《生物化学原理》（英文版）chapter 19 Oxidative phosphorylation

8885ac19690-7503/1/0411:32 AM Page705 6mac76:385 19.2 ATP Synthes FIGURE 19-17 Chemiosmotic model. In this 2H+ 6 theory applied to mitochondria, electrons from Intermembrane NADH and other oxidizable substrates pass through a chain of carriers arranged asymmet- rically in the inner membrane. Electron flow is gradient(△pH) and an electrical gradient(△ Succinate The inner mitochondrial membrane is imper mable to protons; protons can reenter the NADH +H NAD+ ADP P; matrix only through proton-specific channel Matrix (F.). The proton-motive force that drives ATP F1才 rotons back into the matrix provides the ATP lectrical energy for ATP synthesis, catalyzed by the F potential potential omplex associated with Fo proton-motive (inside alkaline Because the energy of substrate oxidation drives hibitors of both ATP synthesis and the transfer of elec- ATP synthesis in mitochondria, we would expect in- trons through the chain of carriers to O2 (Fig. 19-18b) ibitors of the passage of electrons to O2(such as Because oligomycin is known to interact not directly with cyanide, carbon monoxide, and antimycin A) to block the electron carriers but with ATP synthase, it follows ATP synthesis(Fig. 19-18a) More surprising is the find- that electron transfer and ATP synthesis are obligately ing that the converse is also true: inhibition of ATP syn- coupled; neither reaction occurs without the other thesis blocks electron transfer in intact mitochondria Chemiosmotic theory readily explains the depend This obligatory coupling can be demonstrated in isolated ence of electron transfer on ATP synthesis in mitochon- mitochondria by providing O2 and oxidizable substrates, dria. When the flow of protons into the matrix through but not ADP(Fig. 19-18b) Under these conditions, no the proton channel of ATP synthase is blocked(with ATP synthesis can occur and electron transfer to O2 oligomycin, for example), no path exists for the return does not proceed. Coupling of oxidation and phosphor- of protons to the matrix, and the continued extrusion plation can also be demonstrated using oligomycin or of protons driven by the activity of the venturicidin, toxic antibiotics that bind to the ATP syn- generates a large proton gradient. The proton-motive thase in mitochondria. These compounds are potent in- force builds up until the cost (free energy) of pumping Add venturicidin DNP Uncoupled ligomycin ADP+ P o ADP P dd cinate Time FIGURE 19-18 Coupling of electron transfer and ATP synthesis in ATP is synthesized. Addition of cyanide(CN"), which blocks electron mitochondria In experiments to demonstrate coupling, mitochondria transfer between cytochrome oxidase and O2, inhibits both respiration are suspended in a buffered medium and an O2 electrode monitors O2 and ATP synthesis. (b)Mitochondria provided with succinate respire consumption. At intervals, samples are removed and assayed for the and synthesize ATP only when ADP and Pi are added. Subsequent ad- presence of ATP. (a) Addition of ADP and P alone results in little or no dition of venturicidin or oligomycin, inhibitors of ATP synthase, blocks ncrease in either respiration(O2 consumption; black) or ATP synthe- oth ATP synthesis and respiration. Dinitrophenol(DNP) is ar sis(red). When succinate is added, respiration begins immediately and coupler, allowing respiration to continue without ATP synthesis

Because the energy of substrate oxidation drives ATP synthesis in mitochondria, we would expect in￾hibitors of the passage of electrons to O2 (such as cyanide, carbon monoxide, and antimycin A) to block ATP synthesis (Fig. 19–18a). More surprising is the find￾ing that the converse is also true: inhibition of ATP syn￾thesis blocks electron transfer in intact mitochondria. This obligatory coupling can be demonstrated in isolated mitochondria by providing O2 and oxidizable substrates, but not ADP (Fig. 19–18b). Under these conditions, no ATP synthesis can occur and electron transfer to O2 does not proceed. Coupling of oxidation and phosphor￾ylation can also be demonstrated using oligomycin or venturicidin, toxic antibiotics that bind to the ATP syn￾thase in mitochondria. These compounds are potent in￾hibitors of both ATP synthesis and the transfer of elec￾trons through the chain of carriers to O2 (Fig. 19–18b). Because oligomycin is known to interact not directly with the electron carriers but with ATP synthase, it follows that electron transfer and ATP synthesis are obligately coupled; neither reaction occurs without the other. Chemiosmotic theory readily explains the depend￾ence of electron transfer on ATP synthesis in mitochon￾dria. When the flow of protons into the matrix through the proton channel of ATP synthase is blocked (with oligomycin, for example), no path exists for the return of protons to the matrix, and the continued extrusion of protons driven by the activity of the respiratory chain generates a large proton gradient. The proton-motive force builds up until the cost (free energy) of pumping 19.2 ATP Synthesis 705 NADH + H+ NAD+ Succinate Fumarate Cyt c + – ADP + Pi ATP + + + + + + + + + + + + + + + – – – – – – – – – – – – 4H+ 4H+ 2H+ H+ Chemical potential ∆pΗ (inside alkaline) ATP synthesis driven by proton-motive force Electrical potential ∆w (inside negative) + + – O2 + – H2 2H+ O 2 1 II IV I III Fo F1 Intermembrane space Matrix Q FIGURE 19–17 Chemiosmotic model. In this simple representation of the chemiosmotic theory applied to mitochondria, electrons from NADH and other oxidizable substrates pass through a chain of carriers arranged asymmet￾rically in the inner membrane. Electron flow is accompanied by proton transfer across the membrane, producing both a chemical gradient (pH) and an electrical gradient (). The inner mitochondrial membrane is imper￾meable to protons; protons can reenter the matrix only through proton-specific channels (Fo). The proton-motive force that drives protons back into the matrix provides the energy for ATP synthesis, catalyzed by the F1 complex associated with Fo. O2 consumed Add ADP
Pi Add succinate (b) Time ATP synthesized Add venturicidin or oligomycin Add DNP Uncoupled O2 consumed Add ADP
Pi Add succinate (a) Time ATP synthesized Add CN FIGURE 19–18 Coupling of electron transfer and ATP synthesis in mitochondria. In experiments to demonstrate coupling, mitochondria are suspended in a buffered medium and an O2 electrode monitors O2 consumption. At intervals, samples are removed and assayed for the presence of ATP. (a) Addition of ADP and Pi alone results in little or no increase in either respiration (O2 consumption; black) or ATP synthe￾sis (red). When succinate is added, respiration begins immediately and ATP is synthesized. Addition of cyanide (CN), which blocks electron transfer between cytochrome oxidase and O2, inhibits both respiration and ATP synthesis. (b) Mitochondria provided with succinate respire and synthesize ATP only when ADP and Pi are added. Subsequent ad￾dition of venturicidin or oligomycin, inhibitors of ATP synthase, blocks both ATP synthesis and respiration. Dinitrophenol (DNP) is an un￾coupler, allowing respiration to continue without ATP synthesis. 8885d_c19_690-750 3/1/04 11:32 AM Page 705 mac76 mac76:385_reb:

8885dc197063/1/041:59 PM Page706mac76mac76:385reb: Chapter 19 Oxidative Phosphorylation and Photophosphorylation BOX 19-1 THE WORLD OF BIOCHEMISTRY Hot, Stinking Plants and Alternative Respiratory Pathways Many flowering plants attract insect pollinators by re- leasing odorant molecules that mimic an insect,s nat ural food sources or potential egg-laying sites. Plants pollinated by flies or beetles that normally feed on or lay their eggs in dung or carrion sometimes use foul smelling compounds to attract these insects One family of stinking plants is the araceae, which includes philodendrons, arum lilies, and skunk cab- bages. These plants have tiny flowers densely packed on an erect structure, the spadix, surrounded by a modified leaf, the spathe. The spadix releases odors FIGURE 1 Eastern skunk cabbage of rotting flesh or dung. Before pollination the spadix also heats up, in some species to as much as 20 to 40C above the ambient temperature. Heat produc- ATP is instead released as heat. Plant mitochondria tion (thermogenesis) helps evaporate odorant mole- also have an alternative NADH dehydrogenase, insen cules for better dispersal, and because rotting flesh sitive to the Complex I inhibitor rotenone(see Table and dung are usually warm from the hyperactive me- 19-4), that transfers electrons from NADH in the ma- tabolism of scavenging microbes, the heat itself might trix directly to ubiquinone, bypassing Complex I and also attract insects. In the case of the eastern skunk its associated proton pumping And plant mitochon- cabbage(Fig. 1), which flowers in late winter or early dria have yet another NADH dehydrogenase, on the spring when snow still covers the ground, thermogen- external face of the inner membrane, that transfers sis allows the spadix to grow up through the snow. electrons from NADPh or NADH in the intermem How does a skunk cabbage heat its spadix? The brane space to ubiquinone, again bypassing Complex mitochondria of plants, fungi, and unicellular eukary- I. Thus when electrons enter the alternative respira- otes have electron-transfer systems that are essen- tory pathway through the rotenone-insensitive NADH tially the same as those in animals, but they also dehydrogenase, the external Nadh dehydrogenase have an alternative respiratory pathway. A cyanide- or succinate dehydrogenase(Complex D, and pass to resistant QH2 oxidase transfers electrons from the O2 via the cyanide-resistant alternative oxidase, en- ubiquinone pool directly to oxygen, bypassing the two ergy is not conserved as AtP but is released as heat proton-translocating steps of Complexes Ill and Iv A skunk cabbage can use the heat to melt snow, pro- (Fig. 2). Energy that might have been conserved as duce a foul stench, or attract beetles or flies External NAD(P)H Intermembrane NAD(P)*/dehydrogenase NAD(P)H Q NADnative NADH NAD+ Heat genase Matrix FIGURE 2 Electron carriers of the inner membrane of plant mitochondria. Electrons can flow through Complexes I, Ill, and IV, as in animal mitochondria, or through plant-specific alterna- ve carriers by the paths shown with blue arrows

706 Chapter 19 Oxidative Phosphorylation and Photophosphorylation BOX 19–1 THE WORLD OF BIOCHEMISTRY Hot, Stinking Plants and Alternative Respiratory Pathways Many flowering plants attract insect pollinators by re￾leasing odorant molecules that mimic an insect’s nat￾ural food sources or potential egg-laying sites. Plants pollinated by flies or beetles that normally feed on or lay their eggs in dung or carrion sometimes use foul￾smelling compounds to attract these insects. One family of stinking plants is the Araceae, which includes philodendrons, arum lilies, and skunk cab￾bages. These plants have tiny flowers densely packed on an erect structure, the spadix, surrounded by a modified leaf, the spathe. The spadix releases odors of rotting flesh or dung. Before pollination the spadix also heats up, in some species to as much as 20 to 40
C above the ambient temperature. Heat produc￾tion (thermogenesis) helps evaporate odorant mole￾cules for better dispersal, and because rotting flesh and dung are usually warm from the hyperactive me￾tabolism of scavenging microbes, the heat itself might also attract insects. In the case of the eastern skunk cabbage (Fig. 1), which flowers in late winter or early spring when snow still covers the ground, thermogen￾esis allows the spadix to grow up through the snow. How does a skunk cabbage heat its spadix? The mitochondria of plants, fungi, and unicellular eukary￾otes have electron-transfer systems that are essen￾tially the same as those in animals, but they also have an alternative respiratory pathway. A cyanide￾resistant QH2 oxidase transfers electrons from the ubiquinone pool directly to oxygen, bypassing the two proton-translocating steps of Complexes III and IV (Fig. 2). Energy that might have been conserved as ATP is instead released as heat. Plant mitochondria also have an alternative NADH dehydrogenase, insen￾sitive to the Complex I inhibitor rotenone (see Table 19–4), that transfers electrons from NADH in the ma￾trix directly to ubiquinone, bypassing Complex I and its associated proton pumping. And plant mitochon￾dria have yet another NADH dehydrogenase, on the external face of the inner membrane, that transfers electrons from NADPH or NADH in the intermem￾brane space to ubiquinone, again bypassing Complex I. Thus when electrons enter the alternative respira￾tory pathway through the rotenone-insensitive NADH dehydrogenase, the external NADH dehydrogenase, or succinate dehydrogenase (Complex II), and pass to O2 via the cyanide-resistant alternative oxidase, en￾ergy is not conserved as ATP but is released as heat. A skunk cabbage can use the heat to melt snow, pro￾duce a foul stench, or attract beetles or flies. FIGURE 1 Eastern skunk cabbage. FIGURE 2 Electron carriers of the inner membrane of plant mitochondria. Electrons can flow through Complexes I, III, and IV, as in animal mitochondria, or through plant-specific alterna￾tive carriers by the paths shown with blue arrows. Heat I IV Intermembrane space Q Matrix Cyt c NAD+ NAD(P)+ External NAD(P)H dehydrogenase Alternative oxidase Alternative NADH dehydrogenase III NADH H2O NAD(P)H 1 –2 O2 8885d_c19_706 3/1/04 1:59 PM Page 706 mac76 mac76:385_reb:

8885dc19690-7503/1/0411:32 AM Page707mac76mac76:385 19.2 ATP Synthes protons out of the matrix against this gradient equals or A prediction of the chemiosmotic theory is that, be- xceeds the energy released by the transfer of electrons cause the role of electron transfer in mitochondrial ATP from NADH to O2. At this point electron flow must stop; synthesis is simply to pump protons to create the elec- the free energy for the overall process of electron flow trochemical potential of the proton-motive force, an ar- coupled to proton pumping becomes zero, and the sys cially created proton gradient should be able to re- tem is at equilibrium place electron transfer in driving ATP synthesis. This Certain conditions and reagents, however, can un- has been experimentally confirmed (Fig. 19-20). Mito- couple oxidation from phosphorylation. When intact mi- hondria manipulated so as to impose a difference of tochondria are disrupted by treatment with detergent or proton concentration and a separation of charge across oy physical shear, the resulting membrane fragments can the inner membrane synthesize ATP in the absence of still catalyze electron transfer from succinate or NADH an oxidizable substrate; the proton-motive force alone O2, but no ATP synthesis is coupled to this respiration. suffices to drive ATP synthesis. Certain chemical compounds cause uncoupling without disrupting mitochondrial structure. Chemical uncouplers Matrix [H+]=10-9M include 2, 4-dinitrophenol (DNP) and carbonylcyanide-p- trifluoromethoxyphenylhydrazone(FCCP)(table 19-4 K+]=[C1-]=0.1M Fig. 19-19), weak acids with hydrophobic properties that permit them to diffuse readily across mitochondrial membranes. After entering the matrix in the protonated 。P① m, they can release a proton, thus dissipating the proton gradient Ionophores such as valinomycin(see Fig. 11-45) allow inorganic ions to pass easily through Intermembrane H+]=10-9M membranes. ionophores uncouple electron transfer from oxidative phosphorylation by dissipating the electrical contribution to the electrochemical gradient across the mitochondrial membrane H lowered from 9 to 7: O2 H+]=10 +H+ IK]<ICII nO2 NO [HTI 10 N NH FIGURE 19-20 Evidence for the role of a proton gradient in ATP syn- thesis. An artificially imposed electrochemical gradient can drive ATP synthesis in the absence of an oxidizable substrate as electron donor. In this two-step experiment, (a) isolated mitochondria are first incu- bated in a pH 9 buffer containing 0. 1 M KCL. Slow leakage of buffer and KCI into the mitochondria eventually brings the matrix into equi- ibrium with the surrounding medium. No oxidizable substrates are present. (b)Mitochondria are now separated from the pH 9 buffer and resuspended in pH 7 buffer containing valinomycin but no KCl. The Carbonylcyanide-p- trifluoromethoxyphenylhydrazor change in buffer creates a difference of two pH units across the inner (FCCP) mitochondrial membrane. The outward flow of K, carried (by vali- nomycin)down its concentration gradient without a counterion, cre- FIGURE 19-19 Two chemical uncouplers of oxidative phosphoryla- ates a charge imbalance across the membrane (matrix negative). The tion. Both DNP and FCCP have a dissociable proton and are very sum of the chemical potential provided by the pH difference and the hydrophobic. They carry protons across the inner mitochondrial mem- electrical potential provided by the separation of charges is a proton rane, dissipating the proton gradient. Both also uncouple photo- motive force large enough to support ATP synthesis in the absence of phosphorylation(see Fig. 19-57 an oxidizable substrate

protons out of the matrix against this gradient equals or exceeds the energy released by the transfer of electrons from NADH to O2. At this point electron flow must stop; the free energy for the overall process of electron flow coupled to proton pumping becomes zero, and the sys￾tem is at equilibrium. Certain conditions and reagents, however, can un￾couple oxidation from phosphorylation. When intact mi￾tochondria are disrupted by treatment with detergent or by physical shear, the resulting membrane fragments can still catalyze electron transfer from succinate or NADH to O2, but no ATP synthesis is coupled to this respiration. Certain chemical compounds cause uncoupling without disrupting mitochondrial structure. Chemical uncouplers include 2,4-dinitrophenol (DNP) and carbonylcyanide-p￾trifluoromethoxyphenylhydrazone (FCCP) (Table 19–4; Fig. 19–19), weak acids with hydrophobic properties that permit them to diffuse readily across mitochondrial membranes. After entering the matrix in the protonated form, they can release a proton, thus dissipating the proton gradient. Ionophores such as valinomycin (see Fig. 11–45) allow inorganic ions to pass easily through membranes. Ionophores uncouple electron transfer from oxidative phosphorylation by dissipating the electrical contribution to the electrochemical gradient across the mitochondrial membrane. 19.2 ATP Synthesis 707 A prediction of the chemiosmotic theory is that, be￾cause the role of electron transfer in mitochondrial ATP synthesis is simply to pump protons to create the elec￾trochemical potential of the proton-motive force, an ar￾tificially created proton gradient should be able to re￾place electron transfer in driving ATP synthesis. This has been experimentally confirmed (Fig. 19–20). Mito￾chondria manipulated so as to impose a difference of proton concentration and a separation of charge across the inner membrane synthesize ATP in the absence of an oxidizable substrate; the proton-motive force alone suffices to drive ATP synthesis. FIGURE 19–19 Two chemical uncouplers of oxidative phosphoryla￾tion. Both DNP and FCCP have a dissociable proton and are very hydrophobic. They carry protons across the inner mitochondrial mem￾brane, dissipating the proton gradient. Both also uncouple photo￾phosphorylation (see Fig. 19–57). FIGURE 19–20 Evidence for the role of a proton gradient in ATP syn￾thesis. An artificially imposed electrochemical gradient can drive ATP synthesis in the absence of an oxidizable substrate as electron donor. In this two-step experiment, (a) isolated mitochondria are first incu￾bated in a pH 9 buffer containing 0.1 M KCl. Slow leakage of buffer and KCl into the mitochondria eventually brings the matrix into equi￾librium with the surrounding medium. No oxidizable substrates are present. (b) Mitochondria are now separated from the pH 9 buffer and resuspended in pH 7 buffer containing valinomycin but no KCl. The change in buffer creates a difference of two pH units across the inner mitochondrial membrane. The outward flow of K
, carried (by vali￾nomycin) down its concentration gradient without a counterion, cre￾ates a charge imbalance across the membrane (matrix negative). The sum of the chemical potential provided by the pH difference and the electrical potential provided by the separation of charges is a proton￾motive force large enough to support ATP synthesis in the absence of an oxidizable substrate. N NH
H
N 2,4-Dinitrophenol (DNP) Carbonylcyanide-p￾trifluoromethoxyphenylhydrazone (FCCP) OH NO2 O
H
NO2 NO2 NO2 N C C C N N C C C N N O F C F F O F C F F [K
]  [Cl]  0.1 M [H
]  109 M [H
]  109 M FoF1 pH lowered from 9 to 7; valinomycin present; no K
(a) [K
] [Cl] [H
]  109 M (b) [H
]  107 M ADP
Pi K
K







        ATP Matrix Intermembrane space 8885d_c19_690-750 3/1/04 11:32 AM Page 707 mac76 mac76:385_reb:

8885dc19690-7503/1/0411:32 AM Page708mac76mac76:385 708 Chapter 19 Oxidative Phosphorylation and Photophosphorylation ATP Synthase Has Two Functional Domains, Fo and fi but cannot produce a proton gradient: Fo has a proton Mitochondrial ATP synthase is an F-type ATPase(see pore through which protons leak as fast as they are Fig. 11-39: Table 11-3) similar in structure and mech- pumped by electron transfer, and without a proton gra- dient the Fr-depleted vesicles cannot make ATP. Iso- anism to the ATP synthases of chloroplasts and eubac- lated Fi catalyzes ATP hydrolysis( the reversal of syn- teria. This large enzyme complex of the inner mito- chondrial membrane catalyzes the formation of ATP thesis) and was therefore originally called F1ATPase When purified Fi is added back to the depleted vesicles from ADP and Pi, accompanied it reassociates with Fo, plugging its proton pore and by the flow of protons from the restoring the membrane,s capacity to couple electron P to the n side of the mem- brane (eqn 19-10). ATP syn- transfer and ATP synthesis hase, also called Complex V ATP Is Stabilized relative to adP on the has two distinct components FI, a peripheral membrane Surface of F, protein, and Fo (o denoting Isotope exchange experiments with purified F, reveal oligomycin-sensitive), which is a remarkable fact about the enzyme's catalytic mecha- integral to the membrane F1, nism: on the enzyme surface, the reaction ADP +Pi the first factor recognized as AtP H,o is readily reversible--the free-energy Efraim Racker essential for oxidative phos- change for ATP synthesis is close to zero! When AtP 1913-1991 phorylation, was identified and hydrolyzed by F, in the presence of 0-labeled water purified by Efraim Racker and the P released contains an 0 atom. Careful meas- his colleagues in the early 1960s urement of the0 content of Pi formed in vitro by Fr- In the laboratory, small membrane vesicles formed catalyzed hydrolysis of ATP reveals that the Pi has not from inner mitochondrial membranes carry out ATP syn- one, but three or four 0 atoms(Fig. 19-21). This in- thesis coupled to electron transfer. When FI is gently dicates that the terminal pyrophosphate bond in ATP extracted, the "stripped vesicles still contain intact res- is cleaved and re-formed repeatedly before Pi leaves the piratory chains and the Fo portion of ATP synthase. The enzyme surface With Pi free to tumble in its binding vesicles can catalyze electron transfer from NADH to O2 site, each hydrolysis inserts o randomly at one of the ATP+H9 O 18O-P=180 Enzyme FIGURE 19-21 Catalytic mechanism of Fr(a) 8O-exchange exper- synthase(derived from PDB ID 1BMF). The a subunit is shown in iment. F, solubilized from mitochondrial membranes is incubated with green, B in gray. The positively charged residues B-Arg82 and a-Arg 6 ATP in the presence of 1O-labeled water. At intervals, a sample of coordinate two oxygens of the pentavalent phosphate intermediate; B- the solution is withdrawn and analyzed for the incorporation of o Lysinteracts with a third oxygen, and the Mg ion(green sphere into the Pi produced from ATP hydrolysis In minutes, the P; contains further stabilizes the intermediate. The blue sphere represents the leav three or four O atoms, indicating that both ATP hydrolysis and ATP ing group(H2O). These interactions result in the ready equilibration synthesis have occurred several times during the incubation. ( b)The of ATP and ADP P: in the active site likely transition state complex for ATP hydrolysis and synthesis in ATP

ATP Synthase Has Two Functional Domains, Fo and F1 Mitochondrial ATP synthase is an F-type ATPase (see Fig. 11–39; Table 11–3) similar in structure and mech￾anism to the ATP synthases of chloroplasts and eubac￾teria. This large enzyme complex of the inner mito￾chondrial membrane catalyzes the formation of ATP from ADP and Pi , accompanied by the flow of protons from the P to the N side of the mem￾brane (Eqn 19–10). ATP syn￾thase, also called Complex V, has two distinct components: F1, a peripheral membrane protein, and Fo (o denoting oligomycin-sensitive), which is integral to the membrane. F1, the first factor recognized as essential for oxidative phos￾phorylation, was identified and purified by Efraim Racker and his colleagues in the early 1960s. In the laboratory, small membrane vesicles formed from inner mitochondrial membranes carry out ATP syn￾thesis coupled to electron transfer. When F1 is gently extracted, the “stripped” vesicles still contain intact res￾piratory chains and the Fo portion of ATP synthase. The vesicles can catalyze electron transfer from NADH to O2 but cannot produce a proton gradient: Fo has a proton pore through which protons leak as fast as they are pumped by electron transfer, and without a proton gra￾dient the F1-depleted vesicles cannot make ATP. Iso￾lated F1 catalyzes ATP hydrolysis (the reversal of syn￾thesis) and was therefore originally called F1ATPase. When purified F1 is added back to the depleted vesicles, it reassociates with Fo, plugging its proton pore and restoring the membrane’s capacity to couple electron transfer and ATP synthesis. ATP Is Stabilized Relative to ADP on the Surface of F1 Isotope exchange experiments with purified F1 reveal a remarkable fact about the enzyme’s catalytic mecha￾nism: on the enzyme surface, the reaction ADP
Pi ATP
H2O is readily reversible—the free-energy change for ATP synthesis is close to zero! When ATP is hydrolyzed by F1 in the presence of 18O-labeled water, the Pi released contains an 18O atom. Careful meas￾urement of the 18O content of Pi formed in vitro by F1- catalyzed hydrolysis of ATP reveals that the Pi has not one, but three or four 18O atoms (Fig. 19–21). This in￾dicates that the terminal pyrophosphate bond in ATP is cleaved and re-formed repeatedly before Pi leaves the enzyme surface. With Pi free to tumble in its binding site, each hydrolysis inserts 18O randomly at one of the yz 708 Chapter 19 Oxidative Phosphorylation and Photophosphorylation Efraim Racker, 1913–1991 18O 18O ADP ATP
H2 18O
18 P O 18O Enzyme (F1) (a) ADP a-Arg376 b-Arg182 b-Glu181 b-Lys155 Mg2+ FIGURE 19–21 Catalytic mechanism of F1. (a) 18O-exchange exper￾iment. F1 solubilized from mitochondrial membranes is incubated with ATP in the presence of 18O-labeled water. At intervals, a sample of the solution is withdrawn and analyzed for the incorporation of 18O into the Pi produced from ATP hydrolysis. In minutes, the Pi contains three or four 18O atoms, indicating that both ATP hydrolysis and ATP synthesis have occurred several times during the incubation. (b) The likely transition state complex for ATP hydrolysis and synthesis in ATP synthase (derived from PDB ID 1BMF). The subunit is shown in green,
in gray. The positively charged residues
-Arg182 and -Arg376 coordinate two oxygens of the pentavalent phosphate intermediate;
- Lys155 interacts with a third oxygen, and the Mg2
ion (green sphere) further stabilizes the intermediate. The blue sphere represents the leav￾ing group (H2O). These interactions result in the ready equilibration of ATP and ADP
Pi in the active site. (b) 8885d_c19_690-750 3/1/04 11:32 AM Page 708 mac76 mac76:385_reb:

8885ac19690-7503/1/0411:32 AM Page709 6mac76:385 19.2 ATP Synthes FIGURE 19-22 Reaction coordinate ATP (in solution) ATP synthase and for a more typical enzyme. In a typical enzyme-catalyzed reaction(left), reaching the transition state () between substrate and ADP+P oduct is the major energy barrier to overcome E·ATP In the reaction catalyzed by ATP synthase(right), elease of ATP from the enzyme, not formation of E·ADP+P ATP, is the major energy barrier. The free-energy change for the formation of ATP from ADP and Pi n aqueous solution is large and positive, but on E+S provides sufficient binding energy to6.。 the enzyme surface, the very tight binding of ATP energy of the enzyme-bound atP close to that of ADP Pi so the reaction is readily reversible Reaction coordinate ATP synthase The equilibrium constant is near 1. The free Typical enzy energy required for the release of ATP is provided by the proton-motive force four positions in the molecule. This exchange reaction It is the proton gradient that causes the enzyme to re- occurs in unenergized Fo Fi complexes(with no proton lease the ATP formed on its surface. The reaction Co- gradient) and with isolated Fl-the exchange does not ordinate diagram of the process(Fig. 19-22)illustrates require the input of energy the difference between the mechanism of ATP synthase Kinetic studies of the initial rates of ATP synthesis and that of many other enzymes that catalyze ender and hydrolysis confirm the conclusion that AG for atP gonic reactions synthesis on the enzyme is near zero. From the meas- For the continued synthesis of ATP, the enzyme ured rates of hydrolysis (,= 10s)and synthesis must cycle between a form that binds ATP very tightly Ck-1=24s), the calculated equilibrium constant for and a form that releases ATP. Chemical and crystallo- the reaction graphic studies of the ATP synthase have revealed the structural basis for this alternation in function Enz.ATP Enz-(ADP P Each B Subunit of ATP Synthase Can Assume Three Different Conformations 2.4 Mitochondrial FI has nine subunits of five different this Keo, the calculated apparent AG"o is close to types, with the composition a3B3yoe. Each of the three This is much different from the k of about 10 B subunits has one catalytic site for ATP synthesis. The (AG"=-30.5 kJ/mol) for the hydrolysis of ATP free in crystallographic determination of the F, structure by solution(not on the enzyme surface) John E. Walker and colleagues revealed structural de- What accounts for the huge difference? ATP syn tails very helpful in explaining the catalytic mechanism thase stabilizes ATP relative to ADP + Pi by binding ATP of the enzyme. The knoblike portion of Fi is a flattened more tightly, releasing enough energy to counterbalance sphere, 8 nm high and 10 nm across, consisting of al- the cost of making ATP. Careful measurements of the erating a and B subunits arranged like the sections of binding constants show that FoFl binds ATP with ver an orange(Fig. 19-23a-c). The polypeptides that make high affinity(Ka s 10-2 M) and ADP with much lower up the stalk in the Fi crystal structure are asymmetri cally arranged, with one domain of the single y subunit to a difference of about 40 kJ/mol in binding energy, and making up a central shaft that passes through Fi, and his binding energy drives the equilibrium toward for mation of the product ATP. the three B subunits, designated B-empty(Fig. 19-23c) Although the amino acid sequences of the three B sub- The Proton gradient Drives the release of atp units are identical, their conformations differ, in part from the Enzyme Surface because of the association of the y subunit with just one of the three. The structures of the s and e subunits are Although ATP synthase equilibrates ATP with ADP not revealed in these crystallographic studies Pi, in the absence of a proton gradient the newly syn- The conformational differences among B subunits thesized atP does not leave the surface of the enzyme. extend to differences in their ATP/ADP-binding sites

four positions in the molecule. This exchange reaction occurs in unenergized FoF1 complexes (with no proton gradient) and with isolated F1—the exchange does not require the input of energy. Kinetic studies of the initial rates of ATP synthesis and hydrolysis confirm the conclusion that G for ATP synthesis on the enzyme is near zero. From the meas￾ured rates of hydrolysis (k1  10 s1 ) and synthesis (k1  24 s1 ), the calculated equilibrium constant for the reaction Enz-ATP Enz–(ADP
Pi) is Keq   2.4 From this Keq , the calculated apparent G is close to zero. This is much different from the Keq of about 105 (G  30.5 kJ/mol) for the hydrolysis of ATP free in solution (not on the enzyme surface). What accounts for the huge difference? ATP syn￾thase stabilizes ATP relative to ADP
Pi by binding ATP more tightly, releasing enough energy to counterbalance the cost of making ATP. Careful measurements of the binding constants show that FoF1 binds ATP with very high affinity (Kd ≤ 1012 M) and ADP with much lower affinity (Kd ≈ 105 M). The difference in Kd corresponds to a difference of about 40 kJ/mol in binding energy, and this binding energy drives the equilibrium toward for￾mation of the product ATP. The Proton Gradient Drives the Release of ATP from the Enzyme Surface Although ATP synthase equilibrates ATP with ADP
Pi , in the absence of a proton gradient the newly syn￾thesized ATP does not leave the surface of the enzyme. 24 s 1 10 s1 k1 k1 yz It is the proton gradient that causes the enzyme to re￾lease the ATP formed on its surface. The reaction co￾ordinate diagram of the process (Fig. 19–22) illustrates the difference between the mechanism of ATP synthase and that of many other enzymes that catalyze ender￾gonic reactions. For the continued synthesis of ATP, the enzyme must cycle between a form that binds ATP very tightly and a form that releases ATP. Chemical and crystallo￾graphic studies of the ATP synthase have revealed the structural basis for this alternation in function. Each
Subunit of ATP Synthase Can Assume Three Different Conformations Mitochondrial F1 has nine subunits of five different types, with the composition 3
3. Each of the three
subunits has one catalytic site for ATP synthesis. The crystallographic determination of the F1 structure by John E. Walker and colleagues revealed structural de￾tails very helpful in explaining the catalytic mechanism of the enzyme. The knoblike portion of F1 is a flattened sphere, 8 nm high and 10 nm across, consisting of al￾ternating and
subunits arranged like the sections of an orange (Fig. 19–23a–c). The polypeptides that make up the stalk in the F1 crystal structure are asymmetri￾cally arranged, with one domain of the single subunit making up a central shaft that passes through F1, and another domain of associated primarily with one of the three
subunits, designated
-empty (Fig. 19–23c). Although the amino acid sequences of the three
sub￾units are identical, their conformations differ, in part because of the association of the subunit with just one of the three. The structures of the  and  subunits are not revealed in these crystallographic studies. The conformational differences among
subunits extend to differences in their ATP/ADP-binding sites. 19.2 ATP Synthesis 709 G (kJ/mol) Reaction coordinate 80 60 40 20 0 ‡ P ADP
Pi ES E
S E ADP
Pi [E ATP] Typical enzyme ATP synthase ATP (in solution) FIGURE 19–22 Reaction coordinate diagrams for ATP synthase and for a more typical enzyme. In a typical enzyme-catalyzed reaction (left), reaching the transition state (‡) between substrate and product is the major energy barrier to overcome. In the reaction catalyzed by ATP synthase (right), release of ATP from the enzyme, not formation of ATP, is the major energy barrier. The free-energy change for the formation of ATP from ADP and Pi in aqueous solution is large and positive, but on the enzyme surface, the very tight binding of ATP provides sufficient binding energy to bring the free energy of the enzyme-bound ATP close to that of ADP
Pi, so the reaction is readily reversible. The equilibrium constant is near 1. The free energy required for the release of ATP is provided by the proton-motive force. 8885d_c19_690-750 3/1/04 11:32 AM Page 709 mac76 mac76:385_reb: