BIOLOGICAL OXIDATION

Ms.MARIYAM JOUHARA B M
Dept. Of Pharmaceutical Chemistry
CONTENTS

➢ETC and its mechanism

➢Oxidative phosphorylation
➢Substrate level phosphorylation
➢Uncouplers
INTRODUCTION
Biological oxidation:
The transfer of electron from reduced coenzymes through the respiratory
chain to oxygen is known as biological oxidation.
Energy released during this process is trapped as ATP.
ELECTRON TRANSPORT CHAIN
The ETC is a series of five protein complexes that transfer electrons
through a membrane within mitochondria to form a gradient of protons
that drives the formation of ATP.
SITE: Inner mitochondrial membrane and mitochondrial matrix
It is the end of catabolic process.
Structural organisation of ETC

➢The inner mitochondrial membrane consist of 5 distinct

enzyme complexes
➢The complex I to IV are carriers of electrons while
complex V is responsible for ATP synthesis
The ETC is the end of catabolic process and the steps can be
divided in to two as;
1.Proton pumping
2.Oxidative phosphorylation
1.PROTON PUMPING
COMPLEX I
NADH-CoQ reductase/NADH Dehydrogenase
It contains flavoproteins consisting of FMN(Coenzyme) and FeS
containing protein(apoenzyme).
The electrons are accepted from NADH and passed to coenzyme
Q through FMN and Fe-S centers.
During this transfer the energy is released, which is utilised to
transfer 4H+ ion from matrix to intermembrane space.
COMPLEX II
NADH-Succinate-CoQ oxidoreductase/Succinate dehydrogenase
It has two prosthetic group one FAD+ and Fe-S centers.

It transfers electrons from succinate to Coenzyme Q via enzyme

bound FAD and Fe-S centers. The complex II is not involved in proton
transfer.
COMPLEX III
Cytochrome b/c1 Complex
The complex is composed of cytochrome b, cytochrome c1 and
Fe-S centre. Cytochrome b accepts electrons from CoQH2 and
transfers them to Fe-S centre from where they are
transferred to cyt c through cyt c1.

During this process 4 H+ ions are transferred from matrix to

intermembrane space.
COMPLEX IV
Cytochrome C Oxidase
Cytochrome oxidase (Cytochrome a and a3 ) is the only
electron carrier, where the heme iron of which can directly
react with molecular oxygen. Besides heme (with iron), this
oxidase also contains copper atom that undergoes oxidation
reduction (Cu 2+ Cu+) during the transport of electrons.
➢ The Cu atom of complex IV accepts the electrons from
the mobile carrier Cyt c and transfer it to cyt a and then
to cyt a3. Finally, the molecular oxygen accepts the
electrons producing a molecule of water .
➢ In this phase another 2H+ is transferred from the matrix
to Inter membrane space.
2.OXIDATIVE PHOSPHORYLATION
➢ The transport of electrons through the ETC is linked with the
release of free energy.
➢ The process of synthesizing ATP from ADP and Pi coupled with the
electron transport chain is known as oxidative phosphorylation.
➢ The complex V of the inner mitochondrial membrane is the site
for oxidative phosphorylation.
COMPLEX V
ATP Synthetase
➢The enzyme ATP synthase is F0F1 complex, located at the
inner mitochondrial membrane
➢The F0 subcomplex is composed of channel protein ‘C’
subunits to which F1-ATP synthase is attached
➢F1-ATP synthase consists of a central gamma- subunit
surrounded by alternating alpha and beta subunits.
Sites of oxidative phosphorylation in ETC
There are three sites in the ETC that are exergonic to result in
the synthesis of 3 ATP molecules
1.Oxidation of FMNH2 by coenzyme Q
2. Oxidation of cytochrome b by cytochrome c1
3. Cytochrome oxidase reaction
Each one of the above reactions represents a coupling site for
ATP production. There are only two coupling sites for the
oxidation of FADH2, since the first site is bypassed.[Hence FADH2
gives only 2ATP during its oxidation to FAD+, While NADH+H+
gives 3 ATP]
MECHANISM OF OXIDATIVE PHOSPHORYLATION
Several hypotheses have been put forth to explain the
process of oxidative phosphorylation.
Namely,
➢Chemiosmotic hypothesis
➢Rotary motor model for ATP generation
➢Chemical coupling hypothesis
Chemiosmotic hypothesis

Proposed by Peter Mitchell (1961)

It explains how the transport of electrons through the respiratory chain

is effectively utilized to produce ATP from ADP + Pi.

Proton gradient :

The inner mitochondrial membrane is impermeable to protons (H+) and

hydroxyl ions (OH–). In ETC as the electrons are transported, the
translocation of protons (H+) across the inner mitochondrial membrane
from the matrix to the intermembrane space also occurs.
The pumping of protons results in an electrochemical or proton
gradient due to the accumulation of more H+ ions (low pH) on
the outer side of the inner mitochondrial membrane than the
matrix .

The proton gradient developed due to the electron flow in the

respiratory chain is utilized by ATP synthase to generate ATP
from ADP and Pi.
 4H+

H+

H+ H+
Rotary motor model for ATP generation
➢ Proposed by Paul Boyer
➢ The model states that a conformational change in the
mitochondrial membrane proteins leads to the synthesis of ATP.
➢ The proton gradient induces the rotation of gamma-subunit, which
in turn induces conformation changes in the 3 beta subunits.
➢ According to the binding change mechanism, the β subunits can
assume the structural forms O, L and T .
➢ L form binds to the substrate with more affinity but they are
catalytically inactive.
➢ Once the L form binds with the substrate [ADP and Pi] it’s
conformation changes to T form.
➢ The T form is the high energy conformation and the protein reverts
back to the low energy conformation resulting in the formation of
ATP from ADP and Pi.
➢ The T site changes to O conformation, and releases ATP.
➢ This cycle of conformation changes of beta subunits is repeated.
And three ATP are generated for each revolution.
Chemical coupling hypothesis

This hypothesis was put forth by Edward Slater.

According to chemical coupling hypothesis, during the course of
electron transfer in respiratory chain, a series of phosphorylated
high-energy intermediates are first produced which are utilized
for the synthesis of ATP.
Limitation:
Lack of experimental evidence
SIGNIFICANCE
➢ETC serves as the electron carrier in the final stage of cellular
respiration
➢They generate the proton gradients that drives the synthesis of ATP
➢Resupplies the cellular metabolic pool with NAD+ and FAD+
Substrate level phosphorylation
➢Substrate-level phosphorylation refers to the formation of
ATP/GTP from ADP/GDP and a phosphorylated intermediate.
Here the energy released as a result of breaking the phosphate
group from the substrate is utilized to phosphorylate the ADP or
GDP.

➢In living cells, it occurs during glycolysis (in the cytoplasm) and
the Krebs cycle (in the mitochondria)
Substrate level phosphorylation in glycolysis
In glycolysis, the substrate-level phosphorylation happens in two separate
steps.
1. In step 6 where a phosphate gp is transferred from 1, 3-
bisphosphoglycerate to ADP, forming 3-phosphoglycerate (3PG) and
ATP. This step is catalyzed by phosphoglycerate kinase (PGK).

1,3-bisphosphoglycerate + ADP → 3-phosphoglycerate + ATP

2. In step 9, a phosphoryl group is transferred from
phosphoenolpyruvate (PEP) molecules to ADP, forming pyruvate
molecules and ATP. The enzyme pyruvate kinase catalyzes the reaction.
Substrate level phosphorylation in TCA

Here the phosphorylation occurs only in one step.

➢A phosphoryl group is transferred from succinyl-CoA to GDP, forming

succinate and GTP. This step is catalyzed by the enzyme succinyl-CoA
synthetase.
Succinyl-CoA + GDP → Succinate + GTP
Importance of Substrate-level Phosphorylation

➢It is a much quicker and more efficient source of ATP and GTP to the
cell than oxidative phosphorylation during cellular respiration.

➢As the process can occur under aerobic and anaerobic conditions, it is a
vital energy-yielding process. It helps in the respiration of oxygen-
depleted muscle cells, human RBCs that lack mitochondria, and even
during the fermentative growth of microorganisms.
UNCOUPLER
An uncoupler or uncoupling agent is a molecule that inhibit the coupling
between the electron transport and phosphorylation reactions and thus
inhibiting the ATP synthesis without affecting the respiratory chain and ATP
synthase. They are employed as poisons or as a drug.
Mechanism of action
➢Uncouplers increases the permeability of mitochondrial membrane towards
protons and prevents the establishment of proton gradient and thus inhibits
ATP synthesis.
Examples of uncoupler

➢ 2,4-DNP
➢ Dinitrocresol
➢ Chloro carbonyl cyanide Phenylhydrazone
➢ Physiological uncouplers-Tyrosine in higher doses
➢ valinomycin
Inhibitors of ETC
Various chemicals selectively inhibits the transfer of electrons in the
ETC chain. Since the oxidative phosphorylation is coupled with ETC,
the inhibition of ETC will inhibit the ATP synthesis.
• SITE OF ACTION WITH IN ETC
• UNCOUPLING
SITE OFAGENT
ACTION EXAMPLES
COMPLEX 1 Rotenone, Amobarbital, Pericidin A,
Chlorpromazine
COMPLEX 2 Malonate, TTFA, Carboxin

COMPLEX 3 Antimycin A , BAL

COMPLEX 4 Cyanide, Azide, CO, H2S

Inhibitors Of Oxidative Phosphorylation
➢Oligomycin
➢Atractyloside
Blocks translocase responsible for the movement of ADP and
➢Bongregate ATP across the inner mitochondrial membrane
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