Chapter 8 Energy - University of Tokyocsls-text.c.u-tokyo.ac.jp/pdf/Chap_08.pdfChapter 8 Energy CSLS...

Chap t e r 8 E n e r g y

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CSLS / THE UN IVERS ITY OF TOKYO 149

Part II

Principles of Individual Cell Function

Chapter 8Energy

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. Energy 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 Energy

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 high-

energy 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

*2ATP’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

*1Fermentation 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.



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˚’ + 2.3RTlog10[ADP][H3PO4] / [ATP]

Electrochemical potential of H+ across the membrane:

Equation 8-2

ΔμH+ = 2.3RTlog10 [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


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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.

I I . 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 high-

energy 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).

I I I . Oxidation-Reduction Reactions and the Respiration Chain

In terms of the utilization of biological energy, the ability to convert energies

*3The 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.

*4Calculation example: To obtain the standard free energy change of the two-electron reaction NADH + H+ + 1/2 O2 ⇄ NAD+ + H2O, with O2 (E˚’ = +0.815 V) and NAD (E˚’ = -0.315 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



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.

The hydrogen (E˚’ = -0.315 V) in NADH releases 218 kJmol-1 of energy by

reacting directly with oxygen (E˚’ = +0.815 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’ = -30.5 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

*5NADH 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.

Figure 8-1Oxidized and reduced forms of ubiquinoneAlso 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.


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synthesis. The standard reduction potential of other major materials are E˚’ =

+0.031 V for succinic acid, E˚’ = +0.045 V for ubiquinone and E˚’ = + 0.235

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

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

*638 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.

Figure 8-2A 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, c1, b, a and a3 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.



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 8-3Mitochondrial respiration chainGlycolysis takes place in the cytoplasm.


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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 8-4F-ATP synthaseThis consists of F1 and F0, with the F0 further divided into rotor and stator parts.



V. Outline of Photosynthesis

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 Energy

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

*7Antenna pigment: A pigment that, following the absorption of light energy, does not perform photosynthesis and conveys energy to the reaction center.

*8Photosynthetic reaction center: The core site containing the chlorophyll pigment that performs photosynthetic reactions on being excited by light energy.

*9Carotenoid: A fat-soluble material with a long-chain polyene. The material functions as an antenna pigment, and also known as a vitamin A precursor.


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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.

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 video-

observed. 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 Demonstration of ATP Synthase Rotation

Column Figure 8-1Schematic diagram of the video-recorded rotationA modified version of the original diagram created by Toru Hisabori, Associate Professor at Tokyo Institute of Technology.



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 8-5Light reaction system of photosynthesisQ 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.


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also transports H+ using the similar quinone cycle. Plastocyanin is a small, copper-

binding 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 oxidation-

reduction potential, the chlorophyll a+ (E˚’ = +1.1 – 1.2 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.



Reactions regulated by light (→) proceed in one direction. All

reactions other than these and the carbon dioxide fixation

reactions catalyzed by RuBisCO are reversible (↔).

VIII. Dark Reaction: Carbon Dioxide Fixation

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.

*10RuBisCO is an abbreviation of the enzyme name ribulose 1,5-bis phosphate carboxylase/oxygenase.

Column Figure 8-2Simplified diagram of the Calvin cycle

Column Carbon Dioxide Fixation Route (Calvin Cycle)


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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 lit t le 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

Figure 8-6 Two reactions catalyzed by RuBisCOThe 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



(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


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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.

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

Column Photosynthesis and Changes in Carbon Dioxide Concentration in the Earth’s Atmosphere

*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.



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.

• The production patterns of biological energy are divided into substrate-

level 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.

• Energy 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 high-

energy 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.

Summary Chapter 8


08


[1]

ATP is known as “the currency of biological energy.” Explain

the reasons for this in connection with the overall processes

of intracellular metabolism.

[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) Energy 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.

[4]

Explain how material transport in the mitochondrial inner

membrane occurs, and how this mechanism contributes to

mitochondrial functions.

[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 0.168 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.

Problems

(Answers on p.256)

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