CoQ and Three Electron Transport Complexes Pump Protons Out of the Mitochondrial Matrix

The multiprotein complexes responsible for proton pumping coupled to electron transport have been identified by selectively extracting mitochondrial membranes with detergents, isolating each of the complexes in near purity, and then preparing artificial phospholipid vesicles (liposomes) containing each complex (see Figure 7-5). When an appropriate electron donor and electron acceptor are added to such liposomes, a change in pH of the medium will occur if the embedded complex transports protons (Figure 8-19). Studies of this type indicate that the NADH-CoQ reductase complex translocates four protons per pair of electrons transported, whereas the cytochrome c oxidase complex translocates two protons per electron pair transported (or, equivalently, for every two molecules of cytochrome c oxidized).

Current evidence suggests that a total of 10 protons are transported from the matrix space across the inner mito-chondrial membrane for every electron pair that is transferred from NADH to O2 (see Figure 8-17). Since the succinate-CoQ reductase complex does not transport protons, only six protons are transported across the membrane for every electron pair that is transferred from succinate (or FADH2) to O2. Relatively little is known about the coupling of electron flow and proton translocation by the NADH-CoQ reductase complex. More is known about operation of the cytochrome c oxidase complex, which we discuss here. The coupled electron and proton movements mediated by the CoQH2-cytochrome c reductase complex, which involves a unique mechanism, are described separately.

After cytochrome c is reduced by the QH2-cytochrome c reductase complex, it is reoxidized by the cytochrome c oxidase complex, which transfers electrons to oxygen. As noted earlier, cytochrome c oxidase contains three copper ions and two heme groups (see Figure 8-18). The flow of electrons through these carriers is depicted in Figure 8-20. Four molecules of reduced cytochrome c bind, one at a time, to a site on subunit II of the oxidase. An electron is transferred from the heme of each cytochrome c, first to Cua2+ bound to subunit II, then to the heme a bound to subunit I, and finally to the Cub2+ and heme a3 that make up the oxygen reduction center.

The cyclic oxidation and reduction of the iron and copper in the oxygen reduction center of cytochrome c oxidase, together with the uptake of four protons from the matrix space, are coupled to the transfer of the four electrons to oxygen and the formation of water. Proposed intermediates in oxygen reduction include the peroxide anion (O22~) and probably the hydroxyl radical (OH-) as well as unusual complexes of iron and oxygen atoms. These intermediates would be harmful to the cell if they escaped from the reaction center, but they do so only rarely.

< EXPERIMENTAL FIGURE 8-19 Electron transfer from reduced cytochrome c (Cyt c2+) to O2 via the cytochrome c oxidase complex is coupled to proton transport. The oxidase complex is incorporated into liposomes with the binding site for cytochrome c positioned on the outer surface. (a) When O2 and reduced cytochrome c are added, electrons are transferred to O2 to form H2O and protons are transported from the inside to the outside of the vesicles. Valinomycin and K+ are added to the medium to dissipate the voltage gradient generated by the translocation of H+, which would otherwise reduce the number of protons moved across the membrane. (b) Monitoring of the medium pH reveals a sharp drop in pH following addition of O2. As the reduced cytochrome c becomes fully oxidized, protons leak back into the vesicles, and the pH of the medium returns to its initial value. Measurements show that two protons are transported per O atom reduced. Two electrons are needed to reduce one O atom, but cytochrome c transfers only one electron; thus two molecules of Cyt c2+ are oxidized for each O reduced. [Adapted from B. Reynafarje et al., 1986, J. Biol. Chem. 261:8254.]

Intermembrane space

▲ FIGURE 8-20 Schematic depiction of the cytochrome c oxidase complex showing the pathway of electron flow from reduced cytochrome c to O2. Heme groups are denoted by red diamonds. Blue arrows indicate electron flow. Four electrons, sequentially released from four molecules of reduced cytochrome c, together with four protons from the matrix, combine with one O2 molecule to form two water molecules. Additionally, for each electron transferred from cytochrome c to oxygen, one proton is transported from the matrix to the intermembrane space, or a total of four for each O2 molecule reduced to two H2O molecules.

For every four electrons transferred from reduced cy-tochrome c through cytochrome c oxidase (i.e., for every molecule of O2 reduced to two H2O molecules), four protons are translocated from the matrix space to the intermembrane space (two protons per electron pair). However, the mechanism by which these protons are translocated is not known.

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