Plant Mitochondria Have Alternative Mechanisms for Oxidizing NADH

Plant mitochondria supply the cell with ATP during periods of low illumination or darkness by mechanisms entirely analogous to those used by nonphotosynthetic organisms. In the light, the principal source of mitochondrial NADH is a reaction in which glycine, produced by a process known as photorespiration, is converted to serine (see Fig. 20-21):

2 Glycine + NAD+-> serine + CO2 + NH3 + NADH + H+

For reasons discussed in Chapter 20, plants must carry out this reaction even when they do not need NADH for ATP production. To regenerate NAD+ from unneeded NADH, plant mitochondria transfer electrons from NADH directly to ubiquinone and from ubiquinone directly to O2, bypassing Complexes III and IV and their proton pumps. In this process the energy in NADH is dissipated as heat, which can sometimes be of value to the plant (Box 19-1). Unlike cytochrome oxidase (Complex IV), the alternative QH2 oxidase is not inhibited by cyanide. Cyanide-resistant NADH oxidation is therefore the hallmark of this unique plant electron-transfer pathway.

SUMMARY 19.1 Electron-Transfer Reactions in Mitochondria

■ Chemiosmotic theory provides the intellectual framework for understanding many biological energy transductions, including oxidative phosphorylation and photophosphorylation. The mechanism of energy coupling is similar in both cases: the energy of electron flow is conserved by the concomitant pumping of protons across the membrane, producing an electrochemical gradient, the proton-motive force.

■ In mitochondria, hydride ions removed from substrates by NAD-linked dehydrogenases donate electrons to the respiratory (electron-transfer) chain, which transfers the electrons to molecular O2, reducing it to H2O.

■ Shuttle systems convey reducing equivalents from cytosolic NADH to mitochondrial NADH. Reducing equivalents from all NAD-linked dehydrogenations are transferred to mito-chondrial NADH dehydrogenase (Complex I).

■ Reducing equivalents are then passed through a series of Fe-S centers to ubiquinone, which transfers the electrons to cytochrome b, the first carrier in Complex III. In this complex, electrons take two separate paths through two b-type cytochromes and cytochrome c1 to an Fe-S center. The Fe-S center passes electrons, one at a time, through cytochrome c and into

Complex IV, cytochrome oxidase. This copper-containing enzyme, which also contains cytochromes a and a3, accumulates electrons, then passes them to O2, reducing it to H2O.

■ Some electrons enter this chain of carriers through alternative paths. Succinate is oxidized by succinate dehydrogenase (Complex II), which contains a flavoprotein that passes electrons through several Fe-S centers to ubiquinone. Electrons derived from the oxidation of fatty acids pass to ubiquinone via the electron-transferring flavoprotein.

■ Plants also have an alternative, cyanide-resistant NADH oxidation pathway.

19.2 ATP Synthesis

How is a concentration gradient of protons transformed into ATP? We have seen that electron transfer releases, and the proton-motive force conserves, more than enough free energy (about 200 kJ) per "mole" of electron pairs to drive the formation of a mole of ATP, which requires about 50 kJ (see Box 13-1). Mi-tochondrial oxidative phospho-rylation therefore poses no thermodynamic problem. But what is the chemical mechanism that couples proton flux with phosphorylation?

The chemiosmotic model, proposed by Peter Mitchell, is the paradigm for this mechanism. According to the model (Fig. 19-17), the electrochemical energy inherent in the difference in proton concentration and separation of charge across the inner mito-chondrial membrane—the proton-motive force—drives the synthesis of ATP as protons flow passively back into the matrix through a proton pore associated with ATP synthase. To emphasize this crucial role of the protonmotive force, the equation for ATP synthesis is sometimes written

Mitchell used "chemiosmotic" to describe enzymatic reactions that involve, simultaneously, a chemical reaction and a transport process. The operational definition of "coupling" is shown in Figure 19-18. When isolated mitochondria are suspended in a buffer containing ADP, Pi, and an oxidizable substrate such as succinate, three easily measured processes occur: (1) the substrate is oxidized (succinate yields fumarate), (2) O2 is consumed, and (3) ATP is synthesized. Oxygen consumption and ATP synthesis depend on the presence of an oxidizable substrate (succinate in this case) as well as ADP and Pi.

Peter Mitchell, 1920-1992


Chemical potential ApH (inside alkaline)

ATP Electrical


Chemical potential ApH (inside alkaline)

ATP Electrical

► synthesis potential driven by ^^^Q AW proton-motive (inside force negative)

FIGURE 19-17 Chemiosmotic model. In this simple representation of the chemiosmotic theory applied to mitochondria, electrons from NADH and other oxidizable substrates pass through a chain of carriers arranged asymmetrically in the inner membrane. Electron flow is accompanied by proton transfer across the membrane, producing both a chemical gradient (ApH) and an electrical gradient (A^). The inner mitochondrial membrane is impermeable to protons; protons can reenter the matrix only through proton-specific channels (Fo). The proton-motive force that drives protons back into the matrix provides the energy for ATP synthesis, catalyzed by the F1 complex associated with Fo.

Because the energy of substrate oxidation drives ATP synthesis in mitochondria, we would expect inhibitors of the passage of electrons to O2 (such as cyanide, carbon monoxide, and antimycin A) to block ATP synthesis (Fig. 19-18a). More surprising is the finding that the converse is also true: inhibition of ATP synthesis blocks electron transfer in intact mitochondria. This obligatory coupling can be demonstrated in isolated mitochondria by providing O2 and oxidizable substrates, but not ADP (Fig. 19-18b). Under these conditions, no ATP synthesis can occur and electron transfer to O2 does not proceed. Coupling of oxidation and phosphor-ylation can also be demonstrated using oligomycin or venturicidin, toxic antibiotics that bind to the ATP syn-thase in mitochondria. These compounds are potent in hibitors of both ATP synthesis and the transfer of electrons through the chain of carriers to O2 (Fig. 19-18b). Because oligomycin is known to interact not directly with the electron carriers but with ATP synthase, it follows that electron transfer and ATP synthesis are obligately coupled; neither reaction occurs without the other.

Chemiosmotic theory readily explains the dependence of electron transfer on ATP synthesis in mitochondria. When the flow of protons into the matrix through the proton channel of ATP synthase is blocked (with oligomycin, for example), no path exists for the return of protons to the matrix, and the continued extrusion of protons driven by the activity of the respiratory chain generates a large proton gradient. The proton-motive force builds up until the cost (free energy) of pumping

FIGURE 19-18 Coupling of electron transfer and ATP synthesis in mitochondria. In experiments to demonstrate coupling, mitochondria are suspended in a buffered medium and an O2 electrode monitors O2 consumption. At intervals, samples are removed and assayed for the presence of ATP. (a) Addition of ADP and P| alone results in little or no increase in either respiration (O2 consumption; black) or ATP synthesis (red). When succinate is added, respiration begins immediately and

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