16.3Respiration: The Citric Acid Cycle and Respiratory Chain


The citric acid cycle

Under aerobic conditions, the pyruvic acid created by glycolysis is converted to acetyl-CoA and is subsequently broken down completely into carbon dioxide and hydrogen by the citric acid cycle*5 (Figure 16-3).

Figure 16-3 Details of citric acid cycle

All enzymes are soluble and localized in the matrix except succinate dehydrogenase. Pyruvate carboxylase catalyzes anaplerotic reactions. Aconitate hydratase is also known as aconitase, and fumarate hydratase as fumarase.


To give a more detailed description of the breakdown, pyruvic acid is converted to acetyl-CoA through the dehydrogenation reaction accompanying decarboxylation and is condensed with oxaloacetic acid to form citric acid, which then enters the citric cycle. Next, the citric acid is isomerized to isocitric acid and then subjected twice to dehydrogenation reactions accompanying decarboxylation to form succinyl-CoA. The latter half of the citric cycle includes GTP synthesis and two hydrogenation reactions, which regenerate oxaloacetic acid. The hydrogen is generated in these reactions in the form of four NADH molecules and one FADH2 molecule*6 . Oxygen molecules are not involved in these reactions, yet these form an important step in aerobic respiration and the citric acid cycle does not take place under anaerobic conditions. In eukaryotic organisms, these reactions occur in the soluble fraction (matrix*7 , see Figure 16-4) of the mitochondria, however, the development of mitochondria is inhibited under anaerobic conditions. Because the 2-oxoglutaric acid of the citric acid cycle is consumed as a precursor for amino acid synthesis, the anaplerotic reaction producing oxaloacetic acid from pyruvic acid is also crucial for uninterrupted progress of the cycle.

*5 Also called the tricarboxylic acid (TCA) cycle because citric acid has 3 carboxylic acid groups, or the Krebs cycle after its discoverer.

*6 Flavin adenine dinucleotide (reduced form), a coenzyme in redox reactions. See Figure 16-6 for the structural formula. FAD+2e-+2H+ ⇋ FADH2

*7 Nicotinamide adenine dinucleotide (reduced form), a coenzyme in redox reactions. See Selection 2 of Chapter 4 (Figure 4-1) for the structural formula.

Figure 16-4 Mitochondrion (top), and respiration chain and protein complex of ATP synthase in the inner membrane of mitochondria (bottom)

Q, which indicates 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. FMN is a flavin mononucleotide, and FAD is a flavin adenine dinucleotide. Reduced Q generated by Complex II also passes electrons on to Complex III via the quinine cycle. Since the mechanism of H+ transport is not fully understood, the number of H+ ions transported by each complex is expressed as n. Overall, approximately three molecules of ATP are synthesized through the complete oxidation of one NADH molecule, and the sum of n is approximately 9 in this case.


High-energy state of the H+ electrochemical gradient

Column Figure 16-1 H+ electrochemical gradient formed across closed membrane vesicle

The H+ electrochemical gradient (also known as electrochemical potential, ΔμH+) formed by the transport of H+ outside a membrane vesicle can be expressed by the following equation:

where R is the gas constant, T is the absolute temperature, F is the Faraday constant, and ΔΦ is the membrane potential.

Terms of the equation, log10[H+]in and log10[H+]out, can be converted to the pH inside and outside the membrane, respectively, whereas ΔΦ indicates the membrane potential. In other words, membrane potential formed by the transport of ions other than H+ also contribute to the H+ electrochemical gradient (in Column Figure 16-1, Na+ also contributes to membrane potential). This electrochemical gradient is used for ATP synthesis as a high-energy state.

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Oxidative phosphorylation and chemiosmotic theory

Although glucose is completely broken down into carbon dioxide and hydrogen (ten NADH molecules and two FADH2 molecules) by glycolysis and through the citric acid cycle, a mere four ATP molecules per glucose molecule are produced during these processes. The number of GTP molecules synthesized in the citric acid cycle is equivalent to two ATP. However, by coupling to a series of reactions in which hydrogen molecules generated in the process (which are actually high-energy electrons) are completely oxidized by oxygen to form water molecules, a total of 38 ATP molecules are produced*8 . This series of reactions is called oxidative phosphorylation. The following equation shows changes in free energy during the complete oxidization of glucose.

C6H12O6 + 6O2→6CO2 + 6H2O
ΔG°′= -2,870 kJ mol-1

*8 Example of calculation: In the reaction NADH + H+ + 1/2 O2 ⇌ NAD+ + H2O

standard changes in free energy are ΔE°′ = +0.815 V (O2) and ΔE°′ = –0.315 V (NAD+). Considering that it is a 2-electron reaction, substitute n = 2 into equation 16-1 on page 197, then ΔG°′ = –2 × 96.5 × [0.815 – (–0.35)] = 218 kJ mol-1, where the Faraday constant is 96.5 kJ∙V-1∙mol-1.

In the 1950s, researchers initially looked for metabolic intermediates with high-energy phosphate bonds based on the assumption that, as in glycolysis, kinases (enzymes that transfer phosphate groups to or from ATP) were involved in reactions that synthesize large amounts of ATP; however, they found none. Later, the mystery surrounding ATP synthesis was solved by the chemiosmotic theory proposed in 1961 by Peter. Mitchell, who was involved in the investigation of active transport. Essentially, active transport uses ATP energy to transfer H+ ions (and other molecules) across the membrane, against the concentration gradient across the membrane. However, according to Mitchell’s theory, ATP is synthesized using the electrochemical gradient of transported H+ coupled with electron transport reactions. In other words, the H+ electrochemical gradient is a high-energy state that is interconvertible with ATP synthesis. However, there are cases of work done without ATP, a good example of which is the flagellar movement of bacteria (see column selection 1 Chapter 17)

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Redox reaction and redox potential

The hydrogen energy extracted by the dehydrogenation reaction in glycolysis and the citric acid cycle is equivalent to the electron energy accumulated in coenzymes (NADH and FADH2). Given that coenzymes are shared in different redox reactions of numerous metabolic reactions, it can be said that a common high-energy state (i.e., electrons) is being removed from various substances through metabolism. Similar coenzymes are also used in photosynthesis, as discussed later.

Electron energy transported by various metabolic substances and coenzymes can be standardized and understood as redox potential. Because electrons have negative charges, the lower the potential, the larger the reducing power will be*9 . This means that electrons are donated or accepted spontaneously from substances with low potential to those with high potential. Changes in free energy released in the reaction are proportionate to the difference in the standard redox potential between the two reactants (electron donor A and the electron acceptor B). This relationship is expressed by equation 16-1.

ΔG°′= -nF (E°′A -E°′B) Equation 16-1
(where n is the number of electrons donated or accepted, and F is the Faraday constant.)

The direct reaction of NADH hydrogen (E°′ = –0.315 V) with oxygen (E°′ = +0.815 V) will release 218 kJ∙mol-1 energy*8.

*9 For instance, the standard redox potential of an electron is –0.42 V for hydrogen molecules and +0.815 V for water molecules. This means that the reducing power of an electron varies among substances and is much larger for hydrogen than for water.

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Respiratory chain

High-energy electrons (i.e., electrons with low redox potential) from NADH slowly release energy via about 20 types of electron carriers, and eventually react with oxygen under moderate conditions. This process is called the respiratory chain. Of these electron carriers, the low-molecular weight molecule ubiquinone and small protein cytochrome c act as mobile electron carriers, whereas the remaining carriers are categorized in four types of protein complexes (I–IV) as cofactors. Cofactors that bind strongly to proteins are also called prosthetic groups. Focusing on the redox of substrates, the four protein complexes are named as follows: Complex I or NADH dehydrogenase, Complex II or succinate dehydrogenase, Complex III or cytochrome bc1 complex (also called ubiquinone/cytochrome c oxidoreductase), and Complex IV or cytochrome c oxidase (Figure 16-4).

Although free-energy changes of NADH (ΔG°′ = –218 kJ∙mol-1) during the complete oxidation are equivalent to those of about 7 molecules of ATP (ΔG°′ = 30.5 kJ∙mol-1), only 3 ATP molecules are actually produced. On the other hand, the electron (FADH2) that can be removed from succinic acid has lower energy than NADH, and thus, 2 ATP molecules are produced. The electron energy of these substances can be estimated based on their redox potential* The total number of ATP molecules produced per glucose molecule is 38, i.e., 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*11 .

*10 The redox potentials of representative substances 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, in the succinate→ubiquinone reaction catalyzed by complex II (succinate dehydrogenase), there is more or less no free energy change. However, in the succinate→oxygen electron exchange, approx. 69% of the energy produced in NADH→oxygen can be obtained.

*11 A total of 38 ATP molecules are estimated to be synthesized per glucose molecule: 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. However, recent studies on the H+ transport mechanism and ATP synthases, described later, have revealed that these figures are not given definite figures but approximates. Eukaryotic organisms

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Relationship between the respiratory chain and metabolic pathways

Figure 16-5 Relation between mitochondrial respiration chain and metabolic pathways

Glycolysis takes place in the cytoplasm as well as in some of the basic metabolic pathways in Figure 4-3 of Chapter 4.

Aerobic respiration in mitochondria can use the NADH and FADH2 generated by the degradation of amino acids and fatty acids for energy production, in addition to those produced from glucose breakdown (Figure 16-5). Fatty acids are broken down by removing two carbons at a time through dehydrogenation (β-oxidation) and enter the citric acid cycle as acetyl-CoA, where they are completely oxidized to carbon dioxide. The amino groups that constitute amino acids are converted to urea, whereas the remaining carbon skeletons are completely oxidized via glycolysis and the citric acid cycle and used for ATP production. In this way, the mitochondrial respiratory chain serves as the key component for energy production.

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Respiratory chain and H+ transport coupling

The main role of the respiratory chain is to efficiently transport H+ across the inner membrane by coupling to the oxidation-reduction steps mediated by electron carriers. Research on the three-dimensional structures of the proteins involved in this respiratory chain is gradually revealing the molecular mechanism of electron transport and mechanism of H+ transport. To describe the mechanism of H+ transport by the quinone cycle proposed by Mitchell: quinones, such as ubiquinone, are lipophilic coenzymes that transfer H+ through oxidation and reduction (Figure 16-6); thus, they carry out important reactions that create high-energy states by transporting H+ across the membrane. The quinone cycle is a mechanism that transports two H+ ions by the transfer of one electron when electrons are transferred from a ubiquinone molecule to the cytochrome bc1 complex. Cytochrome bc1 complexes have separate sites used for quinone oxidation and reduction, and a reduced ubiquinone bound to an oxidation site releases two H+ ions and two electrons. Of these, 1 electron is transferred to cytochrome c1 via iron-sulfur clusters, and the other reduces another ubiquinone at the quinone reduction site via cytochrome b. As a result, when 1 electron flows on to the acceptor, both H+ ions are released to the intermembrane space. With improvements in spectroscopy and structural biology in recent years, the structure of these protein complexes and the behavior of each electron carrier are gradually being elucidated. In particular, Japan leads the world in the field of research on cytochrome c oxidase.

consume energy equivalent to two ATP molecules during the transportation from cytoplasm to mitochondria of the two NADH molecules generated in glycolysis, making the ATP yield 36.

Figure 16-6 Structure and reactions of important coenzymes related to metabolic reactions

Coenzyme A (CoA) is a coenzyme that activates the acyl group. It should be noted that it has a nucleotide structure in addition to the activating site (-SH). Flavin adenine dinucleotide (FAD), redox coenzyme, and ubiquinone are also known as coenzyme Q. It is unique that they are a lipid soluble compound that can give and receive electrons and H+ separately. They form an H+ gradient by transporting H+ across the membrane.

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ATP synthase

F-ATP synthase synthesizes ATP by coupling to H+ transport down the H+ electrochemical gradient (Figure 16-7). This enzyme consists of F0 (region within the membrane) and F1 (region above the membrane, inside the matrix of the mitochondria). F0 consists of a rotor and a fixed part, between which there are channel-like paths for H+ transport. F1 has ATP synthetic and degradative activities, and it is also known as F-ATPase. If F1 is removed from the membrane, the membrane will not be able to maintain the H+ gradient. Thus, ATP synthesis cannot occur even if electrons are transported. For this reason, F1 was initially known as the coupling factor for electron transport and ATP synthesis. It was later revealed that an enzyme of the same type as mitochondrial ATP synthase exists in chloroplasts and in the cell membranes of eubacteria, and that it is widely involved in ATP synthesis. This enzyme is called F-type, with F standing for “factor.”

Figure 16-7 F-type ATP synthase

F0 consists of the rotating rotor (in brown, residue binding to H+ is indicated by “○”) and the stator (in green), which does not rotate.

One key point in the coupling of H+ transport and the ATP synthesis reactions is that in coupling with H+ transport, the rotor and the stalk rotate in a clockwise direction to supply energy to F1. This means that F1 does not rotate, and it synthesizes ATP using the energy generated by the rotation of the stalk. It has been demonstrated in an elegantly designed experiment that part of the ATP synthase rotates like a motor in the anticlockwise direction in a reverse reaction (hydrolysis of ATP) (see Column at the bottom). Details on the coupling of H+ transport and ATP synthesis are not covered here, but in the past, it used to be thought that one ATP molecule is synthesized by the transport of about three H+ ions. However, in recent years, this has been found to differ depending on the organism*12 .

*12 Stoichiometry analyzing how many H+ ions are transported to synthesize ATP is important for investigating actual cells. The three-dimensional structure of ATP synthase suggests that three active centers of the F1 part synthesize one ATP molecule each during 1 revolution of the rotor. As a rotor subunit binds with one H+, there is a need to transport H+ ions equivalent to the number of subunits (n) comprising the rotor. If n = 9, then n/3 ATPs = 3, which is the experimental result (approx. 3) in the text. However, analysis of the actual structure suggested “n” varying between 10 and 15 depending on the organism’s species.


Demonstration of ATP synthase rotation

Column Figure 16-2 Schematic diagram of video-recorded rotation

It should be noted that the diagram is upside down, compared to Figure 16-7. Adapted from the original diagram created by Toru Hisabori, Professor at Tokyo Institute of Technology.

A Japanese research group demonstrated the rotation of ATP synthase through an elegantly designed experiment. In the experiment, F1 of ATP synthase was fixed on a glass slide, and an actin filament treated with a fluorescent dye was bound to the stalk, thus making the 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 filmed. Interestingly, the complex only rotated in the counterclockwise direction in three 120-degree steps, indicating that ATP is degraded via three stable intermediates, and the energy released from this degradation is transferred to the stalk. It can be considered that ATP synthesis proceeds in the opposite direction. It is believed that when the stalk rotates by coupling to the H+ transport by F0, the rotational energy accumulated as distortion between F0 and F1 (which does not rotate) causes ATP synthesis. Crystal structure analysis of F1 showed that an asymmetric stalk-like γ-subunit is inserted into the base of the three-fold symmetrical sphere containing three α-subunits with ATP synthetic activity, and that these three α-subunits are in different states. This indicates that structural changes in the α-subunits, which are involved in ATP synthesis, are linked to the positional relationship with the stalk, thus causing the stalk to rotate in three 120-degree steps.

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