Biochemistry, 4th Edition P65 docx

20.3 How Is the Electron-Transport Chain Organized? 603 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 20.8; also see Chapter 23). The path of electrons from succinate to UQ is shown in Figure 20.7. Complex III Mediates Electron Transport from Coenzyme Q to Cytochrome c In the third complex of the electron-transport chain, reduced coenzyme Q (UQH 2 ) 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 Fe 2ϩ (ferrous) and oxidized Fe 3ϩ (ferric) states. Cytochromes were first named and classified on the basis of their absorption spectra (Figure 20.9), which depend upon the structure and environment of their heme groups. The b cytochromes contain iron protoporphyrin IX (Figure 20.10), the same heme found in hemoglobin and myoglobin. The c cytochromes contain heme c, derived from iron protoporphyrin IX by the covalent attachment to cysteine residues from the associated protein. (One other heme variation, heme a, contains a 15-carbon iso- prenoid chain on a modified vinyl group and a formyl group in place of one of the methyls [see Figure 20.10]. Cytochrome a is found in two forms in Complex IV of the electron-transport chain, as we shall see.) UQ–cyt c reductase (Figure 20.11) contains a b-type cytochrome, of 30 to 40 kD, with two different heme sites and one c-type cytochrome. The two hemes on the b cytochrome polypeptide in UQ–cyt c reductase are distinguished by their reduction potentials and the wavelength (␭ max ) of the so-called H 3 C O C SCoA [FAD] [FADH 2 ]UQ H 3 C O C SCoA FIGURE 20.8 The fatty acyl-CoA dehydrogenase reaction, emphasizing that the reaction involves reduction of enzyme-bound FAD (indicated by brackets). β (a) (b) 450 500 550 600 650 Wavelength (nm) (a) Cytochrome c: reduced spectrum α (b) Cytochrome c: oxidized spectrum Absorbance FIGURE 20.9 Visible absorption spectra of cytochrome c. N Fe N N N CH CH 2 CH 3 CH 2 CH 2 COO _ CH 2 CH 2 COO _ H 3 C H 3 C CH 2 CH H 3 C N Fe N N N CHCH 3 CH 3 CH 2 CH 2 COO _ CH 2 CH 2 COO _ H 3 C H 3 C H 3 C S S N Fe N N N CH CH 2 CH 3 CH 2 CH 2 COO _ CH 2 CH 2 COO _ H 3 C CH 2 CH H 3 C OCH OH CH 3 CH Iron protoporphyrin IX (found in cytochrome b, myoglobin, and hemoglobin) Heme c (found in cytochrome c) Heme a (found in cytochrome a) FIGURE 20.10 The structures of iron protoporphyrin IX, heme c, and heme a. 604 Chapter 20 Electron Transport and Oxidative Phosphorylation ␣-band. One of these hemes, known as b L or b 566 , has a standard reduction potential, Ᏹ o Ј, of Ϫ0.100 V and a wavelength of maximal absorbance (␭ max ) of 566 nm. The other, known as b H or b 562 , has a standard reduction potential of ϩ0.050 V and a ␭ max of 562 nm. (H and L here refer to high and low reduction potential.) The structure of the UQ–cyt c reductase, also known as the cytochrome bc 1 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 21). 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 ␣-helices in each monomer and a small domain that extends 38 Å into the intermembrane space (Figure 20.11). Most of the Rieske protein (an Fe-S protein named for its discoverer) is mobile in the crystal (only 62 of its 196 residues are shown in the structure in Figure 20.11), and Deisen- hofer 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 20.12. A large pool of UQ and UQH 2 exists in the inner mitochondrial membrane. The Q cycle is initiated when a molecule of UQH 2 from this pool diffuses to a site (called Q p ) on Complex III near the cytosolic face of the membrane. Oxidation of this UQH 2 occurs in two steps. First, an electron from UQH 2 is transferred to the Rieske protein and then to cytochrome c 1 . This releases two H ϩ to the cytosol and leaves UQ и Ϫ , a semiquinone anion form of UQ, at the Q p site. The second electron is then transferred to the b L heme, converting UQ и Ϫ to UQ. The Rieske protein and cytochrome c 1 are similar in structure; each has a globular domain and is anchored to the inner mitochondrial membrane by a hydrophobic segment. However, the hydrophobic segment is N-terminal in the Rieske protein and C-terminal in cytochrome c 1 . FIGURE 20.11 The structure of UQ–cyt c reductase, also known as the cytochrome bc 1 complex.The ␣-helical bundle near the top of the structure defines the transmembrane domain of the protein (pdb id ϭ 1BE3). 20.3 How Is the Electron-Transport Chain Organized? 605 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 (Ᏹ o ЈϭϪ0.100 V) to b H (Ᏹ o Јϭ ϩ0.050 V). 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 и Ϫ . The resulting UQ и Ϫ remains firmly bound to the Q n site. This completes the first half of the Q cycle (Figure 20.12a). The second half of the cycle (Figure 20.12b) is similar to the first half, with a second molecule of UQH 2 oxidized at the Q p site, one electron being passed to cytochrome c 1 and the other transferred to heme b L 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 и Ϫ , at the Q n site. With the addition of two H ϩ from the mitochondrial matrix, this produces a molecule of UQH 2 , 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 other significant feature of this mechanism is that it offers a convenient way for a two-electron carrier, UQH 2 , to interact with the b L and b H hemes, the Rieske protein Fe-S cluster, and cytochrome c 1 , all of which are one-electron carriers. e – e – e – e – e – e – (a) First half of Q cycle Intermembrane space (P-phase) Matrix (N-phase) Matrix (N-phase) UQH 2 UQ Pool UQH 2 UQ – UQ UQ UQ – Q p site H + 2 FeS Cyt c 1 Cyt c Cyt b L Cyt b H Q n site First UQH 2 from pool UQ to pool 2 e – oxidation at Q p site 2 H + out UQ at Q n site Cyt c Synopsis (b) Second half of Q cycle Intermembrane space (P-phase) UQH 2 UQ – UQ Q p site H + 2 FeS Cyt c 1 Cyt c Cyt b L Cyt b H Q n site UQH 2 UQ – H + 2 1 e – 1 e – Second UQH 2 from pool UQH 2 to pool UQ to pool 2 e – oxidation at Q p site 2 H + out 2 H + UQ . – at Q n site Cyt c Synopsis 1 e – 1 e – Net UQH 2 2 e – 4 H + out + 2 Cyt c red + UQ + 2 H + in + 2 Cyt c ox UQH 2 UQ Pool UQH 2 UQH 2 UQ UQH 2 UQ UQ ACTIVE FIGURE 20.12 The Q cycle in mitochondria. (a) The electron-transfer pathway follow- ing oxidation of the first UQH 2 at the Q p site near the cytosolic face of the membrane. (b) The pathway fol- lowing oxidation of a second UQH 2 . Test yourself on the concepts in this figure at www.cengage.com/ login. 606 Chapter 20 Electron Transport and Oxidative Phosphorylation Cytochrome c Is a Mobile Electron Carrier Electrons traversing Complex III are passed through cytochrome c 1 to cytochrome c. Cytochrome c is the only one of the mitochondrial cytochromes that is water soluble. Its structure (Figure 20.13) is globular; the planar heme group lies near the center of the protein, surrounded pre- dominantly by hydrophobic amino acid 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 cytochrome c from hemoglobin (see 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 c 1 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. Complex IV Transfers Electrons from Cytochrome c to Reduce Oxygen on the Matrix Side Complex IV is called cytochrome c oxidase because it accepts electrons from cytochrome c and directs them to the four-electron reduction of O 2 to form H 2 O: 4 cyt c (Fe 2ϩ ) ϩ 4 H ϩ ϩ O 2 ⎯⎯→ 4 cyt c (Fe 3ϩ ) ϩ 2 H 2 O (20.21) Thus, cytochrome c oxidase and O 2 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. The combined processes of oxygen reduction and proton transport involve a total of 8H ϩ in each catalytic cycle—four H ϩ for O 2 reduction and four H ϩ transported from the matrix to the intermembrane space. The total number of subunits in cytochrome c oxidase varies from 2–4 (in bacte- ria) to 13 (in mammals). Three subunits (I, II, and III) are common to most organisms (Figure 20.14). This minimal complex, which contains two hemes (termed a and a 3 ) and three copper ions (two in the Cu A center and one in the Cu B site), is sufficient to carry out both oxygen reduction and proton transport. The total mass of the protein in mammalian Complex IV (Figure 20.15) is 204 kD. In mammals, 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 10 smaller subunits are coded by nuclear DNA, are synthesized in the cytosol, and are presumed to play regulatory roles in the complex. FIGURE 20.13 The structure of mitochondrial cytochrome c. The heme is shown at the center of the structure. It is covalently linked to the protein via two sulfur atoms. A third sulfur from a methionine residue coordi- nates the iron (pdb id ϭ 2B4Z). FIGURE 20.14 Bovine cytochrome c oxidase consists of 13 subunits.The 3 largest subunits—I (purple), II (yellow), and III (blue)—contain the proton channels and the redox centers (pdb id ϭ 2EIJ). FIGURE 20.15 The complete structure of bovine cytochrome c oxidase (pdb id ϭ 2EIJ). 20.3 How Is the Electron-Transport Chain Organized? 607 In the bovine structure, subunit I is cylindrical in shape and consists of 12 transmembrane helices, without any significant extramembrane parts. Hemes a and a 3 , which lie perpendicular to the membrane plane, and Cu B are cradled by the helices of subunit I (Figure 20.16). Subunits II and III lie on opposite sides of subunit I and do not contact each other (see Figure 20.14). Subunit II has an extramembrane domain on the outer face of the inner mitochondrial membrane. This domain consists of a 10-strand ␤-barrel that holds the two copper ions of the Cu A site 7 Å from the nearest surface atom of the subunit. Subunit III consists of seven transmembrane helices with no significant extramembrane domains. Electron Transfer in Complex IV Involves Two Hemes and Two Copper Sites Electron transfer through Complex IV begins with binding of cytochrome c to the ␤-barrel of subunit II. Four electrons are transferred sequentially (one each from four molecules of cytochrome c) first to the Cu A center, next to heme a, and finally to the Cu B /heme a 3 active site, where O 2 is reduced to H 2 O (Figure 20.16): Cyt c ⎯⎯→ Cu A ⎯⎯→ heme a ⎯⎯→ Cu B /heme a 3 ⎯⎯→ O 2 (20.22) A tryptophan residue, which lies 5Å above the Cu A site (Figure 20.17a), is the entry point for electrons from cytochrome c. It lies in a hydrophobic patch on subunit II, surrounded by a ring of negatively charged Asp and Glu residues. Electrons flow rapidly from Cu A to heme a, which is coordinated by a pair of His residues (Figure 20.17b), and then to the Cu B /heme a 3 complex. The Fe atom in heme a 3 is five co- ordinate (Figure 20.17c), with four ligands from the heme plane and one from His 376 . This leaves a sixth position free, and this is the catalytic site where O 2 binds and is reduced. Cu B is about 5Å from the Fe atom of heme a 3 and is coordinated by three histidine ligands, including His 240 , His 290 , and His 291 (Figure 20.17c). An un- usual crosslink between His 240 and Tyr 244 lowers the pK a of the Tyr hydroxyl so that it can participate in proton transport across the membrane. 2 H + 2 H + H 2 O Cyt a 3 Cyt a O 2 + 2 H + 2 – 1 2 ϫ Cyt c Cu B Cu A e – e – e – 2 ϫ 2 ϫ 2 ϫ ACTIVE FIGURE 20.16 The electron- transfer pathway for cytochrome oxidase. Cytochrome c binds on the cytosolic side, transferring electrons through the copper and heme centers to reduce O 2 on the matrix side of the membrane (pdb id ϭ 2EIJ).Test yourself on the concepts in this figure at www.cengage.com/ login. 608 Chapter 20 Electron Transport and Oxidative Phosphorylation Proton Transport Across Cytochrome c Oxidase Is Coupled to Oxygen Reduction Proton transport in R. sphaeroides cytochrome c oxidase takes place via two channels denoted the D- and K-pathways (Figure 20.18a). Both these channels contain water molecules, and they are lined with polar residues that can either protonate and de- protonate or form hydrogen bonds. The D-pathway is named for Asp 132 at the channel opening, and the K-pathway is named for Lys 362 , a critical residue located midway in the channel. These two channels converge at the binuclear Cu B /heme a 3 site midway across the complex and the membrane. Here, Glu 286 serves as a branch point, shuttling protons either to the catalytic site for O 2 reduction (to form H 2 O) or to the exit channel (residues 320 to 340) that leads protons to the intermembrane space (Figure 20.18a). In each catalytic cycle, two H ϩ pass through the K-pathway and six H ϩ traverse the D-pathway. The K-pathway protons and two of the D-pathway protons participate in the reduction of one O 2 to two H 2 O, and the remaining four D-pathway protons are passed across the membrane and released to the intermembrane space. (a) (b) (c) M 207 H 61 H 378 H 376 H 240 H 290 H 291 C 196 C 200 H 161 H 204 E 198 FIGURE 20.17 Structures of the redox centers of bovine cytochrome c oxidase. (a) The Cu A site, (b) the heme a site, and (c) the binuclear Cu B /heme a 3 site (pdb id ϭ 2EIJ). (a) Cu A DЈ 229 KЉ 227 T 337 H 334 Cu B / heme a 3 EЈ 101 T 359 Y 288 K 362 S 365 H 333 D 132 N 139 D- p athway K- p athway S 201 W 172 E 286 E 286 Heme a R 481 R 482 N 121 O O (b) O O C C H H H H C C H + H + FIGURE 20.18 (a) The proton channels of cytochrome c oxidase from R. sphaeroides.Functional residues in the D- and K-pathways are indicated.The D- and K-pathways converge at the Cu B /heme a 3 center.The proton exit channel is lined by residues 320 to 340 of subunit I (pdb id ϭ 1M56). (b) Protons are presumed to “hop”along arrays of water molecules in the proton transport channels of cytochrome c oxidase. Such a chain of protonation and deprotonation events means that the proton eventually released from the exit channel is far removed from the proton that entered the D-pathway and initiated the cascade. 20.3 How Is the Electron-Transport Chain Organized? 609 How are protons driven across cytochrome c oxidase? The mechanism involves three key features: • The pK a values of protein side chains in the proton channels are shifted (by the local environment) to make them effective proton donors or acceptors during transport. For example, the pK a of Glu 286 is unusually high at 9.4. (This is similar to the behavior of Asp 85 and Asp 96 in bacteriorhodopsin; see pages 285–286, Chapter 9.) • Electron transfer events induce conformation changes that control proton transport. For example, redox events at the Cu B /heme a 3 site are sensed by Glu 286 and an adjacent proton-gating loop (residues 169 to 175), controlling H ϩ binding and release by Glu 286 and proton movement through the exit channel. • Protons are “transported” via chains of hydrogen-bonded water molecules in the proton channels (Figure 20.18b). Sequential hopping of protons along these “proton wires” essentially transfers a “positive charge” between distant residues in the channel. (Note that the H ϩ that arrives at an accepting residue is not the same proton that left the donating residue.) The Four Electron-Transport Complexes Are Independent 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 associa- tions 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 reconsti- tuted systems show that electron transport does not operate by means of connected sets of the four complexes. The Model of Electron Transport Is a Dynamic One The model that emerges for electron transport is shown in Figure 20.19. 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 in the intermembrane space, 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. Electron Transfer Energy Stored in a Proton Gradient: The Mitchell Hypothesis 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. In this hypothesis, protons are driven across the membrane from the matrix to the intermembrane space and cytosol by the events of electron transport. This mechanism stores the energy of electron transport in an electrochemical potential. As protons are driven out of the matrix, the pH rises and the matrix becomes negatively charged with respect to the cytosol (Figure 20.20). Electron transport-driven proton pumping thus creates a pH gradient and an electrical gradient across the inner membrane, both of which tend to attract protons back into the matrix from the cytoplasm. Flow of protons down this electrochemical gradient, an energetically favorable process, drives the synthesis of ATP. 610 Chapter 20 Electron Transport and Oxidative Phosphorylation Succinate Fumarate Intermembrane space Matrix 4 H + 4 H + 2 H + 4 H + 2 H + 2 H + III I II IV Cyt c ox Cyt c ox Cyt c red Cyt c red UQ UQ UQH 2 UQH 2 H 2 O NADH + H + NAD + O 2 + 2 H + 2 – 1 FIGURE 20.19 A model for the electron-transport pathway in the mitochondrial inner membrane. UQ/UQH 2 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. Cytosol Intermembrane space (high [H + ], low pH) Matrix (low [H + ], high pH) H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + I F 0 F 1 II III IV FIGURE 20.20 The proton and electrochemical gradients existing across the inner mitochondrial membrane. The electrochemical gradient is generated by the transport of protons across the membrane by Complexes I, III, and IV in the inner mitochondrial membrane. 20.5 How Does a Proton Gradient Drive the Synthesis of ATP? 611 The ratio of protons transported per pair of electrons passed through the chain— the so-called H ؉ /2e ؊ ratio—has been an object of great interest for many years. Nev- ertheless, the ratio has remained extremely difficult to determine. The consensus esti- mate for the electron-transport pathway from succinate to O 2 is 6H ϩ /2e Ϫ . The ratio for Complex I by itself remains uncertain, but recent best estimates place it as high as 4H ϩ /2e Ϫ . On the basis of this value, the stoichiometry of transport for the pathway from NADH to O 2 is 10H ϩ /2e Ϫ . Although this is the value assumed in Figure 20.19, it is important to realize that this represents a consensus drawn from many experiments. 20.4 What Are the Thermodynamic Implications of Chemiosmotic Coupling? 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 ϩ in ⎯⎯→H ϩ out (20.23) 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 ⌬G ϭ RT ln ϩ ZᏲ⌬␺ (20.24) where c 1 and c 2 are the proton concentrations on the two sides of the membrane, Z is the charge on a proton, Ᏺ is Faraday’s constant, and ⌬␺ is the potential difference across the membrane. For the case at hand, this equation becomes ⌬G ϭ RT ln ϩ Z Ᏺ⌬␺ (20.25) In terms of the matrix and cytoplasm pH values, the free energy difference is ⌬G ϭϪ2.303 RT(pH out Ϫ pH in ) ϩ Ᏺ⌬␺ (20.26) Reported values for ⌬␺ and ⌬pH 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 ⌬␺ ϭ 0.18 V and ⌬pH ϭ 1 unit, the free energy change associated with the movement of one mole of protons from inside to outside is ⌬G ϭ 2.3 RT ϩ Ᏺ(0.18 V) (20.27) With Ᏺ ϭ 96.485 kJ/V и mol, the value of ⌬G at 37°C is ⌬G ϭ 5.9 kJ ϩ 17.4 kJ ϭ 23.3 kJ (20.28) which is the free energy change for movement of a mole of protons across an 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 ⌬G for inward flow of protons is Ϫ23.3 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. 20.5 How Does a Proton Gradient Drive the Synthesis of ATP? The great French chemist Antoine Lavoisier showed in 1777 that foods undergo combustion in the body. Since then, chemists and biochemists have wondered how energy from food oxidation is captured by living things. Mitchell paved the way by [H ϩ out ] ᎏ [H ϩ in ] [c 2 ] ᎏ [c 1 ] 612 Chapter 20 Electron Transport and Oxidative Phosphorylation suggesting that a proton gradient across the inner mitochondrial membrane could drive the synthesis of ATP. But how could the proton gradient be coupled to ATP production? The answer lies in a mitochondrial complex called ATP synthase, or sometimes F 1 F 0 –ATPase (for the reverse reaction it catalyzes). The F 1 portion of the ATP synthase was first identified in early electron micrographs of mitochondrial preparations as spherical, 8.5-nm projections or particles on the inner membrane. The purified particles catalyze 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 Is Composed of F 1 and F 0 ATP synthase is a remarkable molecular machine. It is an enzyme, a proton pump, and a rotating molecular motor. Nearly all the ATP that fuels our cellular processes is made by this multifaceted molecular superstar. The spheres observed in electron micrographs make up the F 1 unit, which catalyzes ATP synthesis (Figure 20.21). These F 1 spheres are attached to an integral membrane protein aggregate called the F 0 unit. F 1 consists of five polypeptide chains named ␣, ␤, ␥, ␦, and ⑀, with a subunit stoichiometry ␣ 3 ␤ 3 ␥␦⑀ (Table 20.3 and Figure 20.22). F 0 includes three hydrophobic subunits denoted by a, b, and c, with an apparent stoichiometry of a 1 b 2 c 10–15 . F 0 forms the transmembrane pore or channel through which protons move to drive ATP synthesis. The a and b subunits of F 0 form part of the stator—a stationary component anchored in the membrane—and a ring of 10 to 15 c-subunits (see Table 20.3) constitutes a major component of the rotor of the motor. Protons flowing through the a–c complex cause the c-ring to rotate in the membrane. Each c subunit is a folded pair of ␣-helices joined by a short loop, whereas the a-subunit is presumed to be a cluster of ␣-helices. The b-subunit, together with the d-and h-subunits and the oligomycin sen- sitivity-conferring protein (OSCP), form a long, slender stalk that connects F 0 in the membrane with F 1 , which extends out into the matrix. The b, d, and h subunits form long ␣-helical segments that comprise the stalk, and OSCP adds a helical bundle cap that sits at the bottom of an ␣-subunit of F 1 (Figure 20.21). The stalk is a stable link between F 0 and F 1 , essentially joining the two, both structurally and functionally. The Catalytic Sites of ATP Synthase Adopt Three Different Conformations The F 1 structure appears at first to be a symmetric hexamer of ␣- and ␤-subunits. However, it is asymmetric in several ways. The ␣- and ␤-subunits, arranged in an al- ternating pattern in the hexamer, are similar but not identical. The hexamer con- Stator Rotor shaft Rotor FIGURE 20.21 The ATP synthase, a rotating molecular motor.The c-, ␥-, and ⑀-subunits constitute the rotating portion (the rotor) of the motor. Flow of protons from the a-subunit through the c -subunit turns the rotor and drives the cycle of conformational changes in ␣ and ␤ that synthesize ATP (pdb id ϭ 1C17; 1E79;2A7U;2CLY; and 2BO5). Protein Subunit Mass Complex Function (kD) Stoichiometry F 1 ␣ 55.4 3 Stator ␤ 51.3 3 Stator ␥ 30.6 1 Rotor ␦ 14.6 1 Rotor † ⑀ 6.6 1 Rotor F 0 a 27.9 1 Stator b 23.3 1 Stator c 7.8 10–15* Rotor d 19.7 1 Stator h 10.4 1 Stator OSCP 20.9 1 Stator *The number of c subunits varies among organisms: yeast mitochondria, 10; Ilyobacter tartaricus, 11; Escherichia coli,12; spinach chloroplasts, 14; Spirulina platensis, 15. † The subunit nomenclature can be confusing. E. coli ATP synthase lacks a ␦-subunit in its rotor; its ␦-subunit is analogous structrually and functionally to the mitochondrial OSCP. TABLE 20.3 Yeast F 1 F 0 –ATP Synthase Subunit Organization