19.1 The Mitochondrial Respiratory Chain in Chapter 19 Oxidative Phosphorylation

19.1 The Mitochondrial Respiratory Chain

A photo shows Albert L. Lehninger. — 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 $(M_{r} < 5,000)$ $left-parenthesis upper M Subscript r Baseline less-than 5,000 right-parenthesis$ 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 $(H^{+})$ $left-parenthesis upper H Superscript plus Baseline right-parenthesis$ ; 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.

A four-part figure shows the biochemical anatomy of a mitochondrion by showing a labeled illustration in part a, an endothelial cell containing mitochondrial in part b, mitochondria in heart muscle in part c, and liver mitochondria in part d. — 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 — $1 to 10 μ m$ $1 to 10 mu m$ 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.

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 — $1 to 10 μ m$ $1 to 10 mu m$ 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.

Part a shows a vertical mitochondrion cutaway to show the interior. The overall mitochondrion forms an elongated vertical oval. The outer surface contains many small circles labeled porin channels. An inner membrane is folded extensively to form horizontal pieces labeled cristae that extend into the center of the mitochondrion. The central region, in between the cristae, is the matrix. Blue dots are abundant on the inside of the inner membrane and are labeled A T P synthase (F subscript 0 end subscript F subscript 1 end subscript). In the openings between the cristae, there are a few strands of red spheres labeled ribosomes. Three boxes describe the outer membrane, inner membrane, and matrix. The first box is labeled outer membrane and contains text reading, Freely permeable to small molecules and ions. The second box is labeled inner membrane and contains text reading, impermeable to most small molecules and ions, including H plus. The inner membrane contains: respiratory electron carriers (complexes Roman numerals 1 through 4); A D P – A T P translocase; A T P synthase (F subscript 0 end subscript F subscript 1 end subscript); and other membrane transporters. The third box is labeled matrix. The matrix contains: pyruvate dehydrogenase complex; citric acid cycle enzymes; fatty acid beta-oxidation enzymes; D N A, ribosomes; many other enzymes; A T P, A D P, P subscript i end subscript, M g 2 plus, C a 2 plus, K plus; and many soluble metabolic intermediates. Part b shows a red oval with darker, rounded patches with many long yellow structures around it in a rounded region against a background of thin blue strands. The yellow structures are each about 6 micrometers in length and the red oval is approximately 20 micrometers in length. Part c shows reddish parallel lines forming two thick bands above and below a linear region containing bluish mitochondria. The reddish parallel lines form a pattern of a wide dark region, a thin light vertical band, a dark vertical line, another thin light vertical band, and then a wide dark region. Three mitochondria are visible. Each has a blue double membrane with linear protrusions, the cristae, extending into the center. The membranes are dark blue and the background is light blue. A yellow region is between the mitochondria. The third mitochondrion, at the upper right, is circular. Another mitochondrion is visible at the bottom right corner. Each mitochondrion is approximately 7.5 micrometers in length and 2 micrometers in width except for the circular mitochondrion, which is approximately 2.5 micrometers in diameter. Part d is a micrograph with many circular gray mitochondria that have dark gray outer membranes and dark linear strands extending in. Each mitochondrion is approximately 1.5 micrometers in diameter. All data are approximate.

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 $P_{i}$ $upper P Subscript i$ 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 Universal Electron 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 ( ${NAD}^{+}$ $NAD Superscript plus$ or ${NADP}^{+}$ $NADP Superscript plus$ ) or flavin nucleotides (FMN or FAD).

Nicotinamide nucleotide–linked dehydrogenases catalyze reversible reactions of the following general types:

Reduced substrate + {NAD}^{+} ⇌ oxidized substrate + NADH + H^{+}

$Reduced substrate plus NAD Superscript plus Baseline right harpoon over left harpoon oxidized substrate plus NADH plus upper H Superscript plus$

Reduced {substrate + NADP}^{+} ⇌ oxidized substrate + NADPH + H^{+}

$Reduced substrate plus NADP Superscript plus Baseline right harpoon over left harpoon oxidized substrate plus NADPH plus upper H Superscript plus$

Most dehydrogenases that act in catabolism are specific for ${NAD}^{+}$ $NAD Superscript plus$ 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 ${NAD(P)}^{+}$ $bold NAD left-parenthesis upper P right-parenthesis Superscript bold plus$ -Linked Dehydrogenases
Reaction^a	Location^b
${NAD}^{+}$ $bold NAD Superscript bold plus$ -linked
$α -Ketoglutarate + CoA + {NAD}^{+} ⇌ succinyl-CoA + {CO}_{2} + NADH + H^{+}$ $alpha hyphen Ketoglutarate plus CoA plus NAD Superscript plus Baseline right harpoon over left harpoon succinyl hyphen CoA plus CO Subscript 2 Baseline plus NADH plus upper H Superscript plus$	M
$L -Malate + {NAD}^{+} ⇌ oxaloacetate + NADH + H^{+}$ $upper L hyphen Malate plus NAD Superscript plus Baseline right harpoon over left harpoon oxaloacetate plus NADH plus upper H Superscript plus$	M and C
$Pyruvate + CoA + {NAD}^{+} ⇌ acetyl-CoA + {CO}_{2} + NADH + H^{+}$ $Pyruvate plus CoA plus NAD Superscript plus Baseline right harpoon over left harpoon acetyl hyphen CoA plus CO Subscript 2 Baseline plus NADH plus upper H Superscript plus$	M
$Glyceraldehyde 3 -phosphate + P_{i} + {NAD}^{+} ⇌ 1, 3 -bisphosphoglycerate + NADH + H^{+}$ $Glyceraldehyde 3 hyphen phosphate plus upper P Subscript i Baseline plus NAD Superscript plus Baseline right harpoon over left harpoon 1 comma 3 hyphen bisphosphoglycerate plus NADH plus upper H Superscript plus$	C
$Lactate + {NAD}^{+} ⇌ pyruvate + NADH + H^{+}$ $Lactate plus NAD Superscript plus Baseline right harpoon over left harpoon pyruvate plus NADH plus upper H Superscript plus$	C
$β -Hydroxyacyl-CoA + {NAD}^{+} ⇌ β -ketoacyl-CoA + NADH + H^{+}$ $beta hyphen Hydroxyacyl hyphen CoA plus NAD Superscript plus Baseline right harpoon over left harpoon beta hyphen ketoacyl hyphen CoA plus NADH plus upper H Superscript plus$	M
${NADP}^{+}$ $bold NADP Superscript bold plus$ -linked
$Glucose 6 -phosphate + {NADP}^{+} ⇌ 6 -phosphogluconate + NADPH + H^{+}$ $Glucose 6 hyphen phosphate plus NADP Superscript plus Baseline right harpoon over left harpoon 6 hyphen phosphogluconate plus NADPH plus upper H Superscript plus$	C
$L -Malate + {NADP}^{+} ⇌ pyruvate + {CO}_{2} + NADPH + H^{+}$ $upper L hyphen Malate plus NADP Superscript plus Baseline right harpoon over left harpoon pyruvate plus CO Subscript 2 Baseline plus NADPH plus upper H Superscript plus$	C
${NAD}^{+}$ $bold NAD Superscript bold plus$ - or ${NADP}^{+}$ $bold NADP Superscript bold plus$ -linked
$L -Glutamate + H_{2} O + NAD {(P)}^{+} ⇌ α -ketoglutarate + {NH}_{4}^{+} + NAD (P) H$ $upper L hyphen Glutamate plus upper H Subscript 2 Baseline upper O plus NAD left-parenthesis upper P right-parenthesis Superscript plus Baseline right harpoon over left harpoon alpha hyphen ketoglutarate plus NH Subscript 4 Superscript plus Baseline plus NAD left-parenthesis upper P right-parenthesis upper H$	M
$Isocitrate + NAD {(P)}^{+} ⇌ α -ketoglutarate + {CO}_{2} + NAD (P) H + H^{+}$ $Isocitrate plus NAD left-parenthesis upper P right-parenthesis Superscript plus Baseline right harpoon over left harpoon alpha hyphen ketoglutarate plus CO Subscript 2 Baseline plus NAD left-parenthesis upper P right-parenthesis upper H plus upper H Superscript plus$	M and C
^aThese reactions and their enzymes are discussed in Chapters 14, 16, 17, and 18. ^bM designates mitochondria; C designates cytosol.

${NAD}^{+}$ $NAD Superscript plus$ -linked dehydrogenases remove two hydrogen atoms from their substrates. One of these is transferred as a hydride ion $(: H^{-})$ $left-parenthesis bold colon upper H Superscript minus Baseline right-parenthesis$ to ${NAD}^{+}$ $NAD Superscript plus$ , and the other is released as $H^{+}$ $upper H Superscript plus$ 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.

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 ${FADH}_{2}$ $FADH Subscript 2$ or ${FMNH}_{2}$ $FMNH Subscript 2$ ). 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 of Membrane-Bound Carriers

The mitochondrial respiratory chain consists of a series of sequentially acting electron carriers, most of which are integral proteins with prosthetic groups capable of accepting and donating either one or two electrons. Three types of electron transfers occur in oxidative phosphorylation: (1) direct transfer of electrons, as in the reduction of ${Fe}^{3 +}$ $Fe Superscript 3 plus$ to ${Fe}^{2 +}$ $Fe Superscript 2 plus$ ; (2) transfer as a hydrogen atom $(H^{+} + e^{-})$ $left-parenthesis upper H Superscript plus Baseline plus e Superscript minus Baseline right-parenthesis$ ; and (3) transfer as a hydride ion $(: H^{-})$ $left-parenthesis bold colon upper H Superscript minus Baseline right-parenthesis$ , 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 ( $^{•} QH$ $Superscript bullet Baseline zero width space QH$ or ubisemiquinone) or two electrons to form ubiquinol $({QH}_{2})$ $left-parenthesis QH Subscript 2 Baseline right-parenthesis$ 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.

A figure shows the space-filling model of ubiquinone (Q or coenzyme Q) and the steps involved in the reduction of ubiquinone (Q) to ubiquinol (Q H 2). — 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 space-filling model has a rounded top with gray spheres in the center, two red spheres at the top and slightly to the right, a red sphere to the lower left, and three gray spheres each bonded to three white spheres visible to the upper left, top rear, and lower right. A kinked vertical chain extends down from this rounded portion. The vertical chain has gray spheres in the center and white spheres around the outside. Ubiquinone (Q) (fully oxidized) is shown as a six-membered ring with double bonds on the left and right sides, its upper and lower left vertices each bonded to O C H 3, its top and bottom vertices each double bonded to O, its lower right vertex bonded to C H 3, and its upper right vertex bonded to (C H 2 bonded to C H double bonded to C bonded to C H 3 above and C H 2 to the right) 2 bonded to H. Upward- and downward-pointing arrows indicate a reversible reaction. The downward arrow is joined by a curved line from red highlighted H plus plus e minus to show that these are added. This yields ubisemiquinone (Q H with a dot to the upper left of Q) (semiquinone radical), shown as a benzene ring with the upper and lower left vertices each bonded to O C H 3, the top vertex bonded to O with a red dot, the upper right vertex bonded to R, the lower right vertex bonded to C H 3, and the bottom vertex bonded to O red highlighted H. Upward- and downward-pointing arrows indicate a reversible reaction. The downward arrow is joined by a curved line from red highlighted H plus plus e minus to show that these are added. This yields ubiquinol (Q H 2) (fully reduced), which is a similar benzene ring in which the top vertex is bonded to O red highlighted H.

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 $({Fe}^{2 +} )$ $left-parenthesis Fe Superscript 2 plus Baseline zero width space right-parenthesis$ 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 $b_{562}$ $b 562$ .

A two-part figure shows three prosthetic groups of cytochromes in part a and the absorption spectra of cytochrome c in part b. — 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 ${Fe}^{2 +}$ $Fe Superscript 2 plus$ or ${Fe}^{3 +}$ $Fe Superscript 3 plus$ . 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.

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 ${Fe}^{2 +}$ $Fe Superscript 2 plus$ or ${Fe}^{3 +}$ $Fe Superscript 3 plus$ . 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.

Part a shows three structures. Iron protoporphyrin Roman numeral 9 (in italicized b end italics-type cytochromes) is shown with F e in the center surrounded by eight rings. The chain forming the outer boundary of this ring structure and two attached alkenes are highlighted in brown. All part described are highlighted in brown unless otherwise specified. The top five-membered ring has a double bond at the top, an upper left vertex bonded to nonhighlighted C H 3, an upper right vertex bonded to C H double bonded to C H 2, a nonhighlighted lower right side double bonded to N at the bottom vertex, and a nonhighlighted lower right side single bonded to N at the bottom vertex. The lower left vertex of this ring and lower right double bond of this top ring is shared with a six-membered ring to its lower left. This six-membered ring has a brown horizontal top vertex, a shared, nonhighlighted double bond at its upper right side that joins to N to the lower right, a vertical double bond at its left side, and a nonhighlighted lower left bond that connects to a left-hand N with a red arrow pointing to the central F e. The lower left-corner of this ring is the upper right corner of the left side five-membered ring. This five-membered ring shares a nonhighlighted bond at its upper right side to the left-side N with the six-membered ring above. It has a vertical top side, an upper left vertex bonded to C H double bonded to C H 2, and vertical double-bond at its left side, a lower left vertex bonded to non-highlighted C H 3, a horizontal bottom bond, and a lower right vertex connected by a non-highlighted bond to the left-hand N to its upper right. The lower right vertex of this ring is the upper left vertex of a six-membered ring below. This six-membered ring has a vertical double bond at its left side, a horizontal bond at its bottom side, and a nonhighlighted double bond to N at its top vertex, which has an arrow pointing up to F e. This double bond is shared with a five-membered ring at the bottom center that has a vertical single bond on its left side, a lower left vertex bonded to nonhighlighted C H 3, a double bond at its bottom side, a lower right vertex bonded to nonhighlighted C H 2 C H 2 C O O minus, a single bond at its right side, and a nonhighlighted single bond from its upper right vertex to N at its top vertex. This five-membered ring shares its upper right side with a six-membered ring to its upper right that has a vertical double bond at its bottom side, a vertical single bond at its right side, and a nonhighlighted diagonal bond at its upper right side to the right-hand N at its upper right vertex that has an arrow pointing left to F e. This nonhighlighted diagonal bond is shared with a five-membered ring at the right side that has the right-hand N as its left side vertex, a double bond at its right side, a lower right vertex bonded to nonhighlighted C H 2 C H 2 C O O minus, a vertical single bond at its left side, an upper right vertex bonded to nonhighlighted C H 3, a horizontal double bond at its top side, and a diagonal upper left side bond to N at its left side vertex. This five-membered ring shares its upper left side with a six-membered ring above that has a vertical bond at its right side, a horizontal bond at its top side, and a diagonal bond at its upper left side to the top center N that forms the bottom vertex of the top center five-membered ring. Heme italicized c end italics (in italicized c end italics-type cytochromes) is a similar structure except that the left side five-membered ring has an upper left vertex bonded to C H bonded to C H 3 and to S further bonded to C y s, with the entire new group in a blue box, and the top five-membered ring has an upper right vertex bonded to C H bonded to C H 3 and to S further bonded to C y s, with the entire new group in a blue box. These two substituents in blue boxes both replaced C H double bonded to C H 2 in iron protoporphyrin Roman numeral 9. Heme italicized a end italics (in italicized a end italics-type cytochromes) is also similar to iron protoporphyrin Roman numeral 9 except that the upper left vertex of the left side five-membered ring is bonded to nonhighlighted C H bonded to nonhighlighted O H and to a zigzag chain in a blue box of C H 2 bonded to C H 2 bonded to C H double bonded to C bonded to C H 3 and to C H 2 bonded to C H 2 bonded to C H double bonded to C bonded to C H 3 and further bonded to C H 2 bonded to C H 2 bonded to C H double bonded to C bonded to 2 C H 3. Part b is a graph that plots relative light absorption against wavelength. The horizontal axis is labeled wavelength (n m) and runs from 300 to 600, labeled in increments of 100. The vertical axis is labeled relative light absorption (percent) and ranges from 0 to 100, labeled in increments of 50. A blue curve labeled oxidized cyt italicized c end italics begins at (315, 10), increases to (340, 20), increases slowly to (360, 25), then increases rapidly to a peak at (400, 80), before decreasing rapidly to (450, 5), rising slightly to (540, 7), and then decreasing to (580, 4). A red curve labeled reduced cyt italicized c end italics begins at (315, 20), drops to (340, 10), rises rapidly to a peak labeled gamma at (400, 98), decreases rapidly to (425, 4), decrease to (470, 2), rises to a small peak labeled beta at (505, 6), drops slightly to (508, 5), rises to a peak labeled alpha at (550, 20), then decreases to end at (580, 3). All data is approximate.

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 after their discoverer, John S. Rieske) are a variation on this theme, in which one Fe atom is coordinated to two His residues rather than two Cys residues. All iron-sulfur proteins participate in one-electron transfers in which one iron atom of the 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 $− 0.65 V$ $negative 0.65 upper V$ to $+ 0.45 V$ $plus 0.45 upper V$ , depending on the microenvironment of the iron within the protein.

A four-part figure shows several arrangements of iron sulfur centers, including a single F e iron surrounded by S atoms in part a, a 2 F e – 2 S center in part b, a 4 F e – F S center in part c, and a ribbon structure of ferredoxin with one 2 F e – 2 S center in part d. — 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.]

Part a shows a square invagination in a protein with two C y s vertically along each side, one near the top and one near the bottom. In the center of the opening, there is a light red circle labeled F e that is bonded to orange circles labeled S to the upper left, lower left, upper right, and lower right. Each circle labeled S is further bonded to its adjacent C y s in the surrounding protein. Part b is similar except the invagination is rectangular. A light red circle labeled F e on the left side is solid wedge bonded to an orange sphere labeled S to the upper left that is further bonded to C y s to its left in the protein, hashed wedge bonded to an orange sphere labeled S to the lower left that is further bonded to C y s to its left in the protein, and bonded to yellow spheres labeled S to its upper and lower right. On the right side of the invagination, a similar light red circle labeled F e is bonded to the same yellow spheres to its upper left and right, solid wedge bonded to an orange sphere labeled S to its upper right further bonded to C y s to its right in the protein, and hashed wedge bonded to an orange sphere labeled S to its lower right further bonded to C y s to its right in the protein. Part c shows a similar invagination in a protein that is higher on its right side than on its left side. The opening contains a cube. C y s in the lower left of the protein is bonded to an orange sphere labeled S to its right further bonded to a light red sphere labeled F e at the lower left rear vertex of the cube. C y s in the upper left of the protein is bonded to an orange sphere labeled S further bonded to a light red cube at the upper right front vertex of the cube. C y s in the lower right of the protein is bonded to an orange sphere labeled S to its left further bonded to a light red sphere labeled F e at the lower right front vertex of the cube. C y s in the upper right of the protein is bonded to an orange sphere labeled S further bonded to a light red sphere labeled F e to its lower left at the upper rear right vertex of the cube. The upper rear left vertex of the cube is a yellow sphere labeled S, the upper front left vertex of the cube is a light red sphere labeled F e, the upper front right vertex of the cube is a yellow sphere labeled S, and the upper rear right vertex of the cube is a light red sphere labeled F e. The lower rear left vertex of the cube is a light red sphere labeled F e, the lower front left vertex of the cube is a yellow sphere labeled S, the lower front right vertex of the cube is light red sphere labeled F e, and the lower rear right vertex of the cube is a yellow sphere labeled S. There are hashed wedge bonds between the light red spheres labeled F e at the lower left rear, lower right front, and upper right rear and a yellow sphere labeled S at the lower right rear. Part d shows a short vertical helix at the bottom center with diagonal strands above and a short horizontal helix to the upper right. A small ball-and-stick model with a diamond shape with red at the left and right and yellow at the top and bottom is at the right side just to the right of the helix. Each red sphere is further bonded to two orange spheres to its outside.

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 $O_{2}$ $upper O Subscript 2$ . 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 $E^{'} °$ $upper E prime degree$ to carriers of higher $E^{'} °$ $upper E prime degree$ . The order of carriers deduced by this method is $NADH \to Q \to cytochrome b \to$ $NADH right-arrow upper Q right-arrow cytochrome b right-arrow$ cytochrome $c_{1} \to cytochrome c \to cytochrome a \to cytochrome a_{3} \to O_{2}$ $c 1 right-arrow cytochrome c right-arrow cytochrome a right-arrow cytochrome a 3 right-arrow upper O Subscript 2 Baseline$ . 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 $O_{2}$ $upper O Subscript 2$ ). When $O_{2}$ $upper O Subscript 2$ 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 $O_{2}$ $upper O Subscript 2$ (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)	$E^{'} °$ $bold-italic upper E bold prime degree$ (V)
$2 H^{+} + 2 e^{-} \to H_{2}$ $2 upper H Superscript plus Baseline plus 2 e Superscript minus Baseline right-arrow upper H Subscript 2 Baseline$	−0.414
${NAD}^{+} + H^{+} + 2 e^{-} \to NADH$ $NAD Superscript plus Baseline plus upper H Superscript plus Baseline plus 2 e Superscript minus Baseline right-arrow NADH$	−0.320
${NADP}^{+} + H^{+} + 2 e^{-} \to NADPH$ $NADP Superscript plus Baseline plus upper H Superscript plus Baseline plus 2 e Superscript minus Baseline right-arrow NADPH$	−0.324
$NADH dehydrogenase (FMN) + 2 H^{+} + 2 e^{-} \to NADH dehydrogenase ({FMNH}_{2})$ $NADH dehydrogenase left-parenthesis FMN right-parenthesis plus 2 upper H Superscript plus Baseline plus 2 e Superscript minus Baseline right-arrow NADH dehydrogenase left-parenthesis FMNH Subscript 2 Baseline right-parenthesis$	−0.30
$Ubiquinone + 2 H^{+} + 2 e^{-} \to ubiquinol$ $Ubiquinone plus 2 upper H Superscript plus Baseline plus 2 e Superscript minus Baseline right-arrow ubiquinol$	0.045
$Cytochrome b ({Fe}^{3 +}) + e^{-} \to cytochrome b ({Fe}^{2 +})$ $Cytochrome b left-parenthesis Fe Superscript 3 plus Baseline right-parenthesis plus e Superscript minus Baseline right-arrow cytochrome b left-parenthesis Fe Superscript 2 plus Baseline right-parenthesis$	0.077
$Cytochrome c_{1} ({Fe}^{3 +}) + e^{-} \to cytochrome c_{1} ({Fe}^{2 +})$ $Cytochrome c 1 left-parenthesis Fe Superscript 3 plus Baseline right-parenthesis plus e Superscript minus Baseline right-arrow cytochrome c 1 left-parenthesis Fe Superscript 2 plus Baseline right-parenthesis$	0.22
$Cytochrome c ({Fe}^{3 +}) + e^{-} \to cytochrome c ({Fe}^{2 +})$ $Cytochrome c left-parenthesis Fe Superscript 3 plus Baseline right-parenthesis plus e Superscript minus Baseline right-arrow cytochrome c left-parenthesis Fe Superscript 2 plus Baseline right-parenthesis$	0.254
$Cytochrome a ({Fe}^{3 +}) + e^{-} \to cytochrome a ({Fe}^{2 +})$ $Cytochrome a left-parenthesis Fe Superscript 3 plus Baseline right-parenthesis plus e Superscript minus Baseline right-arrow cytochrome a left-parenthesis Fe Superscript 2 plus Baseline right-parenthesis$	0.29
$Cytochrome a_{3} ({Fe}^{3 +}) + e^{-} \to cytochrome a_{3} ({Fe}^{2 +})$ $Cytochrome a 3 left-parenthesis Fe Superscript 3 plus Baseline right-parenthesis plus e Superscript minus Baseline right-arrow cytochrome a 3 left-parenthesis Fe Superscript 2 plus Baseline right-parenthesis$	0.35
$½ O_{2} + 2 H^{+} + 2 e^{-} \to H_{2} O$ $one half upper O Subscript 2 Baseline plus 2 upper H Superscript plus Baseline plus 2 e Superscript minus Baseline right-arrow upper H Subscript 2 Baseline upper O$	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 $O_{2}$ $upper O Subscript 2$ and an electron donor, carriers that function before the inhibited step become fully reduced, and those that function after this step are completely oxidized (Fig. 19-6). By using several inhibitors that block different steps in the chain, investigators have determined the entire sequence; it is the same as deduced in the first two approaches.

A figure shows a method for determining the sequence of electron carriers by using inhibitors to affect electron transfer. — 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 $O_{2}$ $upper O Subscript 2$ , each inhibitor causes a characteristic pattern of oxidized/reduced carriers: those before the block become reduced (blue), and those after the block become oxidized (light red).

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 $O_{2}$ $upper O Subscript 2$ , each inhibitor causes a characteristic pattern of oxidized/reduced carriers: those before the block become reduced (blue), and those after the block become oxidized (light red).

Three horizontal bands are shown. The top band has a blue rectangle labeled N A D H on the left, then a right-pointing arrow beneath a red circle containing a red X beneath a box labeled rotenone. The band to the right is light red and contains the following sequence from left to right: Q right-pointing arrow C y t italicized b end italics right-pointing arrow C y t italicized c end italics subscript 1 end subscript right-pointing arrow C y t italicized c end italics right-pointing arrow C y t italicized a end italics right-pointing arrow C y t italicized a end italics subscript 3 end subscript. To the right of the light red bar, an arrow points right to O 2. The middle band has a blue portion on the left that reads, N A D H right-pointing arrow Q right-pointing arrow C y t italicized b end italics. An arrow points right beneath a red circle containing a red X beneath a box labeled antimycin A. The band to the right is light red and contains the following sequence from left to right: C y t italicized c end italics subscript 1 end subscript right-pointing arrow C y t italicized c end italics right-pointing arrow C y t italicized a end italics right-pointing arrow C y t italicized a end italics subscript 3 end subscript. To the right of the light red bar, an arrow points right to O 2. The bottom band has a blue portion that reads, N A D H right-pointing arrow Q right-pointing arrow C y t italicized b end italics right-pointing arrow C y t italicized c end italics subscript 1 end subscript right-pointing arrow C y t italicized c end italics right-pointing arrow C y t italicized a end italics right-pointing arrow C y t italicized a end italics subscript 3 end subscript. To the right of the light red bar, a right-pointing arrow is shown beneath a red circle containing a red X beneath a box labeled C N minus or C O. O 2 is to the right of this arrow.

Electron Carriers Function in Multienzyme Complexes

The electron carriers of the respiratory chain are organized into membrane-embedded supramolecular complexes that can be physically separated. Gentle treatment of the inner mitochondrial membrane with detergents allows the resolution of four unique 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 $O_{2}$ $upper O Subscript 2$ .

A figure shows a method for separating the functional complexes of the respiratory chain. — 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 $O_{2}$ $upper O Subscript 2$ , as shown. In vitro, isolated ATP synthase has only ATP-hydrolyzing (ATPase), not ATP-synthesizing, activity.

A mitochondrion is shown to the upper left and is cutaway to show the folded inner membrane and cristae. Rounded structures with oval projections are shown on the inner membrane, as are sets of four shapes in curved horizontal patterns. Many dots are present on the membrane. An arrow pointing to the mitochondrion reads, treatment with digitoxin. The mitochondrion is fragmented to the lower right. An outer piece of membrane folds away and an arrow points away from it reading, outer membrane fragments discarded. Smaller pieces of membrane are shown to the right beneath text that reads, osmotic rupture. Smaller pieces containing parts of the respiratory chain and A T P synthase are visible and one piece has formed a sphere. Arrows point from the broken end of the mitochondrion to a piece of membrane containing a respiratory chain and A T P synthase. The components of the chain are clearly visible and are numbered from right to left as Roman numeral 1, Roman numeral 2, Roman numeral 3, and Roman numeral 4. A T P synthase is to the left. Each component has its characteristic shape and A T P synthase is shown with a rectangular piece in the membrane and a rounded portion above. An arrow points down to text reading, solubilization with detergent followed by ion-exchange chromatograph. An arrow breaks into five parts below, each pointing to a different tube. The first arrow points to Roman numeral 1 above a tube of pink liquid, matching the color of the respiratory chain component labeled Roman numeral 1. Beneath the tube, a curved arrow points from N A D H to Q. The second arrow points to Roman numeral 2 above a tube of beige liquid, matching the color of the respiratory chain component labeled Roman numeral 2. Beneath the tube, a curved arrow points from succinate to Q. The third arrow points to Roman numeral 3 above a tube of blue liquid, matching the color of the respiratory chain component labeled Roman numeral 3. Beneath the tube, a curved arrow points from Q to C y t italicized c end italics. The fourth arrow points to Roman numeral 4 above a tube of green liquid, matching the color of the respiratory chain component labeled Roman numeral 4. Beneath the tube, a curved arrow points from C y t italicized c end italics to O 2. The fifth arrow points to A T P synthase above a tube of orange liquid, matching the color of A T P synthase shown in the membrane above. Beneath the tube, a curved arrow points from A T P to A D P plus P i. Text below reads, reactions catalyzed by isolated fractions in vitro.

TABLE 19-3 The Protein Components of the Mitochondrial Respiratory Chain
Enzyme complex/protein	Mass (kDa)	Number of subunits^a	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^b	250	11	Hemes, Fe-S
Cytochrome c^c	13	1	Heme
IV Cytochrome oxidase^b	204	13 (3–4)	Hemes; ${Cu}_{A}, {Cu}_{B}$ $Cu Subscript upper A Baseline comma Cu Subscript upper B Baseline$
^aNumber of subunits in bacterial complexes is shown in parentheses. ^bMass and subunit data are for the monomeric form. ^cCytochrome 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 polypeptides in the membrane arm and 7 in the matrix arm are conserved and essential (Fig. 19-8).

A figure shows the structure of Complex Roman numeral 1 of the respiratory chain (N A D H: ubiquinone oxidoreductase). — 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 ${QH}_{2}$ $QH Subscript 2$ , 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 (n side negative, p side positive). Three of the membrane subunits (subunits Nqo12, Nqo13, and Nqo14) are structurally related to a known ${Na}^{+} {-H}^{+}$ $Na Superscript plus Baseline hyphen upper H Superscript plus$ 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.]

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 ${QH}_{2}$ $QH Subscript 2$ , 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 (n side negative, p side positive). Three of the membrane subunits (subunits Nqo12, Nqo13, and Nqo14) are structurally related to a known ${Na}^{+} {-H}^{+}$ $Na Superscript plus Baseline hyphen upper H Superscript plus$ 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.]

A horizontal piece of phospholipid membrane is shown with the region above labeled intermembrane space (P side) and the region below labeled matrix (N side). A structure within the membrane is labeled, complex Roman numeral 1. Four roughly spherical structures with rough surfaces are aligned horizontally in the membrane. The left side of this horizontal piece is labeled, membrane arm. The left-hand, light pink structure is labeled N q o 12. A red arrow points upward through it to red highlighted H plus above. The second structure, which is dark pink, is labeled N q o 13. A red arrow points upward through it to red highlighted H plus above. The third, light pink structure is labeled N q o 14. A red arrow points upward through it to red highlighted H plus above. The fourth, dark pink structure is narrow and connects to a vertical piece below. A red arrow points upward through it to red highlighted H plus above. To its right, the horizontal piece ends with another light red structure. Light, dark, and intermediate pink components come together to form an oblong piece that extends down from the right side of the horizontal piece. This is labeled, matrix arm. At the bottom, a curved arrow runs from N A D H plus H plus on the left, up to the bottom of the matrix arm, and down to N A D plus. A blue arrow labeled 2 e minus points upward from the place that the curved arrow meets the matrix arm. The blue arrow points to a space-filling model labeled R M N that has a red portion below, then bends right to meet a longer portion pointing up that is blue and gray at the bottom and gray on top. Rounded structures with two yellow and two orange spheres are to the left and right and are labeled, series of F e – S centers. The blue arrow runs upward along a curved path of these orange and yellow spheres and enters a long, light pink region that joins to the fourth light-pink region of the horizontal portion in the membrane. Accompanying text reads, N – 2. To the right, text reads, 2 e minus. The blue arrow points to Q. A black arrow runs up from Q, curves through the right end of the horizontal part, joins a red arrow from 2 H plus to the lower left of the matrix arm, and exits the horizontal part at Q H 2 within the phospholipid membrane.

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 \to {NAD}^{+} + {QH}_{2}

$NADH plus upper H Superscript plus Baseline plus upper Q right-arrow NAD Superscript plus Baseline plus QH Subscript 2 Baseline$

(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 often written with subscripts that indicate the location of the protons: p for the positive side of the inner membrane (the intermembrane space), n for the negative side (the matrix):

NADH + {5H}_{N}^{+} + Q \to {NAD}^{+} + {QH}_{2} + {4H}_{P}^{+}

$NADH plus 5 upper H Subscript upper N Superscript plus Baseline plus upper Q right-arrow NAD Superscript plus Baseline plus QH Subscript 2 Baseline plus 4 upper H Subscript upper P Superscript plus$

(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^a	Target/mode of action
Inhibition of electron transfer	Cyanide Carbon monoxide	Inhibit cytochrome oxidase
	Antimycin A	Blocks electron transfer from cytochrome b to cytochrome $c_{1}$ $c 1$
	Myxothiazol Rotenone Amytal Piericidin A	Prevent electron transfer from Fe-S center to ubiquinone
Inhibition of ATP synthase	Aurovertin	Inhibit $F_{1}$ $upper F Subscript 1$
	Oligomycin Venturicidin	Inhibit $F_{o}$ $upper F Subscript o$
	DCCD	Blocks proton flow through $F_{o}$ $upper F Subscript o$
Uncoupling of phosphorylation from electron transfer	FCCP DNP	Hydrophobic proton carriers
	Valinomycin	$K^{+}$ $upper K Superscript plus$ 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
^aDCCD, dicyclohexylcarbodiimide; FCCP, cyanide-p-trifluoromethoxyphenylhydrazone; DNP, 2,4-dinitrophenol.

Three of the seven integral protein subunits of the membrane arm are related to a ${Na}^{+} {-H}^{+}$ $Na Superscript plus Baseline zero width space hyphen upper H Superscript plus$ 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 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 ${QH}_{2}$ $QH Subscript 2$ 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.

A figure shows the structure of Complex Roman numeral 2 of the respiratory chain (succinate dehydrogenase). — 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.]

A horizontal piece of phospholipid membrane is shown with the region above labeled intermembrane space (P side) and the region below labeled matrix (N side). A large beige irregular oval labeled A is located beneath the membrane. A smaller purple irregular oval labeled B its across most of the top of this beige oval and is narrower on its left side, where it meets a narrow, curved tail of a green piece labeled C that runs along the upper left of the beige oval labeled A. This green piece has a thin tail that curves to the lower left, then widens to the right above the purple piece labeled B as it extends upward through the phospholipid membrane. Above the left-hand tail of the green piece labeled C, there is a small purple piece of B visible and then a dark purple, roughly triangular piece labeled D that extends into the membrane and narrows about halfway through the membrane, where it runs behind part C so that only a tiny piece is visible to the upper left just below the top of the membrane. A roughly linear, horizontal, space-filling model in part a has black spheres on the left, then a region with many red spheres, then a circular region of gray spheres with blue spheres around the outside on the left labeled F A D. The right side of this horizontal molecule, where it has mostly black spheres with one red sphere behind, is labeled succinate-binding site. A blue arrow labeled e minus points from the black region labeled succinate-binding site to the black and blue rounded region labeled F A D then a blue arrow points from a red sphere to the upper right of F A D to a yellow and orange sphere in part B. This yellow and orange sphere have yellow spheres to the upper left and lower right and orange spheres to the lower left and upper right and are labeled an F e – S center. Other F e – S centers are visible above and blue arrows show that the electron travels through them into the green region to reach a clump of yellow spheres labeled ubiquinone where the green piece meets a small pink piece to its upper left and the corner of dark purple piece D. A blue arrow points away from ubiquinone to Q H 2 in the membrane to the right. Heme italicized b end italics is visible as a roughly rectangular vertical clump of light red spheres above ubiquinone. Just above this clump of red spheres, there is an opening in green structure C that shows chains of yellow spheres below labeled phosphatidylethanolamine and part of purple structure D below. Chains of phosphatidylethanolamine are also visible emerging to the upper left of purple structure D within the bottom half of the membrane.

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 $(H_{2} O_{2})$ $left-parenthesis upper H Subscript 2 Baseline upper O Subscript 2 Baseline right-parenthesis$ and the superoxide radical $(^{•} O_{2}^{-})$ $left-parenthesis Superscript bullet Baseline upper O Subscript 2 Superscript bold minus Baseline right-parenthesis$ , 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 $O_{2}$ $upper O Subscript 2$ 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, ${QH}_{2}$ $QH Subscript 2$ ) pass through two more large protein complexes in the inner mitochondrial membrane before reaching the ultimate electron acceptor, $O_{2}$ $upper O Subscript 2$ . Complex III (also called cytochrome $b c_{1}$ $bold-italic b bold-italic c 1$ 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 $c_{1}$ $c 1$ , 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 $Q_{N}$ $upper Q Subscript upper N$ on one monomer) to the intermembrane space (site $Q_{P}$ $upper Q Subscript upper P$ on the other monomer) as it shuttles electrons and protons across the inner mitochondrial membrane.

FIGURE 19-10 Structure of Complex III (cytochrome $b c_{1}$ $b c Subscript 1 Baseline$ 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 $(b_{H} and b_{L})$ $left-parenthesis b Subscript upper H Baseline and b Subscript upper L Baseline right-parenthesis$ ; the Rieske iron-sulfur protein (purple), with its 2Fe-2S centers; and cytochrome $c_{1}$ $c 1$ (blue), with its heme. Cytochrome $c_{1}$ $c 1$ and the Rieske iron-sulfur protein project from the p 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, $Q_{N}$ $upper Q Subscript upper N$ and $Q_{P}$ $upper Q Subscript upper P$ , 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 $c_{1}$ $c 1$ , specifically from heme $b_{H}$ $b Subscript upper H$ to Q, binds at $Q_{N}$ $upper Q Subscript upper N$ , close to heme $b_{H}$ $b Subscript upper H$ on the n (matrix) side of the membrane. Myxothiazol, which prevents electron flow from ${QH}_{2}$ $QH Subscript 2$ to the Rieske iron-sulfur protein, binds at $Q_{P}$ $upper Q Subscript upper P$ , near the 2Fe-2S center and heme $b_{L}$ $b Subscript upper L$ on the p side. The dimeric structure is essential to the function of Complex III. The interface between monomers forms two caverns, each containing a $Q_{P}$ $upper Q Subscript upper P$ site from one monomer and a $Q_{N}$ $upper Q Subscript upper N$ 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.]

A horizontal piece of phospholipid membrane is shown with the region above labeled intermembrane space (P side) and the region below labeled matrix (N side). Multiple complex structures run vertically through the membrane, forming an overall cylindrical shape with two large oblongs beneath it. An irregular, long light purple piece on the left, labeled a Rieske iron-sulfur-protein, has a long, irregular blue piece between its two halves that is labeled cytochrome italicized c end italics subscript 1 end subscript near the top of the membrane, above which it widens into a pale irregular oval that runs behind the top of the light purple piece to its right. A rounded red molecule partially behind these two structures and a blue structure, where it emerges to touch a dark purple structure to the right, is labeled heme italicized c end italics subscript 1 end subscript. The light blue structure between the dark purple structure to the right and the pale irregular oval is an extension of cytochrome italicized c end italics subscript 1 end subscript below. A dashed circle runs across the top of the light blue piece and dark purple piece and contains a light red square that is uneven, with slightly longer bottom and right sides, and that is labeled cyt italicized c end italics. The purple structure has an irregular oval portion that runs horizontally across the top center of the overall structure, has a cleft containing a dark blue portion, and curves down through the membrane along its right side. This is labeled Rieske iron-sulfur protein. A red oval clump of spheres labeled heme italicized c end italics subscript 1 end subscript is located where the right side of the dark purple oval portion meets the dark blue portion in its cleft. At the lower left corner of the purple oval, there is a structure with two yellow and two orange spheres that has yellow spheres in front and back and orange spheres at the top and bottom and that is labeled 2 F e – 2 S. A green structure labeled cytochrome italicized b end italics is beneath this horizontal purple oval and runs down across the membrane with the long light purple structure to its left. It contains a roughly square orange structure labeled heme italicized b end italics subscript L end subscript near its top, just below the structure labeled 2 F e – 2 S, and has a similar roughly square structure below the first that is labeled heme italicized b end italics subscript H end subscript. A similar roughly square structure to the right of heme italicized b end italics subscript L end subscript is labeled heme italicized b end italics subscript L end subscript and extends into a dark green oval below also labeled cytochrome italicized b end italics that contains another roughly square orange structure labeled heme italicized b end italics subscript H that is to the right of the first heme italicized b end italics subscript H. A long gray lobe is to the right of this dark green structure and extends down to a large, rounded oblong. To its left, there is a similar lighter structure with parts of the light purple and light blue pieces above extending into it below a horizontal piece that runs across the top and across the top of the dark gray piece to the right. The region where the light dark and green pieces of cytochrome italicized b end italics meet above a small opening above the pale horizontal piece of the left-hand large oblong is labeled cavern.

To account for the role of Q in energy conservation, Mitchell proposed the Q cycle (Fig. 19-11). As electrons move from ${QH}_{2}$ $QH Subscript 2$ through Complex III, ${QH}_{2}$ $QH Subscript 2$ is oxidized with the release of protons on one side of the membrane (at $Q_{P}$ $upper Q Subscript upper P$ ), while at the other site $(Q_{N} )$ $left-parenthesis upper Q Subscript upper N Baseline zero width space right-parenthesis$ , Q is reduced and protons are taken up.

A two-part figure shows the first stage of the Q cycle in part a and the second stage of the Q cycle in part b. — 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 n 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 ${QH}_{2}$ $QH Subscript 2$ . Meanwhile, on the p side of the membrane, two molecules of ${QH}_{2}$ $QH Subscript 2$ are oxidized to Q, releasing two protons per Q molecule (four protons in all) into the intermembrane space. Each ${QH}_{2}$ $QH Subscript 2$ donates one electron (via the Rieske Fe-S center) to cytochrome $c_{1}$ $c 1$ , and one electron (via cytochrome b) to a molecule of Q near the n side, reducing it in two steps to ${QH}_{2}$ $QH Subscript 2$ . This reduction also consumes two protons per Q, which are taken up from the matrix (n side). Reduced cytochrome $c_{1}$ $c 1$ 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 $Q_{N}$ $upper Q Subscript upper N$ site is coupled with two oxidations of ${QH}_{2}$ $QH Subscript 2$ at the $Q_{P}$ $upper Q Subscript upper P$ site by consuming two protons from the matrix and releasing four protons into the intermembrane space.

In both parts, a horizontal piece of membrane is shown with the region above labeled intermembrane space (P side) and the region below labeled matrix (N side). A cylindrical structure within the membrane has a large piece in the back with long, roughly oval light blue vertical halves that run across the membrane, extending further above than below with an invagination in the center between them. A dark purple oval labeled cyt italicized c end italics subscript 1 end subscript is at the upper left side of this structure and contains a light red diamond labeled heme italicized c end italics subscript 1 end subscript. A pink oval to its lower right touches the invagination at the top center of the large light blue structure behind and ends just to its right. A rounded green structure with two, roughly oval, vertical halves is in the center of the structure and is labeled cytochrome italicized b end italics. The top of this green structure runs behind the lower left of the pink oval and there is a cleft at the bottom center between the two halves. Two dark green horizontal bars are within this green structure, one near the bottom and a longer one at the top adjacent to the pink oval. Part a is labeled stage 1. A yellow box labeled Q near the bottom of the membrane has an arrow pointing to a similar box labeled Q at the left side of the bottom dark green horizontal bar, just to the left of the center of cytochrome italicized b end italics. An arrow points right from this box to a similar box at the right side of the bar that shows Q with a dot at its upper left and a negative sign. A yellow box labeled Q H 2 near the top of the membrane has an arrow pointing right to a similar box at the left side of the top dark green horizontal bar, from which an arrow points right to a yellow box labeled Q at the right end of the horizontal bar, from which another arrow points out of the cylindrical structure to a yellow box labeled Q in the upper membrane to the right. Blue arrows point up and down from the arrow between Q H 2 and Q in the top dark green horizontal bar. The downward-pointing blue arrow is labeled e minus and curves left to a light red rectangle labeled heme italicized b end italics subscript L end subscript, from which another blue arrow labeled e minus points down to a similar light red rectangle that is slightly extended at its upper left side and labeled heme italicized b end italics subscript H end subscript. A similar blue arrow labeled e minus points from this light red rectangle to the yellow box labeled Q at the left side of the bottom dark green horizontal bar. The blue arrow pointing up from the arrow between Q H 2 and Q in the top dark green horizontal bar is labeled e minus and points to a complex of two yellow and two orange spheres labeled 2 F e – 2 S, with yellow spheres on the left and right and orange spheres above and below, within the pink oval at the top center of the cylindrical structure. A blue arrow labeled e minus points from the structure labeled 2 F e – 2 S to a light red diamond labeled heme italicized c end italics subscript 1 end subscript in the dark purple oval labeled cyt italicized c end italics subscript 1 end subscripts. A blue arrow labeled e minus points from this light red diamond to a light red diamond above the cylindrical structure that is labeled cyt c and enclosed in a dashed circle that touches the upper left of the large cylindrical structure. A pink arrow points from the arrow between Q H 2 and Q in the top dark green horizontal bar through the upper right of the cylindrical structure to end at pink highlighted 2 H plus above. Text below reads, Q H 2 plus Q plus cyt italicized c end italics (oxidized) yields Q plus Q with a dot at the upper left and a negative charge plus 2 H subscript p end subscript with a positive charge plus cyt italicized c end italics (reduced). An arrow points right to part b, which is labeled stage 2. A pink arrow points from pink highlighted 2 H plus at the lower right to Q with a dot to its upper left and a negative charge located in a yellow box at the left of the bottom dark green horizontal bar. An arrow points right to a yellow box labeled Q H 2 at the right side of the bottom dark horizontal bar, from which an arrow points out of the cylindrical structure to a yellow box labeled Q H 2 near the bottom of the membrane. A yellow box labeled Q H 2 near the top of the membrane has an arrow pointing right to a yellow box labeled Q H 2 at the left side of the upper dark green horizontal bar. An arrow points right from this Q H 2 to a yellow box labeled Q on the right side of the upper dark green horizontal bar, from which an arrow points to the right out of the cylindrical structure to Q in a yellow box near the top of the membrane. Blue arrows point up and down from the arrow from Q H 2 to Q in the top dark green horizontal bar. The downward-pointing blue arrow is labeled e minus and curves left to a light red rectangle from which another blue arrow labeled e minus points down to a similar light red rectangle that is slightly extended at its upper left side from which a similar blue arrow labeled e minus points to the yellow box labeled Q with a dot at its upper left and a negative charge located at the left side of the bottom dark green horizontal bar. The blue arrow pointing up from the arrow between Q H 2 and Q in the top dark green horizontal bar is labeled e minus and points to a complex of two yellow and two orange spheres labeled 2 F e – 2 S, with yellow spheres on the left and right and orange spheres above and below, within the pink oval at the top center of the cylindrical structure. A blue arrow labeled e minus points from the structure labeled 2 F e – 2 S to a light red diamond in the dark purple oval, from which a blue arrow labeled e minus points to a light red diamond above the cylindrical structure that is enclosed in a dashed circle that touches the upper left of the large cylindrical structure. Text below reads, Q H 2 plus Q with a dot on the upper left and a negative charge plus 2 H subscript N end subscript with a positive charge plus cyt italicized c end italics (oxidized) yields Q plus 2 H subscript P end subscript with a positive charge plus Q H 2 plus cyt italicized C end italics (reduced). A bracket enclosing the reactions from parts a and b is accompanied by text reading, Net equation: Q H 2 plus 2 cyt italicized c end italics (oxidized) plus 2 subscript N end subscript with a positive charge yields Q plus 2 cyt italicized c end italics (reduced) plus 4 H subscript P end subscript with a positive charge.

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 ${QH}_{2}$ $QH Subscript 2$ is oxidized at active site 1, shedding two $H^{+}$ $upper H Superscript plus$ 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 ${QH}_{2}$ $QH Subscript 2$ , one in each stage. Reducing one Q at one site while oxidizing two ${QH}_{2}$ $QH Subscript 2$ at another may seem counterproductive at first glance. However, the two processes have complementary functions. The oxidation of two ${QH}_{2}$ $QH Subscript 2$ 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 ${QH}_{2}$ $QH Subscript 2$ at site 1) is pulling protons from the matrix, creating a net movement of protons from the matrix to the intermembrane space. The ${QH}_{2}$ $QH Subscript 2$ 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

{QH}_{2} + 2 cyt c (oxidized) + {2H}_{N}^{+} \to Q + 2 cyt c (reduced) + {4H}_{P}^{+}

$QH Subscript 2 Baseline plus 2 cyt c left-parenthesis oxidized right-parenthesis plus 2 upper H Subscript upper N Superscript plus Baseline right-arrow upper Q plus 2 cyt c left-parenthesis reduced right-parenthesis plus 4 upper H Subscript upper P Superscript plus$

(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 $b_{L}$ $b Subscript upper L$ and $b_{H}$ $b Subscript upper H$ of cytochrome b, and cytochromes $c_{1}$ $c 1$ 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: ${QH}_{2}$ $QH Subscript 2$ 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. After 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 $O_{2}$ $bold upper O Subscript bold 2$

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 $H_{2} O$ $upper H Subscript 2 Baseline upper O$ . Complex IV is a large, dimeric enzyme of the inner mitochondrial membrane, each monomer having 13 subunits and $M_{r}$ $upper M Subscript r$ 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).

A two-part figure shows the structure of Complex Roman numeral 4 (cytochrome oxidase) in part a and the binuclear center of C u subscript A end subscript within complex Roman numeral 4 in part b. — 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 $a_{3}$ $a 3$ , near a single copper ion, ${Cu}_{B}$ $Cu Subscript upper B$ (not visible here). Heme $a_{3}$ $a 3$ and ${Cu}_{B}$ $Cu Subscript upper B$ 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, ${Cu}_{A}$ $Cu Subscript upper A$ , 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 p 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 ${Cu}_{A}$ $Cu Subscript upper A$ . The Cu ions (blue spheres) share electrons equally. When the center is reduced, the ions have the formal charges ${Cu}^{1 +} {Cu}^{1 +}$ $Cu Superscript 1 plus Baseline Cu Superscript 1 plus$ ; when oxidized, ${Cu}^{1.5 +} {Cu}^{1.5 +}$ $Cu Superscript 1.5 plus Baseline Cu Superscript 1.5 plus$ . 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.]

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 $a_{3}$ $a 3$ , near a single copper ion, ${Cu}_{B}$ $Cu Subscript upper B$ (not visible here). Heme $a_{3}$ $a 3$ and ${Cu}_{B}$ $Cu Subscript upper B$ 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, ${Cu}_{A}$ $Cu Subscript upper A$ , 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 p 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 ${Cu}_{A}$ $Cu Subscript upper A$ . The Cu ions (blue spheres) share electrons equally. When the center is reduced, the ions have the formal charges ${Cu}^{1 +} {Cu}^{1 +}$ $Cu Superscript 1 plus Baseline Cu Superscript 1 plus$ ; when oxidized, ${Cu}^{1.5 +} {Cu}^{1.5 +}$ $Cu Superscript 1.5 plus Baseline Cu Superscript 1.5 plus$ . 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.]

In part a, a horizontal piece of phospholipid membrane is shown with the region above labeled intermembrane space (P side) and the region below labeled matrix (N side). Multiple complex structures run vertically through the membrane, forming an overall long cylindrical shape. In summary, dark green subunit Roman numeral 4 is on the right with dark yellow subunit Roman numeral 1 visible behind it and behind subunit Roman numeral 2 to its left. To the left of these, between two pieces of dark green subunit Roman numeral 4, a small dark purple piece of subunit Roman numeral 3 is visible. C u ions are visible in a pink piece at the upper left and near the top of subunit Roman numeral 4 to the right. Two pairs of heme molecules are visible in the left and right center. A large light green piece is on the left side. In its upper center, there is an irregularly-shaped pink piece with two adjacent blue spheres labeled C u ions at its bottom center. Just below, there is a light yellow structure visible that extends behind the light green structure and a light purple structure to the lower right. Where the light yellow structure meets these other two structures, there are two roughly rectangular structures made up of red spheres. The left-hand sphere has a long strand of spheres extending down from its left side to meet a small opening in the light green piece where the light yellow piece is visible. Beneath the right side of the red rectangular structures, there is a slightly angled light purple piece that touches a dark green oblong at its top and forms a rectangular structure below with a small piece of yellow visible at its top left. The dark green oblong is part of subunit Roman numeral 4 and runs down along the light purple piece, then up to the right as wo thick strands that join together at their top. Between the left-hand and center strands of the green piece, a long, dark purple piece labeled subunit Roman numeral 2 extends up to the very top. To the left of this dark purple piece, there is a small dark green piece above a small purple piece labeled subunit Roman numeral 3 above the left-hand dark green piece that curves above the light purple piece. Between the middle and right-hand dark green pieces, a piece of yellow is visible that is labeled subunit 1 and that runs behind the middle dark green piece and the dark purple piece of subunit Roman numeral 2 to its left. There is a roughly rectangular structures made up of spheres located at the boundary between the middle dark green structure and the dark purple structure to its left. This is labeled heme italicized a. A similar structure to its right is in the middle dark green piece and has a long strand of spheres that runs down to its right. This structure is labeled heme italicized a end italics subscript 3 end subscript. Two adjacent blue spheres are located in the dark green piece near the top of the membrane adjacent to the dark purple piece to the left. Part b shows a close-up of the C u ions shown as two adjacent blue spheres near the top of subunit Roman numeral 4 adjacent to subunit Roman numeral 2. An arrow shows a 180 degree rotation to the left. The two blue spheres are shown with a vertical orange bar between them. The top sphere has yellow wireframe sticks extending diagonally to the left and right that meet similar yellow sticks that extend diagonally to the bottom blue sphere. The vertex where these yellow sticks met joins with purple sticks on either side and these regions are labeled C y s. A ribbon structure is visible at the upper right and a wireframe model of H i s extends from it with its ring above the top blue sphere. G l u is to the lower right. Another H i s is below with its ring beneath the bottom blue sphere. M e t is to the upper left.

Subunit II of Complex IV contains two Cu ions complexed with the —SH groups of two Cys residues in a binuclear center ( ${Cu}_{A}$ $Cu Subscript upper A$ ; Fig. 19-12b) that resembles the 2Fe-2S centers of iron-sulfur proteins. Subunit I contains two heme groups, designated a and $a_{3}$ $a 3$ , and another copper ion $({Cu}_{B})$ $left-parenthesis Cu Subscript upper B Baseline right-parenthesis$ . Heme $a_{3}$ $a 3$ and ${Cu}_{B}$ $Cu Subscript upper B$ form a second binuclear center that accepts electrons from heme a and transfers them to $O_{2}$ $upper O Subscript 2$ bound to heme $a_{3}$ $a 3$ . 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 ${Cu}_{A}$ $Cu Subscript upper A$ center, to heme a, to the heme $a_{3} – {Cu}_{B}$ $a 3 en-dash Cu Subscript upper B Baseline$ center, and finally to $O_{2}$ $upper O Subscript 2$ (Fig. 19-13a). For every four electrons passing through this complex, the enzyme consumes four “substrate” $H^{+}$ $upper H Superscript plus$ from the matrix (n side) in converting $O_{2}$ $upper O Subscript 2$ to two $H_{2} O$ $upper H Subscript 2 Baseline upper O$ . 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}^{+} + O_{2} \to 4 cyt c (oxidized) + {4H}_{P}^{+} + {2H}_{2} O

$4 cyt c left-parenthesis reduced right-parenthesis plus 8 upper H Subscript upper N Superscript plus Baseline plus upper O Subscript 2 Baseline right-arrow 4 cyt c left-parenthesis oxidized right-parenthesis plus 4 upper H Subscript upper P Superscript plus Baseline plus 2 upper H Subscript 2 Baseline upper O$

(19-4)

A two-part figure shows the path of electrons through complex Roman numeral 4 by showing the structures involved in part a and the chemical reactions in part b. — 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 ${Cu}_{A}$ $Cu Subscript upper A$ . From here, electrons pass through heme a to the Fe-Cu center (heme $a_{3}$ $a 3$ and ${Cu}_{B}$ $Cu Subscript upper B$ ). Oxygen now binds to heme $a_{3}$ $a 3$ and is reduced to its peroxy derivative ( $O_{2}^{2 -}$ $upper O Subscript 2 Superscript 2 minus$ ; 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 $O_{2}^{2 -}$ $upper O Subscript 2 Superscript 2 minus$ 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, $P_{R}$ $upper P Subscript upper R$ , 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 $H_{2} O$ $upper H Subscript 2 Baseline upper O$ is released in two separate steps.

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 ${Cu}_{A}$ $Cu Subscript upper A$ . From here, electrons pass through heme a to the Fe-Cu center (heme $a_{3}$ $a 3$ and ${Cu}_{B}$ $Cu Subscript upper B$ ). Oxygen now binds to heme $a_{3}$ $a 3$ and is reduced to its peroxy derivative ( $O_{2}^{2 -}$ $upper O Subscript 2 Superscript 2 minus$ ; 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 $O_{2}^{2 -}$ $upper O Subscript 2 Superscript 2 minus$ 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, $P_{R}$ $upper P Subscript upper R$ , 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 $H_{2} O$ $upper H Subscript 2 Baseline upper O$ is released in two separate steps.

In part a, a horizontal piece of phospholipid membrane is shown with the region above labeled intermembrane space (P side) and the region below labeled matrix (N side). A large, green structure crosses the membrane. A blue oval that is flattened on its right side is near the left side of this structure and is labeled subunit Roman numeral 3. Immediately to its right, a roughly rectangular yellow structure is labeled Subunit Roman numeral 2. To its right, there is a thin, vertical, purple structure that expands up to a rounded structure above that overlaps the upper right of the yellow structure to its left. O 2 is shown in the membrane with an arrow that runs horizontally through blue subunit Roman numeral 3, then curves downward above a blue sphere labeled C u subscript B end subscript and beneath a light diamond labeled heme italicized a end italics subscript 3 end subscript in yellow subunit Roman 1, then runs vertically downward through yellow subunit 1 and the green bottom of the overall large structure to end at 2 H 2 O beneath the entire complex and membrane. To the left, an arrow runs up from 4 H plus (substrate) into the green structure, curves through the lower right of blue subunit Roman numeral 3, and curves right into yellow subunit Roman numeral 1. Between the arrow from 4 H plus and the arrow to 2 H 2 O, a red arrow points from red highlighted 4 H plus (pumped) almost vertically up through the green bottom of the overall structure, through yellow subunit Roman numeral 1, through the green top of the overall structure, and out to red highlighted 4 H plus above the structure and membrane. To the right of this red highlighted 4 H plus, there is a dashed circle containing 4 C y t italicized c end italics and 4 e minus next to a light red diamond. A blue arrow points down from the light red diamond through the upper left of purple subunit Roman numeral 2 to a blue sphere adjacent to a second blue sphere, labeled C u subscript A end subscript, and then to a light red diamond at the upper right of yellow subunit 1 labeled heme italicized a end italics. The arrow points through a second light red diamond labeled heme italicized a end italics subunit 3 to a blue sphere labeled C u subscript B end subscript. The structure containing C u and hemes is labeled F e – C u center. Part b shows a series of reactions in a cycle. At the upper left, a bolded O is shown above F e 3 plus C u 2 plus. An arrow points right and is joined by a curved line showing the addition of 2 e minus. This yields F e 2 plus C u plus beneath bolded R. An arrow points down and is joined by a curved arrow showing the addition of O in a light red box. This yields F e 2 plus bonded to O 2 C u plus next to bolded A. An arrow points down joined by a curved line showing the addition of e minus. This yields F e 3 plus bonded to O minus bonded to O minus C u plus above bolded P subscript R end subscript. An arrow points left and is accompanied by a curved arrow showing the addition of 2 H plus and release of H 2 O in a blue box. A red arrow points down from red highlighted H plus above through the place where the curved arrow meets the left-pointing arrow. This yields F e 4 plus double bonded to O 2 minus C u 2 plus above bolded F. An arrow points upward joined by a curved line showing the addition of e minus, H plus. A red arrow points from red highlighted H plus to the right left across the upward arrow where e minus and H plus are added. This yields F e 3 plus bonded to O H C u 2 plus next to bolded H. An arrow points up accompanied by a curved arrow showing the addition of H plus and loss of H 2 O in a blue box. Red highlighted H plus to the right has a red arrow pointing left across the point where the upward-pointing arrow and curved arrow meet. This completes the cycle by producing F e 3 plus C u 2 plus under bolded O.

Note that the $O_{2}$ $upper O Subscript 2$ in this reaction is the final acceptor for electrons originating from the many sources already described, and the stoichiometries between electron sources and $O_{2}$ $upper O Subscript 2$ 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 $O_{2}$ $upper O Subscript 2$ . For calculation simplicity, the stoichiometries will be presented in terms of $½ O_{2}$ $one half upper O Subscript 2$ in some examples to come.

At Complex IV, $O_{2}$ $upper O Subscript 2$ 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 in Respirasomes

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.

A two-part figure shows purified supercomplexes containing complexes Roman numerals 3 and 4 in part a and the structure of a respirasome composed of complexes Roman numerals 1, 3, and 4 in part b. — 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.]

Part a shows a curved piece of membrane with the region above labeled intermembrane space (P side) and the region below labeled matrix (N side). The complex Roman numeral 3 dimer is a roughly rectangular vertical structure made up of many blue cylinders connected by strands that extends across the left center of the membrane, with a wider region below that has two rounded halves. To its right, Complex Roman numeral 4 is made up of many green cylinders that are mostly vertical or slightly diagonal within the membrane connected by strands within the membrane and to a few cylinders above and below the membrane. Part b shows a similar piece of membrane. Complex Roman numeral 3 dimer and complex Roman numeral 4 are similar, but a long, pink piece labeled complex Roman numeral 1 is to their left. It has many vertical or slightly diagonal cylinders within the membrane, a few cylinders that are mostly horizontal or slightly angled above the membrane, and a long extension below the membrane that extends below the other two complexes. Several clumps of orange and yellow spheres are visible at the lower left. The view from the matrix shows a pink, roughly horizontal structure made of cylinders that runs across the bottom and expands to the left, where it contains several clumps of orange and yellow spheres. A rounded structure of blue cylinders is above its center and a smaller, oval structure of green cylinders is to the right and extends right beyond the pink structure below.

Other Pathways Donate Electrons to the Respiratory 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 ${QH}_{2}$ $QH Subscript 2$ . 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 ${QH}_{2}$ $QH Subscript 2$ produced enters the pool of ${QH}_{2}$ $QH Subscript 2$ 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 ${QH}_{2}$ $QH Subscript 2$ passes its electrons through Complex III and ultimately to $O_{2}$ $upper O Subscript 2$ .

A figure shows paths of electron transfer to ubiquinone in the respiratory chain. — 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 $Q . {QH}_{2}$ $upper Q period QH Subscript 2 Baseline$ freely diffuses through the membrane (black dashed arrow), and can interact with several additional complexes.

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 $Q . {QH}_{2}$ $upper Q period QH Subscript 2 Baseline$ freely diffuses through the membrane (black dashed arrow), and can interact with several additional complexes.

A horizontal piece of phospholipid membrane is shown with the region above labeled intermembrane space (P side) and the region below labeled matrix (N side). Two complexes of the respiratory chain are shown embedded in the membrane. Complex Roman numeral 1 is shown on the left as a horizontal pink piece that angles down beneath the membrane to the right. A curved arrow shows that N A D H plus H plus is added and N A D plus is produced. At the point where the curved arrow touches the bottom of complex Roman numeral 1, a blue arrow points up to F M N, from which a blue arrow points up to F e – S, from which a blue arrow points up to a yellow box labeled Q. A blue arrow points from Q to a yellow box labeled Q H 2 in the membrane to the right. Above the membrane, Dihydroorotate has a blue arrow pointing to an irregular green oval containing F M N labeled dihydroorotate dehydrogenase. A blue arrow points down to the same yellow box labeled Q H 2. A dashed arrow curves up from the yellow box labeled Q H 2 through the top of the membrane to end at a yellow box labeled Q H 2 to the right side of complex Roman numeral 2. Complex Roman numeral 2 has a narrow vertical portion that extends through the membrane and a rounded portion beneath the membrane. A curved arrow at the bottom of this complex shows that succinate is added and fumarate is lost. At the point where the curved arrow meets the complex, a blue arrow points upward to F A D, from which a blue arrow points up to F e – S, from which a blue arrow points up to a yellow box labeled Q. A blue arrow points from the yellow box labeled Q to the same yellow box labeled Q H 2 in the membrane indicated by the dashed arrow from the left. A blue arrow points from glycerol 3-phosphate (cytosolic) to a purple, roughly triangular structure containing F A D labeled glycerol 3-phosphate dehydrogenase, from which a blue arrow points down to the yellow box labeled Q H 2. To the lower right, a gray oblong containing F A D labeled acyl-Co A dehydrogenase has a curved arrow below showing the addition of fatty acyl-Co A and loss of enoyl-Co A. At the point where the curved arrow meets the gray oblong, a blue arrow points up to F A D inside, from which a blue arrow points up to F A D in a larger irregular gray structure labeled E T F. Another blue arrow points up from here to F e – S, F A D in a gray structure labeled E T F: Q oxidoreductase. From this point, a blue arrow points up to Q H 2.

The Energy of Electron Transfer Is Efficiently Conserved 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}^{+} + O_{2} \to {2 NAD}^{+} + {2H}_{2} O

$2 NADH plus 2 upper H Superscript plus Baseline plus upper O Subscript 2 Baseline right-arrow 2 NAD Superscript plus Baseline plus 2 upper H Subscript 2 Baseline upper O$

(19-5)

This net reaction is highly exergonic. For the redox pair ${NAD}^{+} /NADH$ $NAD Superscript plus Baseline slash NADH$ , $E^{'} °$ $upper E prime degree$ is $− 0.320 V$ $negative 0.320 upper V$ , and for the pair $O_{2} {/H}_{2} O$ $upper O Subscript 2 Baseline slash upper H Subscript 2 Baseline upper O$ , $E^{'} °$ $upper E prime degree$ is $0.816 V$ $0.816 upper V$ . The $Δ E^{'} °$ $upper Delta upper E prime degree$ for this reaction is therefore 1.14 V, and the standard free-energy change (see Eqn 13-7, p. 492) is

\begin{array}{l} Δ G^{'} ° = & - n F Δ E^{'} ° \\ = & - 2 (96.5 kJ/V • mol) (1.14 V) \\ = & - 220 kJ/mol (of NADH) \end{array}

$StartLayout 1st Row 1st Column upper Delta upper G Superscript prime Baseline degree equals 2nd Column minus n upper F upper Delta upper E Superscript prime Baseline degree 2nd Row 1st Column equals 2nd Column minus 2 left-parenthesis 96.5 kJ slash upper V bullet mol right-parenthesis left-parenthesis 1.14 upper V right-parenthesis 3rd Row 1st Column equals 2nd Column negative 220 kJ slash mol left-parenthesis of NADH right-parenthesis EndLayout$

(19-6)

This standard free-energy change is based on the assumption of equal concentrations (1 m) of NADH and ${NAD}^{+}$ $NAD Superscript plus$ . In actively respiring mitochondria, the actions of many dehydrogenases keep the actual $[NADH] / [{NAD}^{+}]$ $left-bracket NADH right-bracket slash left-bracket NAD Superscript plus Baseline right-bracket$ 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 $- 220 kJ/mol$ $negative 220 kJ slash mol$ . A similar calculation for the oxidation of succinate shows that electron transfer from succinate $(E^{'} ° for fumarate/succinate = 0.031 V)$ $left-parenthesis upper E Superscript prime Baseline degree for fumarate slash succinate equals 0.031 upper V right-parenthesis$ to $O_{2}$ $upper O Subscript 2$ has a smaller, but still negative, standard free-energy change of about $- 150 kJ/mol$ $negative 150 kJ slash mol$ .

Much of this energy is used to pump protons out of the matrix. For each pair of electrons transferred to $O_{2}$ $upper O Subscript 2$ , 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

2 NADH + 22 H_{N}^{+} + O_{2} \to 2 {NAD}^{+} + 20 H_{P}^{+} + 2 H_{2} O

$2 NADH plus 22 upper H Subscript upper N Superscript plus Baseline plus upper O Subscript 2 Baseline right-arrow 2 NAD Superscript plus Baseline plus 20 upper H Subscript upper P Superscript plus Baseline plus 2 upper H Subscript 2 Baseline upper O$

(19-7)

A figure summarizes the flow of electrons and protons through the four complexes of the respiratory chain. — 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 $({QH}_{2})$ $left-parenthesis QH Subscript 2 Baseline right-parenthesis$ 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 $O_{2}$ $upper O Subscript 2$ . 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.]

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 $({QH}_{2})$ $left-parenthesis QH Subscript 2 Baseline right-parenthesis$ 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 $O_{2}$ $upper O Subscript 2$ . 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.]

A horizontal piece of phospholipid membrane is shown with the region above labeled intermembrane space (P side) and the region below labeled matrix (N side). The complexes of the respiratory chain are shown embedded in the membrane. Complex Roman numeral 1 is shown on the left with a horizontal piece of alternating light and dark pink pieces that bend down to a thicker, roughly oval piece to the right. Red arrows point from beneath the membrane through four of these alternating light and dark pink pieces, beginning with a light pink piece on the far left, to H plus above the membrane. At the bottom of the piece that extends down, a curved arrow shows that N A D H plus H plus is added and N A D plus is produced. A blue arrow labeled 2 e minus points up into the structure to a complex with many red spheres near the bottom, blue and black spheres in the middle, and a diagonal black piece to the upper left. A diagonal line of four spheres each made up of orange and yellow spheres runs from the lower right to upper left and up through the center of the structure. A blue arrow points from the red, blue, and black structure along these orange and yellow spheres to a top orange and yellow sphere, from which a short blue arrow points to a yellow square labeled Q where the structure meets the lower boundary of the membrane. A red arrow points from red highlighted 2 H plus to the left across the pink structure to join with a blue arrow from the yellow square labeled Q, from which point a blue arrow continues out to point to a yellow square labeled Q H 2 in the membrane to the right. Complex Roman numeral 2 is to the right. A dashed arrow points from the yellow square labeled Q H 2 across the top of the membrane, above complex Roman numeral 2, to a similar yellow square labeled Q H 2 in the membrane to the right of complex Roman numeral 2. Complex Roman numeral 2 has a roughly oval green piece embedded vertically across the membrane with a purple piece on its left side with a light purple piece below above a horizontal piece extending from the right-hand green piece. Two yellow strands extend from the purple piece into the membrane to the left. Beneath there horizontal green piece, there is a light purple piece above a rounded beige piece. An arrow points from succinate to the lower left, up to a structure of red, blue, and black spheres near the top of the beige piece, and out to fumarate to the lower right. The structure of red, blue, and black spheres is blue and black on the left, has more red in the middle, and has more black to the right. A blue arrow extends up from the middle of this red, blue, and black structure across several spheres made up of orange and yellow sphere within the purple structure, across a final orange and yellow sphere in the green structure above, to a yellow square labeled Q. A red, roughly square structure made of spheres is above the yellow square labeled Q beneath two yellow chains of spheres that extend across an opening through which a piece of the dark purple piece that is mostly visible to the left can be seen. A blue arrow points from the yellow square labeled Q to the same yellow square labeled Q H 2 in the membrane to the right indicated by the dashed arrow from complex Roman numeral 1. Complex Roman numeral 3 is to the right. It has a cylinder of relatively linear pieces arranged within the membrane above a lighter gray sphere to the lower left and a darker gray sphere to the lower right. From left to right, the pieces in the cylinder across the membrane are purple on either side of a darker purple piece, green beneath a bright purple piece with a small bit of blue to the left, dark green up to an overhang from the green piece to its left and beneath more of the bright purple piece, then a thin gray protrusion from the gray sphere below that ends below a small bit of bright purple beneath a blue piece, before the cylinder ends with purple along the right side. Red highlighted 2 H plus beneath the structure has an arrow pointing up to a yellow box labeled Q H 2 at the point where the green piece meets the bright purple piece above. Two roughly rectangular structures made of spheres are visible along the path of the red arrow and two similar structures are to the right, one in the light green part and one in the dark green part beneath it. A blue arrow points from the yellow box labeled Q H 2 in the membrane to the left to the same Q H 2 at the point where the green and purple pieces meet. A red arrow points up from Q H 2 to red highlighted 4 H plus above the membrane. A blue arrow points up from Q H 2 to a gray sphere labeled cyt italicized c end italics containing a roughly round red structure made up of spheres. A dashed arrow labeled e plus (2 x) points from this gray sphere to a similar gray sphere above complex Roman numeral 4 to the right. Complex Roman numeral 4 has a light green portion on the left, then a pink portion above a thin yellow piece that runs behind more of the light green piece and is partially visible below. A light purple piece to the right also has an opening showing a bit of yellow. Near the top of the yellow piece, there are two roughly rectangular structures made of spheres and a chain of spheres extends down from the left side of the left-hand one. Two adjacent blue spheres are visible at the bottom of the pink piece above. There is a light green piece to the right of the pink piece above, with dark green beneath above the purple piece previously described. A dark green piece is to the right of the light green piece at the top, with a small piece of purple below between it and more of the dark green piece. Most of the right side is dark green, but a long, thin, vertical purple piece extends down through it with a small bit of yellow between it and the dark green piece adjacent to the light purple piece. There is a yellow region visible in the right center of the green piece with a small piece of purple visible at its top. Two roughly rectangular structures made of spheres are visible with one at the boundary between the narrow vertical purple piece and the green piece to its right and the other within the green piece with a chain of spheres extending down along the boundary of this piece and the yellow piece in its center. A blue arrow points down from the gray sphere above this complex to end next to the red rectangular structure at the boundary of the purple and green pieces, where it meets a curved arrow showing the addition of 2 H plus plus one-half O 2 and loss of H 2 O. A red arrow points from below the lower right side of the entire complex, beneath the green portion, up through the right-hand red rectangular structure, next to the yellow piece to the right of the right-hand red rectangular structure, up to red highlighted 2 H plus above the complex.

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 $(H^{+})$ $left-parenthesis upper H Superscript plus Baseline right-parenthesis$ 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).

A figure shows proton-motive force across the inner mitochondrial membrane. — FIGURE 19-17 Proton-motive force. The inner mitochondrial membrane separates two compartments of different $[H^{+}]$ $left-bracket upper H Superscript plus Baseline right-bracket$ , resulting in differences in chemical concentration $(Δ pH)$ $left-parenthesis upper Delta pH right-parenthesis$ and charge distribution $(Δ ψ)$ $left-parenthesis upper Delta psi right-parenthesis$ across the membrane. The net effect is the proton-motive force $(Δ G)$ $left-parenthesis upper Delta upper G right-parenthesis$ , which can be calculated as shown here.

FIGURE 19-17 Proton-motive force. The inner mitochondrial membrane separates two compartments of different $[H^{+}]$ $left-bracket upper H Superscript plus Baseline right-bracket$ , resulting in differences in chemical concentration $(Δ pH)$ $left-parenthesis upper Delta pH right-parenthesis$ and charge distribution $(Δ ψ)$ $left-parenthesis upper Delta psi right-parenthesis$ across the membrane. The net effect is the proton-motive force $(Δ G)$ $left-parenthesis upper Delta upper G right-parenthesis$ , which can be calculated as shown here.

A horizontal piece of membrane is shown with a vertical beige structure running across it labeled proton pump. Seven red highlighted H plus are lined up horizontally above the membrane. Text reads, P side, [H plus] subscript p end subscript equals italicized C end italics subscript 2 end subscript. Seven O H minus are lined up horizontally below the membrane. A red arrow points from red highlighted H plus below the beige structure to the central red highlighted H plus above the structure. Text reads, N side, [H plus] subscript N end subscript equals italicized C end italics subscript 1 end subscript. Text below reads, delta G equals italicized R T end italics l n (italicized C end italics subscript 2 end subscript / italicized C end italics subscript 1 end subscript) plus italicized Z F delta psi end italics equals 2.3 italicized R T end italics delta p H plus italicized F delta psi.

As we saw in Chapter 11, the free-energy change for the creation of an electrochemical gradient by an ion pump is

Δ G = R T ln(C_{2} / C_{1} ​) + Z F Δ ψ

$upper Delta upper G equals upper R upper T ln left-parenthesis upper C 2 slash upper C 1 zero width space right-parenthesis plus upper Z upper F upper Delta psi$

(19-8)

where $C_{2}$ $upper C 2$ and $C_{1}$ $upper C 1$ are the concentrations of an ion in two regions, and $C_{2} > C_{1}$ $upper C 2 greater-than upper C 1$ ; Z is the absolute value of its electrical charge (1 for a proton); and $Δ ψ$ $upper Delta psi$ is the transmembrane difference in electrical potential, measured in volts.

For protons,

\begin{array}{l} ln (C_{2} / C_{1}) & = 2 .3 {(log[H}^{+}]_{P} - lo ​ {g[H}^{+}]_{N} ​) \\ = 2.3 {(pH}_{N} - {pH}_{P} ​) = 2 .3 Δ pH \end{array}

$StartLayout 1st Row 1st Column ln left-parenthesis upper C 2 slash upper C 1 right-parenthesis 2nd Column equals 2 .3 left-parenthesis log left-bracket upper H Superscript plus Baseline right-bracket Subscript upper P Baseline minus l o zero width space g left-bracket upper H Superscript plus Baseline right-bracket Subscript upper N Baseline zero width space right-parenthesis 2nd Row 1st Column Blank 2nd Column equals 2 period 3 left-parenthesis pH Subscript upper N Baseline minus pH Subscript upper P Baseline zero width space right-parenthesis equals 2 .3 upper Delta pH EndLayout$

and Equation 19-8 reduces to

Δ G = 2.3 R T Δ pH + F Δ ψ

$upper Delta upper G equals 2.3 upper R upper T upper Delta pH plus upper F upper Delta psi$

(19-9)

In actively respiring mitochondria, the measured $Δ ψ$ $upper Delta psi$ 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 of Electron 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 $Δ ψ$ $upper Delta psi$ is 0.15 V and the pH difference is 0.75 unit at body temperature of $37 ° C$ $37 degree upper C$ .

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 $Δ pH$ $upper Delta pH$ (0.75 unit) and $Δ ψ$ $upper Delta psi$ (0.15 V) in this equation gives $Δ G = 19 kJ/mol$ $upper Delta upper G equals 19 kJ slash mol$ (of protons). Because the transfer of two electrons from NADH to $O_{2}$ $upper O Subscript 2$ 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 $P_{i}$ $upper P Subscript i$ . 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 Generated during 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 $(^{•} Q^{-})$ $left-parenthesis Superscript bullet Baseline zero width space upper Q Superscript minus Baseline right-parenthesis$ as an intermediate. The $^{•} Q^{-}$ $Superscript bullet Baseline zero width space upper Q Superscript minus$ radical is formed when a single electron is passed to $O_{2}$ $upper O Subscript 2$ in the reaction

O_{2} + e^{-} \to^{•} ​ O_{2}^{-}

$upper O Subscript 2 Baseline plus e Superscript minus Baseline right-arrow Superscript bullet Baseline zero width space upper O Subscript 2 Superscript minus$

Successive reduction of the superoxide radical with additional electrons produces $H_{2} O_{2}$ $upper H Subscript 2 Baseline upper O Subscript 2$ , hydroxyl radicals $(^{•} OH)$ $left-parenthesis Superscript bullet Baseline zero width space OH right-parenthesis$ , and finally $H_{2} O$ $upper H Subscript 2 Baseline upper O$ . The very reactive hydroxyl radical can be especially damaging (Fig. 19-18).

A figure shows how R O S form in mitochondria and mitochondrial defenses against R O S. — 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 $({}^{•}O_{2}^{-})$ $left-parenthesis Superscript bullet Baseline upper O Baseline Subscript 2 Superscript minus Baseline right-parenthesis$ production increases at Complexes I and III as the partially reduced ubiquinone radical $({}^{•}Q^{-})$ $left-parenthesis Superscript bullet Baseline upper Q Baseline Superscript minus Baseline right-parenthesis$ donates an electron to $O_{2}$ $upper O Subscript 2$ . Superoxide acts on aconitase, a 4Fe-4S protein (not shown), to release $Fe Superscript 2 plus Baseline period$ $Fe Superscript 2 plus Baseline period$ ${Fe}^{2 +}$ $Fe Superscript 2 plus$ in turn will react with hydrogen peroxide (in a process called the Fenton reaction) to produce the highly reactive hydroxyl free radical $({}^{•}O H)$ $left-parenthesis Superscript bullet Baseline upper O Baseline upper H right-parenthesis$ . 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 $H_{2} O_{2}$ $upper H Subscript 2 Baseline upper O Subscript 2$ and of the oxidized Cys residues $(— S — S —)$ $left-parenthesis em-dash upper S em-dash upper S em-dash right-parenthesis$ of enzymes and other proteins; GSH is regenerated from the oxidized form (GSSG) by reduction with NADPH.

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 $({}^{•}O_{2}^{-})$ $left-parenthesis Superscript bullet Baseline upper O Baseline Subscript 2 Superscript minus Baseline right-parenthesis$ production increases at Complexes I and III as the partially reduced ubiquinone radical $({}^{•}Q^{-})$ $left-parenthesis Superscript bullet Baseline upper Q Baseline Superscript minus Baseline right-parenthesis$ donates an electron to $O_{2}$ $upper O Subscript 2$ . Superoxide acts on aconitase, a 4Fe-4S protein (not shown), to release $Fe Superscript 2 plus Baseline period$ $Fe Superscript 2 plus Baseline period$ ${Fe}^{2 +}$ $Fe Superscript 2 plus$ in turn will react with hydrogen peroxide (in a process called the Fenton reaction) to produce the highly reactive hydroxyl free radical $({}^{•}O H)$ $left-parenthesis Superscript bullet Baseline upper O Baseline upper H right-parenthesis$ . 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 $H_{2} O_{2}$ $upper H Subscript 2 Baseline upper O Subscript 2$ and of the oxidized Cys residues $(— S — S —)$ $left-parenthesis em-dash upper S em-dash upper S em-dash right-parenthesis$ of enzymes and other proteins; GSH is regenerated from the oxidized form (GSSG) by reduction with NADPH.

A horizontal phospholipid membrane labeled inner mitochondrial membrane is shown and contains components of a respiratory chain. A gray oval labeled nicotinamide nucleotide transhydrogenase runs vertically across the membrane on the left side. To its right, complex Roman numeral 1 is shown as a pink structure with a horizontal left side that bends down to a rounded portion below the membrane to the lower right. To the right, complex Roman numeral 3 is shown as an irregular blue shape that is wider on top and narrows as it passes through the membrane, then has protrusions to the left and right beneath the membrane, then widens to a rounded portion with a small invagination at the bottom center. To its right, complex Roman numeral 4 is green and almost rectangular but extends down farther to the lower left and right and has a purple circle on its upper left labeled cyt italicized c end italics. N A D H to the lower right of nicotinamide nucleotide transhydrogenase has a curved arrow across the bottom of nicotinamide nucleotide transhydrogenase to N A D plus on the other side. The same N A D H has a curved arrow to the right that meets the lower right side of complex Roman numeral 1, then ends at N A D plus. To its right, red highlighted O 2 has a curved arrow up to a yellow box labeled Q in the membrane and then down to O 2 with a dot to the upper left and a negative charge from which a red arrow points left to O H with a dot on O and a blue arrow labeled superoxide dismutase points right to red highlighted H 2 O 2. A vertical curved arrow labeled glutathione peroxidase points from this red highlighted H 2 O 2 to H 2 O below and meets an upward curved arrow to its left. This left-hand curved arrow begins at 2 G S H below, meets the blue arrow pointing down from H 2 O 2 to H 2 O, and curves up to G S S G above. A curved arrow labeled glutathione reductase points from G S S G back down to 2 G S H and meets a curved arrow to its left running in the opposite direction, from N A D P H below to N A D P plus above. A curved arrow points from this N A D P plus up to nicotinamide nucleotide transhydrogenase down to N A D P H to start the process again. A dashed arrow points down from 2 G S H to 2 G S H below with a curved arrow labeled protein thiol reduction that points down to G S S G below. This curved arrow meets a blue downward curved arrow from a circle labeled E n z bonded to two S that are bonded to each other, forming a triangular shape, labeled inactive to a similar circle bonded to two S H that are not joined together and that is labeled active. A red arrow labeled oxidative stress curves back up from the active form to the inactive form above.

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 $O_{2}$ $upper O Subscript 2$ used in respiration forms $^{•} O_{2}^{-}$ $Superscript bullet Baseline zero width space upper O Subscript 2 Superscript minus$ — 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 $^{•} O_{2}^{-}$ $Superscript bullet Baseline zero width space upper O Subscript 2 Superscript minus$ 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 $O_{2}$ $upper O Subscript 2$ ) and therefore have a large proton-motive force and a high ${QH}_{2} /Q$ $QH Subscript 2 Baseline slash upper Q$ ratio, and (2) there is a high ${NADH/NAD}^{+}$ $NADH slash NAD Superscript plus$ 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 $^{•} O_{2}^{-}$ $Superscript bullet Baseline upper O Subscript 2 Superscript minus$ , cells have the enzyme superoxide dismutase, which catalyzes the reaction

2^{•} O_{2}^{-} + 2 H^{+} \to H_{2} O_{2} + O_{2}

$2 Superscript bullet Baseline upper O Subscript 2 Superscript minus Baseline plus 2 upper H Superscript plus Baseline right-arrow upper H Subscript 2 Baseline upper O Subscript 2 Baseline plus upper O Subscript 2 Baseline$

The hydrogen peroxide $(H_{2} O_{2})$ $left-parenthesis upper H Subscript 2 Baseline upper O Subscript 2 Baseline right-parenthesis$ 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 Mitochondrial Respiratory 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 $O_{2}$ $upper O Subscript 2$ , reducing it to $H_{2} O$ $upper H Subscript 2 Baseline upper O$ .
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 $a_{3}$ $a 3$ , accumulates electrons, then passes them to $O_{2}$ $upper O Subscript 2$ , reducing it to $H_{2} O$ $upper H Subscript 2 Baseline upper O$ .
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 ${QH}_{2}$ $QH Subscript 2$ .
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.