OXIDATIVE PHOSPHORYLATION
The transport of electrons through the ETC is linked with the release of free energy. In
oxidative phosphorylation ATP is produced by combining ADP and Pi with the energy
generated by the flow of electrons from NADH to molecular oxygen in the electron transport
chain. The complex V of the inner mitochondrial membrane is the site of oxidative
phosphorylation.
The transport of electrons through the ETC can be summarized by the following equation
NADH + H+ + O + 3ADP + 3Pi------------ NAD+ + 3ATP + 4H2O
The free energy available for the synthesis of ATP during electron transfer from NADH to
oxygen can be calculated from the difference in the value of the redox potential of the
electron donor system and that of the electron acceptor system. The redox potential of the
NADH/NAD+ is -0.32 V and that of H2O/½O2 is +0.82 V, therefore, the standard potential
difference between them is: electron acceptor- electron donor,
= (+0.82 V) - (-0.32 V) = +1.14 v
The standard free energy is calculated from the expression:
Formation of each ATP requires 7.3 kcal (30.5 kJ), and 21.9 kcal accounts for three ATPs
synthesized. The remainder of the energy is assumed to be dissipated as heat.
OXIDATIVE PHOSPHORYLATION
Dr. Sameer M Mahmood
Mechanism of Oxidative Phosphorylation:
The most accepted theory of oxidative phosphorylation is the chemiosmotic theory (also
known as the Mitchell hypothesis).
The energy from oxidation of components in the respiration chain is coupled to the
translocation of hydrogen ions (Protons, H+) from the mitochondrial matrix to the inner
mitochondrial membrane. Each of the respiratory chain complexes I, III and IV acts as a proton
pump. The inner membrane is impermeable to ions in general but particularly to protons,
which accumulate in the inner membrane, creating an electrochemical potential difference
across the membrane. This consists of a chemical potential (pH gradient, (difference in pH)
the outside of the membrane is at a lower pH than the inside) and an electrical potential (with
more positive charges on the outside of the membrane than on the inside). The
electrochemical potential resulting from the asymmetric distribution of the hydrogen ion is
used to drive the mechanism responsible for the formation of ATP.
ATP SYNTHASE
ATP synthase, present in the complex V, utilizes the proton gradient for the synthesis of ATP.
This enzyme is also known as ATPase since it can hydrolyse ATP to ADP and Pi.
ATP synthase is a complex enzyme and consists of two functional subunits, namely Ft and F0.
F0, spans inner mitochondrial membrane and serves as a proton channel through which
protons enter into mitochondria, while F1 projects into the mitochondrial matrix as spheres
and catalyses the ATP synthesis.
The chemiosmotic hypothesis proposes that after protons have been transferred to the
cytosolic side of the inner mitochondrial membrane, they re-enter the mitochondrial matrix
by passing through a channel in the ATP synthase complex, resulting in the synthesis of ATP
from ADP + Pi.
OXIDATIVE PHOSPHORYLATION
Dr. Sameer M Mahmood
P/O Ratio: The P/O ratio is the ratio of the number of moles of ATP generated to the number
of atoms of oxygen reduced in the electron transport chain. It is used to express the efficiency
of oxidative phosphorylation of the system.
There are 3 sites of generation of ATP in the electron transport chain. Thus P/O ratio is 3 if
reducing equivalents enter electron transport chain through NAD. On the other hand, for the
FAD requiring reactions P/O ratio is 2
INHIBITORS OF OXIDATIVE PHOSPHORYLATION:
The mitochondrial transport of electrons is tightly coupled with oxidative phosphorylation
(ATP synthesis). In other words, oxidation and phosphorylation proceed simultaneously.
There are certain compounds that can uncouple (or delink) the electron transport from
oxidative phosphorylation. Such compounds known as uncouplers, increase the permeability
of inner mitochondrial membrane to protons (H+). The result is that ATP synthesis does not
occur. The energy linked with the transport of electrons is dissipated as heat. The uncouplers
allow (often at accelerated rate) oxidation of substrates (via NADH or FADH2) without ATP
formation.
OXIDATIVE PHOSPHORYLATION
Dr. Sameer M Mahmood
Synthetic uncoupler: The uncoupler, 2,4-dinitrophenol (DNP), is a lipophilic proton-carrier
and can easily diffuse through the inner mitochondrial membrane. This uncoupler causes
electron transport to proceed at a rapid rate without establishing a proton gradient, The
energy produced by the transport of electrons is released as heat rather than being used to
synthesize ATP. In the people seeking to lose weight, DNP was used as a drug. However, this
is now discontinued, as it produces hyperthermia and other side effects. In fact, Food and
Drug Administration (USA) has banned the use of DNP.
The other uncouplers is trifluorocarbonylcyanide phenylhydrazone (FCCP) which is 100 times
more effective as an uncoupler than dinitrophenol. When administered in high dose aspirin
acts as an uncoupler.
Another example is Oligomycin antibiotic, it prevents the mitochondrial oxidation as well as
phosphorylation. lt binds with ATPase and blocks the proton (H+) channels, thus prevents the
translocation (re-entry) of protons into the mitochondrial matrix. Due to this, protons get
accumulated at higher concentration in the intermembrane space. Electron transport
ultimately stops, since protons cannot be pumped out against steep proton gradients.
Physiological Uncouplers: The maintenance of body temperature is particularly important in
hairless animals, hibernating animals and the animals adapted to cold. These animals possess
a specialized tissue called brown adipose tissue. The mitochondria of brown adipose tissue
are rich in electron carriers and are specialized to carry out an oxidation uncoupled from
phosphorylation. This causes liberation of heat when fat is oxidized in the brown adipose
tissue. The presence of active brown adipose tissue in certain individuals is believed to protect
them from becoming obese. The excess calories consumed by these people are burnt and
liberated as heat, instead of being stored as fat.
Thermogenin (or uncoupling protein, UCP) is a natural uncoupler protein located in the inner
mitochondrial membrane of brown adipose tissue. It acts like an uncoupler, create a "proton
leak," that is, allow protons to re-enter the mitochondrial matrix without energy being
captured as ATP thus blocks the formation of ATP, and liberates heat.
Note: humans have little brown fat (except in the new-born), and UCP does not appear to
play a major role in energy balance.
Other examples of physiological Uncouplers are excessive thyroxine hormone, long chain FA
in Brown adipose tissue.
OXIDATIVE PHOSPHORYLATION
Dr. Sameer M Mahmood
Transport of Cytoplasmic NADH to Mitochondria:
Glycolysis produces NADH in the cytoplasm, however, the inner mitochondrial membrane is
impermeable to NADH. Therefore, the NADH produced in the cytosol cannot directly enter
the mitochondria. Two pathways namely glycerol-phosphate shuttle and malate-aspartate
shuttle are operative to do this job. They transport the reducing equivalents from cytosol to
mitochondria and not vice versa.
Glycerol-phosphate shuttle:
Through the glycerophosphate shuttle electrons are transported to the mitochondrial
electron transport chain via FAD.
In the first step, Cytosolic glycerol-3-phosphate dehydrogenase with the help of
dihydroxyacetone phosphate, catalyses the oxidation of cytosolic NADH to NAD+ which reenters glycolysis.
In the next step, mitochondrial glycerol 3-phosphate dehydrogenase present on outer surface
of inner mitochondrial membrane transfer the electrons from glycerol 3-phosphate to a
flavoprotein (i.e., reduction of FAD to FADH2). The produced Dihydroxyacetone phosphate
escapes into the cytosol and the shuttling continues. Finally, the FADH2 supplies electrons
directly to the electron transport (gets oxidized via ETC) to generate 2 ATP
OXIDATIVE PHOSPHORYLATION
Dr. Sameer M Mahmood
Malate- Aspartate Shuttle
ln the cytosol. oxaloacetate accepts the reducing equivalents (NADH) and becomes malate.
Malate then enters mitochondria where it is oxidized by mitochondrial malate
dehydrogenase. In this reaction, NADH and oxaloacetate are regenerated. NADH gets
oxidized via electron transport chain and 3 ATP are produced. This is in contrast to
glycerophosphate shuttle where only 2 ATP are produced. In the mitochondria, oxaloacetate
(which can’t pass through the mitochondrial membrane) participates in transamination
reaction with glutamate to produce aspartate and α-ketoglutarate. The aspartate enters the
cytosol and transaminate with α-ketoglutarate to give oxaloacetate and glutamate.
OXIDATIVE PHOSPHORYLATION
Dr. Sameer M Mahmood