Chapter 8 Energy. I. Biological Energy. Part II Principles of Individual Cell Function

Transcription

1 Part II Principles of Individual Cell Function Chapter 8 This chapter discusses the mechanisms of mitochondrial respiration and photosynthesis by chloroplasts, which are processes involved in the production of biological energy. ATP synthesis by respiration is known as oxidative phosphorylation, and that carried out by photosynthesis is called photophosphorylation. These processes, combined with the substrate-level phosphorylation discussed in the previous chapter, represent almost all the energy production pathways found in cells. This chapter also outlines the production of organic compounds by photosynthesis. Reactions in which organisms use energy to perform activities are diverse, but the production methods for energy and organic compounds are universal and very simple. production systems have been studied as a central pillar of biology since the early 20th century. The energy production system mediated by oxidation-reduction reactions in the biological membrane is particularly well understood from the atomic and molecular levels to the cellular level, and is an active area in which cutting-edge research remains ongoing. I. Biological Biological energy is produced by the breakdown of various organic compounds through fermentation and respiration*1, by respiration using inorganic compounds, and by photosynthesis using light energy. It is controlled by the highenergy phosphate bonds of ATP and other substances and by the concentration gradient of H + for use by organisms in various activities. As outlined in Chapter 7, free energy change (ΔG) resulting from the hydrolysis of ATP s terminal phosphate bond is expressed by Equation 8-1. Interestingly, ATP has two phosphate bonds, and only the terminal one is formed by the processes of respiration and photosynthesis discussed in this chapter; the inner bond is formed by other enzymes*2. The concentration gradient of H + across the membrane can be expressed as the electrochemical potential of H + (ΔμH+) (Equation 8-2). This is the same as the free energy change that occurs when H + is transported across the membrane. Here, log10[h + in] and log10[h + out] can be converted to the ph *1 Fermentation and respiration: In energy metabolism, fermentation refers to reactions that do not accompany oxidation and reduction, while respiration refers to reactions that accompany oxidation by oxygen or other substances. Sulfate respiration and nitrate respiration, which respectively use SO4 2- and NO3 - instead of oxygen, are also known. *2 ATP s terminal high-energy phosphate bond is formed by ATP synthase. High-energy phosphate bonds in ADP are formed by adenylate kinase from AMP and ATP: AMP + ATP 2ADP CSLS / THE UNIVERSITY OF TOKYO 149

2 inside and outside the membrane, and ΔΦ is the membrane potential. In other words, even membrane potential formed by the transport of ions other than H + contributes to the high-energy state of H +. Free energy change by ATP hydrolysis: Equation 8-1 ΔG = ΔG + 2.3RTlog 10 [ADP][H3PO4] / [ATP] Electrochemical potential of H + across the membrane: Equation 8-2 ΔμH+ = 2.3RTlog 10 [H + in] / [H + out] + FΔΦ where R is the gas constant, T is the absolute temperature, ΔG is the free energy change under standard conditions (ph7) and F is the Faraday constant. Organisms have many enzymes that catalyze a range of chemical reactions in their cells. Cells working in normal conditions constantly incorporate nutrients and energy from outside and discard waste materials, thereby keeping the intracellular conditions constant (i.e., in a state of dynamic equilibrium). This state is achieved through a series of metabolic reactions that proceed almost constantly through the catalytic action of enzymes. If this is the case, do all chemical reactions catalyzed by enzymes in living cells reach dynamic equilibrium? The answer is no. If all such reactions reached this state, new reactions would not occur without the entry and exit of new materials, although existing conditions might be maintained. Such cells could no longer be called autonomously living cells. In performing new reactions, actual organisms change equilibrium by putting energy into particular reactions. For this purpose, a high-energy state that deviates from equilibrium needs to be maintained. Such a state involves the concentration gradient of ATP and H +, and the mechanism of putting in energy as appropriate involves the activity regulation of enzymes that use the energy (e.g., the ATPase of myosins that is used for cell movement). The main function of organisms is to convert the energy produced by the fermentation of various materials, as well as by respiration and photosynthesis, into the standardized forms, namely ATP and concentration gradient of H +, and maintain it. The organic compounds and energy that support the activities of almost all organisms on earth are produced and supplied through photosynthesis by plants and other organisms. All oxygen is also produced and released by this process. The mechanism of photosynthesis is completely different from that of fermentation CSLS / THE UNIVERSITY OF TOKYO 150

3 and respiration in that it uses physical energy in the form of light, but the mechanism of ATP synthesis using the physical energy obtained from the concentration gradient of H + created by electron transport is very similar among the three mechanisms. In processes other than photosynthesis, organic compound energy is supplied through autotrophic chemical synthesis by bacteria*3. *3 The sulfur bacteria (Thiomicrospira spp.) found in organisms such as Calyptogena spp. that live in hydrothermal vents on the ocean floor (also known as black smokers) are an example of this. II. Outline of the Respiratory Chain and Oxidative Phosphorylation As outlined in Chapter 7, glucose is fully broken down into carbon dioxide and hydrogen (NADH and FADH2), and only four ATP molecules per glucose are synthesized during the process. However, coupling with a series of reactions through which NADH and FADH2 (generated during the process) are fully oxidized by oxygen to become water molecules generates 38 molecules of ATP*4. The series of oxidation-reduction reactions beginning from NADH and FADH2 are known as electron transport reactions or the respiration chain, and the mechanism of synthesizing ATP coupling with the reactions is called oxidative phosphorylation. In the 1950s, researchers initially looked for metabolic intermediates with highenergy phosphate bonds on the assumption that, as in glycolysis, kinases (enzymes that transfer the phosphate group to ATP) were also involved in reactions that synthesize large amounts of ATP, but these could not be found. Soon thereafter, the mystery surrounding ATP synthesis was solved by the chemiosmotic theory proposed in 1961 by P. Mitchell, who was involved in the investigation of active transport. This is the process that transports H + and other substances across the membrane against the concentration gradient using ATP energy. In Mitchell s hypothesis, ATP is synthesized using the concentration gradient of transported H + coupled with electron transport reactions. In other words, the concentration gradient of H + is a high-energy state that is interconvertible with ATP, and the conversion is catalyzed by F-ATP synthase (discussed later). *4 Calculation example: To obtain the standard free energy change of the two-electron reaction NADH + H + + 1/2 O2 NAD + + H2O, with O2 (E = V) and NAD (E = V), n = 2 is assigned to Equation 8-3: ΔG = -2 x 96.5 x [0.815 (-0.315)] = -218 kj mol -1 where the Faraday constant is 96.5 kjv -1 mol -1 III. Oxidation-Reduction Reactions and the Respiration Chain In terms of the utilization of biological energy, the ability to convert energies CSLS / THE UNIVERSITY OF TOKYO 151

4 Figure 8-1 Oxidized and reduced forms of ubiquinone Also known as coenzyme Q, this is a lipophilic compound that can transfer electrons and H + separately, and is involved in the creation of the H + concentration gradient. generated by various oxidation-reduction reactions to a single form, H + electrochemical potential, is a significant advantage. Oxidation-reduction reactions involve the transfer of electrons between two materials, and are mediated mainly by cofactors (i.e., coenzymes). Since the same cofactors (e.g., NAD + ) tend to be used in different enzymatic oxidation-reduction reactions in cells, energy can be efficiently produced through a common mechanism by converging enzymatic oxidation-reduction reactions into the respiration chain through the mediation of cofactors. Among the common cofactors, NADH and FADH2 are often utilized, and quinones (such as ubiquinone) and cytochrome c are also used (Fig. 8-1). Most of these cofactors are common to all organisms, and photosynthesis (discussed in V in this chapter) also uses very similar cofactors. The free energy change of oxidation-reduction reactions has a linear relationship with the difference in oxidation-reduction potential between two reactants, A and B (Equation 8-3). Equation 8-3 ΔG = -nf(e A E B) where n is the number of electrons transferred and F is the Faraday constant. *5 NADH is a water-soluble coenzyme that is shared by many enzymes. FADH2, on the other hand, binds to certain enzymes, and is a coenzyme (or cofactor) that facilitates the transfer of electrons in enzymatic reactions. Therefore, in Figure 8-2, NADH is in the matrix, and FADH2 (FAD in the figure the oxidized form that receives electrons) binds to the inside of Complex II. The hydrogen (E = V) in NADH releases 218 kjmol -1 of energy by reacting directly with oxygen (E = V)*4, but the respiration chain is separated into electrons and H +. These electrons, which have a high reducing ability (i.e., low oxidation-reduction potential), gradually release energy via around 20 types of electron carrier and finally react with oxygen in mild conditions. Among these electron carriers, ubiquinone (a low molecular weight compound) (Fig. 8-1) and cytochrome c (a small protein) act as mobile electron carriers, and the rest are contained in four types of protein complex (Complexes I IV) as cofactors. These cofactors, which strongly bind to proteins, are known as the prosthetic group. In terms of substrate oxidation and reduction, these protein complexes are also called NADH dehydrogenase (Complex I), succinate dehydrogenase (Complex II), cytochrome bc1 complex (Complex III) (also known as ubiquinone-cytochrome c oxidoreductase) and cytochrome c oxidase (Complex IV) (Fig. 8-2*5). The free energy change during the complete oxidation of NADH (ΔG o = -218 kjmol -1 ) is equivalent to that of around seven ATP molecules (ΔG o = kjmol -1 per molecule); however, only three ATP molecules are actually generated, and the remaining free energy change is used to tilt the equilibrium toward ATP CSLS / THE UNIVERSITY OF TOKYO 152

5 Chapter 8 synthesis. The standard reduction potential of other major materials are E = V for succinic acid, E = V for ubiquinone and E = V for cytochrome c. In other words, the free energy change in the reaction that produces ubiquinone from succinic acid catalyzed by Complex II (succinate dehydrogenase) is insufficient for ATP synthesis, whereas it is large in the *6 38 molecules: This number is for bacteria; the number for mitochondrial ATP synthesis is in fact 36. This is because energy equivalent to that released by two ATP molecules is used to transport two molecules of NADH generated in glycolysis from the cytoplasm to mitochondria. enzymatic reactions of Complex I, III and IV, thus allowing ATP synthesis. The inability of the Complex II reaction to generate enough energy to synthesize ATP is consistent with the fact that in the citric acid cycle, FADH2 is generated only at the stage of succinate dehydrogenase and the number of ATP molecules generated by the oxidation of FADH2 is one fewer than in the oxidation of NADH. It can be said that the reducing power of succinic acid is weaker than that of NADH. The number of ATP molecules generated per glucose molecule is therefore 2 in glycolysis, 2 in the citric acid cycle, 4 in the oxidation of 2 FADH2 molecules, and 30 in the oxidation of 10 NADH molecules, making a total of 38 molecules*6. In the aerobic respiration of mitochondria, glucose can be used for energy production in addition to the NADH and FADH2 generated by the degradation of amino acids and fatty acids (Fig. 8-3). Fatty acids undergo dehydrogenation Figure 8-2 A mitochondrion (top), and the respiration chain and ATP synthase a protein complex found in the inner membrane of mitochondria (bottom) Q (an electron transport component) is a ubiquinone, c, c 1, b, a and a 3 are hemes (cytochrome cofactors), and FeS is an iron-sulfur cluster cofactor. FMD is flavin mononucleotide, and FAD is flavin adenine dinucleotide. Reduced Q generated by Complex II also passes electrons on to Complex III via the quinone + + cycle. Since the mechanism of H transport is not fully understood, the number of H ions transported by each complex is set as n. Overall, approximately three molecules of ATP are synthesized through the complete oxidation of one NADH molecule, and the value of n is approximately 9 in this case. C S L S / T H E U N IV E R S IT Y OF T OK YO 153

6 by two-carbon-atom units (β-oxidation) and enter the citric acid cycle as acetyl- CoA, where they are completely oxidized. Amino groups that constitute amino acids are converted to urea, and the remaining carbon skeletons are completely oxidized in glycolysis and the citric acid cycle for use in ATP production. The mitochondrial respiration chain is thus a key component of energy production. The main role of the respiration chain is to efficiently transport H + across the inner membrane to the outside by coupling with the stepwise oxidation and reduction of electron carriers, and the details of the mechanism, including the quinone cycle proposed by Mitchell, have been gradually revealed. Quinones, such as ubiquinone, are lipophilic materials that transfer H + through oxidation and reduction (Fig. 8-1), and are therefore widely used by various organisms in reactions that create high-energy states by transporting H + across the membrane. The quinone cycle is a system that transports two H + ions by the transfer of one electron when electrons are transferred from a ubiquinone to a cytochrome bc1 complex. Such complexes have separate sites for quinone oxidation and reduction, and a reduced ubiquinone bound to an oxidation site releases two H + ions and two electrons. Of these, one electron is transferred to cytochrome c1 via iron-sulfur clusters, and the other reduces another ubiquinone at the quinone reduction site via cytochrome b. Both H + ions are subsequently released to the outside of the inner membrane. With the progress of spectroscopy and structural biology in recent years, the structure of these protein complexes and the behavior of each electron carrier have gradually been clarified, and Japan leads the world in the field of research into cytochrome c oxidase. Figure 8-3 Mitochondrial respiration chain Glycolysis takes place in the cytoplasm. CSLS / THE UNIVERSITY OF TOKYO 154

7 IV. ATP Synthase F-ATP synthase synthesizes ATP by coupling with the H + transport that follows the concentration gradient of H + (Fig. 8-4). This enzyme consists of F0 (the membrane intrinsic site) and F1 (the membrane superficial site). F0 consists of a rotor and a fixed part, between which channel-like paths for H + transport are found. F1 involves ATP synthetic and degradative activities, and is also known as F-ATPase. If F1 is removed from the membrane, electron transport does not lead to ATP synthesis. For this reason, F1 was initially known as the coupling factor for electron transport and ATP synthesis. It was subsequently revealed that an enzyme of the same type as mitochondrial ATP synthase exists in chloroplasts and the cell membrane of eubacteria, and that it is widely involved in ATP synthesis; this enzyme is called the F-type (F stands for factor ). The important point in the coupling of H + transport and ATP synthesis reactions is that, coupling with H + transport, the rotor and the stalk rotate in a clockwise direction, thus supplying energy to F1. F1 does not rotate, and synthesizes ATP using the energy generated by the rotation of the stalk. The phenomenon of ATP synthase rotating like a motor as a reverse reaction has been demonstrated in an elegantly designed experiment (see the Column on p.157). The details regarding the coupling of H + transport and ATP synthesis are not covered here, but the stoichiometry is 3H + /ATP. Figure 8-4 F-ATP synthase This consists of F1 and F0, with the F0 further divided into rotor and stator parts. CSLS / THE UNIVERSITY OF TOKYO 155

8 V. Outline of Photosynthesis *7 Antenna pigment: A pigment that, following the absorption of light energy, does not perform photosynthesis and conveys energy to the reaction center. *8 Photosynthetic reaction center: The core site containing the chlorophyll pigment that performs photosynthetic reactions on being excited by light energy. Photosynthesis consists of reactions in which light energy is absorbed and converted to chemical energy to produce ATP and reducing power (light reactions), and reactions in which carbon dioxide is fixed as organic compounds using the ATP and reducing power (dark reactions). Photosynthesis proceeds in chloroplasts (organelles unique to plant cells), light reactions occur locally in the thylakoid membrane, and dark reactions occur locally in the stroma (an aqueous space in chloroplasts). In light reactions, light energy is absorbed and transported by antenna pigment*7, and electrons are released in the photochemical reaction center*8, thus driving electron transport. The electron transport reaction of photosynthesis is different from the respiration chain in that NADPH as well as ATP is synthesized through H + transport. The reducing power necessary in the series of reactions is obtained from the oxidation of water molecules, and oxygen that is no longer needed is disposed of. As an example, the atmosphere did not contain any oxygen when the earth was created; all the oxygen that currently exists in the air is derived from photosynthesis. In dark reactions, the carbon dioxide fixation reaction and the Calvin cycle (or saccharometabolic cycle) are driven by ATP and NADPH. A carbon dioxide fixation similar to that in dark reactions of photosynthesis also takes place in chemoautotrophic bacteria. VI. Absorption of Light *9 Carotenoid: A fat-soluble material with a long-chain polyene. The material functions as an antenna pigment, and also known as a vitamin A precursor. Antenna pigments that absorb light energy include chlorophyll a, chlorophyll b and carotenoid*9. Blue and red pigments, such as phycobilin, are also known in red algae and cyanobacteria. When visible light excites these pigments, nearly 100% of the excitation energy moves between the pigments and is conveyed to the photosynthetic reaction center. For this purpose, the antenna pigments need to be located close to each other, and are incorporated into proteins and accumulated within the thylakoid membrane. Although plant leaves may appear evenly green, only the thylakoid membrane of chloroplasts, which contain chlorophyll a and b, is in fact green. Other organelles, such as those found in animal cells, have no color. While most terrestrial plants have green leaves, pigments found in algae vary in color (e.g., phycocyanin is blue, phycoerythrin is red and fucoxanthin is brown) because the spectrum of sunlight deviates in water depending on the conditions of absorption and scattering. On CSLS / THE UNIVERSITY OF TOKYO 156

9 the other hand, anthocyan a water-soluble colorful pigment found in plant tissues such as flowers is located in vacuoles, and cannot use absorbed light energy for photosynthesis. Column Demonstration of ATP Synthase Rotation A Japanese research group demonstrated the rotation of ATP synthase through an elegantly designed experiment. In the setup, F1 of ATP synthase was fixed to a glass slide, and an actin filament treated with a fluorescent dye was bound to the stalk, thus making one complex visible under a fluorescence microscope. When ATP was applied to the glass, the complex started rotating as ATP hydrolysis proceeded, and this action was videoobserved. Interestingly, the rotation took place in an anticlockwise direction in three 120-degree steps, indicating that ATP is degraded via three stable intermediates and the energy is transferred to the stalk. It can be considered that ATP synthesis proceeds in the opposite direction. It is believed that, through H + transport, the energy of H + causes ATP synthesis through the rotation of the stalk. Crystal structure analysis of F1 had already shown that an asymmetric stalk-like γ-subunit is located on the bottom surface of the threefold symmetrical sphere containing three α-subunits with ATP synthetic activity, and that these three subunits are in different states. This indicated that the structural change of the α-subunits, which are involved in ATP synthesis, is linked to the positional relationship with the stalk, thus rotating the complex in three 120-degree steps. Column Figure 8-1 Schematic diagram of the video-recorded rotation A modified version of the original diagram created by Toru Hisabori, Associate Professor at Tokyo Institute of Technology. CSLS / THE UNIVERSITY OF TOKYO 157

10 VII. Photochemical Reaction and Electron Transport In the photosynthetic reaction center, a photochemical reaction occurs in which certain chlorophyll a receives excitation energy and releases electrons (similar to the process seen in photoelectric reactions), thus generating chlorophyll a +. This drives electron transport, creating a concentration gradient of H + (as seen in mitochondrial electron transport reactions) based on which ATP is synthesized and NADP + is reduced to produce NADPH. The pigments and electron carriers involved in these reactions are buried in the thylakoid membrane as three types of protein complex, and the mobile electron carriers that connect these complexes are plastoquinone and plastocyanin (Fig. 8-5). Although not described in detail here, these two photochemical complexes are akin to photoelectric semiconductors, and are designed so that electron transport steadily proceeds without the recombination of electrons and positive charges separated in photochemical reactions. The cytochrome b6f complex, on the other hand, transports H +, and has a structure and functionality similar to those of the mitochondrial cytochrome bc1 complex. The basic skeleton of plastoquinone is the same as that of mitochondrial ubiquinone, and the cytochrome b6f complex Figure 8-5 Light reaction system of photosynthesis Q is plastoquinone, and b6 and f are hemes (i.e., cytochrome cofactors). The cytochrome b6f complex and the quinone cycle have the same basic structure as mitochondrial complex III. CSLS / THE UNIVERSITY OF TOKYO 158

11 also transports H + using the similar quinone cycle. Plastocyanin is a small, copperbinding protein, and is one of the major reasons why plants require copper in particular. When copper is deficient, some algae instead use c-type cytochrome, which bears a close resemblance to mitochondrial cytochrome c. In this way, respiration and photosynthesis use very similar electron transport mechanisms, and the systems are therefore believed to have evolved from the same energy metabolic system. When photosynthetic electron transport is considered in terms of oxidationreduction potential, the chlorophyll a + (E = V) generated in photosystem II deprives electrons from stable water molecules using its high oxidizing power, thereby emitting oxygen. On the other hand, the highly reducing electrons (E = -1.4 V) produced in photosystem I reduce NADP + to produce NADPH. In this way, a series of electron transport from water molecules to NADPH is driven by light energy through the tandem connection of the two photosystems. The reason for these systems being connected in tandem is that the two reactions cannot take place simultaneously with the energy of visible light. Indeed, photosynthetic bacteria which have only one photosystem cannot break down water molecules. Besides the electron transport pathway in which the two photosystems are connected in tandem, a cyclic electron transport pathway also exists, in which electrons flow from photosystem I to near the cytochrome b6f complex. This pathway transports only H + for ATP synthesis. One of these two pathways is used in accordance with the environment and the needs at hand. Also in chloroplasts, F-ATP synthase located in the thylakoid membrane works in the coupling of H + transport and ATP synthesis. This enzyme is essentially the same as those found in mitochondria and eubacteria (Fig. 8-4). Equation 8-4 Photosynthetic light reaction can therefore be summarized as follows: 2H2O + 2NADP + + Light energy O2 + 2NADPH + 2H + (+ natp) The number of ATP molecules synthesized here is n, because it changes depending on the combination of the two electron transport pathways mentioned above. The value of n is 3 (NADPH:ATP = 2:3) in the general photosynthetic dark reactions discussed later (Equation 8-5), and is 4 or 5 in C4 photosynthesis (discussed in IX), which requires more ATP molecules. CSLS / THE UNIVERSITY OF TOKYO 159

12 Column Carbon Dioxide Fixation Route (Calvin Cycle) Reactions regulated by light ( ) proceed in one direction. All reactions other than these and the carbon dioxide fixation reactions catalyzed by RuBisCO are reversible ( ). Column Figure 8-2 Simplified diagram of the Calvin cycle VIII. Dark Reaction: Carbon Dioxide Fixation *10 RuBisCO is an abbreviation of the enzyme name ribulose 1,5-bis phosphate carboxylase/oxygenase. Carbon dioxide fixation reactions in photosynthesis are divided into three reaction groups (Column Fig. 8-2). The first is a group of reactions in which ribulose 1,5-bisphosphate incorporates carbon dioxide (CO2) to produce two molecules of phosphoglycerate (top of Fig. 8-6) and is catalyzed by ribulose 1,5-bisphosphate carboxylase/oxygenase (a.k.a. RuBisCO*10). The second is a group of reactions that use ATP and NADPH to produce and release sugar phosphates from the cycle, consequently synthesizing starch and sucrose. The third is a pathway in which various sugar phosphates are connected by equilibrium reactions, regenerating ribulose 5-phosphate a precursor of ribulose 1,5-phosphate. These reactions are known as the Calvin cycle or the reductive pentose phosphate cycle. CSLS / THE UNIVERSITY OF TOKYO 160

13 Figure 8-6 Two reactions catalyzed by RuBisCO The carbon dioxide fixation reaction (top) and the reaction with oxygen (bottom). It should be noted that two molecules of phosphoglycerate are generated in the above reaction. Column Regulation by Coupling and Light Since ATP synthesis reactions involving kinase (phosphotransferase) are coupled with metabolic reactions at a ratio of 1:1, the ATP synthesis efficiency is 100%. On the other hand, ATP synthesis reactions involving F-ATP synthase are indirectly coupled with electron transport via H + transport, making the story a little more complex. As an example, the appearance of chloroplasts differs between daytime and nighttime. During the day, when light is available, chloroplasts are in a state of high energy with the thylakoid membrane maintaining an H + gradient by electron transport, and are ready to supply ATP when it is used. However, at nighttime, it is difficult to maintain this high-energy state. Although the thylakoid membrane has low permeability for H + and is therefore suited to maintaining the concentration gradient, it cannot stop H + from gradually leaking through the membrane during the night. Generally, enzymatic reactions are reversible, and when the concentration gradient of H + is decreased, the gradient is restored by breaking down ATP, which is a waste of energy. In the F-ATP synthase of chloroplasts, therefore, an activity regulation mechanism involving thioredoxin (an oxidation-reduction protein) has evolved. When thioredoxin with cyctein residue is reduced by photosynthetic electron transport, the cyctein residue of a protein in the stalk of ATP synthase is reduced, thereby activating the enzyme; in dark places, on the other hand, the activity of the enzyme is inhibited through the oxidation of the cyctein residue, thereby reducing the wasteful use of energy. The sum of dark reactions after removing the substances regenerated in the reactions outlined above is expressed as follows: Equation 8-5 CO2 + 3ATP + 2NADPH + 2H + (CH2O) + H2O + 2NADP + + 3ADP + 2H3PO4 CSLS / THE UNIVERSITY OF TOKYO 161

14 (CH2O) corresponds to a sugar. This equation indicates that three ATP molecules and two NADPH molecules are needed to fix one molecule of CO2. A distinct characteristic of photosynthetic carbon dioxide fixation reactions is the unique nature of RuBisCO. As its full name suggests, RuBisCO involves carboxylase and oxygenase activity; when the oxygen concentration is high and the carbon dioxide concentration is low, it incorporates O2 instead of CO2 and wastefully uses ribulose 1,5-bisphosphate (bottom of Fig. 8-6). The reactivity of RuBisCO to CO2 is more than 100 times higher than its reactivity to O2; however, since the oxygen concentration in the atmosphere is 500 times higher than that of CO2, the oxygenase activity in chloroplasts cannot be ignored. A byproduct generated in the oxygenase reaction phosphoglycolate is converted back to phosphoglycerate using ATP and NADPH; this pathway is called photorespiration, since CO2 is released during the process. Additionally, since the rate of reactions catalyzed by RuBisCO is much slower than that of other metabolic enzymes, the amount of RuBisCO required is several hundred times more than that of other enzymes that catalyze the reactions seen before and after those catalyzed by RuBisCO. As a result, RuBisCO proteins account for over half the water-soluble proteins found in chloroplasts, making them the most abundant proteins on earth. These intriguing characteristics of RuBisCO reflect the high CO2 concentration and absence of O2 in the atmosphere of primitive earth when phototrophs first appeared. IX. C4 Photosynthesis For many plants, the initial carbon dioxide fixation product in the Calvin cycle is phosphoglycerate (with three carbon atoms), whereas in other plants, carbon dioxide is fixed as malic acid or aspartic acid (with four carbon atoms). Those in the former group, which includes rice, spinach and trees, are called C3 plants, and those in the latter, which includes corn, are known as C4 plants. In C4 plants, photosynthesis occurs in a roundabout way in which phosphoenolpyruvate carboxylase with a bicarbonate ion (HCO3 ) against which oxygen does not compete as a substrate fixes carbon dioxide, other enzymes are then released in the cell, and finally RuBisCO refixes carbon dioxide. Although this C4 photosynthetic reaction uses extra energy (ATP), oxygenase activity is not induced under conditions of low extracellular carbon dioxide concentration, thus allowing efficient photosynthesis. These C4 plants CSLS / THE UNIVERSITY OF TOKYO 162

15 have thrived over tens of millions of years, reducing the carbon dioxide concentration in the atmosphere from higher levels to the current 0.037%. Practical application of C4 photosynthesis has been investigated with the aim of increasing the productivity of crops such as rice. Column Photosynthesis and Changes in Carbon Dioxide Concentration in the Earth s Atmosphere When the first organisms appeared on earth 3.8 billion years ago, it is believed that the carbon dioxide concentration as a ratio of atmospheric pressure was much higher than today. However, this level was greatly reduced by the emergence of phototrophs to just a few percent in the Paleozoic era, and the growth of terrestrial plants along with the emergence of C4 plants further reduced it to today s levels. Additionally, significant short-term changes have occurred in connection with human activity and climate change. As an example, the carbon dioxide level, which stood at 0.027% in the 17th century, rose sharply after the Industrial Revolution to the current figure of 0.037%. X. Topology of Mitochondria and Chloroplasts As outlined in Chapter 5, mitochondria and chloroplasts originate from certain eubacteria*11 that lived symbiotically in eukaryotic cells (endosymbiotic theory: see the Column on p. 104 in Chapter 5). Unlike other organelles, mitochondria and chloroplasts are therefore surrounded by double membranes one inner and one outer *12. The inner membrane of mitochondria forms cristae, through which H + is transported outside the membrane by electron transport in the respiration chain. In chloroplasts, on the other hand, the photosynthetic system (including chlorophyll) does not exist in the inner envelope. Rather, it is found in the thylakoid membrane further inside, and transports H + into this membrane by photosynthetic electron transport. The directions of this transport seem opposite to each other; however, since the matrix of mitochondria and the stroma of chloroplasts are homologous compartments, the two directions are in fact the same when it is *11 Eubacteria: A type of cyanobacteria (a blue-green algae), which are ancestors of chloroplasts. The major candidate ancestors of mitochondria are rickettsiae (intracellular parasitic pathogens and a type of α-proteobacterium), but due to the marked specialization of mitochondria, their ancestral relationship has not been determined. *12 Inner and outer membranes: In chloroplasts, these are also referred to as the inner and outer envelopes. In both chloroplasts and mitochondria, the outer membrane has non-specific carriers and therefore does not block material transport. Endosymbiotic theory suggests that the outer membrane is derived from a host cell, but another well-accepted theory is that the outer membrane is homologous to a special outer membrane of cyanobacteria, which are ancestors of chloroplasts. CSLS / THE UNIVERSITY OF TOKYO 163

16 considered that H + is emitted from them (i.e., they are topologically homologous). The matrix of mitochondria (in which citric-acid-cycle enzymes work) and the stroma of chloroplasts (in which Calvin-cycle enzymes work) are homologous, and H + movement in electron transport is directed outward from these compartments. Coupling with H + transport based on this H + concentration gradient, F-ATP synthase therefore synthesizes ATP in the same compartment as the citric acid cycle and the Calvin cycle. Summary Chapter 8 The production patterns of biological energy are divided into substratelevel phosphorylation (see Chapter 7), oxidative phosphorylation (respiration) and photophosphorylation (photosynthesis) as discussed in this chapter. In respiration and photosynthesis, through the convergence of a series of oxidation-reduction reactions into a common electron transport chain, all energy is replaced with the concentration gradient of H + (electrochemical potential), and common F-ATP synthase synthesizes ATP using the electrochemical potential of H +. The respiration chain, the electron transport chain in photosynthesis and ATP synthase act as protein complexes incorporated into the biological membrane. These complexes react differently from many enzymes in aqueous solution, and have been studied in detail as typical examples of life s complex systems. changes in various reactions (free energy change (ΔG ), changes in oxidation-reduction potential (ΔE ), changes in the electrochemical potential of H + (ΔμH+) and the formation and dissociation of ATP highenergy phosphate bonds) can be understood uniformly as being interconvertible. In respiration and photosynthesis, a mechanism exists to maintain a state of high energy by regulating ATP production and supply in response to ATP utilization and environmental changes. This mechanism is known as homeostasis. Biological systems can function autonomously because a high-energy state is maintained within cells. CSLS / THE UNIVERSITY OF TOKYO 164

17 Problems [1] ATP is known as the currency of biological energy. Explain the reasons for this in connection with the overall processes of intracellular metabolism. [4] Explain how material transport in the mitochondrial inner membrane occurs, and how this mechanism contributes to mitochondrial functions. [2] In eukaryotic cells, H + released by glycolysis and the citric acid cycle produces energy via oxidative phosphorylation in the electron transport chain. Briefly explain the mechanism of ATP synthesis in mitochondria using the terms below (and drawings if desired). Terms: inner membrane, intermembrane space, matrix, F-ATP synthase [3] The oxidation of sugar molecules in cells follows the equation below: C6H12O6 (glucose) + 6O2 6CO2 + 6H2O + energy Indicate whether the following statements are correct, and provide reason for your answers. A) is produced by the oxidation of carbon atoms. B) The water required by cells is largely supplied by this reaction. C) In cells, this reaction is a several-step process. D) Reaction with oxygen occurs in many of the steps of sugar molecule oxidation. E) In some organisms, the reverse reaction occurs. F) Some cells produce CO2 while growing in an O2-free environment. [5] In chloroplasts, the ph of the external solution is increased by photosynthetic electron transport, whereas in mitochondria it is reduced by electron transport. However, in both cases, ATP synthase is arranged facing the stroma or matrix. Explain the topological differences regarding this H + movement. [6] The energy stored in the mitochondrial inner membrane (H + motive force, pmf) is expressed by the equation below: ΔG = 2.3 RT [ph (inside) ph (outside)] + ZFΔψ where R is the gas constant, T is the absolute temperature, Z is the electric charge (e.g., H + = 1), F is the Faraday constant and ψ is the membrane potential. 1) Calculate the value of ΔG when the membrane potential is V (inside - negative), the ph difference between the inside and outside of the membrane is 0.75 and the temperature is 37 C. 2) The free energy change required to synthesize ATP from ADP in vivo is slightly larger than that required in standard conditions. Assuming that the value of this change is 45 kjmol -1, give the minimum number of H + ions that need to be transported to synthesize one ATP molecule. (Answers on p.256) CSLS / THE UNIVERSITY OF TOKYO 165