrial membrane, dissipating the membrane potential. ATP synthesis decreases, which causes the rate of electron transfer to increase. This results in an increase in the H+ gradient, O2 consumption, and amount of heat released.

22. The Pasteur Effect When investigators add O2 to an anaerobic suspension of cells consuming glucose at a high rate, the rate of glucose consumption declines greatly as the cells consume the O2, and accumulation of lactate ceases. This effect, ﬁrst observed by Louis Pasteur in the 1860s, is characteristic of most cells capable of both aerobic and anaerobic glucose catabolism. a. Why does the accumulation of lactate cease aer the addition of O2? b. Why does the presence of O2 decrease the rate of glucose consumption? c. How does the onset of O2 consumption slow down the rate of glucose consumption? Explain in terms of speciﬁc enzymes.

AThe response to (a), increased [ADP], is faster because the response to (b), reduced pO2, requires protein synthesis. Ba. NADH is reoxidized via electron transfer instead of lactic acid fermentation. b. Oxidative phosphorylation is more efﬁcient. c. The high mass-action ratio of the ATP system inhibits phosphofructokinase-1. CComplete lack of glucokinase (two defective alleles) makes it impossible to carry out glycolysis at a sufﬁcient rate to raise [ATP] to the threshold required for insulin secretion. DThe inner mitochondrial membrane is impermeable to NADH, but the reducing equivalents of NADH are transferred (shuttled) through the membrane indirectly: they are transferred to oxaloacetate in the cytosol, the resulting malate is transported into the matrix, and mitochondrial NAD+ is reduced to NADH.

23. Respiration-Deﬁcient Yeast Mutants and Ethanol Production Researchers can produce respiration-deﬁcient yeast mutants (p−; “petites”) from wild-type parents by treatment with mutagenic agents. The mutants lack cytochrome oxidase, a deﬁcit that markedly affects their metabolic behavior. One striking effect is that fermentation is not suppressed by O2 — that is, the mutants do not experience the Pasteur effect (see Problem 19). Some companies are very interested in using these mutants to ferment wood chips to ethanol for energy use. Why does the absence of cytochrome oxidase eliminate the Pasteur effect? Explain the advantages of using these mutants rather than wild-type yeast for large-scale ethanol production.

ADefects in Complex II result in increased production of ROS, damage to DNA, and mutations that lead to unregulated cell division (cancer; see Section 12.9). It is not clear why the cancer tends to occur in the midgut. BThe citric acid cycle is stalled for lack of an acceptor of electrons from NADH. Pyruvate produced by glycolysis cannot enter the cycle as acetyl-CoA; accumulated pyruvate is transaminated to alanine and exported to the liver. CThe Pasteur effect is not observed, because the citric acid cycle and respiratory chain are inactive. Fermentation to ethanol could be accomplished in the presence of O2, which is an advantage because strict anaerobic conditions are difﬁcult to maintain. Da. The rate of electron transfer necessary to meet the ATP demand increases, and thus the P/O ratio decreases. b. High concentrations of uncoupler produce P/O ratios near zero. The P/O ratio decreases and more fuel must be oxidized to generate the same amount of ATP. The extra heat released by this oxidation raises the body temperature. c. Increased activity of the respiratory chain in the presence of an uncoupler r…

24. Mitochondrial Disease and Cancer Mutations in the genes that encode certain mitochondrial proteins are associated with a high incidence of some types of cancer. How might defective mitochondria lead to cancer?

ASuperoxide dismutase catalyzes the reduction of superoxide to hydrogen peroxide. The hydrogen peroxide can then be eliminated by glutathione peroxidase. This is a major pathway for eliminating the superoxide generated during respiration, helping to ameliorate the damaging effects of reactive oxygen species. BReactive oxygen species react with macromolecules, including DNA. If a mitochondrial defect leads to increased production of ROS, proto-oncogenes in the nuclear chromosomes can be damaged, producing oncogenes and leading to unregulated cell division and cancer (see Section 12.9). CThe response to (a), increased [ADP], is faster because the response to (b), reduced pO2, requires protein synthesis. DThe steady-state concentration of Pi in the cell is much higher than that of ADP. The Pi released by ATP hydrolysis changes total [Pi] very little.

25. Variable Severity of a Mitochondrial Disease Different individuals with a disease caused by the same speciﬁc defect in the mitochondrial genome may have symptoms ranging from mild to severe. Explain why.

AReaction 1: (a), (d) NADH; (b), (e) E-FMN; (c) NAD+/NADH and E-FM N/FM NH2 Reaction 2: (a), (d) E-FM NH2; (b), (e) Fe3+; (c) E-FM N/FM NH2 and Fe3+/Fe2+ Reaction 3: (a), (d) Fe2+; (b), (e) Q; (c) Fe3+/Fe2+ and Q/QH2 BDifferent extents of heteroplasmy for the defective gene produce different degrees of defective mitochondrial function. Ca. The rate of electron transfer necessary to meet the ATP demand increases, and thus the P/O ratio decreases. b. High concentrations of uncoupler produce P/O ratios near zero. The P/O ratio decreases and more fuel must be oxidized to generate the same amount of ATP. The extra heat released by this oxidation raises the body temperature. c. Increased activity of the respiratory chain in the presence of an uncoupler r… Da. All carriers reduced; CN− blocks the reduction of O2 catalyzed by cytochrome oxidase. b. All carriers reduced; in the absence of O2, the reduced carriers are not reoxidized. c. All carriers oxidized d. Early carriers more reduced; later carriers more oxidized