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8885dc19690-7503/1/0411:32 AM Page697 6mac76:385 19.1 Electron-Transfer Reactions in Mitochondria Treatment with digitonin Intermembrane phosphate FAD FeS I FMN Osmotic rupture (FAD) NADH NAD+ ETF: Q Inner ETF fragments (FAD) Outer membrane Matrix II ATP FIGURE 19-8 Path of electrons from NADH, succinate, fatty Solubilization with detergent acyl-CoA, and glycerol 3-phosphate to ubiquinone. Electrons from followed by ion-exchange chromatography NADH pass through a flavoprotein to a series of iron-sulfur proteins (in Complex I)and then to Q. Electrons from succinate pass through centers(in Complex In)on the ATP Q. Glycerol 3-phosphate donates electrons to a flavoprotein(glycerol 3-phosphate dehydrogenase) on the outer face of the inner mito chondrial membrane, from which they pass to Q Acyl-CoA dehydro- genase(the first enzyme of B oxidation) transfers electrons to electron- transferring flavoprotein (ETF), from which they pass to Q ne OxI exergonic transfer to ubiquinone of a hydride ion from NADH Q Suc- QQ Cytc Cytc O2 ATP ADP NADH and a proton from the matrix, expressed by NADH+H++Q→→NAD++QH2(19-1) Reactions catalyzed by isolated vitro and (2)the endergonic transfer of four protons from the matrix to the intermembrane space Complex I is there- FIGURE 19-7 Separation of functional complexes of the respiratory fore a proton pump driven by the energy of electron chain. The outer mitochondrial membrane is first removed by treat- transfer, and the reaction it catalyzes is vectorial: it ment with the detergent digitonin. Fragments of inner membrane are moves protons in a specific direction from one location then obtained by osmotic rupture of the mitochondria, and the frag-(the matrix, which becomes negatively charged with the ments are gently dissolved in a second detergent. The resulting mix- departure of protons) to another (the intermembrane ture of inner membrane proteins is resolved by ion-exchange chro- space, which becomes positively charged). To empha matography into different complexes(I through IV) of the respiratory size the vectorial nature of the process, the overall re- chain, each with its unique protein composition(see Table 19-3),an action is often written with subscripts that indicate the the enzyme ATP synthase(sometimes called Complex V). The isolated Complexes I through IV catalyze transfers between donors (NADH location of the protons: P for the positive side of the in- and succinate), intermediate carriers(Q and cytochrome c), and O2, ner membrane(the intermembrane space), N for the as shown. In vitro, isolated ATP synthase has only ATP-hydrolyzing negative side(the matrix): (ATPase), not ATP-synthesizing, activity. NADH+5H+Q→→NAD++QH2+4H(19-2)exergonic transfer to ubiquinone of a hydride ion from NADH and a proton from the matrix, expressed by NADH H Q On NAD QH2 (19–1) and (2) the endergonic transfer of four protons from the matrix to the intermembrane space. Complex I is therefore a proton pump driven by the energy of electron transfer, and the reaction it catalyzes is vectorial: it moves protons in a specific direction from one location (the matrix, which becomes negatively charged with the departure of protons) to another (the intermembrane space, which becomes positively charged). To emphasize the vectorial nature of the process, the overall reaction is often written with subscripts that indicate the location of the protons: P for the positive side of the inner membrane (the intermembrane space), N for the negative side (the matrix): NADH 5H N Q On NAD QH2 4H P (19–2) 19.1 Electron-Transfer Reactions in Mitochondria 697 Osmotic rupture Inner membrane fragments Outer membrane fragments discarded ATP synthase IV III II I I II III IV ATP synthase NADH Q Succinate Q Q Cyt c Cyt c O2 ATP ADP Pi Reactions catalyzed by isolated fractions in vitro Solubilization with detergent followed by ion-exchange chromatography Treatment with digitonin FIGURE 19–7 Separation of functional complexes of the respiratory chain. The outer mitochondrial membrane is first removed by treatment with the detergent digitonin. Fragments of inner membrane are then obtained by osmotic rupture of the mitochondria, and the fragments are gently dissolved in a second detergent. The resulting mixture of inner membrane proteins is resolved by ion-exchange chromatography into different complexes (I through IV) of the respiratory chain, each with its unique protein composition (see Table 19–3), and the enzyme ATP synthase (sometimes called Complex V). The isolated Complexes I through IV catalyze transfers between donors (NADH and succinate), intermediate carriers (Q and cytochrome c), and O2, as shown. In vitro, isolated ATP synthase has only ATP-hydrolyzing (ATPase), not ATP-synthesizing, activity. I II Intermembrane space Matrix Fe-S Fe-S FAD Glycerol 3-phosphate (cytosolic) glycerol 3-phosphate dehydrogenase FAD FMN NADH NAD+ Succinate ETF:Q oxidoreductase acyl-CoA dehydrogenase ETF (FAD) Fe-S (FAD) Fatty acyl–CoA FAD Q FIGURE 19–8 Path of electrons from NADH, succinate, fatty acyl–CoA, and glycerol 3-phosphate to ubiquinone. Electrons from NADH pass through a flavoprotein to a series of iron-sulfur proteins (in Complex I) and then to Q. Electrons from succinate pass through a flavoprotein and several Fe-S centers (in Complex II) on the way to Q. Glycerol 3-phosphate donates electrons to a flavoprotein (glycerol 3-phosphate dehydrogenase) on the outer face of the inner mitochondrial membrane, from which they pass to Q. Acyl-CoA dehydrogenase (the first enzyme of oxidation) transfers electrons to electrontransferring flavoprotein (ETF), from which they pass to Q via ETF:ubiquinone oxidoreductase. 8885d_c19_690-750 3/1/04 11:32 AM Page 697 mac76 mac76:385_reb: