CITRIC ACID CYCLE AND RESPIRATORY CHAIN
Mitochondria (see textbook of histology and repeat basic knowledge about mitochondrion)
Mitochondria are organelles the size of bacteria. They are found in almost all eukaryotic cells in very
large numbers. Typically, there are about 2000 mitochondria per cell. Mitochondria probably have
envolved from aerobic prokaryotic bacteria living in symbiosis with anaerobic host cells. Mitochondria
have their own DNA = mt DNA and ribosomes.
Structure of mitochondria: (see Fig. 1)
Mitochondria are composed of two membranes: outer membrane (smooth) and inner membrane
(folded) that encloses the matrix space. The folding of the inner membrane results in a very large
surface area. The folds are called as cristae, while the space between the inner and outer membrane is
usually called the intermembrane space.
The proteins of the inner membrane include specific transport proteins ("carriers") for the controlled
exchange of metabolites with the cytoplasm. Outer membrane includes porins which allow small
molecules to be exchanged between cytoplasm and the intermembrane space.
Matrix contains many enzymes of metabolic pathways and mitochondrial genom.
Fig. 1: Structure of mitochondrion.
Figure is found on http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/oxphos.html
Function of mitochondria:
Mitochondria are the biochemical powerhouse of the cell. (i. e. by oxidative degradation of foods, they
produce the majority of ATP required by the cell).
The following processes and pathways are located in the mitochondria:
In matrix space:
● citric acid cycle
● degradation of fatty acids (FA) by β-oxidation
● pyruvate dehydrogenace reaction
● parts of the urea cycle
● parts of the heme synthesis
In the inner membrane:
● respiratory chain coupled to the synthesis of ATP (= oxidative phosphorylation)
The major function of the mitochondria is the uptake of energy - yielding substrates (fatty acids,
pyruvate, carbon skeletons of amino acids) from the cytoplasm and their oxidative degradation to yield
CO2 and H2O coupled to the synthesis of ATP.
Citric acid cycle (CAC)
CAC is known as tricarboxylic acid cycle or Krebs cycle. This cycle was first proposed by Hans Krebs
in 1937. This metabolic pathway is located in the mitochondrial matrix.
Function of CAC:
CAC oxidizes acetyl residues (CH3-CO-) to carbon dioxide (CO2) and extracting energy primarily as the
reduced high energy electron carriers NADH and FADH2. NAD+ and FAD bind reducing equivalents
(= H atoms) and transfer chemical energy from metabolic intermediates to the electron transport chain
(respiratory chain) to form of ATP.
Acetyl-CoA is a key metabolic junction. Sources of acetylcoenzyme A are saccharides, proteins and
triacylglycerols. Acetyl-CoA is mainly derived from the β-oxidation of FA and from the pyruvate
dehydrogenase reaction, both of these processes take place in mitochondrial matrix.
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Reactions of CAC: (see your textbook of biochemistry for more details and Fig. 2)
1. Acetyl residue (2 C) is transferred to a carrier molecule = oxalacetate (4 C). Reaction is catalyzed by
enzyme citrate synthase. Product of this reaction is citrate (6 C).
2. In the next step citrate undergoes isomerization to yield isocitrate by enzyme aconitase (aconitate
hydratase).
3. Next step is the first oxidative one. Hydroxyl group of isocitrate is oxidized to an oxo-group by the
enzyme isocitrate dehydrogenase. At the same time, a carboxyl group is released as CO2, and
2 - oxoglutarate is formed and NADH + H+ is released.
4. The formation of succinyl-CoA also involves an oxidation and a decarboxylation.It is catalyzed by
enzyme 2-oxoglutarate dehydrogenace (a multienzyme complex). NADH + H+ is released.
5. The subsequent cleavage of succinyl-CoA into succinate and coenzyme A is catalyzed by enzyme
succinate-CoA ligase (succinyl-CoA synthetase or succinic thiokinase).
The reaction is strongly exergonic and the energy is bound in phosphoric acid anhydride bond
(= substrate level phosphorylation). ATP is not produced here, but rather guanosine triphosphate
(GTP). GTP can be converted into ATP by enzyme nucleoside diphosphate kinase.
Acetyl residue has been completely oxidized by reactions above. At the same time, the carrier
molecule (= oxaloacetate) has been reduced to succinate.
Three futher reactions of the cycle regenerate oxaloacetate from succinate.
6. Succinate is oxidized to fumarate by enzyme succinate dehydrogenase, FADH2 is released. In
contrast to the other enzymes of the cycle, succinate dehydrogenase is an integral protein of the inner
mitochondrial membrane and this enzyme is a component of a respiratory chain as complex II.
7. Enzyme fumarate hydratase (fumarase) catalyzes the addition of water to the double bond of
fumarate resulting in the production of malate.
8. Malate is oxidized into oxaloacetate by enzyme malate dehydrogenase, once again with the
production of NADH + H+. With this reaction, the cycle is complete and can begin again from the start.
Fig. 2: Scheme of citric acid cycle.
Figure is found on http://web.indstate.edu/thcme/mwking/tca-cycle.html
IDH = isocitrate dehydrogenase, -KGDH = -ketoglutarate dehadrogenase, MDH = malate
dehydrogenase, OAA = oxaloacetate
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Metabolic functions of CAC:
The CAC is the central junction of intermediary metabolism. It has both catabolic and anabolic
functions = it is an amphibolic pathway.
Many catabolic pathways generate CAC intermediates or metabolites, such as pyruvate and acetyl-CoA,
which can be oxidized to CO2 by the cycle. The reducing equivalents thus obtained are used for
oxidative phosphorylation (= synthesis of ATP).
The CAC also supplies important precursors for anabolic pathways:
→ i.e. for the biosynthesis pathways for glucose (oxaloacetate and malate for gluconeogenesis)
→ for the biosynthesis of porphyrins (succinyl-CoA)
→ for the biosynthesis most of the amino acids (2-oxoglutarate, oxaloacetate)
→ for the biosynthesis of fatty acids and isoprenoids (citrate)
The Krebs cycle furnishes the acetyl-CoA required for the biosynthesis of fatty acids in the cytoplasm.
The intermediates of the CAC are present in the mitochondria in very small quantities. They are
constantly regenerated during the oxidation of acetyl residues. But anabolic pathways remove
intermediates of the cycle (e. g. gluconeogenesis). Anaplerotic reactions are processes which supply
intermediates of the cycle if it is necessary.
Examples of anaplerotic reactions:
● degradation of most amino acids produces oxaloacetate, 2-oxoglutarate, fumarate
● pyruvate can be carboxylated into oxaloacetate: Pyr + CO2 + ATP → oxaloacetate + ADP + Pi,
reaction is catalyzed by enzyme pyruvate carboxylase
● succinyl-CoA is produced from propionyl-CoA
Regulation of the CAC:
The most important factor in the regulation of the cycle is the NADH / NAD+ ratio.
Enzymes citrate synthase, α-ketoglutarate dehydrogenase and isocitrate dehydrogenase are all
regulatory steps in the cycle and are each inhibited by an excess of NADH. Abundant energy in the cell
is indicated through high concentrations of NADH. ATP is an allosteric inhibitor of citrate synthase.
The activity of the Krebs cycle is closely linked to the availability of O2, although none of the steps in
pathway directly use O2. Oxygen is required for the electron transport chain to function, which recycles
NADH back to NAD+ and FADH2 to FAD. If the O2 supply to a muscle cell is low, NAD+ and FAD levels
fall, the Krebs cycle cannot proceed forward, and the cell must resort to fermentation to continue making
ATP.
Respiratory chain
Most of the energy of glucose or fatty acids is extracted through oxidation to produce the reduced high
energy electron carriers NADH and FADH2. From there, the energy is transferred next to the electron
transport system (= respiratory chain) associated with the inner mitochondrial membrane.
Components of the respiratory chain:
The respiratory chain consists of four protein complexes (complexes I, II, III, IV) integrated in the inner
mitochondrial membrane and two mobile carrier molecules: ubiquinone (coenzyme Q) and
cytochrome c.
All of the complexes of respiratory chain are made up of polypeptide subunits and contain redox
coenzymes: flavins (FMN and FAD), iron-sulfur centers (Fe/S centers), heme groups.
Function of respiratory chain: (see Fig. 3)
Components of respiratory chain transfer electrons from NADH and FADH2 in series of redox reactions
from carrier to carrier. Oxygen is the final acceptor of electrons at the end of the chain, resulting in
production of water.
Electrons enter the respiratory chain in various different ways. In the oxidation of NADH+H+ by complex
I, in oxidation of FADH2 by complex II.
Complex I (NADH dehydrogenase complex) accepts protons and electrons from NADH + H+ and
transfers them (H and electrons) to ubiquinone (= coenzyme Q) to form ubiquinol.
Complex II (succinate dehydrogenase) accepts reducing equivalents from succinate, again for passage
to coenzyme Q to form ubiquinol.
Ubiqinol transfers electrons from complex I and II on to complex III.
Coenzyme Q is a highly lipophilic molecule and it is quite mobile in the mitochondrial membrane.
Complex III (ubiquinone - cytochrome c oxidoreductase). Cytochrome c then accepts electrons from
complex III for transport to complex IV.
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Complex IV (cytochrome c oxidase) catalyzes the final transfer of electrons to O 2 (O2 is a final electron
acceptor). The strongly basic O2- ion produced by the reduction of O2 immediately binds two H+ and is
converted to water.
Fig. 3: Complexes of respiratory chain
Figure is found on http://web.indstate.edu/thcme/mwking/oxidative-phosphorylation.html
ATP synthesis (see Fig. 4)
Protons (H+) are ejected from mitochondrial matrix into intermembrane space at three points
(complexes I, III and IV) in this sequence of reactions. These protons create an electrochemical
gradient across the inner mitochondrial membrane. Energy of electrochemical gradient (protons) is
used for movement of ATP synthase in the inner mitochondrial membrane. ATP synthase is enzyme
(belongs to hydrolyses) which allows protons to flow back down their concentration gradient across the
membrane and in the process uses energy of the gradient to drive ATP synthesis from ADP and Pi (=
oxidative phosphorylation). This chemiosmotic mechanism was proposed by Peter Mitchell.
The enzyme ATP synthase consists of 2 parts: a proton channel (F0 unit) is integrated into the
membrane and a catalytic unit (F1) – matrix.
Fig. 4: Respiratory chain and oxidative phosphorylation.
Figure is found on http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/oxphos.html
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Result of CAC and respiratory chain:
CAC produces: 2 CO2, 3 NADH + H+, 1 FADH2 and 1 GTP (GTP is converted to ATP) per 1 acetyl
residue.
Energetical result of CAC and respiratory chain and oxidative phosphorylation:
3 NAD+  3 NADH (3 ATP per 1 NAD+)
3 x 3 = 9 ATP
FAD  FADH2 (2 ATP per 1 FAD)
1 x 2 = 2 ATP
GTP  ATP
1 x 1 = 1 ATP
---------Sum
12 ATP per 1 acetyl – CoA
Additional notes:
Uncoupling agents are chemical compounds which allow protons to flow across the mitochondrial
membrane without producing ATP i. e. 2,4-dinitrophenol can transport protons to flow across the inner
mitochondrial membrane, energy is consumed to pump protons out of mitochondria and this energy is
not used in chemical form in ATP. This energy is released as heat.
Proteins can also act as uncoupling agents (UCP = uncoupling proteins) in mitochondria.
Thermogenin, also called the uncoupling protein (UCP-1) is a component of mitochondria of brown
fat. Most mammals (including humans especially newborns) have a type of adipose tissue called brown
fat in which oxidation serves not to produce ATP. The mitochondria of brown fat oxidize fuels (fatty
acids) normally, passing electrons through the respiratory chain to O2. This electron transfer is followed
by proton pumping out of the mitochondrial matrix. This protein (UCP-1) provides a path for protons to
return to the matrix without passing through the ATP synthase. The energy of oxidation is not conserved
by ATP formation but is released as heat, which serves to maintaining the body temperature.
Hibernating mammals (for example: grizzly bears) also have large amounts of brown fat.
Clinical correlation:
Cyanide poisoning
Inhalation of HCN (hydrogen cyanide gas) or ingestion of KCN causes a rapid and extensive inhibition of
mitochondrial electron transport chain at the cytochrome oxidase step. Cytochrome oxidase contains
cytochrome a, a3 which is the terminal step in the respiratory chain. Cyanide (CN-) binds to the Fe3+ in
the heme of the cytochrome a, a3. Mitochondrial respiration and energy production is stopped and cell
death occurs rapidly.
Hypoxia is a lack of O2 in the inner mitochondrial membrane. Hypoxia is caused by i. e. myocardial
infarction or atherosclerosis. Cell death from lack of O2 can be very rapid because synthesis of ATP
(oxidative phosphorylation) is decreased.
References:
Koolman, J., Rőhm, K-H.: Color Atlas of Biochemistry, Thieme, Stuttgart (1996)
Smith, C., Marks, A., D., Lieberman, M.: Marks´ Basic Medical Biochemistry A Clinical Approach,
2nd edition, Lippincott Williams & Wilkins, Baltimore, USA (2005)
Pavla Balínová
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