ATP, biological oxidation, bioenergetics and oxidative phosphorylation for undergraduate medical students

1. R. C. Gupta
Professor and Head
Dept. of Biochemistry
National Institute of Medical Sciences
Jaipur, India

2. Energy
We require energy for
various activities:
Muscle contraction
Nerve conduction
Synthetic reactions
Active transport
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3. The ultimate source of energy is the
food that we consume
Carbohydrates, lipids and proteins
present in food provide us energy
These are present in food in the
form of large complex molecules

4. Complex
molecules
in food
Mono-
saccharides,
fatty acids
and amino
acids
Oxidation in
catabolic
pathways
The carbon atoms are oxidized to carbon
dioxide
Hydrogen atoms are transferred to
coenzymes e.g. NAD+, FMN, FAD etc
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5. Reduced coenzymes transfer the hydrogen
atoms to the mitochondrial respiratory chain
wherein these are oxidized to water
The energy released during this oxidation is
used to phosphorylate adenosine diphosphate
(ADP) to adenosine triphosphate (ATP)

6. Oxidation coupled with phosphorylation of ADP
is known as oxidative phosphorylation
This is the mechanism by which the energy
present in various nutrients is captured in an
easily utilizable form

7. Energy released during oxidation of nutrients
may not be required immediately
High-energy phosphates
The energy released during catabolism is
captured in the form of “high-energy phosphates”
There must be some way of storing energy so
that it may be readily available when needed

8. The most important high-energy phosphate
is ATP
Hydrolysis of ATP into ADP and Pi
liberates 7.3 kcal of energy per mol
Hydrolysis of ADP into AMP and Pi also
releases nearly the same amount of energy

9. However, hydrolysis of AMP into
adenosine and Pi liberates much less
energy (3.4 kcal/mol)
This difference is because of the nature
of bonds by which phosphate is
attached

10. In ATP:
Third phosphate is attached
to the second by acid
anhydride bond
Second phosphate is attached
to the first by acid
anhydride bond
The first phosphate is attached
to ribose by an ester bond

11. H
CH2— O —P — O — P — O — P — OH
OH
H
OH
H
H
O
||
O
||
O
||
|
OH
|
OH
|
OH
Acid anhydride
bond
Ester
bond
Acid anhydride
bond
Adenine
O
Adenosine triphosphate

12. Hydrolysis of acid anhydride bonds
releases much more energy than
hydrolysis of ester bonds
Lipmann suggested a curved line (~) to
denote a high-energy bond
Thus, ATP may be represented as:
Adenosine− P ~ P ~ P

13. Compound D Go Compound D Go
Phosphoenol pyruvate – 14.8 AMP (Adenosine + Pi) – 3.4
Carbamoyl phosphate – 12.3 Glucose-1-phosphate – 5.0
1,3-Biphosphoglycerate – 11.8 Fructose-6-phosphate – 3.8
Creatine phosphate – 10.3 Glucose-6-phosphate – 3.3
ATP (ADP + Pi) – 7.3 Glycerol-3-phosphate – 2.2
Standard free energy (DGo) of hydrolysis of some
important organic phosphates (kcal/mol)*
*These are the values of DGo obtained in standard laboratory
conditions of 1M reactant concentration at pH 7.0 at 25°C;
values obtained in living cells (DG’o) are different as the
reactant concentrations, pH and temperature are different

14. Compounds which liberate 6 kcal/mol or
more on hydrolysis of the phosphate group
are known as high-energy phosphates
Organic phosphates which release less than
6 kcal/mol on hydrolysis of the phosphate
group are known as low-energy phosphates

15. High-energy phosphates
ATP and the related
nucleotides
Phosphoenol
pyruvate
Carbamoyl
phosphate
1,3-Biphospho-
glycerate
Creatine phosphate
Low-energy phosphates
AMP
Glucose-6-phosphate
Glucose-1-phosphate
Fructose-6-
phosphate
Glycerol-3-phosphate
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16. Compounds having DGo above that of ATP
can transfer their phosphate to ADP
forming ATP
The compounds having DGo below that of
ATP cannot transfer their phosphate
groups to ADP

17. Adenosine triphosphate
Energy currency of the cells
Captures energy from
exergonic reactions
Can transfer it to endergonic
reactions
Storage form of energy
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18. Besides high-energy phosphates, some
sulphur compounds having thio-ester
bonds are also high-energy compounds
In acetyl CoA, succinyl CoA, acyl CoA etc,
CoA is attached by a high-energy thio-
ester bond
R‒C~S‒CoA
O

19. Oxidation was defined in the past as
addition of oxygen to or removal of
hydrogen from a substance
The reverse was termed as reduction
These definitions have now been
supplanted by a more comprehensive
concept
Oxidation and reduction

20. Oxidation is now defined as removal of
electrons and reduction as addition of
electrons
The electron being removed or added may
be a free electron or it may be associated
with a proton as in a hydrogen atom

21. Fe+2
Oxidation of Fe+2
Reduction of Fe+3
Fe+3 + e‒ (electron)
AH2 + NAD+
AH2 + FAD
Oxidation of AH2
Oxidation of AH2
Reduction of A
Reduction of A
A + NADH + H+
A + FADH2

22. Our earliest concepts of oxidation in living
beings (biological oxidation) originated
from the work of Lavoisier
He proposed that respiration in animals is
an oxidative process
In this, atmospheric oxygen is used to
oxidize carbon atoms to carbon dioxide

23. Pasteur showed later that the presence of
oxygen is not essential for oxidation
He showed that living organisms can
oxidize substrates even in the absence of
oxygen
Wieland proposed that biological oxidation
occurred by dehydrogenation of activated
substrates

24. With the discovery of cytochromes by Keilin,
it became clear that the substrates are
dehydrogenated
The reducing equivalents ( H or e–) are taken
up by cytochromes
They are finally transferred to oxygen in the
presence of cytochrome oxidase (Warburg’s
enzyme) to be converted into water

25. Electron transfer chain (ETC)
Also known as respiratory chain
Present in inner mitochondrial membrane
Sequence of carriers of reducing equivalents
Consists of enzymes and coenzymes
Transfers reducing equivalents from
substrates to oxygen
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26. Substrate Nicotinamide-linked
dehydrogenase
Flavin-linked
dehydrogenase
Cytochrome b Cytochrome c1
Cytochrome cCytochrome a
Cytochrome a3 Oxygen
Coenzyme Q

27. A reactant undergoing reduction-oxidation
can exist in reduced form and oxidized form
The reduced and the oxidized forms of the
reactant constitute a redox couple
Every redox couple has a redox potential ‒ a
measure of its affinity of for electrons
Redox potential

28. A redox couple having high redox potential
has a high affinity for electrons
It readily accepts electrons from a redox
couple having a lower redox potential
The redox potential of a reactant can be
measured in the laboratory

29. Reduced and oxidised forms of reactant at 1M
concentration each are taken in sample cell
1M solution of H+ in equilibrium with H2 gas is
taken in a reference cell
The two are connected by an agar bridge
through which electrons can flow
Measurement of redox potential

30. Electrodes dipping in each solution are
connected to a voltmeter
The potential difference between the two
solutions is the redox potential of the
reactant

31. EMB-RCG
M
reactant

32. For biological systems, redox potential (Eo) is
measured at pH 7.0, and is denoted by E’o
Components of respiratory chain are arranged
in increasing order of redox potential
The electrons move from a relatively
electro-negative component to a relatively
electro- positive component at every site

33. Redox couple E´o(volts)
2H+ - H2 – 0.42
NAD+ - NADH – 0.32
FAD - FADH2 – 0.22
Cyt b(Fe+3) - Cyt b(Fe+2) – 0.08
CoQ - CoQH2 + 0.04
Cyt c1 (Fe+3) - Cyt c1 (Fe+2) + 0.22
Cyt c (Fe+3) - Cyt c (Fe+2) + 0.25
Cyt a (Fe+3) - Cyt a (Fe+2) + 0.29
Cyt a3 (Fe+3) - Cyt a3 (Fe+2) + 0.38
½ O2 - H2O + 0.82
Redox potential (E’o) of carriers in the
respiratory chain

34. The enzymes concerned with biological
oxidation are oxido-reductases
They can be sub-divided into: (i) oxidases,
(ii) dehydrogenases (iii) hydroperoxidases
(iv) oxygenases
Enzymes involved in biological oxidation

35. Oxidases transfer hydrogen from a substrate to
oxygen forming water or hydrogen peroxide
The enzymes forming water are metallo-
enzymes that usually contain copper e.g.
cytochrome oxidase and tyrosinase
The general reaction catalysed by oxidases is:
AH2 + ½ O2 → A + H2O
Oxidases

36. The enzymes forming hydrogen peroxide
are flavoproteins containing FMN or FAD
Examples are L-amino acid oxidase and
xanthine oxidase
These enzymes catalyse the general
reaction:
AH2 + O2 → A + H2O2

37. These are conjugated proteins containing
nicotinamide nucleotides or flavin
nucleotides or iron-porphyrin
They remove hydrogen from a substrate,
and transfer it to another substrate
The prosthetic group acts as carrier of
hydrogen atoms
Dehydrogenases

38. AH2
A
BH2
BCarrier‒H2
Carrier

39. Prosthetic group Examples
NAD Lactate dehydrogenase, malate
dehydrogenase etc
NADP Glucose-6-phosphate dehydro-
genase, 6-phosphogluconate
dehydrogenase etc
Flavin
nucleotides
Succinate dehydrogenase, acyl
CoA dehydrogenase etc
Iron-
porphyrin
Cytochrome a, cytochrome b
etc

40. These enzymes convert H2O2 into H2O, and
include peroxidase and catalase
Peroxidase catalyses the reaction:
H2O2 + AH2  A + 2 H2O
Catalase catalyses the reaction:
2 H2O2  O2 + 2 H2O
Hydroperoxidases

41. These enzymes incorporate oxygen into a
substrate
They can be sub-divided into:
Oxygenases
Mono-oxygenasesDi-oxygenases

42. These enzymes catalyse the incorporation
of both the atoms of O2 into the substrate:
A + O2 → AO2
Examples include homogentisate oxidase
and tryptophan pyrrolase

43. These enzymes incorporate one atom of
oxygen molecule into the substrate
The other atom of oxygen oxidises
another reduced substrate to water:
AH + O2 + BH2 → A–OH + B + H2O
Mono-oxygenases

44. Mono-oxygenases are also known as
hydroxylases or mixed function oxidases
They are used to metabolize xenobiotics
(foreign compounds), for example drugs
like morphine, rifampicin etc
They also hydroxylate endogenous
substrates e.g. phenylalanine, tryptophan,
cholecalciferol, steroids etc

45. Two slightly different hydroxylase systems
are present in the cells:
Microsomal
hydroxylase
system
Mitochondrial
hydroxylase
system

46. Microsomal hydroxylase system
Complete system for hydroxylation
of xenobiotics and some steroids
Present in microsomes (membrane
of endoplasmic reticulum)
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47. Microsomal hydroxylase system consists of:
Cytochrome P-450 (CYP) which
possesses mono-oxygenase activity
NADPH
NADPH:cytochrome P-450 reductase
(a flavoprotein containing FAD and FMN)
Molecular oxygen
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48. NADPH ‒ Cytochrome
P ‒ 450 reductase
Cytochrome P ‒ 450
(mono-oxygenase)
Microsomal hydroxylase system
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50. A‒OH + H2O
e-
e-2 H+
AH
AH
AH
AH
AH
AH

51. Present in inner mitochondrial membrane
Catalyses hydroxylation of endogenous
substrates
The reaction catalysed by this system is
similar to microsomal hydroxylation reaction
Mitochondral hydroxylase system

52. The enzyme is NADPH:adrenodoxin reductase
(FAD-containing flavoprotein) instead of
NADPH:cytochrome P-450 reductase
The reducing equivalents accepted by FAD are
transferred to cytochrome P-450 via adreno-
doxin (iron-sulphur protein)

53. Iron-sulphur centres
Adrenodoxin possesses an iron-sulphur
centre having catalytic activity
Cys
Cys
Cys
Cys
Cys Cys
CysCys

54. NADPH‒
Adrenodoxin
reductase
Cytochrome
P‒450 (mono-
oxygenase)
Iron-
sulphur
protein
AH + O2 A-OH + H2O
Mitochondral hydroxylase system
FAD Fe+3FeS
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NADPH
+
H+
NADP+

55. Oxidative phosphorylation
Process by which energy released
during oxidation of energy-rich substrates
is used to phosphorylate ADP
Can occur at the substrate level or at the
level of the respiratory chain
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56. Substrate
level
Oxidation of
substrate is
linked with
formation of a
high-energy bond
in the product
which, in turn, is
used to convert
ADP into ATP
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Respiratory
chain
ADP is
phosphorylated
when the
reducing
equivalents
removed from
various substrates
are oxidized in the
respiratory chain

57. • Also known as
substrate-linked
oxidative
phosphorylation
• Examples are
found in the
glycolytic pathway
and citric acid cycle
Oxidative
phosphorylation
at
substrate
level
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58. H ‒ C = O
H ‒ C ‒ OH
CH2 ‒ O ‒ P
O
II
Glyceraldehyde-3-phosphate
Glyceraldehyde-3-
phosphate
dehydrogenase
NAD+ + Pi
NADH + H+
C ‒ O ~ P
CH2 ‒ O ‒ P
H ‒ C ‒ OH
1, 3-Biphosphoglycerate
I
I
In glycolysis,
oxidation of
glyceraldehyde-3-
phosphate is
linked
with introduction of
a high-energy
phosphate in the
product

59. 1, 3-Biphosphoglycerate
3-Phosphoglycerate
Phospho-
glycerate
kinase
In the next
reaction,
the high energy
phosphate is
transferred to
ADP forming ATP

60. In another glycolytic reaction, energy released
during dehydration of 2-phosphoglycerate
converts a low-energy phosphate into a high-
energy phosphate
The high-energy phosphate is transferred to
ADP in the next reaction

61. H
COOH
C O P
CH2OH
COOH
H2O
C O
CH2 CH2
COOH
OHC
ADP ATP
Enolase
Enol
pyruvate
Phosphoenol
pyruvate
2-Phospho-
glycerate
Pyruvate
kinase
P

62. CH2 — COOH
|
CH2 — C — COOH
CH2 — COOH
|
NADH+H+
+
CO2
a-Ketoglutarate
dehydrogenase
CH2 — C ~ S — CoA
O
a-Ketoglutarate Succinyl CoA
NAD+
+
CoA‒SHO
In citric acid cycle, the energy released
during oxidation of a-ketoglutarate is used
to form a high-energy bond between
succinate and CoA

63. CH2 — COOH
CH2 — COOH|
|
CH2 — COOHGDP
+
Pi
GTP
+
CoA–SH
Succinate
thiokinase
CH2 — C ~ S — CoA
O
Succinyl CoA Succinate
In the next reaction, CoA is split off and the
energy released is used to phosphorylate
GDP to GTP

64. Only a small fraction of energy is captured by
substrate-linked oxidative phosphorylation
For example, complete oxidation of one glucose
molecule phosphorylates 38 molecules of ADP
Of these, only six are phosphorylated at the
substrate level
The rest of the energy is captured in the
respiratory chain

65. Energy-giving nutrients are oxidized
stepwise by a series of reactions in various
metabolic pathways
In many reactions, reducing equivalents are
removed from the substrates, and are taken
up by coenzymes like NAD and FAD
Oxidative phosphorylation at the level of
respiratory chain

66. The reduced coenzymes transfer the
reducing equivalents to respiratory chain
Reducing equivalents are oxidized in the
respiratory chain
Their oxidation is coupled with the
phosphorylation of ADP to ATP

67. This is the most important mechanism for
capturing the energy present in various
nutrients
As the respiratory chain is located in
mitochondria, the mitochondria are called
the power-house of the cells

68. Substrate Nicotinamide-linked
dehydrogenase
Flavin-linked
dehydrogenase
Cytochrome b Cytochrome c1
Cytochrome cCytochrome a
Cytochrome a3 Oxygen
Coenzyme Q
Sequence of carriers in the respiratory chain

69. Most of the substrates transfer the reducing
equivalents to NAD
Reduced NAD transfers them to a flavoprotein
containing FMN and iron-sulphur (FeS) centre
Reduced flavoprotein transfers the reducing
equivalents to coenzyme Q (ubiquinone)
Transport of reducing equivalents in
respiratory chain

70. Coenzyme Q is a fat-soluble compound
resembling vitamin K in structure
It contains 6-10 isoprenoid units, and
can be reduced to ubiquinol

71. ||
O
O
||
CH3—O
CH3—O
CH3
(CH2—CH=C—CH2)n—H
[2H]
CH3
|
|
OH
OH
|
CH3—O
CH3—O
CH3
(CH2—CH=C—CH2)n—H
CH3
|
Ubiquinol
Ubiquinone (n = 6-10)

72. Reduced coenzyme Q transfers the
reducing equivalents to cytochrome b
Cytochrome b is associated with iron-
sulphur protein
The subsequent carriers, cytochromes c1,
c and a, are typical iron-porphyrin proteins

73. The cytochromes differ from each other in
their protein portions and in the side
chains attached to the porphyrin nucleus
The cytochromes transport electrons

74. Prosthetic group of cytochrome a

75. Prosthetic group of
cytochrome b
Prosthetic group of
cytochrome c

76. The electrons are taken up and transferred
by the iron portion of the cytochromes
Iron oscillates between Fe+3 and Fe+2 forms
The last cytochrome (cyt a3) is an oxidase
It catalyses the transfer of reducing equi-
valents to oxygen forming water

77. QAH2
A
NAD+
NADH
+ H+
(FMN,
FeS)
Fp
(Cyt b,
FeS)
(Cyt c1) (Cyt c) (Cyt a) (Cyt a3)
½ O2
H2O
Succinate
Fp (FAD, FeS)
2H+
FpH2
QH2
2Fe+2
2Fe+3 2Fe+3
2Fe+3
2Fe+2
2Fe+2
2Fe+2
2Fe+3
2Fe+2
2Fe+3

78. Components of respiratory chain do not
function as discrete carriers of reducing
equivalents
They are organized into four complexes
Each complex acts as a specific oxido-
reductase

79. Coenzyme Q and cytochrome c are not
parts of any complex
They are not fixed in the inner
mitochondrial membrane
The other components are fixed in the
membrane

80. Succinate
FAD, FeS
Complex II
NADH FMN,FeS Q Cyt b,FeS,Cyt c Cyt c Cyt aa3 O2
Complex I Complex III Complex IV

81. Complex I, II, III and IV in respiratory chain
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82. EMB-RCG

83. Complex I acts as NADH:ubiquinone
oxidoreductase, and transfers reducing
equivalents from NADH to CoQ

84. Complex II acts as succinate:ubiquinone
oxidoreductase, and transfers reducing
equivalents from succinate to CoQ

85. Complex III acts as ubiquinol:ferricytochrome c
oxidoreductase, and transfers reducing
equivalents from reduced CoQ to cytochrome c

86. Complex IV acts as ferrocytochrome c:oxygen
oxidoreductase,and transfers reducing equivalents
from reduced cytochrome c to oxygen

87. The proximal end of the respiratory
chain has a negative redox potential
The distal end has a positive redox
potential
Electrons move from a relatively electro-
negative component to a relatively electro-
positive component
Energy is released during this movement

88. Quantum of energy released at any site
is proportional to the difference in redox
potentials of:
Thus, different quanta of energy are
released at different sites
Component accepting the
reducing equivalents
Component donating the
reducing equivalents

89. Hydrolysis of the terminal phosphate of
ATP releases 7.3 kcal of energy per mol
in standard laboratory conditions
But in conditions prevailing in living cells,
the energy required for phosphorylation
of ADP to ATP is about 10 kcal/mol

90. Quantum of energy released exceeds 10
kcal/mol if the difference in the redox
potentials of the donor and the acceptor is
0.3 volts or more
ADP is phosphorylated at these sites
There are three such sites in the respiratory
chain

91. Site I is between NAD and CoQ
Site II is between Co Q and cytochrome c
Site III is between cytochrome c and oxygen
These sites correspond to complexes I, III and
IV respectively in the respiratory chain

92. All the substrates undergoing
dehydrogenation do not transfer the
reducing equivalents to NAD
Reducing equivalents are accepted by a
carrier having a redox potential just above
that of the substrate

93. Substrates that transfer reducing
equivalents to NAD can phosphorylate
three molecules of ADP
Examples are isocitrate, malate,
glutamate etc

94. Substrates that transfer reducing
equivalents to FAD can phosphorylate
only two molecules of ADP
Examples are succinate, glycerol-3-
phosphate, acyl CoA etc

95. The ratio of ADP molecules phospho-
rylated to number of oxygen atoms
reduced is known as P:O ratio
P:O ratio is three when the reducing
equivalents are accepted by NAD
P:O ratio is two when the reducing
equivalents are accepted by FAD

96. Mechanism by which oxidation and phospho-
rylation are coupled remained unclear for
long
Several hypotheses were advanced to explain
the mechanism of this coupling
The chemiosmotic hypothesis is the most
plausible
Mechanism of oxidative phosphorylation

97. Proposed by Mitchell
Inner mitochondrial membrane is
impermeable to protons (H+)
Energy released during transport of
electrons is used to actively eject H+ from
the matrix of mitochondria
This ejection establishes an electro-
chemical gradient across the membrane
Chemiosmotic hypothesis

99. Complexes I, III and IV in the respiratory chain act
as proton pumps ejecting hydrogen ions from the
mitochondrial matrix to the inter-membrane space
Succinate
Fumarate

100. The concentration of H+ on the outer
side becomes higher as compared to the
inner side
The outer side also becomes electro-
positive as compared to the inner side

101. This electrochemical gradient increases
up to a certain limit
When this limit is reached, the hydrogen
ions re-enter the matrix releasing energy

102. Re-entry of protons releases energy

103. The energy released during influx of protons is
used to activate a membrane-bound enzyme
This enzyme, vectorial ATP synthetase,
converts ADP and Pi into ATP

104. Efraim Racker showed that:
Vectorial ATP synthetase is made
up of F0 and F1 components
F0 component is embedded in
the inner mitochondrial membrane
F1 component projects into the
matrix

105. EMB-RCG

106. F1 component
F0 component
Inner
mitochondrial
membrane
EMB-RCG

107. F0 component acts as a channel for
the passage of hydrogen ions
F1 component has got ATP synthetase
activity
This activity is switched on when hydrogen
ions pass through the F0 component

108. John Walker deciphered the genes
encoding the F0 and F1 components
The F0 component is made up of a, b and
c subunits
The F1 component is made up of a, b, g, d
and e subunits (a3b3gde)

109. The subunits of F0 and F1 components of
vectorial ATP synthetase
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110. This form readily combines with ADP by a
high-energy bond
Water is formed and inorganic phosphate is
converted into a highly reactive form
Two hydrogen ions combine with two electrons
and one oxygen atom of inorganic phosphate
When hydrogen ions flow back into the matrix:
EMB-RCG

111. O
||
||
O
P‒O‒
O
||
O
||
| |
–O –O
H2O H2O3
O
||
O‒P‒O‒ –
O
||
O
||
|
O
| |
–O –O
Pi ADP
2H+ 4
|
ATP
2H+ O‒P‒O‒P‒O‒ A‒
‒
‒
O‒P‒O‒P‒O‒P‒O‒ A
O
||
O
||
| |
–O –O
||
O
O–
–
O‒P‒O‒P‒O‒ A

112. Paul Boyer has shown that:
F1 complex has got three sites
having specific conformations
These are O site (open site), L site
(loose-binding site) and T site (tight-
binding site)

113. Each site is made up of one a and
one b subunit
The three sites are inter-convertible
In the absence of an electrochemical
gradient:
T site is occupied by ATP
L site is occupied by ADP & Pi

114. T site is converted into O site, the O site
into L site and the L site into T site
The energy released during the influx of
H+ inter-converts the three sites
Boyer proposed the binding-change
mechanism
EMB-RCG

115. Conversion of T site into O site
releases the bound ATP
Conversion of L site into T site converts
the bound ADP & Pi into ATP
Another pair of ADP & Pi enters the
new L site, and the cycle is repeated
EMB-RCG

116. EMB-RCG

117. The inter-conversion of sites occurs because
of rotation of a and b subunits of F1 component
The rotation occurs in steps of 120o

118. There is strong experimental evidence in
support of chemiosmotic hypothesis
Building up of H+ gradient is the basic
premise of the hypothesis
It is seen that on bathing mitochondria in
a fluid having a relatively high H+ concen-
tration, phosphorylation occurs in the
absence of oxidation

119. The hypothesis envisages a membrane-
bound vectorial ATP synthetase
It has been shown that disruption of the
mitochondrial membrane leads to loss of
ATP synthetase activity

120. The P:H+ and H+:O ratios of various
substrates are also in agreement with the
experimental evidence
Thus, the chemiosmotic hypothesis has
now become the accepted theory to
explain oxidative phosphorylation in the
respiratory chain

121. Certain agents are known to inhibit
oxidative phosphorylation at specific sites
in the respiratory chain
Amobarbitone, rotenone and piericidin A
inhibit oxidative phosphorylation at site I
These have been shown to inhibit the
oxidoreductase activity of complex I
Inhibitors of oxidative phosphorylation

122. Dimercaprol and antimycin inhibit the
oxidoreductase activity of complex III
H2S, carbon monoxide and cyanide inhibit
the oxidoreductase activity of complex IV
When oxidation is inhibited, phospho-
rylation also cannot occur

123. Oligomycin inhibits oxidative
phosphorylation at all the sites
It binds to and inhibits F0 component of ATP
synthetase
The subscript ‘o’ derives from the tendency
of this component to bind oligomycin

124. Inhibitors of Oxidative Phosphorylation

125. Some compounds are known to uncouple
oxidation and phosphorylation
Examples are dinitrophenol, dinitrocresol
and dicoumarol
In their presence, phosphorylation is
inhibited but oxidation goes on
Uncouplers of oxidative phosphorylation

126. The uncouplers make the inner mito-
chondrial membrane permeable to H+
This doesn’t allow electrochemical
gradient to build up
Therefore, ATP cannot be synthesized
even though oxidation is going on

127. Thermogenin is a protein present in brown
adipose tissue
Brown adipose tissue is rich in mitochondria
Thermogenin is present in inner mito-
chondrial membrane
It acts as a channel for entry of hydrogen
ions into mitochondria
An endogenous uncoupler

128. Thermogenin
Inner
mitochondrial
membrane
Inter-
membrane
space
Mitochondrial
matrix
H+

129. As hydrogen ion gradient can not build up,
phosphorylation does not occur
Oxidation occurs in brown adipose tissue
without generation of ATP resulting in
production of heat
Brown adipose tissue is present in
significant amount in infancy and
decreases with age

130. Oxidative phosphorylation in the respiratory
chain results in consumption of oxygen
This is also known as tissue respiration
When oxidizable substrates and oxygen are
available, the rate of tissue respiration is
regulated mainly by concentration of ADP
Regulation of oxidative phosphorylation

131. Increased utilization of ATP raises ADP
concentration
This stimulates tissue respiration resulting
in increased phosphorylation of ADP into
ATP

132. Most of the NADH is produced in mitochondria
Some NADH is produced in cytosol also
Mitochondrial membrane is impermeable to
NADH
Special mechanisms are required to transport
NADH from cytosol into mitochondria
Oxidation of extra-mitochondrial NADH

133. Two important mechanisms are:
Malate
shuttle
Glycero-
phosphate
shuttle
EMB-RCG

134. Cytosolic NADH is used to reduce
dihydroxyacetone phosphate to glycerol-3-
phosphate
The latter goes into mitochondria as the
mitochondrial membrane is permeable to it
In mitochondria, glycerol-3-phosphate is
oxidized to dihydroxyacetone phosphate
Glycerophosphate shuttle

135. Dihydroxyacetone phosphate comes out
into the cytosol
In the mitochondrial reaction, the
hydrogen atoms are accepted by FAD
Only two ADP molecules are
phosphorylated when FADH2 is oxidized in
the respiratory chain

136. Cytosol Mitochondrial matrix
NADH
+
H+
NAD+
FADH2
FAD
Dihydoxyacetone
phosphate
Glycerol-3-
phosphate
Dihydoxyacetone
phosphate
Glycerol-3-
phosphate
Glycerol-3-
phosphate
dehydrogenase
Glycerol-3-
phosphate
dehydrogenase
Glycerophosphate shuttle

137. This is quantitatively more significant
Cytosolic malate dehydrogenase (MDH)
reduces oxaloacetate to malate
NADH is converted into NAD in this
reaction
Malate shuttle

138. Malate goes into mitochondria, and is oxidized
to oxaloacetate by mitochondrial MDH
The reducing equivalents are accepted by
NAD; hence there is no loss of energy
But mitochondrial membrane is not
permeable to oxaloacetate

139. For transporting oxaloacetate back to
cytosol, a special mechanism is needed
Mitochondrial glutamate oxaloacetate
transaminase (GOT) catalyses a trans-
amination reaction
It transfers an amino group from
glutamate to oxaloacetate forming a-
ketoglutarate and aspartate

140. a-Ketoglutarate and aspartate come out
of mitochondria
They are reconverted into glutamate and
oxaloacetate by cytosolic GOT
Glutamate goes back into mitochondria

141. Cytosol Mitochondrial Matrix
NADH+H+
MDH
Oxalo-
acetate
Oxalo-
acetateAspartate Aspartate NADH+H+
3
3
GOT
Glutamate
NAD+ NAD+Malate
a-Keto-
glutarate
a-Keto-
glutarate
MDH
Glutamate
Malate4
GOT
5
Malate shuttle

142. The mitochondrial membrane is selectively
permeable
Small uncharged molecules can pass
through the membrane easily
Monocarboxylic acids can also pass
through the membrane
Transport across mitochondrial membrane

143. Mitochondrial membrane is impermeable
to:
Special transport mechanisms are
required to transport these
Amino acids
Tricarboxylic acids
Dicarboxylic acids

144. Two compounds traversing
the membrane in the same
direction
Two compounds traversing
the membrane in opposite
directions
The transport mechanisms may
operate as:

145. Transport of pyruvate and H+ into
mitochondria is an example of a symport
Membrane Mitochondrial matrixCytosol
Pyruvate Pyruvate
H+ H+

146. Memb MatrixCytosol
ADP ADP
ATP ATP
Malate
a-Ketoglutarate
Malate Malate
Citrate + H+
+ +
Glutamate Glutamate
Aspartate Aspartate
Some important antiports
Malate
a-Ketoglutarate
Citrate + H+