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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 substrateprotons 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 system is at equilibrium. Certain conditions and reagents, however, can uncouple oxidation from phosphorylation. When intact mitochondria 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-ptrifluoromethoxyphenylhydrazone (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, because the role of electron transfer in mitochondrial ATP synthesis is simply to pump protons to create the electrochemical potential of the proton-motive force, an artificially created proton gradient should be able to replace electron transfer in driving ATP synthesis. This has been experimentally confirmed (Fig. 19–20). Mitochondria 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 phosphorylation. Both DNP and FCCP have a dissociable proton and are very hydrophobic. They carry protons across the inner mitochondrial membrane, dissipating the proton gradient. Both also uncouple photophosphorylation (see Fig. 19–57). FIGURE 19–20 Evidence for the role of a proton gradient in ATP synthesis. 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 incubated in a pH 9 buffer containing 0.1 M KCl. Slow leakage of buffer and KCl into the mitochondria eventually brings the matrix into equilibrium 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 valinomycin) down its concentration gradient without a counterion, creates 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 protonmotive force large enough to support ATP synthesis in the absence of an oxidizable substrate. N NH H N 2,4-Dinitrophenol (DNP) Carbonylcyanide-ptrifluoromethoxyphenylhydrazone (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: