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 ATPATP 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: