20.7 How Are the Electrons of Cytosolic NADH Fed into Electron Transport? 623 other hand, if these 2 NADH take part in the malate–aspartate shuttle, each yields 2.5 ATP, giving a total (in this case) of 32 ATP formed per glucose oxidized. Most of the ATP—26 out of 30 or 28 out of 32—is produced by oxidative phosphorylation; only 4 ATP molecules result from direct synthesis during glycolysis and the TCA cycle. The situation in bacteria is somewhat different. Prokaryotic cells need not carry out ATP–ADP exchange. Thus, bacteria have the potential to produce approxi- mately 38 ATP per glucose. 3.5 Billion Years of Evolution Have Resulted in a Very Efficient System Hypothetically speaking, how much energy does a eukaryotic cell extract from the glucose molecule? Taking a value of 50 kJ/mol for the hydrolysis of ATP under cel- lular conditions (see Chapter 3), the production of 32 ATPs per glucose oxidized yields 1600 kJ/mol of glucose. The cellular oxidation (combustion) of glucose yields ⌬G ϭϪ2937 kJ/mol. We can calculate an efficiency for the pathways of gly- colysis, the TCA cycle, electron transport, and oxidative phosphorylation of 1600/2937 ϫ 100% ϭ 54%. ATP Yield per Glucose Glycerol– Malate– Phosphate Aspartate Pathway Shuttle Shuttle Glycolysis: glucose to pyruvate (cytosol) Phosphorylation of glucose Ϫ1 Ϫ1 Phosphorylation of fructose-6-phosphate Ϫ1 Ϫ1 Dephosphorylation of 2 molecules of 1,3-BPG ϩ2 ϩ2 Dephosphorylation of 2 molecules of PEP ϩ2 ϩ2 Oxidation of 2 molecules of glyceraldehyde-3- phosphate yields 2 NADH Pyruvate conversion to acetyl-CoA (mitochondria) 2 NADH Citric acid cycle (mitochondria) 2 molecules of GTP from 2 molecules ϩ2 ϩ2 of succinyl-CoA Oxidation of 2 molecules each of isocitrate, ␣-ketoglutarate, and malate yields 6 NADH Oxidation of 2 molecules of succinate yields 2 [FADH 2 ] Oxidative phosphorylation (mitochondria) 2 NADH from glycolysis yield 1.5 ATPs each if NADH ϩ3 ϩ5 is oxidized by glycerol–phosphate shuttle; 2.5 ATP by malate–aspartate shuttle Oxidative decarboxylation of 2 pyruvate to 2 acetyl-CoA: 2 NADH produce 2.5 ATPs each ϩ5 ϩ5 2 [FADH 2 ] from each citric acid cycle produce ϩ3 ϩ3 1.5 ATPs each 6 NADH from citric acid cycle produce 2.5 ATPs each ϩ15 ϩ15 Net Yield 30 32 Note:These P/O ratios of 2.5 and 1.5 for mitochondrial oxidation of NADH and [FADH 2 ] are “consensus values.”Because they may not reflect actual values and because these ratios may change depending on metabolic conditions,these esti- mates of ATP yield from glucose oxidation are approximate. TABLE 20.4 Yield of ATP from Glucose Oxidation 624 Chapter 20 Electron Transport and Oxidative Phosphorylation 20.8 How Do Mitochondria Mediate Apoptosis? Mitochondria not only are the home of the TCA cycle and oxidative phosphoryla- tion but also are a crossroads for several cell signaling pathways. Mitochondria take up Ca 2ϩ ions released from the endoplasmic reticulum, thus helping control intra- cellular Ca 2ϩ signals. They produce reactive oxygen species (ROS) that play signal- ing roles in cells, although ROS can also cause cellular damage. Mitochondria also participate in the programmed death of cells, a process known as apoptosis (the sec- ond “p” is silent in this word). Apoptosis is a mechanism through which certain cells are eliminated from higher organisms. It is central to the development and homeostasis of multicellular organisms, and it is the route by which unwanted or harmful cells are eliminated. Under normal circumstances, apoptosis is suppressed through compartmentation of the involved activators and enzymes. Mitochondria play a major role in this sub- cellular partitioning of the apoptotic activator molecules. One such activator is cyto- chrome c, which normally resides in the intermembrane space, bound tightly to a lipid chain of cardiolipin in the membrane (Figure 20.33). A variety of triggering agents, including Ca 2ϩ , ROS, certain lipid molecules, and certain protein kinases, can induce the opening of pores in the mitochondrial membrane. For example, us- ing hydrogen peroxide as a substrate, cytochrome c can oxidize its bound cardio- lipin chain, releasing itself from the membrane. When the outer membrane is made permeable by other apoptotic signals, cytochrome c can enter the cytosol. Permeabilization events, which occur at points where outer and inner mito- chondrial membranes are in contact, involve association of the ATP–ADP translo- case in the inner membrane and the voltage-dependent anion channel (VDAC) in the outer membrane. This interaction leads to the opening of protein-permeable pores. Cytochrome c, as well as several other proteins, can pass through these pores. Cyt c OOH (a) CL CO - POX H 2 O 2 Cardiolipin OOH CO - POX (b) CC CC C C C C FIGURE 20.33 (a) Cytochrome c is anchored at the inner mitochondrial membrane by association with cardio- lipin (diphosphatidylglycerol).The peroxidase activity of cytochrome c oxidizes a cardiolipin lipid chain, releasing cytochrome c from the membrane. (b) The opening of pores in the outer membrane, induced by a variety of triggering agents, releases cytochrome c to the cytosol, where it initiates the events of apoptosis. ROS: Reactive oxygen species, such as oxygen ions, free radicals, and peroxides. 20.8 How Do Mitochondria Mediate Apoptosis? 625 Pore formation is carefully regulated by the BCL-2 family of proteins, which in- cludes both proapoptotic members (proteins known as Bax, Bid, and Bad) and antiapoptotic members (BCL-2 itself, as well as BCL-X L and BCL-W). Cytochrome c Triggers Apoptosome Assembly But how is the release of cytochrome c translated into the activation of the death cascade, a point of no return for the cell? The answer lies in the assembly, in the cy- tosol, of a signaling platform called the apoptosome (Figure 20.34). The function of the apoptosome is to activate a cascade of proteases called caspases. (Here, “c” is for cysteine and “asp” is for aspartic acid. Caspases have Cys at the active site and cleave their peptide substrates after Asp residues.) The apoptosome is a wheel- shaped, heptameric platform that looks like (and in some ways behaves like) an earth-orbiting space station. It is assembled from seven subunits of the apoptotic protease-activating factor 1 (Apaf-1), a multidomain protein. Apaf-1 contains an ATPase domain (which prefers dATP over ATP in some organisms), a caspase- recruitment domain (CARD), and a WD40 repeat domain. Normally (before the death-signaling cytochrome c is released from mitochondria), these three domains are folded against each other (Figure 20.34b), with dATP tightly bound, and Apaf- 1 is “locked” in an inactive monomeric state. Binding of cytochrome c to the WD40 domain, followed by dATP hydrolysis, converts Apaf-1 to an extended conforma- tion. Then, exchange of dADP for a new molecule of dATP prompts assembly of the heptameric platform (Figure 20.34), which goes on to activate the death-dealing caspase cascade. CARD CARD NOD (a) Apaf-1 NOD WD40 WD40 Cytochrome c Cytochrome c binding dATP hydrolysis WD40 (b) Locked form Semi-open, autoinhibited form Apoptosome dATP-dADP exchange (c) FIGURE 20.34 (a) Apaf-1 is a multidomain protein, consisting of an N-terminal CARD, a nucleotide-binding and oligomerization domain (NOD), and several WD40 domains. (b) Binding of cytochrome c to the WD40 domains and ATP hydrolysis unlocks Apaf-1 to form the semiopen conformation. Nucleotide exchange leads to oligomerization and apoptosome formation. (c) A model of the apoptosome, a wheellike structure with molecules of cytochrome c bound to the WD40 domains, which extend outward like spokes. 626 Chapter 20 Electron Transport and Oxidative Phosphorylation Mitochondria-mediated apoptosis is the mode of cell death for many neurons in the brain during strokes and other brain-trauma injuries. When a stroke occurs, the neurons at the site of oxygen deprivation die within minutes by a nonspecific process of necrosis, but cells adjacent to the immediate site of injury die more slowly by apoptosis. These latter cells have been saved by a variety of therapeutic inter- ventions that suppress apoptosis in laboratory studies, raising the hope that strokes and other neurodegenerative conditions may someday be treated clinically in simi- lar ways. SUMMARY 20.1 Where in the Cell Do Electron Transport and Oxidative Phospho- rylation Occur? The processes of electron transport and oxidative phosphorylation are membrane associated. In prokaryotes, the conver- sion of energy from NADH and [FADH 2 ] to the energy of ATP via elec- tron transport and oxidative phosphorylation is carried out at (and across) the plasma membrane. In eukaryotic cells, electron transport and oxidative phosphorylation are localized in mitochondria. Mito- chondria are surrounded by a simple outer membrane and a more com- plex inner membrane. The space between the inner and outer mem- branes is referred to as the intermembrane space. 20.2 What Are Reduction Potentials, and How Are They Used to Ac- count for Free Energy Changes in Redox Reactions? Just as the group transfer potential is used to quantitate the energy of phosphoryl transfer, the standard reduction potential, denoted by Ᏹ o Ј, quantitates the ten- dency of chemical species to be reduced or oxidized. Standard reduction potentials are determined by measuring the voltages generated in reac- tion half-cells. A half-cell consists of a solution containing 1 M concen- trations of both the oxidized and reduced forms of the substance whose reduction potential is being measured and a simple electrode. 20.3 How Is the Electron-Transport Chain Organized? The com- ponents of the electron-transport chain can be purified from the mito- chondrial inner membrane as four distinct protein complexes: (I) NADH–coenzyme Q reductase, (II) succinate–coenzyme Q reduc- tase, (III) coenzyme Q–cytochrome c reductase, and (IV) cytochrome c oxidase. In complexes I, II, and IV, electron transfer drives the move- ment of protons from the mitochondrial matrix to the intermembrane space. Complex I (NADH dehydrogenase) involves more than 45 polypep- tide chains, 1 molecule of flavin mononucleotide (FMN), and as many as nine Fe-S clusters, together containing a total of 20 to 26 iron atoms. The complex transfers electrons from NADH to FMN, then to a series of FeS proteins, and finally to coenzyme Q. Complex II (succinate dehydrogenase) oxidizes succinate to fumarate, with concomitant reduction of bound FAD to FADH 2 . This FADH 2 trans- fers its electrons immediately to Fe-S centers, which pass them on to UQ. Electrons flow from succinate to UQ. Complex III drives electron transport from coenzyme Q to cytochrome c via a unique redox pathway known as the Q cycle. UQ–cytochrome c reductase (UQ–cyt c reductase), as this complex is known, involves three different cytochromes and an Fe-S protein. In the cytochromes of these and similar complexes, the iron atom at the center of the porphyrin ring cycles between the reduced Fe 2ϩ (ferrous) and oxidized Fe 3ϩ (ferric) states. Complex IV transfers electrons from cytochrome c to reduce oxygen on the matrix side. Complex IV (cytochrome c oxidase) accepts elec- trons from cytochrome c and directs them to the four-electron reduc- tion of O 2 to form 2H 2 O via Cu A sites, the heme iron of cytochrome a, Cu B , and the heme iron of a 3 . 20.4 What Are the Thermodynamic Implications of Chemiosmotic Coupling? Peter Mitchell was the first to propose that electron transport leads to formation of a proton gradient that drives ATP synthesis. The free energy difference for protons across the inner mitochondrial mem- brane includes a term for the concentration difference and a term for the electrical potential. It is this energy that drives the synthesis of ATP, in ac- cord with Mitchell’s model. 20.5 How Does a Proton Gradient Drive the Synthesis of ATP? The mi- tochondrial complex that carries out ATP synthesis is ATP synthase (F 1 F 0 –ATPase). ATP synthase consists of two principal complexes, desig- nated F 1 and F 0 . Protons taken up from the cytosol by one of the proton access channels in the a-subunit of F 0 ride the rotor of c-subunits until they reach the other proton access channel on a, from which they are re- leased into the matrix. Such rotation causes the ␥-subunit of F 1 to turn relative to the three -subunit nucleotide sites of F 1 , changing the con- formation of each in sequence, so ADP is first bound, then phosphory- lated, then released, according to Boyer’s binding change mechanism. The inhibitors of oxidative phosphorylation include rotenone, a common insecticide that strongly inhibits the NADH–UQ reductase. Complex IV is specifically inhibited by cyanide (CN Ϫ ), azide (N 3 Ϫ ), and carbon monoxide (CO). Cyanide and azide bind tightly to the ferric form of cytochrome a 3 , whereas carbon monoxide binds only to the ferrous form. Uncouplers disrupt the coupling of electron transport and ATP syn- thase. Uncouplers share two common features: hydrophobic character and a dissociable proton. They function by carrying protons across the inner membrane, acquiring protons on the outer surface of the mem- brane (where the proton concentration is high) and carrying them to the matrix side. Uncouplers destroy the proton gradient that couples electron transport and the ATP synthase. ATP–ADP translocase mediates the movement of ATP and ADP across the mitochondrial membrane. The ATP–ADP translocase is an inner membrane protein that tightly couples the exit of ATP with the entry of ADP so that the mitochondrial nucleotide levels remain approximately constant. For each ATP transported out, one ADP is transported into the matrix. ATP–ADP translocase binds ATP on the matrix side, reorients to face the intermembrane space, and exchanges ATP for ADP, with subse- quent reorientation back to the matrix face of the inner membrane. 20.6 What Is the P/O Ratio for Mitochondrial Oxidative Phosphoryla- tion? The P/O ratio is the number of molecules of ATP formed in ox- idative phosphorylation per two electrons flowing throug h a defined segment of the electron-transport chain. The consensus value for the mitochondrial P/O ratio is 10/4, or 2.5, for electrons entering the elec- tron-transport chain as NADH. For succinate to O 2 , the P/O ratio in this case would be 6/4, or 1.5. 20.7 How Are the Electrons of Cytosolic NADH Fed into Electron Trans- port? Eukaryotic cells have a number of shuttle systems that collect the electrons of cytosolic NADH for delivery to mitochondria without actu- ally transporting NADH across the inner membrane. In the glyc- erophosphate shuttle, two different glycerophosphate dehydrogenases, one in the cytosol and one on the outer face of the mitochondrial inner membrane, work together to carry electrons into the mitochondrial ma- trix. In the malate–aspartate shuttle, oxaloacetate is reduced in the cy- tosol, acquiring the electrons of NADH (which is oxidized to NAD ϩ ). Malate is transported across the inner membrane, where it is reoxidized by malate dehydrogenase, converting NAD ϩ to NADH in the matrix. Problems 627 20.8 How Do Mitochondria Mediate Apoptosis? Mitochondria are a crossroads for several cell signaling pathways. Mitochondria take up Ca 2ϩ ions released from the endoplasmic reticulum, helping control in- tracellular Ca 2ϩ signals. They produce ROS that play signaling roles in cells. They also participate in apoptosis, the programmed death of cells. Triggering agents, including Ca 2ϩ , ROS, and certain lipid molecules and protein kinases, can induce the opening of pores in the mitochon- drial membrane, releasing cytochrome c, which then binds to the WD40 domain of Apaf-1, activating formation of the heptameric apoptosome platform. Mitochondria-mediated apoptosis is the mode of cell death of many neurons in the brain during strokes and other brain-trauma in- juries, and interventions that suppress apoptosis may eventually be use- ful in clinical settings. PROBLEMS Preparing for an exam? Create your own study path for this chapter at www.cengage.com/login. 1. For the following reaction, [FAD] ϩ 2 cyt c (Fe 2ϩ ) ϩ 2 H ϩ ⎯⎯→[FADH 2 ] ϩ 2 cyt c (Fe 3ϩ ) determine which of the redox couples is the electron acceptor and which is the electron donor under standard-state conditions, calcu- late the value of ⌬Ᏹ o Ј, and determine the free energy change for the reaction. 2. Calculate the value of ⌬Ᏹ o Ј for the glyceraldehyde-3-phosphate dehydrogenase reaction, and calculate the free energy change for the reaction under standard-state conditions. 3. For the following redox reaction, NAD ϩ ϩ 2 H ϩ ϩ 2 e Ϫ ⎯⎯→NADH ϩ H ϩ suggest an equation (analogous to Equation 20.12) that predicts the pH dependence of this reaction, and calculate the reduction potential for this reaction at pH 8. 4. Sodium nitrite (NaNO 2 ) is used by emergency medical personnel as an antidote for cyanide poisoning (for this purpose, it must be administered immediately). Based on the discussion of cyanide poisoning in Section 20.5, suggest a mechanism for the lifesaving ef- fect of sodium nitrite. 5. A wealthy investor has come to you for advice. She has been ap- proached by a biochemist who seeks financial backing for a com- pany that would market dinitrophenol and dicumarol as weight-loss medications. The biochemist has explained to her that these agents are uncouplers and that they would dissipate metabolic energy as heat. The investor wants to know if you think she should invest in the biochemist’s company. How do you respond? 6. Assuming that 3 H ϩ are transported per ATP synthesized in the mi- tochondrial matrix, the membrane potential difference is 0.18 V (negative inside), and the pH difference is 1 unit (acid outside, basic inside), calculate the largest ratio of [ATP]/[ADP][P i ] under which synthesis of ATP can occur. 7. Of the dehydrogenase reactions in glycolysis and the TCA cycle, all but one use NAD ϩ as the electron acceptor. The lone exception is the suc- cinate dehydrogenase reaction, which uses covalently bound FAD of a flavoprotein as the electron acceptor. The standard reduction poten- tial for this bound FAD is in the range of 0.003 to 0.091 V (see Table 20.1). Compared with the other dehydrogenase reactions of glycolysis and the TCA cycle, what is unique about succinate dehydrogenase? Why is bound FAD a more suitable electron acceptor in this case? 8. a. What is the standard free energy change (⌬G°Ј) for the reduction of coenzyme Q by NADH as carried out by Complex I (NADH–coenzyme Q reductase) of the electron-transport path- way if Ᏹ o Ј (NAD ϩ /NADH) ϭϪ0.320 V and Ᏹ o Ј (CoQ/CoQH 2 ) ϭ ϩ0.060 V. b. What is the equilibrium constant (K eq ) for this reaction? c. Assume that (1) the actual free energy release accompanying the NADH–coenzyme Q reductase reaction is equal to the amount released under standard conditions (as calculated in part a), (2) this energy can be converted into the synthesis of ATP with an efficiency ϭ 0.75 (that is, 75% of the energy re- leased upon NADH oxidation is captured in ATP synthesis), and (3) the oxidation of 1 equivalent of NADH by coenzyme Q leads to the phosphorylation of 1 equivalent of ATP. Under these conditions, what is the maximum ratio of [ATP]/ [ADP] attainable by oxidative phosphorylation when [P i ] ϭ 1 mM? (Assume ⌬G°Ј for ATP synthesis ϭϩ30.5 kJ/mol.) 9. Consider the oxidation of succinate by molecular oxygen as carried out via the electron-transport pathway Succinate ϩ ᎏ 1 2 ᎏ O 2 ⎯⎯→fumarate ϩ H 2 O a. What is the standard free energy change (⌬G°Ј) for this reaction if Ᏹ o Ј (Fum/Succ) ϭϩ0.031 V and Ᏹ o Ј ( ᎏ 1 2 ᎏ O 2 /H 2 O) ϭϩ0.816 V. b. What is the equilibrium constant (K eq ) for this reaction? c. Assume that (1) the actual free energy release accompanying succinate oxidation by the electron-transport pathway is equal to the amount released under standard conditions (as calculated in part a), (2) this energy can be converted into the synthesis of ATP with an efficiency ϭ 0.7 (that is, 70% of the energy released upon succinate oxidation is captured in ATP synthesis), and (3) the oxi- dation of 1 succinate leads to the phosphorylation of 2 equivalents of ATP. Under these conditions, what is the maximum ratio of [ATP]/ [ADP] attainable by oxidative phosphorylation when [P i ] ϭ 1 mM? (Assume ⌬G°Ј for ATP synthesis ϭϩ30.5 kJ/mol.) 10. Consider the oxidation of NADH by molecular oxygen as carried out via the electron-transport pathway NADH ϩ H ϩ ϩ ᎏ 1 2 ᎏ O 2 ⎯⎯→NAD ϩ ϩ H 2 O a. What is the standard free energy change (⌬G°Ј) for this reac- tion if Ᏹ o Ј (NAD ϩ /NADH) ϭϪ0.320 V and Ᏹ o Ј (O 2 /H 2 O) ϭ ϩ0.816 V. b. What is the equilibrium constant (K eq ) for this reaction? c. Assume that (1) the actual free energy release accompanying NADH oxidation by the electron-transport pathway is equal to the amount released under standard conditions (as calculated in part a), (2) this energy can be converted into the synthesis of ATP with an efficiency ϭ 0.75 (that is, 75% of the energy released upon NADH oxidation is captured in ATP synthesis), and (3) the oxi- dation of 1 NADH leads to the phosphorylation of 3 equivalents of ATP. Under these conditions, what is the maximum ratio of [ATP]/ [ADP] attainable by oxidative phosphorylation when [P i ] ϭ 2 mM? (Assume ⌬G°Ј for ATP synthesis ϭϩ30.5 kJ/mol.) 11. Write a balanced equation for the reduction of molecular oxygen by reduced cytochrome c as carried out by Complex IV (cytochrome oxidase) of the electron-transport pathway. a. What is the standard free energy change (⌬G°Ј) for this reaction if ⌬Ᏹ o Ј cyt c(Fe 3ϩ )/cyt c(Fe 2ϩ ) ϭϩ0.254 volts and Ᏹ o Ј ( ᎏ 1 2 ᎏ O 2 /H 2 O) ϭ 0.816 volts b. What is the equilibrium constant (K eq ) for this reaction? c. Assume that (1) the actual free energy release accompanying cytochrome c oxidation by the electron-transport pathway is equal to the amount released under standard conditions (as calculated 628 Chapter 20 Electron Transport and Oxidative Phosphorylation in part a), (2) this energy can be converted into the synthesis of ATP with an efficiency ϭ 0.6 (that is, 60% of the energy released upon cytochrome c oxidation is captured in ATP synthesis), and (3) the reduction of 1 molecule of O 2 by reduced cytochrome c leads to the phosphorylation of 2 equivalents of ATP. Under these conditions, what is the maximum ratio of [ATP]/ [ADP] attainable by oxidative phosphorylation when [P i ] ϭ 3 mM? (Assume ⌬G°Ј for ATP synthesis ϭϩ30.5 kJ/mol.) 12. The standard reduction potential for (NAD ϩ /NADH) is Ϫ0.320 V, and the standard reduction potential for (pyruvate/lactate) is Ϫ0.185 V. a. What is the standard free energy change (⌬G°Ј) for the lactate de- hydrogenase reaction: NADH ϩ H ϩ ϩ pyruvate⎯⎯→lactate ϩ NAD ϩ b. What is the equilibrium constant (K eq ) for this reaction? c. If [pyruvate] ϭ 0.05 mM and [lactate] ϭ 2.9 mM and ⌬G for the lactate dehydrogenase reaction ϭϪ15 kJ/mol in erythrocytes, what is the [NAD ϩ ]/[NADH] ratio under these conditions? 13. Assume that the free energy change (⌬G) associated with the move- ment of 1 mole of protons from the outside to the inside of a bac- terial cell is Ϫ23 kJ/mol and 3 H ϩ must cross the bacterial plasma membrane per ATP formed by the bacterial F 1 F 0 –ATP synthase. ATP synthesis thus takes place by the coupled process: 3 H ϩ out ϩ ADP ϩ P i 343 H ϩ in ϩ ATP ϩ H 2 O a. If the overall free energy change (⌬G overall ) associated with ATP synthesis in these cells by the coupled process is Ϫ21 kJ/mol, what is the equilibrium constant (K eq ) for the process? b. What is ⌬G synthesis , the free energy change for ATP synthesis, in these bacteria under these conditions? c. The standard free energy change for ATP hydrolysis (⌬G°Ј hydrolysis ) is Ϫ30.5 kJ/mol. If [P i ] ϭ 2 mM in these bacterial cells, what is the [ATP]/[ADP] ratio in these cells? 14. Describe in your own words the path of electrons through the Q cycle of Complex III. 15. Describe in your own words the path of electrons through the cop- per and iron centers of Complex IV. 16. In the course of events triggering apoptosis, a fatty acid chain of car- diolipin undergoes peroxidation to release the associated cyto- chrome c. Lipid peroxidation occurs at a double bond. Suggest a mechanism for the reaction of hydrogen peroxide with an unsatura- tion in a lipid chain, and identify a likely product of the reaction. 17. In problem 18 at the end of Chapter 19, you might have calculated the number of molecules of oxaloacetate in a typical mitochon- drion. What about protons? A typical mitochondrion can be thought of as a cylinder 1 m in diameter and 2 m in length. If the pH in the matrix is 7.8, how many protons are contained in the mitochondrial matrix? 18. Considering that all other dehydrogenases of glycolysis and the TCA cycle use NADH as the electron donor, why does succinate de- hydrogenase, a component of the TCA cycle and the electron trans- fer chain, use FAD as the electron acceptor from succinate, rather than NAD ϩ ? Note that there are two justifications for the choice of FAD here—one based on energetics and one based on the mecha- nism of electron transfer for FAD versus NAD ϩ . Preparing for the MCAT Exam 19. Based on your reading on the F 1 F 0 –ATPase, what would you con- clude about the mechanism of ATP synthesis: a. The reaction proceeds by nucleophilic substitution via the S N 2 mechanism. b. The reaction proceeds by nucleophilic substitution via the S N 1 mechanism. c. The reaction proceeds by electrophilic substitution via the E1 mechanism. d. The reaction proceeds by electrophilic substitution via the E2 mechanism. 20. Imagine that you are working with isolated mitochondria and you manage to double the ratio of protons outside to protons inside. In or- der to maintain the overall ⌬G at its original value (whatever it is), how would you have to change the mitochondria membrane potential? FURTHER READING Apoptosis Cereghetti, G. M., and Scorrano, L., 2006. The many shapes of mito- chondrial death. Oncogene 25:4717–4724. Cerveny, K. L., Tamura, Y., et al., 2007. Regulation of mitochondrial fu- sion and division. Trends in Cell Biology 17:563–569. Chan, D. C., 2006. Mitochondrial fusion and fission in mammals. An- nual Review of Cell and Developmental Biology 22:79–99. Orrenius, S., 2007. Reactive oxygen species in mitochondria-mediated cell death. Drug Metabolism Reviews 39:443–455. Orrenius, S., and Zhivotovsky, B., 2005. Cardiolipin oxidation sets cyto- chrome c free. Nature Chemical Biology 1:188–189. Riedl, S. J., and Salvesen, G. S., 2007. The apoptosome: Signalling plat- form of cell death. Nature Reviews Molecular Cell Biology 8:405–413. ATP–ADP Translocase Nury, H., Dahout-Gonzalez, C., et al., 2006. Relations between structure and function of the mitochondrial ADP/ATP carrier. Annual Review of Biochemistry 75:713–741. Bioenergetics Babcock, G. T., and Wikstrom, M., 1992. Oxygen activation and the con- servation of energy in cell respiration. Nature 356:301–309. Merz, S., Hammermeister, M., et al., 2007. Molecular machinery of mito- chondrial dynamics in yeast. Biological Chemistry 388:917–926. Mitchell, P., 1979. Keilin’s respiratory chain concept and its chemi- osmotic consequences. Science 206:1148–1159. Mitchell, P., and Moyle, J., 1965. Stoichiometry of proton translocation through the respiratory chain and adenosine triphosphatase sys- tems of rat mitochondria. Nature 208:147–151. Electron Transfer Belevich, I., and Verkhovsky, M. I., 2008. Molecular mechanism of pro- ton translocation by cytochrome c oxidase. Antioxidants and Redox Signaling 10:1–29. Boekema, E. J., and Braun, H-P., 2007. Supramolecular structure of the mitochondrial oxidative phosphorylation system. Journal of Biologi- cal Chemistry 282:1–4. Brandt, U., 2006. Energy converting NADH:quinone oxidoreductase (Complex I). Annual Review of Biochemistry 75:69–72. Brzezinski, P., and Adelroth, P., 2006. Design principles of proton- pumping haem-copper oxidases. Current Opinion in Structural Biology 16:465–472. Busenlehner, L. S., Branden, G., et al., 2008. Structural elements in- volved in proton translocation by cytochrome c oxidase as revealed by backbone amide hydrogen–deuterium exchange of the E286H mutant. Biochemistry 47:73–83. Cecchini, G., 2003. Function and structure of Complex II of the respi- ratory chain. Annual Review of Biochemistry 72:77–109. Hunte, C., Koepke, J., et al., 2000. Structure at 2.3 Å resolution of the cytochrome bc 1 complex from the yeast Sacchar omyces cerevisiae co- crystallized with an antibody Fv fragment. Structure 8:669–684. Further Reading 629 Iwata, S., Ostermeier, C., et al., 1995. Structure at 2.8 Å resolution of cyto- chrome c oxidase from Paracoccus denitrificans. Nature 376:660–669. Lenaz, G., Fato, R., et al., 2006. Mitochondrial Complex I: Structural and functional aspects. Biochimica et Biophysica Acta 1757:1406–1420. Sazanov, L. A., 2007. Respiratory Complex I: Mechanistic and structural insights provided by the crystal structure of the hydrophilic domain. Biochemistry 46:2275–2288. Seibold, S. A., Mills, D. A., et al., 2005. Water chain formation and pos- sible proton pumping routes in Rhodobacter sphaeroides cytochrome c oxidase: A molecular dynamics comparison of the wild type and R481K mutant. Biochemistry 44:10475–10485. Slater, E. C., 1983. An ubiquitous mechanism of electron transfer. Trends in Biochemical Sciences 8:239–242. Sun, F., Huo, X., et al., 2005. Crystal structure of mitochondrial respira- tory membrane protein Complex II. Cell 121:1043–1057. Trumpower, B. L., 1990. The protonmotive Q cycle: Energy transduction by coupling of proton translocation to electron transfer by the cyto- chrome bc 1 complex. Journal of Biological Chemistry 265:11409–11412. Tsukihara, T., Aoyama, H., et al., 1996. The whole structure of the 13- subunit oxidized cytochrome c oxidase at 2.8 Å. Science 272:1136–1144. Wikstrom, M., and Verkhovsky, M. I., 2007. Mechanism and energetics of proton translocation by the respiratory heme-copper oxidases. Biochimica et Biophysica Acta 1767:1200–1214. Xia, D., Yu, C A., et al., 1997. The crystal structure of the cytochrome bc 1 complex from bovine heart mitochondria. Science 277:60–66. Yoshikawa, S., Muramoto, K., et al., 2006. Reaction mechanism of bovine heart cytochrome c oxidase. Biochimica et Biophysica Acta 1757:395–400. F 1 F 0 –ATPase Adachi, K., Oiwa, K., et al., 2007. Coupling of rotation and catalysis in F 1 -ATPase revealed by single-molecule imaging and manipulation. Cell 130:309–321. Aksimentiev, A., Balabin, I. A., et al., 2004. Insights into the molecular mechanism of rotation in the F 0 sector of ATP synthase. Biophysical Journal 66:1332–1344. Boyer, P. D., 2002. A research journey with ATP synthase. Journal of Bio- logical Chemistry 277:39045–39061. Dickson, V. K., Silvester, J. A., et al., 2006. On the structure of the stator of the mitochondrial ATP synthase. EMBO Journal 25:2911–2918. Rastogi, V. K., and Girvin, M. E., 1999. Structural changes linked to pro- ton translocation by subunit c of the ATP synthase. Nature 402: 263–268. Senior, A. E., 2007. ATP synthase: Motoring to the finish line. Cell 130: 220–221. Senior, A. E., and Weber, J., 2004. Happy motoring with ATP synthase. Nature Structural and Molecular Biology 11:110–112. Stock, D., Leslie, A. G. W., et al., 1999. Molecular architecture of the ro- tary motor in ATP synthase. Science 286:1700–1705. Weber, J., 2007. ATP synthase: The structure of the stator stalk. Trends in Biochemical Sciences 32:53–55. Wilkins, S., 2005. Rotary molecular motors. Advances in Protein Chemistry 71:345–382. Uncouplers Fogelman, A. M., 2005. When pouring water on the fire makes it burn brighter. Cell Metabolism 2:6–7. Nedergaard, J., Ricquier, D., et al., 2005. Uncoupling proteins: Current status and therapeutic prospects. EMBO Reports 6:917–921. © Richard Hamilton Smith/CORBIS 21 Photosynthesis The vast majority of energy consumed by living organisms stems from solar energy captured by the process of photosynthesis. Only chemolithotrophic prokaryotes are independent of this energy source. Of the 1.5 ϫ 10 22 kJ of energy reaching the earth each day from the sun, 1% is absorbed by photosynthetic organisms and transduced into chemical energy. 1 This energy, in the form of biomolecules, be- comes available to other members of the biosphere through food chains. The transduction of solar, or light, energy into chemical energy is often expressed in terms of carbon dioxide fixation, in which hexose is formed from carbon dioxide and oxygen is evolved: Light 6 CO 2 ϩ 6 H 2 O ⎯⎯→ C 6 H 12 O 6 ϩ 6 O 2 (21.1) Estimates indicate that 10 11 tons of carbon dioxide are fixed globally per year, of which one-third is fixed in the oceans, primarily by photosynthetic marine micro- organisms. Although photosynthesis is traditionally equated with CO 2 fixation, light energy (or rather the chemical energy derived from it) drives all endergonic processes in phototrophic cells. The assimilation of inorganic forms of nitrogen and sulfur into organic molecules (see Chapter 25) represents two other metabolic conversions closely coupled to light energy in green plants. Our previous considerations of aer- obic metabolism (Chapters 18 through 20) treated cellular respiration (precisely the reverse of Equation 21.1) as the central energy-releasing process in life. It nec- essarily follows that the formation of hexose from carbon dioxide and water, the products of cellular respiration, must be endergonic. The necessary energy comes from light. Note that in the carbon dioxide fixation reaction described, light is used to drive a chemical reaction against its thermodynamic potential. 21.1 What Are the General Properties of Photosynthesis? Photosynthesis Occurs in Membranes Organisms capable of photosynthesis are very diverse, ranging from simple prokaryotic forms to the largest organisms of all, Sequoia gigantea, the giant red- wood trees of California. Despite this diversity, we find certain generalities regard- ing photosynthesis. An important one is that photosynthesis occurs in membranes. In photosynthetic prokaryotes, the photosynthetic membranes fill up the cell interior; in photosynthetic eukaryotes, the photosynthetic membranes are localized in large organelles known as chloroplasts (Figures 21.1 and 21.2). Chloroplasts are one Field of goldenrod. In a sun-flecked lane, Beside a path where cattle trod, Blown by wind and rain, Drawing substance from air and sod; In ruggedness, it stands aloof, The ragged grass and puerile leaves, Lending a hand to fill the woof In the pattern that beauty makes. What mystery this, hath been wrought; Beauty from sunshine, air, and sod! Could we thus gain the ends we sought- Tell us thy secret, Goldenrod. Rosa Staubus Oklahoma pioneer (1886–1966) KEY QUESTIONS 21.1 What Are the General Properties of Photosynthesis? 21.2 How Is Solar Energy Captured by Chlorophyll? 21.3 What Kinds of Photosystems Are Used to Capture Light Energy? 21.4 What Is the Molecular Architecture of Photosynthetic Reaction Centers? 21.5 What Is the Quantum Yield of Photosynthesis? 21.6 How Does Light Drive the Synthesis of ATP? 21.7 How Is Carbon Dioxide Used to Make Organic Molecules? 21.8 How Does Photorespiration Limit CO 2 Fixation? ESSENTIAL QUESTIONS Photosynthesis is the primary source of energy for all life forms (except chemolitho- trophic prokaryotes). Much of the energy of photosynthesis is used to drive the syn- thesis of organic molecules from atmospheric CO 2 . How is solar energy captured and transformed into metabolically useful chemi- cal energy? How is the chemical energy produced by photosynthesis used to create organic molecules from carbon dioxide? Create your own study path for this chapter with tutorials, simulations, animations, and Active Figures at www.cengage.com/login. 1 Of the remaining 99%, two-thirds is absorbed by the earth and oceans, thereby heating the planet; the remaining one-third is lost as light reflected back into space. 21.1 What Are the General Properties of Photosynthesis? 631 member in a family of related plant-specific organelles known as plastids. Chloro- plasts themselves show a range of diversity, from the single, spiral chloroplast that gives Spirogyra its name to the multitude of ellipsoidal plastids typical of higher plant cells (Figure 21.3). Characteristic of all chloroplasts, however, is the organization of the inner mem- brane system, the so-called thylakoid membrane. The thylakoid membrane is orga- nized into paired folds that extend throughout the organelle, as in Figure 21.2. These paired folds, or lamellae, give rise to flattened sacs or discs, thylakoid vesicles (from the Greek thylakos, meaning “sack”), which occur in stacks called grana. A sin- gle stack, or granum, may contain dozens of thylakoid vesicles, and different grana are joined by lamellae that run through the soluble portion, or stroma, of the or- ganelle. Chloroplasts thus possess three membrane-bound aqueous compartments: the intermembrane space, the stroma, and the interior of the thylakoid vesicles, the so-called thylakoid space (also known as the thylakoid lumen). As we shall see, this third compartment serves an important function in the transduction of light energy into ATP formation. The thylakoid membrane has a highly characteristic lipid com- position and, like the inner membrane of the mitochondrion, is impermeable to FIGURE 21.1 Electron micrograph of a representative chloroplast. James Dennis/CNRI/Phototake NYC Intermembrane space Granum (stack of thylakoids) Stroma Thylakoid lumen Lamella Inner membrane Outer membrane Thylakoid vesicle FIGURE 21.2 Schematic diagram of an idealized chloroplast. 632 Chapter 21 Photosynthesis most ions and molecules. Chloroplasts, like their mitochondrial counterparts, pos- sess DNA, RNA, and ribosomes and consequently display a considerable amount of autonomy. However, many critical chloroplast components are encoded by nuclear genes, so autonomy is far from absolute. Photosynthesis Consists of Both Light Reactions and Dark Reactions If a chloroplast suspension is illuminated in the absence of carbon dioxide, oxygen is evolved. Furthermore, if the illuminated chloroplasts are now placed in the dark and supplied with CO 2 , net hexose synthesis can be observed (Figure 21.4). Thus, the evolution of oxygen can be temporally separated from CO 2 fixation and also has a light dependency that CO 2 fixation lacks. The light reactions of photosynthesis, of which O 2 evolution is only one part, are associated with the thylakoid membranes. In contrast, the light-independent reactions, or so-called dark reactions, notably CO 2 fixation, are located in the stroma. A concise summary of the photosynthetic process is that radiant electromagnetic energy (light) is transformed by a specific photochemical system located in the thylakoids to yield chemical energy in the form of reducing potential (NADPH) and high-energy phosphate (ATP). NADPH and ATP can then be used to drive the endergonic process of hexose formation from (a) (b) FIGURE 21.3 (a) Spirogyra—a freshwater green alga. (b) A higher plant cell. © Perennou Nuridsany/Photo Researchers, Inc. Biophoto Associates/Science Source O 2 O 2 O 2 O 2 CO 2 CO 2 CO 2 CO 2 CO 2 Chloroplast suspension Light Into dark Absence of CO 2 CO 2 fixation into sugars O 2 evolved ANIMATED FIGURE 21.4 The light-dependent and light-independent reactions of photosyn- thesis. See this figure animated at www.cengage.com/login.