Chapter 21

Electron Transport and Oxidative Phosphorylation

Wall Piece #IV(1985), a kinetic sculpture by George Rhoads. This complex mechanical art
form can be viewed as a metaphor for the molecular apparatus underlying electron transport
and ATP synthesis by oxidative phoshorylation. (1985 by George Rhoads)

Living cells save up metabolic energy predominantly in the form of fats and carbohydrates, and they "spend" this energy for biosynthesis, membrane transport, and movement. In both directions, energy is exchanged and transferred in the form of ATP. In Chapters 19 and 20 we saw that glycolysis and the TCA cycle convert some of the energy available from stored and dietary sugars directly to ATP. However, most of the metabolic energy that is obtainable from substrates entering glycolysis and the TCA cycle is funneled via oxidation-reduction reactions into NADH and reduced flavoproteins, the latter symbolized by [FADH₂]. We now embark on the discovery of how cells convert the stored metabolic energy of NADH and [FADH₂] into ATP.

     Whereas ATP made in glycolysis and the TCA cycle is the result of substrate-level phosphorylation, NADH-dependent ATP synthesis is the result of oxidative phosphorylation. Electrons stored in the form of the reduced coenzymes, NADH or [FADH₂], are passed through an elaborate and highly organized chain of proteins and coenzymes, the so-called electron transport chain, finally reaching O₂ (molecular oxygen), the terminal electron acceptor. Each component of the chain can exist in (at least) two oxidation states, and each component is successively reduced and reoxidized as electrons move through the chain from NADH (or [FADH₂]) to O₂. In the course of electron transport, a proton gradient is established across the inner mitochondrial membrane. It is the energy of this proton gradient that drives ATP synthesis.

21.1 · Electron Transport and Oxidative Phosphorylation Are Membrane-Associated Processes

The processes of electron transport and oxidative phosphorylation are membrane-associated. Bacteria are the simplest life form, and bacterial cells typically consist of a single cellular compartment surrounded by a plasma membrane and a more rigid cell wall. In such a system, the conversion of energy from NADH and [FADH₂] to the energy of ATP via electron transport and oxidative phosphorylation is carried out at (and across) the plasma membrane. In eukaryotic cells, electron transport and oxidative phosphorylation are localized in mitochondria, which are also the sites of TCA cycle activity and (as we shall see in Chapter 24) fatty acid oxidation. Mammalian cells contain from 800 to 2500 mitochondria; other types of cells may have as few as one or two or as many as half a million mitochondria. Human erythrocytes, whose purpose is simply to transport oxygen to tissues, contain no mitochondria at all. The typical mitochondrion is about 0.5 ± 0.3 microns in diameter and from 0.5 micron to several microns long; its overall shape is sensitive to metabolic conditions in the cell.
      Mitochondria are surrounded by a simple outer membrane and a more complex inner membrane (Figure 21.1). The space between the inner and outer membranes is referred to as the intermembrane space. Several enzymes that utilize ATP (such as creatine kinase and adenylate kinase) are found in the intermembrane space. The smooth outer membrane is about 30 to 40% lipid and 60 to 70% protein, and has a relatively high concentration of phosphatidylinositol. The outer membrane contains significant amounts of porin¾a transmembrane protein, rich in b-sheets, that forms large channels across the membrane, permitting free diffusion of molecules with molecular weights of about 10,000 or less. Apparently, the outer membrane functions mainly to maintain the shape of the mitochondrion. The inner membrane is richly packed with proteins, which account for nearly 80% of its weight; thus, its density is higher than that of the outer membrane. The fatty acids of inner membrane lipids are highly unsaturated. Cardiolipin and diphosphatidylglycerol (Chapter 8) are abundant. The inner membrane lacks cholesterol and is quite impermeable to molecules and ions. Species that must cross the mitochondrial inner membrane¾ions, substrates, fatty acids for oxidation, and so on¾are carried by specific transport proteins in the membrane. Notably, the inner membrane is extensively folded (Figure 21.1). The folds, known as cristae, provide the inner membrane with a large surface area in a small volume. During periods of active respiration, the inner membrane appears to shrink significantly, leaving a comparatively large intermembrane space.

Figure 21.1 · (a) An electron micrograph of a mitochondrion. (b) A drawing of a mitochondrion with components labelled. (a, B. King/BPS) 

The Mitochondrial Matrix Contains the Enzymes of the TCA Cycle

The space inside the inner mitochondrial membrane is called the matrix, and it contains most of the enzymes of the TCA cycle and fatty acid oxidation. (An important exception, succinate dehydrogenase of the TCA cycle, is located in the inner membrane itself.) In addition, mitochondria contain circular DNA molecules, along with ribosomes and the enzymes required to synthesize proteins coded within the mitochondrial genome. Although some of the mitochondrial proteins are made this way, most are encoded by nuclear DNA and synthesized by cytosolic ribosomes.

21.2 · Reduction Potentials An Accounting Device for Free Energy Changes in Redox Reactions

On numerous occasions in earlier chapters, we have stressed that NADH and reduced flavoproteins ([FADH₂]) are forms of metabolic energy. These reduced coenzymes have a strong tendency to be oxidized¾that is, to transfer electrons to other species. The electron transport chain converts the energy of electron transfer into the energy of phosphoryl transfer stored in the phosphoric anhydride bonds of ATP. Just as the group transfer potential was used in Chapter 3 to quantitate the energy of phosphoryl transfer, the standard reduction potential, denoted by E_o', quantitates the tendency of chemical species to be reduced or oxidized. The standard reduction potential describing electron transfer between two species,

                  (21.1)

is related to the free energy change for the process by

DG^o' = -nÁDE_o'                                (21.2)

where n represents the number of electrons transferred; Á is Faraday's constant, 96,485 J/V × mol; and DE_o' is the difference in reduction potentials between the donor and acceptor. This relationship is straightforward, but it depends on a standard of reference by which reduction potentials are defined.

Figure 21.2 · Experimental apparatus used to measure the standard reduction potential of the indicated redox couples: (a) the acetaldehyde/ethanol couple, (b) the fumarate/succinate couple, (c) the Fe³⁺/Fe²⁺ couple.

 Measurement of Standard Reduction Potentials

Standard reduction potentials are determined by measuring the voltages generated in reaction half-cells (Figure 21.2). A half-cell consists of a solution containing 1 M concentrations of both the oxidized and reduced forms of the substance whose reduction potential is being measured, and a simple electrode. (Together, the oxidized and reduced forms of the substance are referred to as a redox couple.) Such a sample half-cell is connected to a reference half-cell and electrode via a conductive bridge (usually a salt-containing agar gel). A sensitive potentiometer (voltmeter) connects the two electrodes so that the electrical potential (voltage) between them can be measured. The reference half-cell normally contains 1 M H⁺ in equilibrium with H₂ gas at a pressure of 1 atm. The H⁺/H₂ reference half-cell is arbitrarily assigned a standard reduction potential of 0.0 V. The standard reduction potentials of all other redox couples are defined relative to the H⁺/H₂ reference half-cell on the basis of the sign and magnitude of the voltage electromotive force, emf) registered on the potentiometer (Figure 21.2).
      If electron flow between the electrodes is toward the sample half-cell, reduction occurs spontaneously in the sample half-cell, and the reduction potential is said to be positive. If electron flow between the electrodes is away from the sample half-cell and toward the reference cell, the reduction potential is said to be negative because electron loss (oxidation) is occurring in the sample half-cell. Strictly speaking, the standard reduction potential, E_o' , is the electromotive force generated at 25^oC and pH 7.0 by a sample half-cell (containing 1 M concentrations of the oxidized and reduced species) with respect to a reference half-cell. (Note that the reduction potential of the hydrogen half-cell is pH-dependent. The standard reduction potential, 0.0 V, assumes 1 M H⁺. The hydrogen half-cell measured at pH 7.0 has an E_o' of -0.421 V.)

 Several Examples

Figure 21.2a shows a sample/reference half-cell pair for measurement of the standard reduction potential of the acetaldehyde/ethanol couple. Because electrons flow toward the reference half-cell and away from the sample half-cell, the standard reduction potential is negative, specifically -0.197 V. In contrast, the fumarate/succinate couple and the Fe³⁺/Fe²⁺ couple both cause electrons to flow from the reference half-cell to the sample half-cell; that is, reduction occurs spontaneously in each system, and the reduction potentials of both are thus positive. The standard reduction potential for the Fe³⁺/Fe²⁺ half-cell is much larger than that for the fumarate/succinate half-cell, with values of +0.771 V and +0.031 V, respectively. For each half-cell, a half-cell reaction describes the reaction taking place. For the fumarate/succinate half-cell coupled to a H⁺H₂ reference half-cell, the reaction occurring is indeed a reduction of fumarate.

      Fumarate + 2 H⁺ + 2 e^- ® succinate                      E_o' = +0.031 V      ( 21.3)

Similarly , for the Fe³⁺/Fe²⁺ half-cell,

      Fe³⁺ + e^- ® Fe²⁺                              E_o' = +0.771 V      ( 21.4)

However , the reaction occurring in the acetaldehyde/ethanol half-cell is the oxidation of ethanol:

      Ethanol ® acetaldehyde + 2 H⁺ + 2 e^-                      E_o' = -0.197 V      ( 21.5)

The Significance of E_o'

Some typical half-cell reactions and their respective standard reduction potentials are listed in Table 21.1. Whenever reactions of this type are tabulated, they are uniformly written as reduction reactions, regardless of what occurs in the given half-cell. The sign of the standard reduction potential indicates which reaction really occurs when the given half-cell is combined with the reference hydrogen half-cell. Redox couples that have large positive reduction potentials have a strong tendency to accept electrons, and the oxidized form of such a couple (O₂, for example) is a strong oxidizing agent. Redox couples with large negative reduction potentials have a strong tendency to undergo oxidation (that is, donate electrons), and the reduced form of such a couple (NADPH, for example) is a strong reducing agent.

Coupled Redox Reactions

The half-reactions and reduction potentials in Table 21.1 can be used to analyze energy changes in redox reactions. The oxidation of NADH to NAD^- can be coupled with the reduction of a-ketoglutarate to isocitrate:

NAD^- + isocitrate ® NADH + H⁺ + a-ketoglutarate + CO₂                  (21.6)

This is the isocitrate dehydrogenase reaction of the TCA cycle. Writing the two half-cell reactions, we have

NAD⁺ + 2 H⁺ + 2 e^- ® NADH + H⁺    
E_o' = -0.38V                         (21.7) a-Keroglutarate + CO₂ + 2 H⁺ + 2 e^- ® isocitrate
E_o' = -0.32V                         (21.8)

In a spontaneous reaction, electrons are donated by (flow away from) the half-reaction with the more negative reduction potential and are accepted by (flow toward) the half-reaction with the more positive reduction potential. Thus, in the present case, isocitrate donates electrons and NAD^- accepts electrons. The convention defines DE_o' as

E_o' = E_o' (acceptor) - E_o' (donor)                                     (21.9)

In the present case, isocitrate is the donor and NAD^- the acceptor, so we write

E_o' = -0.32V - (-0.38V) = +0.06V                         (21.10)

From Equation 21.2, we can now calculate DG^o' as

DG^o' = -(2)(96.485 kJ/V × mol)(0.06V)                         (21.11)
DG^o' = -11.58 kJ/mol

Note that a reaction with a net positive DE_o' yields a negative DG^o', indicating a spontaneous reaction.

The Dependence of the Reduction Potential on Concentration

We have already noted that the standard free energy change for a reaction, DG^o', does not reflect the actual conditions in a cell, where reactants and products are not at standard-state concentrations (1 M). Equation 3.12 was introduced to permit calculations of actual free energy changes under non-standard-state conditions. Similarly, standard reduction potentials for redox couples must be modified to account for the actual concentrations of the oxidized and reduced species. For any redox couple,

(21.12)

the actual reduction potential is given by

(21.13)

      Reduction potentials can also be quite sensitive to molecular environment. The influence of environment is especially important for flavins, such as FAD/FADH₂ and FMN/FMNH₂. These species are normally bound to their respective flavoproteins; the reduction potential of bound FAD, for example, can be very different from the value shown in Table 21.1 for the free FAD-FADH₂ couple of -0.219 V. A problem at the end of the chapter addresses this case.

21.3 · The Electron Transport Chain¾An Overview

As we have seen, the metabolic energy from oxidation of food materials¾sugars, fats, and amino acids¾is funneled into formation of reduced coenzymes (NADH) and reduced flavoproteins ([FADH₂]). The electron transport chain reoxidizes the coenzymes, and channels the free energy obtained from these reactions into the synthesis of ATP. This reoxidation process involves the removal of both protons and electrons from the coenzymes. Electrons move from NADH and [FADH₂] to molecular oxygen, O₂, which is the terminal acceptor of electrons in the chain. The reoxidation of NADH,

      NADH( reductant) + H⁺ + ½O₂(oxidant) ® NAD^- + H₂O           (21.14)

involves the following half-reactions:

NAD⁺ + 2H⁺ + 2 e^- ® NADH + H⁺          E_o' = -0.32V           ( 21.15)

½O₂ + 2H^{+ +} 2 e^- ® H₂O                        E^o' = +0.816V         (21.16)

Here, half-reaction (21.16) is the electron acceptor and half-reaction (21.15) is the electron donor. Then

DE_o' = 0.816 - (-0.32) = 1.136 V

and, according to Equation (21.2), the standard-state free energy change, DG^o', is -219 kJ/mol. Molecules along the electron transport chain have reduction potentials between the values for the NAD⁺/NADH couple and the oxygen/H₂O couple, so that electrons move down the energy scale toward progressively more positive reduction potentials (Figure 21.3).

Figure 21.3 · E_o' and E values for the components of the mitochondrial electron transport chain. Values indicated are consensus values for animal mitochondria. Black bars represent E_o'; red bars,E'.

      Although electrons move from more negative to more positive reduction potentials in the electron transport chain, it should be emphasized that the electron carriers do not operate in a simple linear sequence. This will become evident when the individual components of the electron transport chain are discussed in the following paragraphs.

The Electron Transport Chain Can Be Isolated in Four Complexes

The electron transport chain involves several different molecular species, including:

(a) Flavoproteins, which contain tightly bound FMN or FAD as prosthetic groups, and which (as noted in Chapter 20) may participate in one- or two-electron transfer events.

(b) Coenzyme Q, also called ubiquinone (and abbreviated CoQ or UQ) (Figure 8.18), which can function in either one- or two-electron transfer reactions.

(c) Several cytochromes (proteins containing heme prosthetic groups [see Chapter 5], which function by carrying or transferring electrons), including cytochromes b, c, c₁, a, and a₃. Cytochromes are one-electron transfer agents, in which the heme iron is converted from Fe²⁺ to Fe³⁺ and back.

(d) A number of iron-sulfur proteins, which participate in one-electron transfers involving the Fe²⁺ and Fe³⁺ states.

(e) Protein-bound copper, a one-electron transfer site, which converts between Cu^- and Cu²⁺.

      All these intermediates except for cytochrome c are membrane-associated (either in the mitochondrial inner membrane of eukaryotes or in the plasma membrane of prokaryotes). All three types of proteins involved in this chain¾flavoproteins, cytochromes, and iron-sulfur proteins¾possess electron-transferring prosthetic groups.

      The components of the electron transport chain can be purified from the mitochondrial inner membrane. Solubilization of the membranes containing the electron transport chain results in the isolation of four distinct protein complexes, and the complete chain can thus be considered to be composed of four parts: (I) NADH-coenzyme Q reductase, (II) succinate-coenzyme Q reductase, (III) coenzyme Q-cytochrome c reductase, and (IV) cytochrome c oxidase (Figure 21.4). Complex I accepts electrons from NADH, serving as a link between glycolysis, the TCA cycle, fatty acid oxidation, and the electron transport chain. Complex II includes succinate dehydrogenase and thus forms a direct link between the TCA cycle and electron transport. Complexes I and II produce a common product, reduced coenzyme Q (UQH₂), which is the substrate for coenzyme Q-cytochrome c reductase (Complex III). As shown in Figure 21.4, there are two other ways to feed electrons to UQ: the electron-transferring flavoprotein, which transfers electrons from the flavoprotein-linked step of fatty acyl-CoA dehydrogenase, and sn-glycerophosphate dehydrogenase. Complex III oxidizes UQH₂ while reducing cytochrome c, which in turn is the substrate for Complex IV, cytochrome c oxidase. Complex IV is responsible for reducing molecular oxygen. Each of the complexes shown in Figure 21.4 is a large multisubunit complex embedded within the inner mitochondrial membrane.

Figure 21.4 · An overview of the complexes and pathways in the mitochondrial electron transport chain. (Adapted from Nicholls, D. G., and Ferguson, S. J., 1992. Bioenergetics 2. London: Academic  Press.)

21.4 · Complex I: NADH-Coenzyme Q Reductase

As its name implies, this complex transfers a pair of electrons from NADH to coenzyme Q, a small, hydrophobic, yellow compound. Another common name for this enzyme complex is NADH dehydrogenase. The complex (with an estimated mass of 850 kD) involves more than 30 polypeptide chains, one molecule of flavin mononucleotide (FMN), and as many as seven Fe-S clusters, together containing a total of 20 to 26 iron atoms (Table 21.2). By virtue of its dependence on FMN, NADH-UQ reductase is a flavoprotein.

      Although the precise mechanism of the NADH-UQ reductase is not known, the first step involves binding of NADH to the enzyme on the matrix side of the inner mitochondrial membrane, and transfer of electrons from NADH to tightly bound FMN:

      NADH + [ FMN] + H⁺ ® [FMNH₂] + NAD⁺                   (21.17)

      The second step involves the transfer of electrons from the reduced [FMNH₂] to a series of Fe-S proteins, including both 2Fe-2S and 4Fe-4S clusters (see Figures 20.8 and 20.16). The unique redox properties of the flavin group of FMN are probably important here. NADH is a two-electron donor, whereas the Fe-S proteins are one-electron transfer agents. The flavin of FMN has three redox states¾the oxidized, semiquinone, and reduced states. It can act as either a one-electron or a two-electron transfer agent and may serve as a critical link between NADH and the Fe-S proteins.

      The final step of the reaction involves the transfer of two electrons from iron-sulfur clusters to coenzyme Q. Coenzyme Q is a mobile electron carrier. Its isoprenoid tail makes it highly hydrophobic, and it diffuses freely in the hydrophobic core of the inner mitochondrial membrane. As a result, it shuttles electrons from Complexes I and II to Complex III. The redox cycle of UQ is shown in Figure 21.5, and the overall scheme is shown schematically in Figure 21.6.

Figure 21.5 · (a) The three oxidation states of coenzyme Q.
(b) A space-filling model of coenzyme Q.

Figure 21.6 · Proposed structure and electron transport pathway for Complex I. Three protein complexes have been isolated, including the flavoprotein (FP), iron-sulfur protein (IP), and hydrophobic protein (HP). FP contains three peptides (of mass 51, 24, and 10 kD) and bound FMN and has 2 Fe-S centers (a 2Fe-2S center and a 4Fe-4S center). IP contains six peptides and at least 3 Fe-S centers. HP contains at least seven peptides and one Fe-S center.

Complex I Transports Protons from the Matrix to the Cytosol

The oxidation of one NADH and the reduction of one UQ by NADH-UQ reductase results in the net transport of protons from the matrix side to the cytosolic side of the inner membrane. The cytosolic side, where H⁺ accumulates, is referred to as the P (for positive) face; similarly, the matrix side is the N (for negative) face. Some of the energy liberated by the flow of electrons through this complex is used in a coupled process to drive the transport of protons across the membrane. (This is an example of active transport, a phenomenon examined in detail in Chapter 10.) Available experimental evidence suggests a stoichiometry of four H⁺ transported per two electrons passed from NADH to UQ.

21.5 · Complex II: Succinate-Coenzyme Q Reductase

Complex II is perhaps better known by its other name¾succinate dehydrogenase, the only TCA cycle enzyme that is an integral membrane protein in the inner mitochondrial membrane. This enzyme has a mass of approximately 100 to 140 kD and is composed of four subunits: two Fe-S proteins of masses 70 kD and 27 kD, and two other peptides of masses 15 kD and 13 kD. Also known as flavoprotein 2 (FP₂), it contains an FAD covalently bound to a histidine residue (see Figure 20.15), and three Fe-S centers: a 4Fe-4S cluster, a 3Fe-4S cluster, and a 2Fe-2S cluster. When succinate is converted to fumarate in the TCA cycle, concomitant reduction of bound FAD to FADH₂ occurs in succinate dehydrogenase. This FADH₂ transfers its electrons immediately to Fe-S centers, which pass them on to UQ. Electron flow from succinate to UQ,

                    Succinate ® fumarate + 2 H⁺ + 2 e^-         (21.18)

                   UQ + 2 H⁺ + 2 e^- ® UQH₂                     (21.19)

Net rxn:      Succinate + UQ ® fumarate + UQH₂DE_o' = 0.029V                   (21.20)
                                                                          DG^o' = -5.6 kJ/mol

yields a net reduction potential of 0.029 V. (Note that the first half-reaction is written in the direction of the e^- flow. As always, DE_o' is calculated according to Equation 21.9.) The small free energy change of this reaction is not sufficient to drive the transport of protons across the inner mitochondrial membrane.

      This is a crucial point because (as we will see) proton transport is coupled with ATP synthesis. Oxidation of one FADH₂ in the electron transport chain results in synthesis of approximately two molecules of ATP, compared with the approximately three ATPs produced by the oxidation of one NADH. Other enzymes can also supply electrons to UQ, including mitochondrial sn-glycerophosphate dehydrogenase, an inner membrane-bound shuttle enzyme, and the fatty acyl-CoA dehydrogenases, three soluble matrix enzymes involved in fatty acid oxidation

Figure 21.7 · The fatty acyl-CoA dehydrogenase reaction, emphasizing that the reaction involves reduction of enzyme-bound FAD (indicated by brackets).

(Figure 21.7; also see Chapter 24). The path of electrons from succinate to UQ is shown in Figure 21.8.

Figure 21.8 · A probable scheme for electron flow in Complex II. Oxidation of succinate occurs with reduction of [FAD]. Electrons are then passed to Fe-S centers and then to coenzyme Q (UQ). Proton transport does not occur in this complex.

21.6 · Complex III: Coenzyme Q-Cytochrome c Reductase

In the third complex of the electron transport chain, reduced coenzyme Q (UQH₂) passes its electrons to cytochrome c  via a unique redox pathway known as the Q cycle. UQ-cytochrome c reductase (UQ-cyt c reductase), as this complex is known, involves three different cytochromes and an Fe-S protein. In the cytochromes of these and similar complexes, the iron atom at the center of the porphyrin ring cycles between the reduced Fe²⁺ (ferrous) and oxidized Fe³⁺ (ferric) states.

Figure 21.9 · Typical visible absorption spectra of cytochromes. (a) Cytochrome c, reduced spectrum; (b) cytochrome c, oxidized spectrum; (c) the difference spectrum: (a) minus (b); (d) beef heart mitochondrial particles: room temperature difference (reduced minus oxidized) spectrum; (e) beef heart submitochondrial particles: same as (d) but at 77 K. a- and b- bands are labeled, and in (d) and (e) the bands for cytochromes a, b and c are indicated.

 
      Cytochromes were first named and classified on the basis of their absorption spectra (Figure 21.9), which depend upon the structure and environment of their heme groups. The b cytochromes contain iron-protoporphyrin IX (Figure 21.10), the same heme found in hemoglobin and myoglobin.

Figure 21.10 · The structures of iron protoporphyrin IX, heme c, and heme a.

The c cytochromes contain heme c, derived from iron-protoporphyrin IX by the covalent attachment of cysteine residues from the associated protein. UQ-cyt c reductase contains a b-type cytochrome, of 30 to 40 kD, with two different heme sites (Figure 21.11) and one c-type cytochrome. (One other variation, heme a, contains a 15-carbon isoprenoid chain on a modified vinyl group, and a formyl group in place of one of the methyls [see Figure 21.10]. Cytochrome a is found in two forms in Complex IV of the electron transport chain, as we shall see.) The two hemes on the b cytochrome polypeptide in UQ-cyt c reductase are distinguished by their reduction potentials and the wavelength (l_max) of the so-called a band (see Figure 21.9). One of these hemes, known as b_L or b₅₆₆, has a standard reduction potential, E_o' , of -0.100 V and a wavelength of maximal absorbance (l_max) of 566 nm. The other, known as b_H or b₅₆₂ has a standard reduction potential of +0.050 V and a l_max of 562 nm. (H and L here refer to high and low reduction potential.)

Figure 21.11 · The structure of UQ-cyt c reductase, also known as the cytochrome bc₁ complex. The alpha helices of cytochrome b (pale green) define the transmembrane domain of the protein. The bottom of the structure as shown extends approximately 75 Å into the mitochondrial matrix, and the top of the structure as shown extends about 38 Å into the intermembrane space. (Photograph kindly provided by Di Xia and Johann Deisenhofer [From Xia, D., Yu,C.-A., Kim, H., Xia, J.-Z., Kachurin, A. M., Zhang, L., Yu, L., and Deisenhofer, J., 1997. The crystal structure of the cytochrome bc₁ complex from bovine heart mitochondria. Science 277:60-66.])

      The structure of the UQ-cyt c reductase, also known as the cytochrome bc₁ complex, has been determined by Johann Deisenhofer and his colleagues. (Deisenhofer was a co-recipient of the Nobel Prize in Chemistry for his work on the structure of a photosynthetic reaction center [see Chapter 22]). The complex is a dimer, with each monomer consisting of 11 protein subunits and 2165 amino acid residues (monomer mass, 248 kD ). The dimeric structure is pear-shaped and consists of a large domain that extends 75 Å into the mitochondrial matrix, a transmembrane domain consisting of 13 transmembrane a-helices in each monomer and a small domain that extends 38 Å into the intermembrane space (Figure 21.11). Most of the Rieske protein ( an Fe-S protein named for its discoverer) is mobile in the crystal (only 62 of 196 residues are shown in the structure in Figure 21.11), and Deisenhofer has postulated that mobility of this subunit could be required for electron transfer in the function of this complex.

Complex III Drives Proton Transport

As with Complex I, passage of electrons through the Q cycle of Complex III is accompanied by proton transport across the inner mitochondrial membrane. The postulated pathway for electrons in this system is shown in Figure 21.12. A large pool of UQ and UQH₂ exists in the inner mitochondrial membrane. The Q cycle is initiated when a molecule of UQH₂ from this pool diffuses to a site (called Q_p) on Complex III near the cytosolic face of the membrane.

Figure 21.12 · The Q cycle in mitochondria. (a) The electron transfer pathway following oxidation of the first UQH₂ at the Q_p site near the cytosolic face of the membrane. (b) The pathway following oxidation of a second UQH₂.

      Oxidation of this UQH₂ occurs in two steps. First, an electron from UQH₂ is transferred to the Rieske protein and then to cytochrome c₁. This releases two H⁺ to the cytosol and leaves UQ ×^- , a semiquinone anion form of UQ, at the Q_p site. The second electron is then transferred to the b_L heme, converting UQ×^- to UQ. The Rieske protein and cytochrome c₁ are similar in structure; each has a globular domain and is anchored to the inner membrane by a hydrophobic segment. However, the hydrophobic segment is N-terminal in the Rieske protein and C-terminal in cytochrome c₁.
      The electron on the b_L heme facing the cytosolic side of the membrane is now passed to the b_H heme on the matrix side of the membrane. This electron transfer occurs against a membrane potential of 0.15 V and is driven by the loss of redox potential as the electron moves from b_L (E_o' = -0.100V) to b_H(E_o' = +0.050V). The electron is then passed from b_H to a molecule of UQ at a second quinone-binding site, Q_n, converting this UQ to UQ×^-. The resulting UQ×^- remains firmly bound to the Q_n site. This completes the first half of the Q cycle (Figure 21.12a).
      The second half of the cycle (Figure 21.12b) is similar to the first half, with a second molecule of UQH₂ oxidized at the Q_p site, one electron being passed to cytochrome c₁ and the other transferred to heme b_H and then to heme b_H. In this latter half of the Q cycle, however, the b_H electron is transferred to the semiquinone anion, UQ×^- at the Q_n site. With the addition of two H⁺ from the mitochondrial matrix, this produces a molecule of UQH₂, which is released from the Q_n site and returns to the coenzyme Q pool, completing the Q cycle.

The Q Cycle Is an Unbalanced Proton Pump

Why has nature chosen this rather convoluted path for electrons in Complex III? First of all, Complex III takes up two protons on the matrix side of the inner membrane and releases four protons on the cytoplasmic side for each pair of electrons that passes through the Q cycle. The apparent imbalance of two protons in for four protons out is offset by proton translocations in Complex IV, the cytochrome oxidase complex. The other significant feature of this mechanism is that it offers a convenient way for a two-electron carrier, UQH₂, to interact with the b_L and b_H hemes, the Rieske protein Fe-S cluster, and cytochrome c₁, all of which are one-electron carriers.

Cytochrome c Is a Mobile Electron Carrier

Figure 21.13 · The structure of mitochondrial cytochrome c. The heme is shown at the center of the structure, covalently linked to the protein via its two sulfur atoms (yellow). A third sulfur from a methionine residue coordinates the iron.

Electrons traversing Complex III are passed through cytochrome c₁ to cytochrome c. cytochrome c is the only one of the cytochromes that is water-soluble. Its structure, determined by X-ray crystallography (Figure 21.13), is globular; the planar heme group lies near the center of the protein, surrounded predominantly by hydrophobic protein residues. The iron in the porphyrin ring is coordinated both to a histidine nitrogen and to the sulfur atom of a methionine residue. Coordination with ligands in this manner on both sides of the porphyrin plane precludes the binding of oxygen and other ligands, a feature that distinguishes the cytochromes from hemoglobin (Chapter 15).
      Cytochrome c, like UQ, is a mobile electron carrier. It associates loosely with the inner mitochondrial membrane (in the intermembrane space on the cytosolic side of the inner membrane) to acquire electrons from the Fe-S-cyt c₁ aggregate of Complex III, and then it migrates along the membrane surface in the reduced state, carrying electrons to cytochrome c oxidase, the fourth complex of the electron transport chain.

21.7 · Complex IV: Cytochrome c Oxidase

Figure 21.14 · An electrophoresis gel showing the complex subunit structure of bovine heart cytochrome c oxidase. The three largest subunits, I, II, and III, are coded for by mitochondrial DNA. The others are encoded by nuclear DNA. (Photo kindly provided by Professor Roderick Capaldi)

Complex IV is called cytochrome c oxidase because it accepts electrons from cytochrome c and directs them to the four-electron reduction of O₂ to form H₂O:

      4 cyt c (Fe²⁺) + 4 H⁺ + O₂ ® 4 cyt c (Fe³⁺) + 2 H₂O   (21.21)

Thus, O₂ and cytochrome c oxidase are the final destination for the electrons derived from the oxidation of food materials. In concert with this process, cytochrome c oxidase also drives transport of protons across the inner mitochondrial membrane. These important functions are carried out by a transmembrane protein complex consisting of more than 10 subunits (Table 21.2).
      An electrophoresis gel of the bovine heart complex is shown in Figure 21.14. The total mass of the protein in the complex, composed of 13 subunits, is 204 kD. Subunits I through III, the largest ones, are encoded by mitochondrial DNA, synthesized in the mitochondrion, and inserted into the inner membrane from the matrix side. The smaller subunits are coded by nuclear DNA and synthesized in the cytosol.
Figure 21.15 · Molecular graphic image of subunits I, II, and III of cytochrome c oxidase.

      The structure of cytochrome c oxidase has been solved. The essential Fe and Cu sites are contained entirely within the structures of subunits I, II, and III. None of the 10 nuclear DNA-derived subunits directly impinges on the essential metal sites. The implication is that subunits I to III actively participate in the events of electron transfer, but that the other 10 subunits play regulatory roles in this process. Subunit I is cylindrical in shape and consists of 12 transmembrane helices, without any significant extramembrane parts (Figure 21.15). Hemes a and a₃, which lie perpendicular to the membrane plane, are cradled by the helices of subunit I. Subunits II and III lie on opposite sides of subunit I and do not contact each other. Subunit II has an extramembrane domain on the cytosolic face of the mitochondrial membrane. This domain consists of a 10-strand b-barrel that holds Cu_A 7 Å from the nearest surface atom of the subunit. Subunit III consists of 7 transmembrane helices with no significant extramembrane domains. Figure 21.16 presents a molecular graphic image of cytochrome c oxidase.

Figure 21.16 · Molecular graphic image of cytochrome c oxidase. Seven of the 10 nuclear DNA-derived subunits (IV, VIa, VIc, VIIa, VIIb, VIIc, and VIII) possess transmembrane segments. Three (Va, Vb, and VIb) do not. Subunits IV and VIc are transmembrane and dumbbell-shaped. Subunit Va is globular and bound to the matrix side of the complex, whereas VIb is a globular subunit on the cytosolic side of the membrane complex. Vb is globular and matrix-side associated as well, but it has an N-terminal extended domain. VIa has a transmembrane helix and a small globular domain. Subunit VIIa consists of a tilted transmembrane helix, with another short helical segment on the matrix side of the membrane. Subunits VIIa, VIIb, and VIII consist of transmembrane segments with short extended regions outside the membrane. 

Electron Transfer in Complex IV Involves Two Hemes and Two Copper Sites

Figure 21.17 · The electron transfer pathway for cytochrome oxidase. Cytochrome c binds on the cytosolic side, transferring electrons through the copper and heme centers to reduce O₂ on the matrix side of the membrane.

Cytochrome c oxidase contains two heme centers (cytochromes a and a₃) as well as two copper atoms (Figure 21.17). The copper sites, Cu_A and Cu_B, are associated with cytochromes a and a₃, respectively. The copper sites participate in electron transfer by cycling between the reduced (cuprous) Cu^- state and the oxidized (cupric) Cu²⁺ state. (Remember, the cytochromes and copper sites are one-electron transfer agents.) Reduction of one oxygen molecule requires passage of four electrons through these carriers¾one at a time (Figure 21.17).
 Figure 21.18 · (a) The Cu_A site of cytochrome oxidase. Copper ligands include two histidine imidazole groups and two cysteine side chains from the protein. (b) The coordination of histidine imidazole ligands to the iron atom in the heme a center of cytochrome oxidase.

     Electrons from cytochrome c are transferred to Cu_A sites and then passed to the heme iron of cytochrome a. Cu_A is liganded by two cysteines and two histidines (Figure 21.18). The heme of cytochrome a is liganded by imidazole rings of histidine residues (Figure 21.18). The Cu_A and the Fe of cytochrome a are within 1.5 nm of each other.

Figure 21.19 · The binuclear center of cytochrome oxidase. A ligand, L (probably a cysteine S), is shown bridging the Cu_B and Fe_a3 metal sites.

      Cu_B and the iron atom of cytochrome a₃ are also situated close to each other and are thought to share a ligand, which may be a cysteine sulfur (Figure 21.19). This closely associated pair of metal ions is referred to as a binuclear center.
Figure 21.20 · A model for the mechanism of O₂ reduction by cytochrome oxidase.
(Adapted from Nicholls, D. G., and Ferguson, S. J., 1992. Bioenergetics 2. London: Academic Press; and Babcock, G. T., and Wikström, M., 1992. Nature 356:301-309.)

      As shown in Figure 21.20, the electron pathway through Complex IV continues as Cu_B accepts a single electron from cytochrome a (state O ® state H). A second electron then reduces the iron center to Fe²⁺ (H ® R), leading to the binding of O₂ (R ® A) and the formation of a peroxy bridge between heme a₃and Cu_B (A ® P). This amounts to the transfer of two electrons from the binuclear center to the bound O₂. The next step involves uptake of two H⁺ and a third electron (P ® F), which leads to cleavage of the O-O bond and generation of Fe⁴⁺ at the heme. Uptake of a fourth e^- facilitates formation of ferric hydroxide at the heme center (F ® O'). In the final step of the cycle (O' ® O), protons from the mitochondrial matrix are accepted by the coordinated hydroxyl groups, and the resulting water molecules dissociate from the binuclear center.

Complex IV Also Transports Protons Across the Inner Mitochondrial Membrane

It should be emphasized here that the four major complexes of the electron transport chain operate quite independently in the inner mitochondrial membrane. Each is a multiprotein aggregate maintained by numerous strong associations between peptides of the complex, but there is no evidence that the complexes associate with one another in the membrane. Measurements of the lateral diffusion rates of the four complexes, of coenzyme Q, and of cytochrome c in the inner mitochondrial membrane show that the rates differ considerably, indicating that these complexes do not move together in the membrane. Kinetic studies with reconstituted systems show that electron transport does not operate by means of connected sets of the four complexes.

A Dynamic Model of Electron Transport

Figure 21.21 · A model for the electron transport pathway in the mitochondrial inner membrane. UQ/UQH₂ and cytochrome c are mobile electron carriers and function by transferring electrons between the complexes. The proton transport driven by Complexes I, III, and IV is indicated.

The model that emerges for electron transport is shown in Figure 21.21. The four complexes are independently mobile in the membrane. Coenzyme Q collects electrons from NADH-UQ reductase and succinate-UQ reductase and delivers them (by diffusion through the membrane core) to UQ-cyt c reductase. cytochrome c is water-soluble and moves freely, carrying electrons from UQ-cyt c reductase to cytochrome c oxidase. In the process of these electron transfers, protons are driven across the inner membrane (from the matrix side to the intermembrane space). The proton gradient generated by electron transport represents an enormous source of potential energy. As seen in the next section, this potential energy is used to synthesize ATP as protons flow back into the matrix.

The H⁺/2e^- Ratio for Electron Transport Is Uncertain

In 1961, Peter Mitchell, a British biochemist, proposed that the energy stored in a proton gradient across the inner mitochondrial membrane by electron transport drives the synthesis of ATP in cells. The proposal became known as Mitchell's chemiosmotic hypothesis. The ratio of protons transported per pair of electrons passed through the chain¾the so-called H⁺/2e^- ratio ¾ has been an object of great interest for many years. Nevertheless, the ratio has remained extremely difficult to determine. The consensus estimate for the electron transport pathway from succinate to O₂ is 6 H⁺/2 e^-. The ratio for Complex I by itself remains uncertain, but recent best estimates place it as high as 4 H⁺/2e^-. On the basis of this value, the stoichiometry of transport for the pathway from NADH to O₂ is 10 H⁺/2e^-. Although this is the value assumed in Figure 21.21, it is important to realize that this represents a consensus drawn from many experiments.

21.8 · The Thermodynamic View of Chemiosmotic Coupling

Peter Mitchell's chemiosmotic hypothesis revolutionized our thinking about the energy coupling that drives ATP synthesis by means of an electrochemical gradient. How much energy is stored in this electrochemical gradient? For the transmembrane flow of protons across the inner membrane (from inside [matrix] to outside), we could write

      H⁺_in ® H⁺_out       ( 21.26)

The free energy difference for protons across the inner mitochondrial membrane includes a term for the concentration difference and a term for the electrical potential. This is expressed as

+ ZÁDy      (21.27)

where c₁ and c₂are the proton concentrations on the two sides of the membrane, Z is the charge on a proton, Á is Faraday's constant, and Dy is the potential difference across the membrane. For the case at hand, this equation becomes

     (21.28)

In terms of the matrix and cytoplasm pH values, the free energy difference is

      DG = -2.303RT( pH_out - pH_in) + ÁDy   (21.29)

Reported values for DC and DpH vary, but the membrane potential is always found to be positive outside and negative inside, and the pH is always more acidic outside and more basic inside. Taking typical values of DC = 0.18 V and DpH = 1 unit, the free energy change associated with the movement of one mole of protons from inside to outside is

      DG = 2.3RT + ( 0.18 V)  (21.30)

With = 96.485 kJ/V×mol, the value of DG at 37^oC is

      DG = 5.9 kJ + 17.4 kJ = 23.3 kJ      ( 21.31)

which is the free energy change for movement of a mole of protons across a typical inner membrane. Note that the free energy terms for both the pH difference and the potential difference are unfavorable for the outward transport of protons, with the latter term making the greater contribution. On the other hand, the DG for inward flow of protons is -23.3 kJ/mol. It is this energy that drives the synthesis of ATP, in accord with Mitchell's model. Peter Mitchell was awarded the Nobel Prize in chemistry in 1978.

21.9 · ATP Synthase

Figure 21.23 · Electron micrograph of submitochondrial particles showing the 8.5-nm projections or particles on the inner membrane, eventually shown to be F₁-ATP synthase. (Parsons, D. F., 1963. Science 140:985)

 The mitochondrial complex that carries out ATP synthesis is called ATP synthase or sometimes F₁F₀-ATPase (for the reverse reaction it catalyzes). ATP synthase was observed in early electron micrographs of submitochondrial particles (prepared by sonication of inner membrane preparations) as round, 8.5-nm-diameter projections or particles on the inner membrane (Figure 21.23). In micrographs of native mitochondria, the projections appear on the matrix-facing surface of the inner membrane. Mild agitation removes the particles from isolated membrane preparations, and the isolated spherical particles cata-lyze ATP hydrolysis, the reverse reaction of the ATP synthase. Stripped of these particles, the membranes can still carry out electron transfer but cannot synthesize ATP. In one of the first reconstitution experiments with membrane proteins, Efraim Racker showed that adding the particles back to stripped membranes restored electron transfer-dependent ATP synthesis.

ATP Synthase Consists of Two Complexes¾F₁ and F₀

ATP synthase actually consists of two principal complexes. The spheres ob-served in electron micrographs make up the F₁ unit, which catalyzes ATP synthesis. These F₁ spheres are attached to an integral membrane protein aggregate called the F_o unit. F₁ consists of five polypeptide chains named a, b, g, d, and e, with a subunit stoichiometry a₃b₃gde (Table 21.3). F_o consists of three hydrophobic subunits denoted by a, b, and c, with an apparent stoichiometry of a₁b₂c_9-12. F_o forms the transmembrane pore or channel through which protons move to drive ATP synthesis. The a, b, g, d, and e subunits of F₁ contain 510, 482, 272, 146, and 50 amino acids, respectively, with a total molecular mass for F₁ of 371 kD. The a and b subunits are homologous, and each of these subunits bind a single ATP. The catalytic sites are in the b subunits; the function of the ATP sites in the a subunits is unknown (deletion of the sites does not affect activity).

  Figure 21.24 · Molecular graphic images (a) side view and (b) top view of the F₁-ATP synthase showing the individual component peptides. The g-subunit is the pink structure visible in the center of view (b).

    John Walker and his colleagues have determined the structure of the F₁ complex (Figure 21.24). The F₁ ATPase is an inherently asymmetrical structure, with the three b subunits having three different conformations. In the structure solved by Walker, one of the b-subunit ATP sites contains AMP-PNP (a nonhydrolyzable analog of ATP), and another contains ADP, with the third site being empty. This state is consistent with the binding change mechanism for ATP synthesis proposed by Paul Boyer, in which three reaction sites cycle concertedly through the three intermediate states of ATP synthesis (take a look at Figure 21.28 on page 697).

Figure 21.25 · A model of the F₁ and F₀ components of the ATP synthase, a rotating molecular motor. The a, b, a, b, and d subunits constitute the stator of the motor, and the c, g, and e subunits form the rotor. Flow of protons through the structure turns the rotor and drives the cycle of conformational changes in a and b that synthesize ATP.

      How might such cycling occur? Important clues have emerged from several experiments that show that the g subunit rotates with respect to the ab complex. How such rotation might be linked to transmembrane proton flow and ATP synthesis is shown in Figure 21.25. In this model, the c subunits of F_o are arranged in a ring. Several lines of evidence suggest that each c subunit consists of a pair of antiparallel transmembrane helices with a short hairpin loop on the cytosolic side of the membrane. A ring of c subunits could form a rotor that turns with respect to the a subunit, a stator consisting of five transmembrane a-helices with proton access channels on either side of the membrane. The g subunit is postulated to be the link between F₁ and F_o. Several experiments have shown that g rotates relative to the (ab)₃ complex during ATP synthesis. If g is anchored to the c subunit rotor, then the c rotor-g complex can rotate together relative to the (ab)₃ complex. Subunit b possesses a single transmembrane segment and a long hydrophilic head domain, and the complete stator may consist of the b subunits anchored at one end to the a subunit and linked at the other end to the (ab)₃ complex via the d subunit, as shown in Figure 21.25.
      What, then, is the mechanism for ATP synthesis? The c rotor subunits each carry an essential residue, Asp⁶¹. (Changing this residue to Asn abolishes ATP synthase activity.) Rotation of the c rotor relative to the stator may depend upon neutralization of the negative charge on each c subunit Asp⁶¹ as the rotor turns. Protons taken up from the cytosol by one of the proton access channels in a could protonate an Asp⁶¹ and then ride the rotor until they reach the other proton access channel on a, from which they would be released into the matrix. Such rotation would cause the g subunit to turn relative to the three b-subunit nucleotide sites of F₁, changing the conformation of each in sequence, so that ADP is first bound, then phosphorylated, then released, according to Boyer's binding change mechanism. Paul Boyer and John Walker shared in the 1997 Nobel Prize for chemistry for their work on the structure and mechanism of ATP synthase.

Boyer's ¹⁸O Exchange Experiment Identified the Energy-Requiring Step

Figure 21.26 · ATP production in the presence of a proton gradient and ATP/ADP exchange in the absence of a proton gradient. Exchange leads to incorporation of ¹⁸O in phosphate as shown.

The elegant studies by Paul Boyer of ¹⁸O exchange in ATP synthase have provided other important insights into the mechanism of the enzyme. Boyer and his colleagues studied the ability of the synthase to incorporate labeled oxygen from H₂¹⁸O into P_i. This reaction (Figure 21.26) occurs via synthesis of ATP from ADP and P_i, followed by hydrolysis of ATP with incorporation of oxygen atoms from the solvent. Although net production of ATP requires coupling with a proton gradient, Boyer observed that this exchange reaction occurs readily, even in the absence of a proton gradient. His finding indicated that the formation of enzyme-bound ATP does not require energy. Indeed, movement of protons through the F_o× channel causes the release of newly synthesized ATP from the enzyme. Thus, the energy provided by electron transport creates a proton gradient that drives enzyme conformational changes resulting in the binding of substrates on ATP synthase, ATP synthesis, and the release of products. The mechanism involves catalytic cooperativity between three interacting sites (Figure 21.27).

Figure 21.27 · The binding change mechanism for ATP synthesis by ATP synthase. This model assumes that F₁ has three interacting and conformationally distinct active sites. The open (O) conformation is inactive and has a low affinity for ligands; the L conformation (with “loose” affinity for ligands) is also inactive; the tight (T) conformation is active and has a high affinity for ligands. Synthesis of ATP is initiated (step 1) by binding of ADP and P_i to an L site. In the second step, an energy-driven conformational change converts the L site to a T conformation and also converts T to O and O to L. In the third step, ATP is synthesized at the T site and released from the O site. Two additional passes through this cycle produce two more ATPs and return the enzyme to its original state.

Racker and Stoeckenius Confirmed the Mitchell Model in a Reconstitution Experiment

When Mitchell first described his chemiosmotic hypothesis in 1961, little evidence existed to support it, and it was met with considerable skepticism by the scientific community. Eventually, however, considerable evidence accumulated to support this model. It is now clear that the electron transport chain generates a proton gradient, and careful measurements have shown that ATP is synthesized when a pH gradient is applied to mitochondria that cannot carry out electron transport. Even more relevant is a simple but crucial experiment reported in 1974 by Efraim Racker and Walther Stoeckenius, which provided specific confirmation of the Mitchell hypothesis. In this experiment, the bovine mitochondrial ATP synthase was reconstituted in simple lipid vesicles with bacteriorhodopsin, a light-driven proton pump from Halobacterium halobium.

Figure 21.28 · The reconstituted vesicles containing ATP synthase and bacteriorhodopsin used by Stoeckenius and Racker to confirm the Mitchell chemiosmotic hypothesis.

As shown in Figure 21.28, upon illumination, bacteriorhodopsin pumped protons into these vesicles, and the resulting proton gradient was sufficient to drive ATP synthesis by the ATP synthase. Because the only two kinds of proteins pres-ent were one that produced a proton gradient and one that used such a gradient to make ATP, this experiment essentially verified Mitchell's chemiosmotic hypothesis.

21.10 · Inhibitors of Oxidative Phosphorylation

Figure 21.29 · The structures of several inhibitors of electron transport and oxidative phosphorylation.

The unique properties and actions of an inhibitory substance can often help to identify aspects of an enzyme mechanism. Many details of electron transport and oxidative phosphorylation mechanisms have been gained from studying the effects of particular inhibitors. Figure 21.29 presents the structures of some electron transport and oxidative phosphorylation inhibitors. The sites of inhibition by these agents are indicated in Figure 21.30.

Figure 21.30 · The sites of action of several inhibitors of electron transport and/or oxidative phosphorylation.

Inhibitors of Complexes I, II, and III Block Electron Transport

Rotenone is a common insecticide that strongly inhibits the NADH-UQ reductase. Rotenone is obtained from the roots of several species of plants. Tribes in certain parts of the world have made a practice of beating the roots of trees along riverbanks to release rotenone into the water, where it paralyzes fish and makes them easy prey. Ptericidin, Amytal, and other barbiturates, mercurial agents, and the widely prescribed painkiller Demerol also exert inhibitory actions on this enzyme complex. All these substances appear to inhibit reduction of coenzyme Q and the oxidation of the Fe-S clusters of NADH-UQ reductase.
      2-Thenoyltrifluoroacetone and carboxin and its derivatives specifically block Complex II, the succinate-UQ reductase. Antimycin, an antibiotic produced by Streptomyces griseus, inhibits the UQ-cytochrome c reductase by blocking electron transfer between b_H and coenzyme Q in the Q_n site. Myxothiazol inhibits the same complex by acting at the Q_p site.

Cyanide, Azide, and Carbon Monoxide Inhibit Complex IV

Complex IV, the cytochrome c oxidase, is specifically inhibited by cyanide (CN^-), azide (N₃^-), and carbon monoxide (CO). Cyanide and azide bind tightly to the ferric form of cytochrome a₃, whereas carbon monoxide binds only to the ferrous form. The inhibitory actions of cyanide and azide at this site are very potent, whereas the principal toxicity of carbon monoxide arises from its affinity for the iron of hemoglobin. Herein lies an important distinction between the poisonous effects of cyanide and carbon monoxide. Because animals (including humans) carry many, many hemoglobin molecules, they must inhale a large quantity of carbon monoxide to die from it. These same organisms, however, possess comparatively few molecules of cytochrome a₃. Consequently, a limited exposure to cyanide can be lethal. The sudden action of cyanide attests to the organism's constant and immediate need for the energy supplied by electron transport.

Oligomycin and DCCD Are ATP Synthase Inhibitors

Inhibitors of ATP synthase include dicyclohexylcarbodiimide (DCCD) and oligomycin (Figure 21.29). DCCD bonds covalently to carboxyl groups in hydrophobic domains of proteins in general, and to a glutamic acid residue of the c subunit of F_o×, the proteolipid forming the proton channel of the ATP synthase, in particular. If the c subunit is labeled with DCCD, proton flow through F_o× is blocked and ATP synthase activity is inhibited. Likewise, oligomycin acts directly on the ATP synthase. By binding to a subunit of F_o×, oligomycin also blocks the movement of protons through F_o×.

21.11 · Uncouplers Disrupt the Coupling of Electron Transport and ATP Synthase

Figure 21.31 · Structures of several uncouplers, molecules that dissipate the proton gradient across the inner mitochondrial membrane and thereby destroy the tight coupling between electron transport and the ATP synthase reaction.

Another important class of reagents affects ATP synthesis, but in a manner that does not involve direct binding to any of the proteins of the electron transport chain or the F₁F₀-ATPase. These agents are known as uncouplers because they disrupt the tight coupling between electron transport and the ATP synthase. Uncouplers act by dissipating the proton gradient across the inner mitochondrial membrane created by the electron transport system. Typical examples include 2, 4-dinitrophenol, dicumarol, and carbonyl cyanide-p-trifluoro-methoxyphenyl hydrazone (perhaps better known as fluorocarbonyl-cyanide phenylhydrazone or FCCP) (Figure 21.31). These compounds share two common features: hydrophobic character and a dissociable proton. As uncouplers, they function by carrying protons across the inner membrane. Their tendency is to acquire protons on the cytosolic surface of the membrane (where the proton concentration is high) and carry them to the matrix side, thereby destroying the proton gradient that couples electron transport and the ATP synthase. In mitochondria treated with uncouplers, electron transport continues, and protons are driven out through the inner membrane. However, they leak back in so rapidly via the uncouplers that ATP synthesis does not occur. Instead, the energy released in electron transport is dissipated as heat.

Endogenous Uncouplers Enable Organisms To Generate Heat

Ironically, certain cold-adapted animals, hibernating animals, and newborn animals generate large amounts of heat by uncoupling oxidative phosphorylation. Adipose tissue in these organisms contains so many mitochondria that it is called brown adipose tissue for the color imparted by the mitochondria. The inner membrane of brown adipose tissue mitochondria contains an endogenous protein called thermogenin (literally, "heat maker"), or uncoupling protein, that creates a passive proton channel through which protons flow from the cytosol to the matrix. Certain plants also use the heat of uncoupled proton transport for a special purpose. Skunk cabbage and related plants contain floral spikes that are maintained as much as 20 degrees above ambient temperature in this way. The warmth of the spikes serves to vaporize odiferous molecules, which attract insects that fertilize the flowers.

21.12 · ATP Exits the Mitochondria via an ATP-ADP Translocase

Figure 21.32 ·  Outward transport of ATP (via the ATP/ADP translocase) is favored by the membrane electrochemical potential.

ATP, the cellular energy currency, must exit the mitochondria to carry energy throughout the cell, and ADP must be brought into the mitochondria for reprocessing. Neither of these processes occurs spontaneously because the highly charged ATP and ADP molecules do not readily cross biological membranes. Instead, these processes are mediated by a single transport system, the ATP-ADP translocase. This protein tightly couples the exit of ATP with the entry of ADP so that the mitochondrial nucleotide levels remain approximately constant. For each ATP transported out, one ADP is transported into the matrix. The translocase, which accounts for approximately 14% of the total mitochondrial membrane protein, is a homodimer of 30-kD subunits. Transport occurs via a single nucleotide-binding site, which alternately faces the matrix and the cytosol (Figure 21.32). It binds ATP on the matrix side, reorients to face the cytosol, and exchanges ATP for ADP, with subsequent movement back to the matrix face of the inner membrane.

Outward Movement of ATP Is Favored over Outward ADP Movement

The charge on ATP