Most of the newly synthesized ATP that is released into the mitochondrial matrix must be transported out of the mitochondria, where it is used for energy-requiring processes such as active ion transport, muscle contraction, or biosynthetic reactions.
Likewise, ADP, phosphate, pyruvate, and other metabolites must be transported into the matrix. This requires transport of compounds through both the inner and outer mitochondrial membranes.
A. Transport through the Inner Mitochondrial Membrane
The inner mitochondrial membrane forms a tight permeability barrier to all polar molecules, including ATP, ADP, Pi; anions such as pyruvate; and cations such as Ca2⫹, H⫹, and K⫹. Yet, the process of oxidative phosphorylation depends on rapid and continuous transport of many of these molecules across the inner mitochondrial membrane (Fig. 18.16). Ions and other polar molecules are transported across the inner mitochondrial membrane by specifi c protein translocases that nearly balance charge during the transport process. Most of the exchange transport is a form of ac- tive transport that generally uses energy from the electrochemical potential gradient, either the membrane potential or the proton gradient.
H+
VD AC
Ca2+
Antiport
ATP–ADP translocase ATP
ADP
Citrate HK
ADP + Pi O2
ATP H+
H+
H+
H+ Symport
COO– C CH3
O
O P O–
O– HO ATP
ANT ADP Membrane potential
Mitochondrial matrix
Symport Proton
gradient Electrochemical potential gradient
Innermem bran
e
Oute r m
em brane – – – –
– – – – + + + + +
– – –
Pi, pyruvate
+ + + –––
FIG. 18.16. Transport of compounds across the inner and outer mitochondrial membranes. The electrochemical potential gradient drives the transport of ions across the inner mitochondrial membrane on specifi c translocases. Each translocase is composed of specifi c membrane-spanning helices that bind only specifi c compounds (adenine nucleotide translocase [ANT]). In contrast, the outer membrane contains relatively large un- specifi c pores called VDAC through which a wide range of ions diffuse. These bind cytosolic proteins such as hexokinase (HK), which enables HK to have access to newly exported ATP.
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ATP-ADP translocase (also called ANT for adenine nucleotide translocase) transports ATP formed in the mitochondrial matrix to the intermembrane space in a specifi c 1:1 exchange for ADP produced from energy-requiring reactions outside of the mitochondria (see Fig. 18.16). Because ATP contains four negative charges and ADP contains only three, the exchange is promoted by the electrochemical potential gradient because the net effect is the transport of one negative charge from the ma- trix to the cytosol. Similar antiports exist for most metabolic anions. In contrast, Pi
and pyruvate are transported into the mitochondrial matrix on specifi c transporters called symports together with a proton. A specifi c transport protein for Ca2⫹uptake, called the Ca2⫹uniporter, is driven by the electrochemical potential gradient, which is negatively charged on the matrix side of the membrane relative to the cytosolic side. Other transporters include the dicarboxylate transporter (malate-phosphate exchange), the tricarboxylate transporter (citrate-malate exchange), the aspartate- glutamate transporter, and the malate-α-ketoglutarate transporter.
B. Transport through the Outer Mitochondrial Membrane Whereas the inner mitochondrial membrane is highly impermeable, the outer mi- tochondrial membrane is permeable to compounds with a molecular weight up to approximately 6,000 daltons because it contains large nonspecifi c pores called volt- age-dependent anion channels (VDACs) that are formed by mitochondrial porins (see Fig. 18.16). These channels are “open” at low transmembrane potential, with a preference for anions such as phosphate, chloride, pyruvate, citrate, and adenine nucleotides. VDACs thus facilitate translocation of these anions between the inter- membrane space and the cytosol. Several cytosolic kinases, such as the hexokinase that initiates glycolysis, bind to the cytosolic side of the channel, where they have ready access to newly synthesized ATP.
C L I N I CA L CO M M E N T S
A summary of the diseases discussed in this chapter is presented in Table 18.3.
Cora N. Thrombolysis stimulated by intravenous recombinant TPA re- stored O2 to Cora N.’s heart muscle and successfully decreased the extent of ischemic damage. The rationale for the use of TPA within 4 to 6 hours after the onset of a myocardial infarction is based on the function of the normal intrinsic fi brinolytic system (see Chapter 7). This system is designed to dissolve un- wanted intravascular clots through the action of the enzyme plasmin, a protease that digests the fi brin matrix within the clot. TPA stimulates the conversion of plasmino- gen to its active form, plasmin. The result is a lysis of the thrombus and improved blood fl ow through the previously obstructed vessel, allowing fuels and O2 to reach the heart cells. The human TPA protein administered to Mrs. N. is produced by re- combinant DNA technology (see Chapter 14). This treatment rapidly restored O2
supply to the heart.
Isabel S. In the case of Isabel S., a diffuse myopathic process was su- perimposed on her AIDS and her pulmonary tuberculosis, either of which could have caused progressive weakness. In addition, she could have been suffering from a congenital mtDNA myopathy, symptomatic only as she ages. A systematic diagnostic process, however, fi nally led her physician to conclude that her myopathy was caused by a disorder of oxidative phosphorylation induced by her treatment with zidovudine (AZT). Fortunately, when AZT was discontinued, Ivy’s myopathic symptoms gradually subsided. A repeat skeletal muscle biopsy performed 4 months later showed that her skeletal muscle cell mtDNA had been restored to normal and that she had experienced a reversible drug-induced disorder of oxidative phosphorylation.
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CHAPTER 18 ■ OXIDATIVE PHOSPHORYLATION, MITOCHONDRIAL FUNCTION, AND OXYGEN RADICALS 295
Table 18.3 Diseases Discussed in Chapter 18 Disease or
Disorder
Environmental
or Genetic Comments Myocardial
infarction
Both The lack of oxygen in the anterior and lateral walls of the heart is caused by severe ischemia due to clots formed within certain coronary arteries at the site of ruptured atherosclerotic plaques. The limited availability of oxygen to act as an electron acceptor decreases the proton motive force across the inner mitochondrial membrane of ischemic cells. This leads to reduced ATP generation, trig- gering events that lead to irreversible cell injury.
Further damage to the heart muscle can occur due to free radical generation after oxygen is reintroduced to the cells that were temporarily ischemic, a pro- cess known as ischemic reperfusion injury.
AIDS treatment complication
Environmental AZT, a component of AIDS treatment cocktails, can act as an inhibitor of mitochondrial DNA poly- merase. Under rare conditions, it can lead to a depletion of mitochondrial DNA in cells, leading to a severe mitochondrial myopathy.
Iron defi ciency anemia
Environmental Lack of iron for heme synthesis, leading to reduced oxygen delivery to cells, and reduced iron in the electron transfer chain, leading to muscle weakness.
Cyanide poi- soning
Environmental Cyanide binds to the Fe3⫹ in the heme of cytochrome aa3, a component of cytochrome oxidase. Mito- chondrial respiration and energy production cease, and cell death rapidly occurs.
Mitochondrial disorders
Genetic Many types of mutations, leading to altered mitochondrial function and reduced energy production, due to mutations in the mitochondrial DNA. See Table 18.2 for a partial listing of these disorders.
Free radical disease
Both Damage caused to proteins and lipids due to free radical generation may lead to cellular dysfunction.
Amyotrophic lateral scle- rosis (ALS)
Both The genetic form of ALS is due to mutations in superoxide dismutase, leading to diffi culty in disposing of superoxide radicals, leading to cell damage due to excessive ROS.
Age-related macular degeneration
Both Oxidative damage occurs in the retinal pigment epi- thelium, leading to fi rst, reduced vision, and sec- ond, to blindness.
ATP, adenosine triphosphate; AZT, azidothymidine; ROS, reactive oxygen species.
R E V I E W Q U E ST I O N S - C H A P T E R 18
1. Consider the following experiment. Carefully isolated liver mitochondria are incubated in the presence of a lim- iting amount of malate. Three minutes after adding the substrate, cyanide is added and the reaction allowed to proceed for another 7 minutes. At this point in time, which one of the following components of the electron transfer chain will be in an oxidized state?
A. Complex I B. Complex II C. Complex III D. Coenzyme Q E. Cytochrome c
2. Consider the following experiment. Carefully isolated liver mitochondria are placed in a weakly buffered solu- tion. Malate is added as an energy source, and an increase in oxygen consumption confi rms that the electron transfer chain is functioning properly within these organelles. Valin- omycin and potassium are then added to the mitochondrial suspension. Valinomycin is a drug that allows potassium ions to freely cross the inner mitochondrial membrane.
What is the effect of valinomycin on the proton-motive force that had been generated by the oxidation of malate?
A. The proton-motive force will be decreased but to a value greater than zero.
B. The proton-motive force will be reduced to a value of zero.
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C. There will be no change in the proton-motive force.
D. The proton-motive force will be increased.
E. The proton-motive force will be decreased to a value less than zero.
3. A 25-year-old female presents with chronic fatigue. A series of blood tests are ordered, and the results suggest that her red blood cell count is low due to iron defi ciency anemia.
Such a defi ciency may play a part in leading to fatigue due to which one of the following?
A. She is not producing as much H2O in the electron transport chain, leading to dehydration, which has resulted in fatigue.
B. Iron forms a chelate with NADH and FAD(2H) that is necessary for them to donate their electrons to the electron transport chain.
C. Iron acts as a cofactor for α-ketoglutarate DH in the TCA cycle, a reaction required for the fl ow of electrons through the electron transport chain.
D. Iron accompanies the protons that are pumped from the mitochondrial matrix to the cytosolic side of the inner mitochondrial membrane. Without iron, the proton gradient cannot be maintained to produce adequate ATP.
E. Her decrease in Fe-S centers is impairing the trans- fer of electrons in the electron transport chain.
4. Rotenone, an inhibitor of NADH dehydrogenase, was originally used for fi shing. When it was sprinkled on a lake, fi sh would absorb it through their gills and die. Until recently, it was used in the United States as an organic pesticide and was recommended for tomato plants. It was considered nontoxic to mammals and birds, which cannot readily absorb it. What effect would rotenone have on ATP production by heart mitochondria, if it could be absorbed?
A. There would be a 10% reduction in ATP production.
B. There would be a 50% reduction in ATP production.
C. There would be a 95% reduction in ATP production.
D. There would be no reduction in ATP production.
5. The level of oxidative damage to mitochondrial DNA is 10 times greater than that to nuclear DNA. This could be due, in part, to which one of the following?
A. Superoxide dismutase is present in the mitochon- dria.
B. The nucleus lacks glutathione.
C. The nuclear membrane presents a barrier to reactive oxygen species.
D. Mitochondrial DNA lacks histones.
E. The mitochondrial membrane is permeable to reactive oxygen species.
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297
19 Generation of ATP from Glucose: Glycolysis
C H A P T E R O U T L I N E
K E Y P O I N T S
■ Glycolysis is the pathway in which glucose is oxidized and cleaved to form pyruvate.
■ The enzymes of glycolysis are in the cytosol.
■ Glucose is the major sugar in our diet; all cells can utilize glucose for energy.
■ Glycolysis generates two molecules of ATP through substrate-level phosphorylation and two molecules of NADH.
■ The cytosolic NADH generated via glycolysis transfers its reducing equivalents to mitochondrial NAD⫹ via shuttle systems across the inner mitochondrial membrane.
■ The pyruvate generated during glycolysis can enter the mitochondria and be oxidized completely to CO2 by pyruvate dehydrogenase and the TCA cycle.
■ Anaerobic glycolysis will generate energy in cells with a limited supply of oxygen or few mitochondria.
■ Under anaerobic conditions, pyruvate is reduced to lactate by NADH, thereby regenerating the NAD⫹ required for glycolysis to continue.
■ Glycolysis is regulated to ensure that ATP homeostasis is maintained.