This is a detailed overview of the electron transport chain and oxidative phosphorylation, both of which take place in the mitochondria.

1. SYED MUHAMMAD KHAN (BS HONS. ZOOLOGY)
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Electron Transport Chain & Oxidative
Phosphorylation
Oxidative phosphorylation is the production of ATP using the energy of oxidation
obtained from electron transport. All of the oxidative steps in the degradation of
carbohydrates, fats, and amino acids converge at this final step of cellular
respiration, in which energy of oxidation derives the synthesis of ATP. It occurs in the
mitochondria of eukaryotes and involves the reduction of oxygen to water with
electrons donated by NADH and FADH2.
Another similar process is photophosphorylation which is how photosynthetic
organisms capture the energy of sunlight; and harness it to make ATP. It occurs in
the chloroplast. Oxidative phosphorylation and photophosphorylation are similar in
three aspects:
1. Both involve the flow of electrons through a chain of membrane-bound
carriers.
2. The free energy made available by this downhill (exergonic) electron flow is
coupled to the uphill transport of protons across a proton impermeable
membrane, conserving the free energy of fuel oxidation as a transmembrane
electrochemical potential.
3. The trans-membrane flow of protons down their conc. gradient through
specific protein channels provides the free energy for synthesis of ATP,
catalyzed by a membrane protein complex (ATP synthase) that couples the
proton flow to the phosphorylation of ADP.
Mitochondria
Mitochondria have two membranes, the outer membrane is permeable to small
molecules and ions. These move freely across the trans-membrane channels formed
by a family of integral membrane proteins called porins. The inner membrane is
impermeable to most small molecules and ions including protons, the only species
that cross this membrane do so through specific transporters.
The inner membrane bears the components of the respiratory chain and the ATP
synthase. The mitochondrial matrix enclosed by the inner membrane contains the
pyruvate dehydrogenase complex and the enzymes of the TCA cycle, the fatty acid
beta-oxidation pathway, and the AA oxidation (except glycolysis).
The inner membrane segregates the intermediates and enzymes of the cytosolic
metabolic pathways from those occurring in the matrix. Some specific transporters
carry the pyruvate, fatty acids, amino acids, etc. into the matrix for access to the

2. SYED MUHAMMAD KHAN (BS HONS. ZOOLOGY)
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machinery to the citric acid cycle. ADP and Pi are also specifically transported into
the matrix and ATP is transported out.
Universal Electron Acceptors
Oxidative phosphorylation begins with the entry of electrons into the respiratory
chain. Most of the electrons arise from the action of dehydrogenases that collect the
electrons from the catabolic pathways and funnel them to the universal electron
acceptors, i.e. NAD, NADP, FMN, FAD, etc.
1. Nicotinamide Nucleotides
Nicotinamide nucleotide – linked dehydrogenases catalyze reversible reactions
of the following general types:
Reduced Substrate + NAD+ ⇋ Oxidized Substrate + NADH + H+ (1)
Reduced Substrate + NADP+ ⇋ Oxidized Substrate + NADPH + H+ (2)
NAD-linked dehydrogenases remove two hydrogen atoms from their substrates. One
of the electrons is transferred as a hydride ion: H- to NAD, the other is released as
H+ (proton) in the medium. NADH and NADPH are water-soluble electron carriers
that associate reversibly with dehydrogenases. NADH carries electrons from
catabolic reactions to their point of entry into the respiratory chain. NADPH supplies
electrons to anabolic reactions. Cells maintain separate pools of NADPH and NADH,
with different redox potentials. Neither NADPH nor NADH can cross the inner
mitochondrial membrane, but the electrons they carry can be shuttled across the
indirectly.
2. Flavoproteins
Flavoproteins – proteins that contain a very tightly, covalently bound nucleic acid
derivative of riboflavin, i.e. FAD or FMN. FAD can be reduced to FADH2; it accepts
two hydrogen atoms, i.e. a net gain of two electrons. FAD – quinone (oxidized) form
accepts two electrons and two protons to become FADH2 – hydroquinone
(reduced) from. FADH2 can then be oxidized to FADH – semiquinone (semi-
reduced) form by donating one electron and one proton. The semiquinone is then
oxidized once more by losing an electron and a proton and is returned to the initial
quinone form (FAD). Electron transfer occurs because flavoproteins have a higher
reduction potential than the compound oxidized. The standard reduction potential of
a flavin nucleotide, unlike that of a NAD or NADPH, depends on the protein with
which it is associated and not on the isolated FAD or FMN.
Membrane-Bound Carriers
Mitochondrial respiratory chain consists of a series of sequentially acting electron
carriers, most of which are integral proteins with prosthetic groups capable of
accepting and donating either one or two electrons. Three types of electron transfers

3. SYED MUHAMMAD KHAN (BS HONS. ZOOLOGY)
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occur in oxidative phosphorylation: (1) direct transfer of electrons; (2) transfer as a
hydrogen atom; (3) transfer as a hydride ion which bears two electrons. In addition to
NAD and flavoproteins, three other types of electron carrying molecules function in
the respiratory chain: A hydrophobic quinone (ubiquinone) and two different types
of iron-containing proteins (cytochromes and iron-sulfur proteins).
1. Ubiquinone
Ubiquinone also called as co-enzyme Q is a lipid-soluble benzoquinone with a long
isoprenoid side chain. They can accept one electron to become semi-quinone
radical or two electrons from ubiquinol, which can act as a junction between a two-
electron donor and one electron acceptor. It is small and hydrophobic, freely
diffusible within the lipid bilayer of the inner mitochondrial membrane, and can
shuttle reducing equivalents between other less mobile electron carriers in the
membrane. It is also important in coupling electron flow to proton movement.
2. Cytochromes
These are proteins with strong absorption of visible light because of their heme
(iron) prosthetic groups. There are three types of cytochromes because of their
characteristics different light absorption spectra: Cytochrome a, Cytochrome b,
and Cytochrome c. The heme cofactors of Cyt a and b are tightly/non-covalently
bound to their associated proteins but this isn’t the case with Cyt c (attachment via
Cysteine residues). Cyt a, b and some types of c are integral proteins of the inner
mitochondrial membrane. Cyt c (a soluble protein) associates through electrostatic
interactions with the outer surface of the inner membrane.
3. Iron-Sulfur Proteins
In iron-sulfur proteins, the iron is present not in heme but association with inorganic
sulfur atoms or with the sulfur atoms of the Cysteine residues in the proteins or both.
Fe-S range from a simple structure having a single Fe atom coordinated to four Cys-
SH groups, to more complex Fe-S centers with two or four Fe atoms. All iron-sulfur
proteins participate in electron transfer, in which one iron atom of the iron-sulfur
cluster is oxidized or reduced. At least eight Fe-S proteins participate in
mitochondrial electron transfer.
Electron Transport Chain
In the overall reaction catalyzed by the mitochondrial respiratory chain, electrons
move from NADH, succinate, or some other primary electron donor through
flavoproteins, ubiquinone, iron-sulfur proteins, cytochromes, and finally to oxygen the
final electron acceptor. The order can be summarized as follows:
(1) NADH → (2) Ubiquinone → (3) Cytochrome b → (4) Cytochrome c1 → (5)
Cytochrome c → (6) Cytochrome a → (7) Cytochrome a3 → (8) Oxygen
Electron carriers of the respiratory chain (electron transport chain) are organized into
membrane-embedded supra-molecular complexes that can be physically separated.

4. SYED MUHAMMAD KHAN (BS HONS. ZOOLOGY)
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There are four unique electron carrier complexes, each capable of catalyzing
electron transfer through a portion of chains.
Complex I and II catalyze electron transfer to ubiquinone from two different electron
donors; NADH (complex I) and succinate (complex II). Complex III carries electrons
from reduced ubiquinone to cytochrome c, and Complex IV completes the sequence
by transferring electrons from cytochrome c to oxygen.
Figure: Electrons from NADH pass through a flavoprotein to a series of iron-sulfur
proteins in complex I. Then they are passed on to ubiquinone (Q). Electrons from
succinate pass through a flavoprotein and several Fe-S centers in complex II and are
then passed on to Q. Glycerol 3-phosphate donates its electrons to a flavoprotein on
the outer face of the inner mitochondrial membrane, from which they are passed on
to Q. Acyl-CoA dehydrogenase transfers electrons to electron-transferring
flavoprotein, from where they are passed on to Q via ETF: ubiquinone
oxidoreductase.
1. Complex I: NADH to Ubiquinone
Complex I also called the NADH: ubiquinone oxidoreductase or NADH
dehydrogenase is a large enzyme composed of 42 different polypeptide chains

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including an FMN containing flavoprotein and six iron-sulfur centers. The complex is
found to be L-shaped, with one arm in the membrane and the other extending into
the matrix. Complex I catalyzes two coupled and simultaneous processes. Firstly, it
transfers a hydride ion from NADH to ubiquinone and a proton from the matrix.
Secondly, it transfers four protons from the matrix to the inter-membrane space.
NADH + H+ + Q → NAD+ + QH2 (1)
NADH + 5H+
MATRIX (N) + Q → NAD+ + QH2 + 4H+
INTER-MEMBRANE SPACE (P) (2)
Complex I is therefore a proton pump driven by the energy of electron transfer. A
proton moves from the matrix to the inter-membrane space, the matrix becomes
negatively charged and the inter-membrane space becomes positively charged.
Ubiquinol (fully reduced form of ubiquinone) diffuses in the inner mitochondrial
membrane from complex I to complex III, where it is oxidized to Q (ubiquinone) in a
process that also involves the outward movement of H+.
Figure: NADH: ubiquinone oxidoreductase (Complex I) contains an FMN containing
flavoprotein and multiple Fe-S proteins. The electrons are extracted from NADH and
transferred to FMN. From there, they are passed on through a series of Fe-S
centers, and finally, they are given to ubiquinone.

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2. Complex II: Succinate to Ubiquinone
Complex II also designated as succinate dehydrogenase; the only membrane-
bound enzyme in the citric acid cycle, smaller and simpler than complex I, contains
five prosthetic groups of two types and four different protein subunits.
Subunits C and D are integral membrane proteins, each with three transmembrane
helices. They contain a heme group, heme b, and binding site for ubiquinone, the
final electron acceptor in the reaction catalyzed by complex II. Subunits A and B
extend into the matrix; they contain three 2Fe-2S centers, bound FAD, and a binding
site for the substrate: succinate.
Electrons pass from succinate to FAD then through the three iron-sulfur centers to
ubiquinone. Heme b of the complex II is not involved in the direct electron transfer
but to prevent the leakage of the electron i.e., from succinate to molecular oxygen to
produce the reactive oxygen species (ROS) hydrogen peroxide and the superoxide
radical.
Figure: Succinate dehydrogenase (Complex II) consists of four subunits: A, B, C,
and D. Subunits C and D are embedded in the inner mitochondrial membrane they
contain heme b and Q binding site. Subunits A and B are in the matrix and contain
2Fe-2S centers, FAD, and succinate binding site. Electrons pass from succinate to
FAD and then to ubiquinone via 3 Fe-S centers.

7. SYED MUHAMMAD KHAN (BS HONS. ZOOLOGY)
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3. Complex III: Ubiquinone to Cytochrome c
It is also called cytochrome bc1 complex or ubiquinone: cytochrome c
oxidoreductase. It transfers electrons from ubiquinol (QH2) to cytochrome c and
also transports protons from the matrix to the intermembrane space. The complex is
a dimer of two identical monomers, each with 11 different subunits. The functional
core is three subunits, cytochrome b with its two hemes bH and bL, the Rieske
iron-sulfur proteins with its 2Fe-2S centers, and cytochrome c1 with its heme.
Cytochrome c1 and the Rieske iron-sulfur protein project from the P (Inter-
membrane space) surface and can interact with the cytochrome c (not part of the
functional complex) in the inter-membrane space. Complex has two distinct binding
sites for the ubiquinone QN and QP.
Figure: Cytochrome bc1 complex (Complex III). (a) The functional core consists of
cytochrome b (with Heme bL and Heme bH), Rieske sulfur proteins, and cytochrome
c1 (with its heme). (b) Simplified view of Complex III showing all of its components.
The Q cycle: On the P side (towards inter-membrane space) of the membrane, two
molecules of QH2 are oxidized to Q, releasing two protons per Q (in total four
protons) all into the intermembrane space. Each QH2 donates two electrons; one to
the cytochrome c1 via the Rieske iron-sulfur proteins and the other via the
cytochrome b to a molecule of Q near the N side (towards matrix), reducing it in two
steps to QH2 (two protons are also taken up in this reduction from the matrix). The
net result is that QH2 is oxidized to Q and two molecules of cytochrome are reduced.
4. Complex IV: Cytochrome c to Oxygen
Cytochrome c is a soluble protein of the intermembrane space, after its heme
accepts an electron from complex III, it moves to complex IV to donate the electron.
Complex IV also called the cytochrome oxidase carries electrons from the

8. SYED MUHAMMAD KHAN (BS HONS. ZOOLOGY)
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cytochrome c to molecular oxygen reducing it to water. Complex IV is a large
enzyme of the inner mitochondrial membrane having 13 subunits, three are
important: Subunit I has two heme groups a and a3, and a copper ion, CuB. Heme
a3 and CuB form a binuclear iron-sulfur center (Heme a3-CuB center). Mitochondrial
subunit II contains two Cu ions (CuA) complexed with the SH groups of the two
Cysteine residues in a binuclear center. The binuclear center and the cytochrome c
binding site are located in a domain of subunit II that protrudes from the P side of the
inner membrane. Subunit III seems to be essential for complex IV function but its
role is not well understood.
Electron transfer begins when two molecules of reduced cytochrome c, each donate
an electron to the binuclear center CuA. From here electrons pass through heme a to
the Fe- Cu center (Cytochrome a3 and CuB). Oxygen now binds to the heme a3 and
is reduced to its peroxy derivative by two electrons from the Fe-Cu center. Delivery
of two more electrons from the cytochrome c converts the peroxy oxygen to two
molecules of water, with the consumption of four protons from the matrix.
Figure: Cytochrome oxidase (Complex IV). Two reduced molecules of cytochrome c
donate electrons to CuA. From here they are passed to Heme a3-CuB center.
Oxygen binds with heme a3 and is reduced to peroxy form. Later it is further reduced
to water, when more electrons are received; hydrogen ions are pumped from the
matrix for this purpose.

9. SYED MUHAMMAD KHAN (BS HONS. ZOOLOGY)
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Oxidative Phosphorylation
For each pair of electrons transferred to Oxygen, four protons are pumped out by
complex I, four by complex III, and two by complex IV (Total = 12). The
electrochemical energy inherent in this difference in proton concentration and
separation of charge represents a temporary conservation of much of the energy of
electron transfer. The energy stored in such a gradient is termed as proton motive
force, it has two components: (1) The chemical potential energy due to the
difference in the concentration of a chemical species H+. (2) The electrical potential
energy that results from the separation of charge when a proton moves across a
membrane without a counter-ion.
NADH + 11H+
N + ½ O2 → NAD+ + 10H+
P + H2O
1. Chemiosmotic Model
The chemiosmotic model was proposed by Peter Mitchell, in this regard.
According to this model, the electrochemical energy due to the difference in proton
concentration and the separation of charge across the inner mitochondrial
membrane - the proton-motive force drives the synthesis of ATP as protons flow
passively (passive transport) back into the matrix through a proton pore associated
with ATP synthase. To emphasize this crucial role of the proton-motive force, the
equation for ATP synthesis is sometimes written:
ADP + Pi + nH+
P → ATP + H2O + nH+
N
Figure: Chemiosmotic Model – Electrons from NADH, etc. pass through a chain of
carriers in the inner membrane. Electron flow is accompanied by proton transfer
across the membrane, producing a chemical and electrical gradient. The inner
mitochondrial membrane is impermeable to protons; protons can reenter the matrix
only through proton-specific channels (Fo). The proton-motive force that drives
protons back into the matrix provides the energy for ATP synthesis, catalyzed by the
F1 complex associated with Fo.

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2. Generation of Reactive Oxygen Species
Several paths in the reduction of oxygen in mitochondria have the potential to
produce highly reactive free radicals that can damage cells. The passage of
electrons from QH2 to complex III, and passage of electrons from complex I to QH2,
involves the radical Q- (semiquinone) as an intermediate. This radical can, with a low
probability, pass an electron to O2 in the reaction:
O2 + e- → O-
2
The superoxide thus generated is highly reactive; its formation also leads to the
production of even more reactive free hydroxyl radicals. These reactive oxygen
species can react and damage the enzymes, membrane lipids, and nucleic acids. To
prevent the oxidative damage by oxygen radicals, cells have several forms of the
enzyme superoxide dismutase, which catalyzes the reaction:
2O-
2 + H+ → H2O2 + O2
The hydrogen peroxide thus generated is rendered harmless by the action of
glutathione peroxidase. Glutathione reductase recycles the oxidized glutathione
to its reduced form, using electrons from NADPH generated by nicotinamide
nucleotide transhydrogenase (in mitochondria) or by the pentose phosphate
pathway. Reduced glutathione also serves to keep protein sulfhydryl groups in their
reduced state, preventing some of the deleterious effects of oxidative stress.
ATP Synthesis
The ATP synthase complex, often dubbed as Complex V, catalyzes ATP synthesis;
it is present on the inner mitochondrial membrane. It has two major components: F1
(peripheral protein above the membrane, inside the matrix of the membrane) and Fo
(an integral membrane protein which provides a trans-membrane pore for protons).
F1 ATPase (so-called because of its ability to hydrolyze ATP, i.e. reverse of
synthesis) has nine subunits of five different types: 3-alpha, 3-beta, gamma,
epsilon, and delta subunits. The beta subunit has one catalytic site for ATP
synthesis. The knob-like portion of F1 is a flattened sphere, 8nm high and 10nm
across consisting of alternating alpha and beta subunits arranged like the sections of
an orange. One domain of the gamma subunit makes up the central shaft that
passes through F1 and the other domain is associated with one of the beta subunits.
Since the gamma subunit is linked to only one of the three beta subunits, so their
conformation is different.
Fo proton pore consists of three subunits a, b, and c, occurring in the proportion of
ab2c10-12. Subunit c is a small hydrophobic polypeptide, consisting of two
transmembrane helices with a small loop extending from the matrix side of the
membrane. This membrane-embedded cylinder of c subunits is attached to the shaft

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made up of F1 gamma and epsilon subunits. The two b subunits associate firmly with
the alpha and beta subunits of F1 holding them fix.
Figures: Diagrams of the FoF1 complex (ATP synthase). The two b subunits (b2) of
Fo associate firmly with the alpha and beta subunits of F1, holding them fixed relative
to the membrane. In Fo, the membrane-embedded cylinder of c subunits (c10) is
attached to the shaft made up of F1 subunits gamma and epsilon. As protons flow
through the membrane from the P side to the N side through Fo, the cylinder and
shaft rotate, and the beta subunits of F1 change conformation as the gamma subunit
associates with each in turn.
The three active sites of F1 take turns to synthesize ATP. The proton motive force
causes rotation of the central shaft about 120 degrees which comes into contact with
a different beta subunit. A given beta subunit starts in the beta-ADP conformation;
that binds ADP and Pi from the surrounding medium, it then changes conformation
assuming the beta-ATP conformation that tightly binds and stabilizes the ATP, and
finally, the subunit changes to a beta-empty conformation which has very low
affinity for ATP and the newly synthesized ATP leaves the enzyme surface.