8885dc196963/1/041:58 PM Page696mac76mac76:385reb Chapter 19 Oxidative Phosphorylation and Photophosphorylation FIGURE 19-6 Method for determining the sequence of electron carriers. This method NADH Q→cytb→)cytc1→)cytc→Cyt(a+a3)→>O2 measures the effects of inhibitors of electron ransfer on the oxidation state of each carrier. In the presence of an electron donor and O2, each inhibitor causes a characteristic pattern of oxidized/reduced carriers: those before the NADH 2→cytb→ Cyt cI→)Cytc t(a+ as) block become reduced(blue), and those after the block become oxidized(pink) cN or CO NADH—Q→cytb→Cytc1→}cytc→Cyt(a+a3) of electron carriers involves reducing the entire chain complexes that can be physically separated. Gentle of carriers experimentally by providing an electron treatment of the inner mitochondrial membrane with source but no electron acceptor (no O,). When Oe is detergents allows the resolution of four unique electron suddenly introduced into the system, the rate at which carrier complexes, each capable of catalyzing electron each electron carrier becomes oxidized (measured transfer through a portion of the chain (Table 19-3; Fig spectroscopically) reveals the order in which the car- 19-7. Complexes I and Il catalyze electron transfer to riers function. The carrier nearest O2 (at the end of the ubiquinone from two different electron donors: NADH chain) gives up its electrons first, the second carrier (Complex D and succinate(Complex ID). Complex Ill from the end is oxidized next, and so on. Such exper- carries electrons from reduced ubiquinone to cyto- iments have confirmed the sequence deduced from chrome c, and Complex Iv completes standard reduction potentials transferring electrons from cytochrome c to O In a final confirmation, agents that inhibit the flow We now look in more detail at the structure and of electrons through the chain have been used in com- function of each complex of the mitochondrial respira bination with measurements of the degree of oxidation tory chain of each carrier. In the presence of O, and an electron donor, carriers that function before the inhibited step Complex I: NADH to Ubiquinone Figure 19-8 illustrates the become fully reduced, and those that function after this relationship between Complexes I and II and ubiquinone Complex I, also called NADH: ubiquinone oxidore- eral inhibitors that block different steps in the chain, in- ductase or NADH dehydrogenase, is a large enzyme vestigators have determined the entire sequence; it is composed of 42 different polypeptide chains, including the same as deduced in the first two approaches an FMN-containing flavoprotein and at least six iron- ulfur centers. High-resolution electron microscopy Electron Carriers Function in Multienzyme Complexes shows Complex I to be L-shaped, with one arm of the L in the membrane and the other extending into the ma- The electron carriers of the respiratory chain are or- trix. As shown in Figure 19-9, Complex I catalyzes two ganized into membrane-embedded supramolecular simultaneous and obligately coupled processes: (1) the TABLE 19-3 The Protein Components of the Mitochondrial Electron-Transfer Chain Enzyme complex/protein Mass(kDa) Number of subunits Prosthetic group(s) I NADH dehydrogenase 850 43(14) MN. Fe-S lI Succinate dehydrogenase 140 4 FAD. Fe-S Ill Ubiquinone cytochrome C oxidoreductase mes Fe-s Cytochrome ct Heme Iv Cytochrome oxidase 13(3-4) Hemes: CUA, CuB Numbers of subunits in the bacterial equivalents in parentheses. Cytochrome c is not part of an enzyme complex, it moves between Complexes ll and iv as a freely soluble protein.of electron carriers involves reducing the entire chain of carriers experimentally by providing an electron source but no electron acceptor (no O2). When O2 is suddenly introduced into the system, the rate at which each electron carrier becomes oxidized (measured spectroscopically) reveals the order in which the car￾riers function. The carrier nearest O2 (at the end of the chain) gives up its electrons first, the second carrier from the end is oxidized next, and so on. Such exper￾iments have confirmed the sequence deduced from standard reduction potentials. In a final confirmation, agents that inhibit the flow of electrons through the chain have been used in com￾bination with measurements of the degree of oxidation of each carrier. In the presence of O2 and an electron donor, carriers that function before the inhibited step become fully reduced, and those that function after this step are completely oxidized (Fig. 19–6). By using sev￾eral inhibitors that block different steps in the chain, in￾vestigators have determined the entire sequence; it is the same as deduced in the first two approaches. Electron Carriers Function in Multienzyme Complexes The electron carriers of the respiratory chain are or￾ganized into membrane-embedded supramolecular complexes that can be physically separated. Gentle treatment of the inner mitochondrial membrane with detergents allows the resolution of four unique electron￾carrier complexes, each capable of catalyzing electron transfer through a portion of the chain (Table 19–3; Fig. 19–7). Complexes I and II catalyze electron transfer to ubiquinone from two different electron donors: NADH (Complex I) and succinate (Complex II). Complex III carries electrons from reduced ubiquinone to cyto￾chrome c, and Complex IV completes the sequence by transferring electrons from cytochrome c to O2. We now look in more detail at the structure and function of each complex of the mitochondrial respira￾tory chain. Complex I: NADH to Ubiquinone Figure 19–8 illustrates the relationship between Complexes I and II and ubiquinone. Complex I, also called NADH:ubiquinone oxidore￾ductase or NADH dehydrogenase, is a large enzyme composed of 42 different polypeptide chains, including an FMN-containing flavoprotein and at least six iron￾sulfur centers. High-resolution electron microscopy shows Complex I to be L-shaped, with one arm of the L in the membrane and the other extending into the ma￾trix. As shown in Figure 19–9, Complex I catalyzes two simultaneous and obligately coupled processes: (1) the 696 Chapter 19 Oxidative Phosphorylation and Photophosphorylation NADH Q Cyt c1 Cyt (a
a3) O2 rotenone antimycin A CN or CO NADH Q O Cyt b Cyt c 2 NADH Q O2 Cyt b Cyt c1 Cyt c Cyt b Cyt c1 Cyt Cyt c Cyt (a
a3) a
a3 ( ) FIGURE 19–6 Method for determining the sequence of electron carriers. This method measures the effects of inhibitors of electron transfer on the oxidation state of each carrier. In the presence of an electron donor and O2, each inhibitor causes a characteristic pattern of oxidized/reduced carriers: those before the block become reduced (blue), and those after the block become oxidized (pink). TABLE 19–3 The Protein Components of the Mitochondrial Electron-Transfer Chain Enzyme complex/protein Mass (kDa) Number of subunits* Prosthetic group(s) I NADH dehydrogenase 850 43 (14) FMN, Fe-S II Succinate dehydrogenase 140 4 FAD, Fe-S III Ubiquinone cytochrome c oxidoreductase 250 11 Hemes, Fe-S Cytochrome c † 13 1 Heme IV Cytochrome oxidase 160 13 (3–4) Hemes; CuA, CuB * Numbers of subunits in the bacterial equivalents in parentheses. † Cytochrome c is not part of an enzyme complex; it moves between Complexes III and IV as a freely soluble protein. 8885d_c19_696 3/1/04 1:58 PM Page 696 mac76 mac76:385_reb: