BIOENERGETICS

OXIDATIVE
PHOSPHORYLATION

Lecture # 23
Presented by: Dr. Farzana Khan
Lecturer: Bakhtawar Amin college Pharmaceutical Sciences Multan
Reference: U. Satyanarayana, Biochemistry ,4rth edition,
(Page No. 221-234)
 BIOENERGETICS
Bioenergetics or biochemical thermodynamics deals with the study
of energy changes (transfer and utilization) in biochemical reactions.
The reactions are broadly classified as exergonic (energy releasing)
and endergonic (energy consuming).
Bioenergetics is concerned with the initial and final states of energy
component of the reactants and not the mechanism of chemical
reactions.
 BIOLOGICAL OXIDATION
Oxidation is defined as the loss of electrons and reduction as the gain of electrons.
 This may be illustrated by the interconversion of ferrous ion (Fe2+) to ferric ion
(Fe3+).
The electron lost in the oxidation is accepted by an acceptor which is said to be
reduced.
Thus the oxidation-reduction is a tightly coupled process.
The general principle of oxidation reduction is applicable to biological systems also.
The oxidation of NADH to NAD+ coupled with the reduction of FMN to FMNH2
is illustrated

In the above illustration, there are two redox pairs NADH/NAD+ and FMN/FMNH2.
The redox pairs differ in their tendency to lose or gain electrons.
CLASSIFICATION OF HIGH-ENERGY COMPOUNDS
There are at least 5 groups of high-energy compounds.
1. Pyrophosphates e.g. ATP.
2. Acyl phosphates e.g. 1,3-bisphosphoglycerate.
3.Enol phosphates e.g. phosphoenolpyruvate.
4.Thioesters e.g. acetyl CoA.
5.Phosphagens e.g. phosphocreatine.
 High-energy bonds :
 The high-energy compounds possess acid anhydride bonds
(mostly phosphoanhydride bonds) which are formed by the condensation of two
acidic groups or related compounds.
These bonds are referred to as high energy bonds, since the free energy is liberated
when these bonds are hydrolysed.
Lipmann suggested use of the symbol ~ (Tilde) to represent high- energy bond.
For instance, ATP is written as AMP~P~P
High-energy compounds
ATP THE MOST IMPORTANT HIGH ENERGY COMPOUND

Adenosine triphosphate (ATP) is a unique and the most important

high energy molecule in the living cells.
It consists of an adenine, a ribose and a triphosphate moiety.
ATP is a high energy compound due to the presence of two
phosphoanhydride bonds in the triphosphate unit.
STRUCTURE OF ATP
 SYNTHESIS OF ATP
ATP can be synthesized in two ways;
1.Oxidative phosphorylation :
This is the major source of ATP in aerobic organisms.
It is linked with the mitochondrial electron transport chain.
2.Substrate level phosphorylation :
ATP may be directly synthesized during substrate oxidation in the metabolism.
The high energy compounds such as phosphoenol pyruvate and 1,3-
bisphosphoglycerate (intermediates of glycolysis) and succinyl CoA (of citric acid
cycle) can transfer high-energy phosphate to ultimately produce ATP.
 ATP-ADP CYCLE

The hydrolysis of ATP is associated with the release of large amount of

energy.
ATP + H2O ADP + Pi + 7.3 Cal.
The energy liberated is utilized for various processes like muscle contraction,
active transport etc.
ATP can also act as a donor of high energy phosphate to low energy
compounds, to make them energy rich.
On the other hand, ADP can accept high energy phosphate from the
compounds possessing higher free energy content to form ATP.
ATP serves as an immediately available energy currency of the cell
which is constantly being utilized and regenerated.
This is represented by ATP ADP cycle, the fundamental basis of energy
exchange reactions in living system.
ATP acts as an energy link between the catabolism (degradation of
molecules) and anabolism (synthesis) in the biological system.
ATP-ADP CYCLE ALONG WITH SOURCES AND UTILIZATION OF ATP (NOTE THAT ~P
DOES NOT EXIST IN FREE FORM, BUT IS ONLY TRANSFERRED).
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 of oxidative
phosphorylation
MECHANISM OF OXIDATIVEPHOSPHORYLATION
Several hypotheses have been put forth to explain the process of oxidative
phosphorylation.
The most important among them namely, chemical coupling, and chemiosmotic
are discussed below.
1. Chemical coupling hypothesis
This hypothesis was put forth by Edward Slater (1953).
 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.
These reactions are believed to be analogous to the substrate level phosphorylation that
occurs in glycolysis or citric acid cycle.
However, this hypothesis lacks experimental evidence, since all attempts, so far, to
isolate any one of the high-energy intermediates have not been successful.
2. Chemiosmotic hypothesis
This mechanism, originally proposed by Peter Mitchell (1961), is now widely
accepted.
 It explains how the transport of electrons through the respiratory chain is effectively
utilized to produce ATP from ADP + Pi.
The concept of chemiosmotic hypothesis is comparable with energy stored in a battery
separated by positive and negative charges.
 Proton gradient :
The inner mitochondrial membrane, as such, is impermeable to protons(H+) and hydroxyl
ions (OH–).
The transport of electrons through ETC is coupled with the translocation of protons
(H+) across the inner mitochondrial membrane (coupling membrane)from the matrix to the
inter membrane space.
 The pumping of protons results in an electrochemical or proton gradient.
This is due to the accumulation of more H+ ions (low pH) on the outer side of the inner
mitochondrial membrane than the inner side.
 The proton gradient developed due to the electron flow in the respiratory chain is
sufficient to result in the synthesis of ATP from ADP and Pi.
OUTLINE OF CHEMIOSMOTIC HYPOTHESIS FOR OXIDATIVE
PHOSPHORYLATION
Enzyme system for ATP synthesis :
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 F1and F0.
Its structure is comparable with ‘lollipops’.
The protons that accumulate on the intermembrane space re-enter the
mitochondrial matrix leading to the synthesis of ATP.
DIAGRAMMATIC REPRESENTATION OF CHEMIOSMOTIC HYPOTHESIS FOR
OXIDATIVE PHOSPHORYLATION (I, III, IV AND V–RESPIRATORY CHAIN
COMPLEXES; F0 , F 1–PROTEIN SUBUNITS FOR PHOSPHORYLATION).
INHERITED DISORDERS OF OXIDATIVE
PHOSPHORYLATION
It is estimated that about 100 polypeptides are required for oxidative phosphorylation.
Of these, 13 are coded by mitochondrial DNA (mt DNA) and synthesized in the
mitochondria, while the rest are produced in the cytosol (coded by nuclear DNA) and
transported.
Mitochondrial DNA is about 10 times mores susceptible to mutations than nuclear
DNA.
Mt DNA mutations are more commonly seen in tissues with high rate of oxidative
phosphorylation (e.g. central nervous system, skeletal and heart muscle, liver).
Leber’s hereditary optic neuropathy is an example for mutations in mtDNA.
This disorder is characterized by loss of bilateral vision due to neuroretinal
degeneration.
INHIBITORS OF OXIDATIVE PHOSPHORYLATION
1. Uncouplers
The mitochondiral transport of electrons is tightly coupled with
oxidative phosphorylation (ATP synthesis).
There are certain compounds that can uncouple (or delink) the electron
transport from oxidative phosphorylation.
 Such compounds, known as uncouplers, increase the permeability of
innermitochondrial 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.
The uncoupler,2,4-dinitrophenol (DNP) , has been extensively studied.
It is a small lipophilic molecule.
DNP is a proton-carrier and can easily diffuse through the inner mitochondrial
membrane.
 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 include dinitrocresol, pentachlorophenol, trifluorocarbonyl
cyanidephenylhydrazone (FCCP).
The last compound (FCCP) is said to be 100 times more effective as an uncoupler than
dinitrophenol.
When administered in high doses, the drug aspirin acts as an uncoupler.
Physiological uncouplers :
Certain physiological substances which act as uncouplers at higher concentration have
been identified.
 These include thermogenin , thyroxine and long chain free fatty acids.
The unconjugated bilirubin is also believed to act as an uncoupler.
OTHER INHIBITORS OF OXIDATIVE PHOSPHORYLATION

1. Oligomycin :
This antibiotic prevents the mitochondrial oxidation as well as phosphorylation.
It binds with the enzyme ATP synthase and blocks the proton (H+) channels.
It 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 (respiration) ultimately stops, since protons cannot be pumped out
against steep proton gradients.
2. Atractyloside :
This is a plant toxin and inhibits oxidative phosphorylation by an indirect mechanism.
 Adenine nucleotide carrier system facilitates the transport of ATP and ADP.
Atractyloside inhibits adenine nucleotide carrier and, thus, blocks the adequate supply
of ADP, thereby preventing phosphorylation.
Reference:
U. Satyanarayana, Biochemistry ,4rth edition.