11. Mitochondrial oxidation.

Electron transfer chain: Molecular structure, Coupling of

Electron transfer Chain and Oxidative Phosphorylation, Regulation.
The mitochondrion is a “power station of the cell”, most of the cell energy is produced there. The catabolic
processes such as oxidative decarboxylation, Krebs cycle, β -oxidation take place in mitochondria. The
electron transfer chain collects the reducing equivalents (H) from all the processes mentioned above and
direct them to their final reaction with oxygen. The obtained energy is captured by ATP.
Mitochondrion is an ellipse like organelle 0.5 - 1µm long. It contains two membranes: the outer permeable
for almost all metabolites, and the inner, which is not permeable for hydrophilic substances or metabolites.
(It is permeable for O 2 , H 2 O, CO 2 ). The inner membrane contains a large number of invagination - Cristi
(Cristae). Their quantity depends on cellular metabolism specificity. For example liver mitochondria has
undeveloped Cristi, whereas cardiomuscle mitochondria has max developed Cristi - and extremely aerobic
metabolism.
1. An electron transfer/transport chain (ETC) is a series of enzymes and coenzymes
that transfer electrons from electron donors to electron acceptors via redox reactions, and couples this
electron transfer with the transfer of protons (H+ ions) across an inner mitochondrial membrane. This
creates an electrochemical proton gradient that drives ATP synthesis, or the generation chemical energy in
the form of ATP.
Electron transfer chain. The electron transfer chain carries out the main biologic redox-reaction on
stages, and storages the energy in ATP. All the components of the electron transfer chain are found in the
inner mitochondrial membrane. The electron transport chain consists of four independent complexes. Their
order approximates the increase in standard reduction potential. The transfer of electrons from reductant
to oxidant (from NADH 2 to O 2 ) is exergonic, it proceeds with a decrease in free energy. Part of the free
energy of electron transport (oxidation) is used for ATP synthesis by process of oxidative phosphorylation.
NADH 2 collects the hydrogen atoms (electrons) from different substrates and donates its reducing
equivalents to complex I - NADH dehydrogenase.
Complex I consists of 26 polipeptide chains and contains FMN, FeS iron-sulfur complexes. Its molecular
weight is 860 kD. Complex I transfers the reducing equivalents to the CoQ, to produce CoQH 2 . The latter
transports the electrons to complex III, and then via cytochrome C to complex IV - cytochrome oxidase.
Cytochrome oxidase transfers the electrons to an oxygen molecule producing oxygen anion. The redox
systems are ordered in such a way, that each system can accept electrons from the previous system and to
donate them to the next system. Redox systems with higher redox potential oxidate those with lower
potential.

Substrates possessing higher redox potential enter the electron transfer chain through flavoprotein
dehydrogenase - complex II. It transports the electrons to coenzyme Q. Complex II contains FAD and Fe-S
(iron-sulfur) complexes. Complex III is Co-Q - cytochrome C reductase. It consist of cytochromes b and c1,
and 2Fe-2S iron sulfur clusters.
The cytochrom C is a soluble small (12 kD) hemoprotein. It carries electrons to complex IV - cytochrome
oxidase.
Cytochrome oxidase contains two cytochromes: a and a3 and two copper ions. The cytochrome oxidase
reaction accounts for more than 90% of all oxygen consumed by humans. The reduction of oxygen by
cytochrome oxidase accounts for production of metabolic water of about 300 ml per day.
The enzymes or complexes of electron transfer chain are distributed in anisothropic way: the first, third and
fourth complexes are incorporated transmembraneously. The second complex is distributed on the inner
side.
The connection between the first and the third complexes is performed by Co-Q, lipophylic molecule which
moves easily inside the inner membrane.
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The hydrogen transport between complexes II and III is mediated in the same way, by CoQ. The soluble
cyt.C transports electrons from third complex to the fourth. It is the only soluble and non-fixed cytochrome
in electron transfer chain.
The transfer of hydrogen atoms and electrons
proceeds with energy release. These energy is
released in portions and sustains a synthesis of
ATP, which occurs in complex V - ATP-synthase.
Both processes are coupled: oxidation insures the
energy for ATP synthesis.
2. ATP-synthase structure. ATP - synthase is an
aggregate of polypeptides that form a mushroom-
shaped molecule on the inner side of the inner
mitochondrial membrane.
The enzyme abbreviation is FoF1 - ATP -ase. Fo is
oligomycin sensitive subunit. It is embedded in
the membrane and functions as a proton pump. F1
subunit is composed of several polypeptide chains,
designated αβδεγ. It forms a mushroom top.
When separated, F1 possesses ATP hydrolysing activity, i.e. ATP-ase activity. The composition of mammalian
mitochondrial enzyme is α 3 β3γδε1-2. The triplet of α3β3 subunits provides three ATP binding sites per
molecule.
3. Inhibition of electron transfer chain.
The first complex is inhibited by rotenone, amytal and barbiturates. Rotenone is an exotic poison which was
used by Indians in fishing, and barbiturates are in sleeping pill-soporific.
Inhibitors accept electrons and stop their flow through electron transfer chain. The consumption of oxygen
stops. If the electron transfer chain is procured with
substrates for complex II, its function will be
restored.
Complex II is inhibited by carboxin and TTFA [4,4,4-
trifluoro-1-(2-thienyl)-1,3-butanedione].
The third complex is inhibited by the antibiotic
antimycin. The block in vitro may be recovered by
vit.C which serves as electron donor for complex IV -
cytochrome oxidase. Cytochrome oxidase is inhibited
by (CN-) cyanides, carbonmonooxide and by azides.
All these compounds bind (Fe) ferrous ions in the
cytochrome and prevent electron transfer.
The ATP - synthase is inhibited by the antibiotic
oligomycin. It completely inhibits oxidative
phosphorylation. Atractyloside is another antibiotic
which inhibits ADP/ATP transport through
mitochondrial membrane.
The ATP production. When electron transfer chain
works from the first complex i.e. NADH -DH, it produces 3 ATP molecules on transferring of 2 hydrogen
atoms or electrons through electron transfer chain.
In case it uses the other “entrance” - complex II,
and starts from FADH2, the electron transfer chain
produces 2ATP molecules only. When electrons
enter the complex IV, the electron transfer chain
produces only one ATP molecule (physiological
substrates for complex IV are not known).
The difference in ATP production may be explained
by the difference in total redox potential between
substrate and oxygen pairs.
For NADH and O2 pair the redox potential equals 1.24
V (Ered. = + 1.24 V).
For FADH2 and O2 pair the redox potential equals
1.06 (Ered. = +1.06).
For cyt.oxidase and O2 pair the redox potential -
Ered. = 0.60 v.
In fact, the number of ATP molecules formed from ADP and Pi per electron pair transported to an atom of
oxygen ( 1/2 O2 ) is termed P /O ratio. With NADH as electron donor the P/O ratio is between 3 and 2.
Current data suggest that it is 2.5.

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With succinate as electron donor, P/O ratio is 1.5, that is from a mole of oxidated succinate 1.5 moles ATP
are produced.

4 Coupling of Electron transfer Chain and Oxidative Phosphorylation.

4.1. Mitchell’s chemiosmotic theory suggests that the exergonic reactions of electron transport provide
energy for the translocation of protons from the mitochondrial matrix through the inner mitochondrial
membrane to the exterior. Electron transport generates and maintains an electrochemical potential. The
movement of charged proton from the mitochondrial matrix establishes both a concentration gradient and an
electrical potential on the inner membrane. Once an electrochemical potential is established it is coupled to
ATP synthesis by ATP-synthase.
4.2 The concept of Mitchell’s theory is that hydrogen atoms: protons and electrons are transported from the
mitochondrial interior (matrix) and protons are released at the exterior surface. Electrons are transported
back to the internal part of the membrane.
The first complex takes hydrogen atoms from NADH, transports them to the outer membrane and
splits them into protons and electrons. Protons are released into the intermembrane matrix, the electrons
are transferred by 2Fe-2S (iron–sulfur) complex to the inner surface of the inner mitochondrial membrane,
where two protons are bound from the matrix and hydrogen atoms are formed. They are transported by CoQ
to the outer surface.
Reduced CoQ translocates the protons outside and transfers the electrons to the third complex -
cytochrome bc1. The electrons are transferred by cytochrome bc1 and two protons are taken from
mitochondrial matrix again.
They are transported by Cit. C to cytochrome oxidase.
The final electron acceptor is oxygen molecule, it is converted into oxygen ion. Thus the
electrochemical potential is created: ∆µH+.
Electrochemical potential consists of electrical potential: positively charged outer side and
negatively charged inner side of the inner mitochondrial membrane, and chemical potential - created by
different proton concentration in side and out side. Now it is known that complex I translocates 3-4 protons,
complex III translocates 4 protons, and complex IV translocates two protons from mitochondrial matrix
outside.
4.2 The electrochemical potential produced across the membrane is used to drive a membrane located ATP-
synthase to form ATP from ADP and Pi.
The mechanism of coupling of proton translocation from out side to in side and ATP synthesis is conjectural.
Protons passed from out side through ATP-synthase complex. Fo- domain function as proton channel. Protons
pass through Fo and bind F1 these lead to conformational change in ATP - synthase and F1 “activation”.
Enzyme obtains energized conformation which insures ATP synthesis from ADP and Pi. After ATP synthesis an
other conformational change occurs to produce the initial state. Protons in mitochondria bind oxygen anion
or hydroxyl anion to produce water.
Efficiency coefficient is about 40% 3 x 30.5 / 230 kJ/mol. But at real cellular concentration of substrates it
is about 70% . The efficiency of oxidative phosphorylation is measured by P/O ratio.
The total quantity of ATP in human organism is low, but the quantity necessary for vita minima 6.500 - 9.300
kJ/mol is significant. Therefore it is assumed that ATP synthesis
occurs all the time - constantly. Different cells compartments Energy [ATP] + 0.5 [ADP]
maintain the concentrations/quantity of ATP+ADP+AMP and =
NADH/NAD. The relation of adenine nucleotide is named energy charge [ATP] + [ADP] + [AMP]
charge.
The energy charge plays an important role in the regulation
of electron transfer chain and of catabolic processes. The
energy charge reflects the molar fraction of the total
adenine nucleotide pool, which contains energy rich bounds
(The factor 0.5 reflects the reaction ADP → 0.5 ATP + 0.5
AMP). The range of possible values for the energy charge
changes from 0 ( all AMP ) to 1 ( all ATP).
Under physiological conditions in all organisms and cells the
energy charge value hovers around 0.85. Under conditions
of low energy charge the catabolism is favored to provide
chemical energy charge. Maintenance of the energy charge
is an important principle in bioenergetics and biochemistry.
High energy charge stopped phosphorylation, when EC<
0.85, oxidative phosphorylation is activated. The relation ATP NADH cyt. Fe2+
of NADH/NAD in mitochondria is 0.1-0.2, in cytoplasma is ADP NAD cyt. Fe3+
0.001.
If the relation is higher than 0.1-0.2, ATP can act as an
allosteric inhibitor of cytochrome oxidase by binding to

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complex IV and cyt c the electron transfer chain is active, if its relation lower, the electron transfer chain
is regulated by feedback control. At high ATP levels, ADP is insufficient for ATP-ase, the phosphorylation
stops. High levels of transmembrane potential impede/block up the oxidation of NADH in electron transfer
chain.
Substrate oxidation in the TCA cycle activates the respiratory chain, leading to the generation of ATP, which
is subsequently translocated to the cytosol.
ANT, adenine nucleotide translocator; GDH, glutamate dehydrogenase; Glu, glutamate; KG, ketoglutarate;
OAA, oxaloacetate; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; Suc-CoA, succinyl-CoA; SDH,
succinate dehydrogenase; TCA, tricarboxylic acid; UCP2, uncoupling protein 2.

Uncouplers of oxidative phosphorylation.

Uncouplers are compounds that increase the permeability of inner mitochondrial membranes for protons.
They are usually lipophylic acids, which can permeate the membrane and dissociate a proton inside.
Historically first uncoupler is 2,4-DNF-dinitrophenol-phosphate.
Uncouplers reduce the electrochemical potential, the oxidation proceeds, but without phosphorylation.
Physiological uncouplers: free fatty acid, thyronines,
bilirubin.
The hyper function of thyroid gland leads to
hyperproduction of thyronines, which activate
metabolism and uncouple the oxidative
phosphorylation. These leads to increased heat
production, perspiration (sweat) and reduced ATP
production.
Prolonged ATP deficiency leads to cardiomuscle
atrophy/dystrophy.
Uncoupling of oxidative phosphorylation is one of the
thermogenesis mechanisms.
Cardio-vascular diseases. Decreasing in oxygen
consumption leads to decreasing of cellular activity and reducing of ATP level . It is pivotal for
cardiomusculature, as it lacks ATP for ion pumps. Changes in ions contain leads to its swelling and death.
Transport of metabolites per mitochondrial membrane.
The relative impermeability of the inner mitochondrial
OH
membrane necessitates exchange transporters. the inner H3PO4
mitochondrial membrane is permeable only for intermembrane Mitochondrial
hydrophobic molecules and small uncharged molecules space
ATP
matrix
ADP
such as H2O, CO2, O2, NH3 and ketone bodies. However
OH
most of the metabolites require specific carrier-
pyruvate
transporter.
1) The adenine nucleotide transporter allows the malate
malate
exchange of ATP and ADP, but not AMP. ATP is citarte
synthesized in mitochondria and widely used in malate
cytoplasm. The ATP/ADP carrier is inhibited by α-ketoglutarate
atractyloside.
H3PO4
2)The transport of di and tricarboxylate anions is closely
malate
related to that of phosphate. Malate may be antiported
by citarte or α-ketoglutarate as well. Phosphate is pyruvate
replaced by hydroxyl. Pyruvate is cotransported with H+
proton, and antiported with hydroxyl.

OXPHOS diseases. Clinical diseases involving components of oxidative phosphorylation (referred to as

OXPHOS diseases) are among the most commonly encountered degenerative diseases.
Examples of OXPHOS Diseases Arising from mtDNA Mutations
1. Leigh disease - mtDNA missense mutations in OXPHOS
Mean age of onset, 1.5–5 years; clinical manifestations (subacute necrotizing included optic atrophy,
opththalmoplegia, nystagmus,.encephaolpathy) respiratory abnormalities, ataxia, hypotonia, spasticity, and
developmental delay or regression.
7–20 % of cases have mutations in Fo subunits of F0-F1-ATPase polypeptides
2.Kearns-Sayre syndrome - mtDNA rearrangements in which genes are deleted or duplicated
Onset before 20 years of age, characterized by opthalmoplegia, atypical retinitis pigmentosa, mitochondrial
myopathy, and one of the following:
cardiac conduction defect, cerebellar syndrome, or elevated CSF proteins.
Deletion of contiguous segments of tRNA and OXPHOS polypeptides, or duplication mutations consisting of
tandemly arranged normal mtDNA and an mtDNA with a deletion mutation.

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