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: