.
Electrons are transferred to O2 only if ADP is concomitantly phosphorylated to ATP.
 |
Because ATP is the end product of cellular respiration, its concentration is the ultimate determinant of the rate of all of the components of respiratory pathways.
| Reaction sequence | ATP yield per glucose molecule |
|---|---|
| Glycolysis: Conversion of glucose into pyruvate (in the cytosol) | |
 Phosphorylation of glucose | - 1 |
| Phosphorylation of fructose 6-phosphate | - 1 |
| Dephosphorylation of 2 molecules of 1,3-BPG | + 2 |
| Dephosphorylation of 2 molecules of phosphoenolpyruvate | + 2 |
| 2 molecules of NADH are formed in the oxidation of 2 molecules of glyceraldehyde 3-phosphate | |
| Conversion of pyruvate into acetyl CoA (inside mitochondria) | |
| 2 molecules of NADH are formed | |
| Citric acid cycle (inside mitochondria) | |
| 2 molecules of guanosine triphosphate are formed from 2 molecules of succinyl CoA | + 2 |
| 6 molecules of NADH are formed in the oxidation of 2 molecules each of isocitrate, α-ketoglutarate, and malate | |
| 2 molecules of FADH2 are formed in the oxidation of 2 molecules of succinate | |
| Oxidative phosphorylation (inside mitochondria) | |
| 2 molecules of NADH formed in glycolysis; each yields 1.5 molecules of ATP (assuming transport of NADH by the glycerol 3-phosphate shuttle) | + 3 |
| 2 molecules of NADH formed in the oxidative decarboxylation of pyruvate; each yields 2.5 molecules of ATP | + 5 |
| 2 molecules of FADH2 formed in the citric acid cycle; each yields 1.5 molecules of ATP | + 3 |
| 6 molecules of NADH formed in the citric acid cycle; each yields 2.5 molecules of ATP | + 15 |
| NET YIELD PER MOLECULE OF GLUCOSE | + 30 |
Source: The ATP yield of oxidative phosphorylation is based on values given in P. C. Hinkle, M. A. Kumar, A. Resetar, and D. L. Harris, Biochemistry 30(1991):3576.
Note: The current value of 30 molecules of ATP per molecule of glucose supersedes the earlier one of 36 molecules of ATP. The stoichiometries of proton pumping, ATP synthesis, and metabolite transport should be regarded as estimates. About two more molecules of ATP are formed per molecule of glucose oxidized when the malate-aspartate shuttle rather than the glycerol 3-phosphate shuttle is used.
Electrons are transferred to O2 only if ADP is concomitantly phosphorylated to ATP.
).
The regulation of the rate of oxidative phosphorylation by the ADP level is called respiratory control or acceptor control. The level of ADP likewise affects the rate of the citric acid cycle because of its need for NAD+ and FAD. The physiological significance of this regulatory mechanism is evident. The ADP level increases when ATP is consumed, and so oxidative phosphorylation is coupled to the utilization of ATP. Electrons do not flow from fuel molecules to O2 unless ATP needs to be synthesized. We see here another example of the regulatory significance of the energy charge.
Oxidative phosphorylation is susceptible to inhibition at all stages of the process. Specific inhibitors of electron transport were invaluable in revealing the sequence of electron carriers in the respiratory chain. For example, rotenone and amytal block electron transfer in NADH-Q oxidoreductase and thereby prevent the utilization of NADH as a substrate (Figure 18.43
). In contrast, electron flow resulting from the oxidation of succinate is unimpaired, because these electrons enter through QH2, beyond the block. Antimycin A interferes with electron flow from cytochrome bH in Q-cytochrome c oxidoreductase. Furthermore, electron flow in cytochrome c oxidase can be blocked by cyanide (CN-), azide (N3-), and carbon monoxide (CO). Cyanide and azide react with the ferric form of heme a3, whereas carbon monoxide inhibits the ferrous form. Inhibition of the electron-transport chain also inhibits ATP synthesis because the proton-motive force can no longer be generated.
ATP synthase also can be inhibited. Oligomycin and dicyclohexylcarbodiimide (DCCD) prevent the influx of protons through ATP synthase. If actively respiring mitochondria are exposed to an inhibitor of ATP synthase, the electron-transport chain ceases to operate. Indeed, this observation clearly illustrates that electron transport and ATP synthesis are normally tightly coupled.
2,4-Dinitrophenol, a lipid-soluble substance, can carry protons across the inner mitochondrial membrane. The dissociable proton is shown in red.
) and certain other acidic aromatic compounds. These substances carry protons across the inner mitochondrial membrane. In the presence of these uncouplers, electron transport from NADH to O2 proceeds in a normal fashion, but ATP is not formed by mitochondrial ATP synthase because the proton-motive force across the inner mitochondrial membrane is dissipated. This loss of respiratory control leads to increased oxygen consumption and oxidation of NADH. Indeed, in the accidental ingestion of uncouplers, large amounts of metabolic fuels are consumed, but no energy is stored as ATP. Rather, energy is released as heat. DNP and other uncouplers are very useful in metabolic studies because of their specific effect on oxidative phosphorylation. The regulated uncoupling of oxidative phosphorylation is a biologically useful means of generating heat.
ATP-ADP translocase is specifically inhibited by very low concentrations of atractyloside (a plant glycoside) or bongkrekic acid (an antibiotic from a mold). Atractyloside binds to the translocase when its nucleotide site faces the cytosol, whereas bongkrekic acid binds when this site faces the mitochondrial matrix. Oxidative phosphorylation stops soon after either inhibitor is added, showing that ATP-ADP translocase is essential.
Uncoupling protein-1 (UCP-1) generates heat by permitting the influx of protons into the mitochondria without the synthesis of ATP.
).
In addition to UCP-1, two other uncoupling proteins have been identified. UCP-2, which is 56% identical in sequence with UCP-1, is found in a wide variety of tissues. UCP-3 (57% identical with UCP-1 and 73% identical with UCP-2) is localized to skeletal muscle and brown fat. This family of uncoupling proteins, especially UCP-2 and UCP-3, may play a role in energy homeostasis. In fact, the genes for UCP-2 and UCP-3 map to regions of the human and mouse chromosomes that have been linked to obesity, substantiating the notion that they function as a means of regulating body weight. The use of uncoupling proteins is not limited to animals, however. The skunk cabbage uses an analogous mechanism to heat its floral spikes, increasing the evaporation of odoriferous molecules that attract insects to fertilize its flowers.
As befitting an organelle that is so central to energy metabolism, mitochondrial malfunction can lead to pathological conditions. The number of diseases that can be attributed to mitochondrial mutations is steadily growing in step with our growing understanding of the biochemistry and genetics of mitochondria. The first mitochondrial disease to be understood was Leber hereditary optic neuropathy (LHON), a form of blindness that strikes in midlife as a result of mutations to the NADH-Q oxidoreductase component of Complex I. Some of these mutations impair NADH utilization, whereas others block electron transfer to Q. The accumulation of mutations in mitochondrial genes in the course of several decades may contribute to aging, degenerative disorders, and cancer.
A human egg harbors several hundred thousand molecules of mitochondrial DNA, whereas a sperm contributes only a few hundred and thus has little effect on the mitochondrial genotype. Because the maternally inherited mitochondria are present in large numbers and not all of the mitochondria may be affected, the pathologies of mitochondrial mutants can be quite complex. Even within a single family carrying an identical mutation, chance fluctuations in the percentage of mitochondria with the mutation lead to large variations in the nature and severity of the symptoms of the pathological condition as well as the time of onset. As the percentage of defective mitochondria increases, energy-generating capacity diminishes until, at some threshold, the cell can no longer function properly. Defects in cellular respiration are doubly dangerous. Not only does energy transduction decrease, but also the likelihood that reactive oxygen species will be generated increases. Organs that are highly dependent on oxidative phosphorylation, such as the nervous system and the heart, are most vulnerable to mutations in mitochondrial DNA.
In the course of development or in cases of significant cell damage, individual cells within multicellular organisms undergo programmed cell death, or apoptosis. Mitochondria act as control centers regulating this process. Although the details have not yet been established, a pore called the mitochondrial permeability transition pore (mtPTP) forms in damaged mitochondria. This pore appears to consist of VDAC (the adenine nucleotide translocator) and several other mitochondrial proteins, including members of a family of proteins (Bcl family) that were initially discovered because of their role in cancer. One of the most potent activators of apoptosis is cytochrome c. Its presence in the cytosol activates a cascade of proteolytic enzymes called caspases. These cysteine proteases (Section 9.1.6) are conserved in evolution, being found in organisms ranging from hydra to human beings. Cytochrome c, in conjunction with other proteins, initiates the cascade by activating procaspase 9 to form caspase 9, which then activates other caspases. Activation of the caspase cascade does not lead to generalized protein destruction. Rather, the caspases have particular targets. For instance, the proteins that maintain cell structure are destroyed. Another example is the degradation of a protein that inhibits an enzyme that destroys DNA (caspase-activated DNAse, CAD), freeing CAD to cleave the genetic material. This cascade of proteolytic enzymes has been called “death by a thousand tiny cuts.”
The main concept presented in this chapter is that mitochondrial electron transfer and ATP synthesis are linked by a transmembrane proton gradient. ATP synthesis in bacteria and chloroplasts (Section 19.4) also is driven by proton gradients. In fact, proton gradients power a variety of energy-requiring processes such as the active transport of calcium ions by mitochondria, the entry of some amino acids and sugars into bacteria, the rotation of bacterial flagella, and the transfer of electrons from NADP+ to NADPH. Proton gradients can also be used to generate heat, as in hibernation. It is evident that proton gradients are a central interconvertible currency of free energy in biological systems (Figure 18.46
). Mitchell noted that the proton-motive force is a marvelously simple and effective store of free energy because it requires only a thin, closed lipid membrane between two aqueous phases.
