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BIOLOGICAL
OXIDATION
Synopsis
What is Biological Oxidation?
Enzymes and Coenzymes of Biological
Oxidation Reactions.
Electron Transport Chain(ETC)
Oxidative Phosphorylation Mechanisms
Inhibitors of ETC and Oxidative
Phosphorylation
Uncouplers
Shuttle Systems
High Energy Compounds
Substrate Level Phosphorylation.
What Is Biological Oxidation?
Biological oxidation
are oxidation
reactions taking place
in living systems.
Importance Of Biological Oxidation
Biological Oxidation
reactions are associated with
metabolism.
Vital for functioning of
cells, survival and
existence of human body.
Definition Of Oxidation Reactions
Oxidation reactions are
biochemical reactions where there is
either:
Removal / Loss of Hydrogen
(Dehydrogenation)
Removal or Loss of Electrons
Addition of Oxygen
(Oxygenation)
Most predominant type of
Oxidation reaction in body
is:
Dehydrogenation Reaction
Catalyzed by Dehydrogenases
Dehydrogenases
remove Hydrogen from
substrates.
Which are temporarily
accepted by Coenzymes.
Coenzymes involved in
Oxidation/Dehydrogenation
reactions.
NAD+
NADP+
FAD
FMN
Coenzymes temporarily accept the
hydrogen from substrates and get
transformed to reduced coenzymes.
NADH+H+
FADH2
NADPH+H+
FMNH2
Enzymes
and
Coenzymes
of
Biological Oxidation
Reactions
5 Enzymes of Biological Oxidation
1. AEROBIC
DEHYDROGENASES
2. ANAEROBIC
DEHYDROGENASES
3. OXYGENASES
4. OXIDASES
5. HYDROPEROXIDASES
All 5 Enzymes of
Biological Oxidation
reactions are classified
in
Class I
Oxido Reductases
Coenzymes
and
Inorganic Cofactors
Of
Biological Oxidation
Reactions
FMN
FAD
NAD+
NADP+
THBP (Tetra Hydro Biopterin)
Cu++
Fe+++
AEROBIC DEHYDROGENASES
Aerobic Dehydrogenases are Flavoproteins.
Enzymes covalently bound to coenzymes FMN or FAD
MH2 Aerobic DH M
FMN FMNH2 (Auto
oxidizable)
Catalase
H2 +O2 H2O2 H2O + O2
FMN/FAD are acceptors
of removed Hydrogen
Reduced Coenzymes
(FMNH2/FADH2) formed
are auto oxidizable
Reduced coenzymes get
reoxidized at reaction
level.
Oxygen gets directly
involved at reaction level
to reoxidize the reduced
coenzymes.
H2O2 is a byproduct of
Aerobic Dehyrogenase
activity.
Catalase then detoxify
the H2O2 to H2O and O2.
Specific Examples Of
Aerobic Dehydrogenases
L Amino acid Oxidase
(Oxidative Deamination of A.A)
Xanthine Oxidase
(Purine Catabolism)
Glucose Oxidase
(Glucose oxidation to Gluconic acid)
Aldehyde Dehydrogenase
( Alcohol Metabolism)
ANAEROBIC DEHYDROGENASES
Anaerobic Dehydrogenases
catalyzes to remove
hydrogen from substrates.
With the help of coenzymes
NAD+/NADP+/FAD.
MXH2 Anaerobic Dehydrogenase MX
NAD+ NADH+ H+ (Non auto oxidizable)
Enter Electron Transport Chain
For its reoxidation
Coenzymes temporarily accept the
hydrogen from substrates and get
reduced to
NADH+ H+
FADH2
NADPH+H+
FMNH2
Reduced coenzymes formed in
Anaerobic Dehydrogenase
reactions are :
Non autoxidizable/not reoxidized
at reaction level.
Reduced coenzymes
NADH+H+ and FADH2
formed at Anaerobic
Dehydrogenase reaction
enter ETC for its
reoxidation.
Oxygen is involved
indirectly at the end of
ETC as electron and
proton acceptor .
Metabolic water is end
product of ETC.
Reduced coenzyme
NADPH+H+ do not enter
ETC
NADPH+H+ is utilized as
reducing equivalent for
reduction reactions
catalyzed by Reductases.
NAD+ Dependent Anaerobic
Dehydrogenases
Glyceraldehyde -3-PO4 Dehydrogenase
Pyruvate Dehydrogenase
Isocitrate Dehydrogenase
Ketoglutarate Dehydrogenase
Malate Dehydrogenase
Lactate Dehydrogenase
Glutamate Dehydrogenase
Hydroxy Acyl Dehydrogenase
NADP+ Dependent
Dehydrogenases
Glucose -6-Phosphate Dehydrogenase
( HMP Shunt)
Phospho Gluconate Dehydrogenase
(HMP Shunt)
Note NADPH+H+does not enter ETC
for its reoxidation instead they are
involved in reduction reactions.
FAD Dependent Anaerobic
Dehydrogenases
Succinate Dehydrogenase
(TCA Cycle)
Acyl CoA Dehydrogenase
( Oxidation Of Fatty Acids)
FMN Dependent Anaerobic
Dehydrogenase
NADH Dehydrogenase
(Warburg's Yellow
Enzyme)
First Component of ETC/
Complex I of ETC
OXYGENASES
Oxygenases add Oxygen
atom from molecular oxygen
(O2) to the substrate.
Form Oxidized Product.
Mono Oxygenases
Mono Oxygenases add one oxygen
atom from molecular oxygen to the
substrate.
Forms Hydroxyl group (-OH )
Monoxygenases are also termed as
Hydroxylases or Mixed Function
Oxidase.
AH + O2+BH2 Mono Oxygenase AOH+ B+H2O
Tyrosine+O2+THBP Tyrosine
DOPA+DHBP+H2O
Hydroxylase
Examples Of Mono Oxygenases
Phenylalanine Hydroxylase
(Phenylalanine to Tyrosine)
Tryptophan Hydroxylase
(Tryptophan to 5HydroxyTryptophan)
25 Hydroxylase
(Vitamin D - Cholecalciferol activation)
1 Hydroxylase
(Vitamin D - Cholecalciferol activation)
Di Oxygenases
Dioxygenases are true Oxygenases
Incorporates two Oxygen atoms
from O2.
A+ O2 Dioxygenase AO2
Examples Of Dioxygenases
Tryptophan Di Oxygenase/
Tryptophan Pyrrolase
(Tryptophan NFormyl Kynurenine )
PHPP Dioxygenase
Cysteine Dioxygenase
Homogentisate Oxidase
(Homogentisate to 4 Maleyl Acetoacetate)
Oxidases
Oxidases involve molecular
Oxygen as Hydrogen (electron
and proton ) acceptor.
Oxidases reduces
molecular Oxygen to Water
(H2O)
AH2 + ? O2 Oxidase A+ H2O
Tyrosine+ O2 Tyrosinase -Cu++ DOPA +
H2O
Examples Of Oxidases
Cytochrome Oxidase
(ETC enzyme) Classic Example
Ascorbate Oxidase
Mono Amine Oxidase
Catechol Oxidase
Hydroperoxidases
Hydroperoxidases detoxify
Hydrogen Peroxide in body.
H2O2 is a substrate/reactant
for Hydroperoxidases.
Hydroperoxidases are
Hemoproteins.
Contains loosely
bound Heme as
prosthetic group.
Hydroperoxidases prevent
accumulation of H2O2 in cells.
H2O2 if accumulated in cells is
toxic
Leads to disruption of
membranes(Hemolysis).
Increases risk of cancer and
atherosclerosis.
Specific Examples Of
Hydroperoxidases
Peroxidases
Catalase
Peroxidases
Indirectly react with H2O2.
Glutathione Peroxidase
(In R.B.C's)
Leukocyte Peroxidase
(In W.B.C's)
H2O2 + 2 GSH Glutathione Peroxidase 2H2O + GSSG
(Reduced (Oxidized
active Form)
inactive Form)
Catalase
Directly reacts with H2O2.
Associated with Aerobic
Dehydrogenase catalyzed
reaction.
2H2O2 Catalase 2H2O +O2
Biological Oxidation Process
Electron Transport Chain
(ETC)
Synonyms
Electron Transport Chain (ETC)
Electron Transport System (ETS)
Respiratory Chain
Internal/Cellular Respiration
Tertiary metabolism
Fate of reduced Coenzymes
NADH+H+/FADH2
Final Oxidative Pathway
Oxidative Phosphorylation
What is Electron Transport Chain?
Electron Transport chain
Vital biological oxidation
process.
Carried out in aerobic
condition.
Located at inner membrane
of Mitochondria.
Fate of ETC is to
reoxidize the reduced
coenzymes
NADH+H+/FADH2.
Formed during Anaerobic
Dehydrogenase reactions.
Electron Transport Chain
Transports Electrons and
Protons
Through a series of ETC
components and
Finally to activated molecular
oxygen.
Generates ATP and metabolic
water.
What is Oxidative Phosphorylation?
Oxidative process (ETC)
is tightly coupled with
Phosphorylation of ADP
with pi to generate ATP.
Oxidative
Phosphorylation is a
major mode of ATP
generation.
Location Of ETC
ETC is carried out in all cells
which contain mitochondria
(Power house of Cell).
(Except mature Erythrocytes which
are devoid of mitochondria)
Components and Enzymes of
ETC are arranged towards inner
surface of inner membrane of
mitochondria.
In vectorial conformation
In increased order of positive
redox potential
Location of Mitochondrial ETC Complexes
? Inner membrane of mitochondria
Condition Of ETC Operation
ETC operates in truly aerobic
condition.
Oxygen unloaded at cellular level
by HbO2 gets utilized at the
end of ETC process.
(Respiratory Chain)
ETC depends on
Respiration Process
Oxygen Concentration
Hemoglobin Structure and
Function
Metabolic Status
Components Of ETC
Flavo Protein- (First Component)
NADH Dehydrogenase-FMN and FeS
centers(Warburg's Yellow Enzyme)
Coenzyme Q/ Ubiquinone
Series of Cytochromes-
Cytochrome b-Cytochrome c1-
Cytochrome c- Cytochrome aa3
Iron-sulfur centers (Fe-S) are prosthetic groups
containing 2, 3 , 4 or 8 iron atoms complexed to elemental
& cysteine S.
4-Fe centers have a tetrahedral structure, with Fe & S
atoms alternating as vertices of a cube.
Cysteine residues provide S ligands to the iron, while also
holding these prosthetic groups in place within the protein.
Electron transfer proteins
may contain multiple Fe-S
centers.
Iron-sulfur centers
transfer only one electron,
even if they contain two or
more iron atoms, because
of the close proximity of
the iron atoms.
Fe+++ (oxidized) + 1 e- Fe++ (reduced)
Coenzyme Q / Ubiquinone
Coenzyme Q (CoQ)/ Ubiquinone)
is located in the lipid core of the
mitochondrial membrane.
It is a Quinone derivative
Lipophilic dissolves in the
hydrocarbon core of a membrane.
Cytochrome has a long Poly
isoprenoid tail, with multiple
units of isoprene.
In human cells, most often n = 10.
Q10 isoprenoid tail is longer than
the width of a bilayer.
Coenzyme Q is very
hydrophobic.
Coenzyme Q functions as a
mobile e- carrier within the
mitochondrial inner
membrane.
Its role in trans-membrane
H+ transport coupled to e-
transfer (Q Cycle).
The Quinone
ring of coenzyme
Q can be reduced
to the Quinol in
a 2e- reaction:
Q + 2 e- + 2 H+ QH .
2
When bound to special sites in respiratory complexes,
CoQ can accept 1 e- to form a semiquinone radical (Q?
-).
Thus CoQ, like FMN, can mediate between 1 e- & 2 e-
donors/acceptors.
Cytochromes
Cytochromes are Hemoproteins
conjugated proteins in ETC
Carrier of electrons
Contain heme as prosthetic
group.
Cytochromes absorb light at
characteristic wavelengths.
Absorbance changes upon
oxidation/reduction of the
Heme Iron
Cytochrome Heme
Cytochrome heme Iron is in
transitional state
Carries electrons only:
Fe (III) + e- Fe (II)
Only one electron is transferred
at a time.
Cytochrome heme iron
can undergo a 1 e-
transition between ferric
and ferrous states:
Fe+++ + e- Fe++
(oxidized) (reduced)
Series of Cytochromes b, c , c, aa
1
3
relay electrons (one at a time, in this
order
Cytochrome c is a small,
water soluble protein with a
single heme group.
Cytochromes a & a3 are
often referred to as
Cytochrome Oxidase
/complex IV.
Cytochrome aa3 has Fe and
Cu.
All Cytochromes
except Cytochrome
Oxidase are Anaerobic
Dehydrogenases.
ETC Complexes
ETC complexes are
combination of one or two
components of ETC.
There are 5 ETC complexes.
Complex I- NADH CoQ Reductase
NADH Dehydrogenase FMN and FeS centre
Complex II ? Succinate CoQ Reductase
Succinate Dehydrogenase FAD and FeS centre.
Complex III?CoQ Cytochrome C Reductase
Cytochrome b ? Cytochrome c1
Complex IV- Cytochrome Oxidase
Cytochrome aa3
Complex V ? ATP Synthetase
F0 and F1 of ATP Synthase
Composition of Respiratory Chain Complexes
No. of
Prosthetic Groups
Complex
Name
Proteins
Complex I
NADH
46
FMN,
Dehydrogenase
9 Fe-S centers
Complex II
Succinate-CoQ
5
FAD, cyt b ,
560
Reductase
3 Fe-S centrs.
Complex III
CoQ-cyt c Reductase
11
cyt b , cyt b , cyt c ,
H
L
1
Fe-SRieske
Complex IV
Cytochrome Oxidase
13
cyt a, cyt a , Cu , Cu
3
A
B
Electrons are transferred from
NADH O via multisubunit
2
inner membrane complexes I, III
& IV, plus CoQ & Cytochrome c.
Within each complex, electrons
pass sequentially through a series
of electron carriers.
Complex I catalyzes
oxidation of NADH+H+ with
reduction of coenzyme Q:
NADH + H+ + FP NAD+ + FPH2
Coenzyme Q accepts 2 e- and picks
up 2 H+ from FPH2 to yield the fully
reduced QH2.
Succinate Dehydrogenase of the Krebs Cycle is
also called complex II or Succinate-CoQ
Reductase.
FAD is the initial e- acceptor.
FAD is reduced to FADH2 during oxidation of
Succinate to Fumarate.
FADH2 is then reoxidized by
transfer of electrons through a
series of 3 iron-sulfur centers to
CoQ, yielding QH2.
The QH2 product may be
reoxidized via complex III.
Providing a pathway for transfer of
electrons from Succinate into the
respiratory chain.
CoQ accepts electrons via
ETC complexes I and II.
Complex III/ Cytochrome b-c1
complex accepts electrons from
coenzyme QH2 that is generated
by electron transfer in complexes
I & II.
Cytochrome c resides in the
intermembrane space.
It alternately binds to
complex III or IV during e-
transfer.
COMPLEX IV
Cytochrome a-a3/ Cytochrome Oxidase
large protein
Both a and a3 contain heme and Cu
a3 Cu binds to oxygen and donates
electrons to oxygen
Cytochrome a3 - only component of
ETC that can interact with O2
Cytochrome oxidase (complex IV) carries out
the following irreversible reaction:
O + 4
O
2
H+ + 4 e- 2 H2
The four electrons are transferred into the
complex one at a time from Cytochrome c.
Complex IV/Cytochrome
Oxidase reduces
molecular Oxygen to
water.
Cytochrome Oxidase
Cu(II) Cu(I)
e- from cyt c to a
Heme A and Cu act together to
transfer electrons to oxygen
Metal centers of cytochrome oxidase (complex IV):
heme a & heme a3,
CuA (2 adjacent Cu atoms) & CuB.
O2 reacts at a binuclear center consisting of heme
a and Cu .
3
B
An Iron-Copper Center in
Cytochrome Oxidase
Catalyzes Efficient O2
Reduction
Complex V
ATP Synthase
Two units, Fo and F1
("knob-and-stalk"; "bal on a stick")
F1 contains the catalytic subunits
where ADP and Pi are brought
together for combination.
F0 spans the membrane and serves as
a proton channel.
? F1 contains 5 types of
polypeptide chains - a3b3gde
? Fo - a1b2c10-14
(c subunits form cylindrical,
membrane-bound base)
? Fo and F1 are connected by a ge
stalk and by exterior column
(a1b2 and d)
? The proton channel ? between c
ring and a subunit.
Complex V ATP Synthase
ATPase is a Rotating Motor
Complex I ,III and IV act as a
Proton Pump.
Pump out the protons from
matrix side to inter membrane
space of mitochondria.
Develop a proton gradient in
inter membrane space.
This supports the mechanism of
Oxidative Phosphorylation.
F F couples ATP synthesis to H+
1 o
transport into the mitochondrial matrix.
Transport of at least 3 H+ per ATP is
required.
Electron Transport is coupled to
Oxidative Phosphorylation
Salient Features Of
ETC/ETS/Oxidative Phosphorylation
Reduced coenzymes NADH and
FADH2 are formed in matrix from:
Oxidative Decarboxylation of Pyruvate to
Acetyl CoA by PDH complex.
Oxidation of acetyl CoA by the citric acid
cycle
Beta Oxidation of fatty acids
Amino acids metabolism
Reduced coenzymes
NADH+H+/FADH2
Generated during Anaerobic
Dehydrogenase reactions of
Carbohydrates, Lipids metabolic
pathways.
Get reoxidized on entering
E.T.C
The NADH+H+ and
FADH2 are energy rich
molecules
Each contains a pair of
electrons having a high
transfer potential.
Glycolysis, Fatty
acid oxidation
TCA cycle
supplies NADH
and FADH2 to
the Electron
Fatty Acids
Acetyl Co A
Transport Chain
Pyruvate
Amino Acids
Glucose
The reduced and oxidized forms of NAD
The reduced and oxidized forms of FAD
Redox Couples and Redox
Potentials
The components of ETC has
capacity to exist in oxidant and
reductant forms.
This pair is known as redox
couple.
CoQ/CoQH2
Cyt b Fe+++/Cyt b Fe++
Redox Potential is a
measure of the tendency of
a redox couple to accept
or donate electrons under
standard condition.
Components that have
the most negative redox
potentials have the
weakest affinity for
electrons.
Redox couple with most
positive redox potentials
have
The strongest affinity for
electrons therefore
Possess strongest tendency to
accept electrons.
In ETC electrons flow from most
electro negative potential
NADH+H+ (-0.32) to most
electro positive potential
(+0.82) ? O2 .
In E.T.C both Protons and Electrons
are transferred up to Coenzyme Q
level.
At coenzyme Q level protons (2H+)are
released in the medium.
From Coenzyme Q onwards only
electrons are transferred through a
series of Cytochromes in E.T.C.
Electrons get transfer through
series of Cytochromes
Cytochrome Fe is in transitional
state (Ferric/Ferrous ).
In E.T.C there are
alternate reduction
and oxidation
reactions.
During E.T.C there is transfer
of reducing equivalents from
low redox potential to high
redox potential.
This exhibit free energy
change there by liberating
heat energy.
Electrons move spontaneously
from one component of ETC to
another with a
low redox potential (a low
affinity for electrons) to a
component with a
high redox potential (a high
affinity for electrons)
In ETC electrons move from a
carrier with
low redox potential
(high tendency to donate
electrons) toward carriers with
higher redox potential
(high tendency to accept
electrons).
The proton gradient
runs downhill to drive
the synthesis of ATP
Oxygen is the terminal
acceptor of electrons in
the electron transport
chain.
At the end of E.T.C by the catalytic
activity of Cytochrome Oxidase
Protons released at Coenzyme Q
and electrons transported by
Cytochromes are
Accepted by activated molecular
oxygen (1/2 O2) to form metabolic
water.
Oxygen has the highest (most
positive) standard redox
potential .
Most likely to accept electrons
from other carriers.
Electrons ultimately reduce Oxygen
to water (metabolic water)
2 H+ + 2 e- + ? O2 -- H2O
Cytochrome oxidase
controls the rate of O2
uptake which
Means this enzyme
determines how rapidly
we breathe.
F F couples ATP synthesis to H+
1 o
transport into the mitochondrial matrix.
Transport of at least 3 H+ per ATP is
required.
The respired Oxygen
transported by Hb
unloaded at tissue/
cellular level is
utilized during E.T.C.
WHY ETC OPERATES ?
During E.T.C operation the
total energy change is
released in small increments
So that energy can be trapped
as chemical bond energy to
form ATP.
When two redox couples of ETC
differ from each other by 0.22 volts
in standard redox potential.
The free energy in the form of heat
released is more than 7.3 Kcal.
This free heat energy is conserved
to chemical form of energy-ATP.
The sites in E.T.C at
which energy liberated
is less than 7.3 Kcal is
simply dissipiated in
the form of heat.
Certain sites in E.T.C
where the heat energy
liberated more than 7.3
Kcal is utilized for
phosphorylation
reaction of ADP with pi
to form ATP.
Thus heat energy is
transformed to
chemical form of
energy (ATP) in E.T.C.
THE ELECTRON TRANSPORT CHAIN
Series of enzyme complexes (electron carriers)
embedded in the inner mitochondrial membrane,
which oxidize NADH+H+ and FADH2 and
transport electrons to oxygen is cal ed
Respiratory Electron-Transport Chain (ETC).
The sequence of electron carriers in ETC
NADH FMN-Fe-S Co-Q Fe- cyt b
S cyt c1 cyt c cyt a cyt a3 O2
Succinate FAD Fe-
S
Electrons of NADH or FADH2 are used to
reduce molecular oxygen to water.
A large amount of free energy is liberated.
The electrons from NADH+H+ and FADH2 are not
transported directly to O2 but are transferred
through series of electron carriers that undergo
reversible reduction and oxidation.
A PROTON GRADIENT POWERS
THE SYNTHESIS OF ATP
The transport of electrons from NADH or FADH2 to
O2 via the electron-transport chain is exergonic
process:
NADH + ?O2 + H+ H2O + NAD+
FADH2 + ?O2 H2O + FAD+
Go' = -52.6 kcal/mol for NADH
-36.3 kcal/mol for FADH2
This process is coupled to the synthesis of ATP
(endergonic process)
ADP + Pi ATP + H2O Go'=+7.3 kcal/mol
The flow of electrons through ETC
complexes leads to the pumping of
protons out of the mitochondrial
matrix in intermembrane space.
The resulting distribution of protons
generates a pH/Proton gradient and
a transmembrane electrical potential
that creates a proton motive force.
A Large Drop in Redox
Potential across each of the
three Respiratory Enzyme
Complexes (I,III,IV).
Provides the Energy for H+
Pumping
ATP is synthesized when
protons flow back from
intermembrane space of
mitochondria to the
mitochondrial matrix
through an enzyme complex
ATP synthase.
The oxidation of fuels
and the phosphorylation
of ADP are coupled by a
proton gradient across
the inner mitochondrial
membrane.
Oxidative phosphorylation is
the process in which ATP is
formed
As a result of the transfer of
electrons from NADH or
FADH2 to O2 by a series of
electron carriers.
E.T.C is a Mode For Free Radical
Generation
During E.T.C operation there
occurs leakage of small amounts
of electrons
Which are transferred directly
to oxygen to form super oxide
ion (Free radicals).
What is a Free Radical ?
Any chemical species with one of
more unpaired electrons.
Unstable/Highly Reactive
Powerful Oxidant
Short half life (nanoseconds)
Can exist freely in the environment
Significance Of ETC
Reduced coenzymes gets reoxidized
to NAD+ /FAD in ETC for its
reutilization in metabolic oxidation
reactions.
Reduced coenzymes NADH+
H+/FADH2 give its reducing
equivalents to E.T.C components and
get reoxidized.
E.T.C generates chemical form of
energy ATP as a valuable by product.
P/O Ratio
The ratio of ATPs formed
per oxygen reduced
OR
Number of ATPs generated
per Oxygen atom used in
ETC process.
To make 1 ATP need 30
kJ/mole.
There needs more than one
proton to translocate during
ETC process to generate 1 ATP.
Ten protons are pumped out of
the matrix during the two electrons
flowing from NADH+H+ to O2
(Complex I, III and IV).
Six protons are pumped out of the
matrix during the two electrons
flowing from FADH2 to O2
(Complex III and IV).
Spontaneous
electron flow
through each
of complexes I,
III, & IV
is coupled to
H+ ejection
from the
matrix.
A total of 10 H+ are ejected from the mitochondrial matrix per
2 e- transferred from NADH to oxygen via the respiratory
chain.
A total of 6 H+ are ejected from the mitochondrial matrix per
2 e- transferred from FADH2 to oxygen via the respiratory
chain.
Complex I and Complex III transports 4H+
out of the mitochondrial matrix per 2e-
transferred from NADH.
Thus there are 2H+ per 2e- that are
effectively transported by complex
IV.
ATP Yield
3
4
4
2
ETC translocates 10
protons per NADH+H+
ETC translocates 6
protons per FADH2
Proton gradient created
as electrons transferred to
oxygen forming water
10 H+ / NADH+H+
6 H+ / FADH2
Translocation of 3H+ required
by ATP Synthase for each ATP
produced
1 H+ needed for transport of Pi.
Net: 4 H+ transported for
each ATP synthesized
through ATP Synthase.
F F couples ATP synthesis to H+
1 o
transport into the mitochondrial matrix.
Transport of least 3 H+ per ATP is
required.
P:O Ratio for NADH+H+
10 H+ X 1 ATP = 2.5 ATP
4 H+
P.O Ratio for FADH2
6 H+ X 1 ATP = 1.5 ATP
4 H+
P:O ratio for NADH: 10 H+/4H+ =
2.5 ATP
P:O ratio for FADH2: 6 H+/ 4H+ =
1.5 ATP
ATP Is A Valuable Byproduct Of
E.T.C/ Oxidative Phosphorylation.
ATP is a high energy phosphate
compound
Biologically important free
nucleotide
ATP has 2 high energy Phospho
anhydride bonds.
ATP is energy currency of
cell.
Predominantly generated
through Oxidative
Phosphorylation.
Sites Of ATP Production In ETC
3 sites Of ATP Generation in ETC
Site I/Complex I-
Electrons transferred from Complex I to
CoQ
Site II/Complex III-
Electrons transferred from Cyt b to Cyt c1.
Site III/Complex IV-
Electrons transferred from Cytochrome
aa3/Complex IV/Cytochrome Oxidase to ?
O2
Uses of ATP generated in
Oxidative Phosphorylation
?Synthetic/Anabolic reactions
?Active transport mechanism.
?Muscular contraction
?Nerve impulse conduction.
ATP allows the coupling
of thermodynamically
unfavorable reactions to
favorable reactions.
ATP is continually being
hydrolyzed and
regenerated
A person at rest
consumes and regenerate
3 ATP/ sec
Staying Alive Energy Wise
We need 2000 Cal/day or 8,360 kJ of energy per day
Each ATP gives 30.5 kJ/mole of energy on hydrolysis
We need 246 moles of ATP
Body has less than 0.1 moles of ATP at any one time
We need to make 245.9 moles of ATP
Each mole of Glucose yields 38 ATPs or 1160 kJ
We need 7.2 moles of Glucose (1.3 kg or 2.86 pounds)
Each mole of Stearic acid yields 147 ATPs or 4,484 kJ
We need 1.86 moles of stearic acid (0.48 kg or 1.0
pound of fat)
Shuttling Electron Carriers from
Cytosol into the Mitochondrion
NADH+H+ carrier of reducing
equivalents generated in the cytosol via
Glycolysis should make its entry into
mitochondrial ETC.
Since the inner mitochondrial membrane
is impermeable to NAD+ and NADH.
This reducing equivalents must be
shuttled into the mitochondrial matrix
before they can enter the ETC.
NADH+H+ is
generated in
the cytosol in
Glycolysis
Reducing Equivalents
from
Cytosolic NADH+H+
Enter Mitochondria
via
Shuttle Systems
NADH itself is not carried
across the mitochondrial
membrane.
Protons and Electrons of
Cytosolic NADH+H+ are carried
through shuttle systems.
Two shuttles Involved:
Glycerol 3-phosphate Shuttle
Malate-Aspartate Shuttle
Glycerol-3-Phosphate Shuttle
Glycerol Phosphate Shuttle
Glycerol Phosphate Shuttle is
active in Skeletal muscles and
Brain.
FADH2 formed enter the
electron-transport chain
through CoQ.
Therefore only 1.5 molecules of
ATP are produced.
Malate-Aspartate Shuttle
Malate/Aspartate Shuttle System
Malate Aspartate Shuttle
Active in Heart and Liver.
2.5 molecules of ATP are
produced.
Summary
Total ATP Generated / 1 Glucose Oxidation
Muscle and Brain
30.0 ATP
Uses Glycerol phosphate Shuttle
Heart and Liver
32.0 ATP
Uses Malate Aspartate Shuttle
E.T.C Inhibitors
Chemical substances having
affinity for ETC components.
Chemically interact with ETC
complexes and functionally
inactivate them.
ETC Inhibitors
Enemies of ETC components who
stop its normal operation.
No ETC and No ATP generation.
Site I/Complex I- E.T.C Inhibitors.
vAmobarbital /Amytal
vRotenone (Fish/Rat Poison)
vMercurials
vPiercidin -A
Site II/Complex III- E.T.C Inhibitors.
vBritish Anti Lewisite ( BAL)
vAntimycin ?A
vDimercaprol
Site III/Complex IV- E.T.C
Inhibitors/Cytochrome Oxidase
Inhibitors.
vCyanide
vCarbon Monoxide
vH2S
vAzide
These chemicals
arrest respiration
by inhibition of the
ETC.
Sequence of
Respiratory
Electron
Carriers
Inhibitors
in green
Specific inhibitors of
Electron Transport
Chain
and
ATP-Synthase
Mechanism Of
Oxidative Phosphorylation
Oxidation tightly coupled
with Phosphorylation
E.T.C Process coupled with
phosphorylation of ADP+pi
to generate ATP.
Chemical Coupling Hypothesis
Conformational Coupling Hypothesis
Chemiosmotic Theory
Chemical Coupling Hypothesis:
Put forward by Edward Slater
(1953)
Proposed series of high energy
phosphorylated intermediates
are produced during E.T.C
operation.
Which are used to produce ATP.
Conformational Coupling
Hypothesis
Paul Boyer 1964
Mitochondrial Cristae undergo
conformational change in the
components of E.T.C.
E.T.C components attain high
energy state which are
responsible for the ATP production.
Chemiosmotic Theory
Put forward by Peter Mitchell (1961)
(Nobel Prize, 1978)
E.T.C process and ATP synthesis are
coupled by a proton gradient
developed in intermembrane space of
mitochondria.
Mitchell's Postulates for
Chemiosmotic Theory
Intact inner mitochondrial
membrane is required
Electrons are pumped through ETC
complex I,III and IV.
Generates a proton gradient in
intermembrane space of
mitochondria.
Proton gradient in inter
membrane space creates
Proton Motive Force.
Due to electrochemical
potential difference.
The proton gradient
have a thermodynamic
tendency.
Proton Motive Force drives the
Protons from mitochondrial
intermembrane space back to
matrix side
Through a specific site of F0
and F1 particle of ATP
Synthase.
ATP Synthase catalyzes the
phosphorylation of ADP with pi
In a reaction driven by
movement of H+ across the
inner membrane back into the
matrix.
Translocation of protons
through ATP Synthase
Stimulates and activates ATP
Synthase
For the catalytic action of
phosphorylation- ADP with pi to
form ATP.
Supports the mechanism of
Oxidative Phosphorylation.
The flow of three H+ through
an ATP Synthase complex
Causes a conformational
change, which causes the
ATP synthase to synthesize
ATP from ADP + Pi.
This process of producing ATP
is known as oxidative
phosphorylation.
The entire process of using the
proton motive force to make
ATP is called Chemiosmosis.
During oxidative
phosphorylation the total
energy change is released
in small increments.
So that energy can be
trapped as chemical bond
energy and form ATP.
Coupling of ATP synthesis to
respiration is indirect, via a H+
electrochemical gradient.
The proton pumps are Complexes I,
III and IV.
Protons return through ATP synthase
ATP synthesis at F1 results from
repetitive comformational changes
as rotates
rotates 1/3 turn-
energy for ATP release
ATP Synthase, a molecular mill.
Overview of Oxidative Phosphorylation
+
+
+
+ +
+
-
-
-
-
As electrons flow through complexes of ETC, protons are translocated
from matrix into the intermembrane space.
The free energy stored in the proton concentration gradient is tapped
as protons reenter the matrix via ATP synthase.
As result ATP is formed from ADP and Pi.
ATP molecules are
transported out of the
mitochondrial matrix
through specific
transporters.
REGULATION OF OXIDATIVE
PHOSPHORYLATION
Important substrates:
NADH/FADH2
O2
ADP
Electron transport is tightly coupled
to phosphorylation.
ATP cannot be synthesized by
oxidative phosphorylation unless
there is energy from electron
transport.
Electrons do not flow through the
electron-transport chain to O2 unless
ADP is phosphorylated to ATP.
ADP and pi is required for ETC
process.
Intramitochondrial ratio
ATP/ADP is a control
mechanism
At low ADP levels oxidative
phosphorylation low.
ADP levels reflect rate of ATP
consumption and energy state
of the cell.
At high ATP/ADP
ATP acts as an allosteric
inhibitor for Complex
IV (Cytochrome Oxidase)
Inhibition is reversed by
increasing ADP levels.
Respiratory Control
The most important factor in determining the
rate of oxidative phosphorylation is the level of
ADP.
The regulation of the rate of oxidative
phosphorylation by the ADP level is cal ed
respiratory control
Inhibitors Of
Oxidative
Phosphorylation
Oligomycin
An antibiotic binds with Fo particle
of ATP Synthase
Fo particle serve as proton channel.
Inhibits phosphorylation of ADP to
ATP.
Atractyloside
A Glycoside prevents the
translocation of ADP across
mitochondrial membrane.
Make it unavailable for
phosphorylation reaction.
Bongregate
Pseudomonas toxin has
inhibitory action similar
like Atractyloside.
Uncouplers Of
Oxidative
Phosphorylation
What are Uncouplers?
Uncouplers are chemical agents
That uncouple the two tightly
coupled processes E.T.C
(Oxidation) from Phosphorylation.
Uncouplers break the
connection between
Electron Transport Chain and
Phosphorylation
Electron transport is a motor
Phosphorylation is the transmission
Uncouplers put the car in NEUTRAL
?Uncouplers are mostly
lipid soluble aromatic
weak acids.
Uncouplers deplete proton
gradient of intermembrane
space during ETC operation.
Uncouplers just bring oxidation
(E.T.C) without phosphorylation.
Uncouplers inhibit generation
of ATP.
Physiological Uncouplers
Thermogenin /UCP-1
Excess of Thyroxine
Long Chain Fatty acids
Unconjugated Hyperbilirubinemia
Chemical Uncouplers
2,4 Di Nitro Phenol
Di Nitro Cresol
Dicumarol
Aspirin
FCCP
Valinomycin
Mode Of Action Of Uncouplers
Certain Uncouplers are ionophores,
lipophilic substances.
They carry protons from
intermembrane space across
mitochondrial membrane to matrix
From the site other than specific
site. (i.e through F0 and F1 particles of
ATP Synthase).
Certain Uncouplers changes
the permeability of the
mitochondrial membrane to
protons.
Translocate protons easily
through mitochondrial
membrane.
2,4 DNP dissolve in the membrane and function as
carriers for H+.
Do not allow to develop proton
gradient and proton motive force
in the intermembrane space of
mitochondria.
Causes no stimulation or
activation of ATP Synthase
No catalysis of Phosphorylation of
ADP with pi to generate ATP.
During uncoupling phenomena
Heat energy dissipiated as it is and
causes swelling of mitochondria.
Uncouplers block oxidative phosphorylation by
dissipating the H+ electrochemical gradient.
Protons pumped out leak back into the mitochondrial
matrix,
preventing development of proton gradient and
proton motive force.
Respiration proceeds in the
presence of an uncoupler,
whether or not ADP is present.
The ATP Synthase reaction runs
backward in presence of an uncoupler.
w Hydrolysis of ATP is spontaneous.
Physiological Uncoupling
By
Uncoupling Protein (UCP-1)
An Uncoupling Protein (UCP-1)/
Thermogenin is produced in brown
adipose tissue of newborn mammals and
hibernating mammals.
This protein of the inner mitochondrial
membrane functions as a H+carrier.
The uncoupling protein blocks
development of a H+ electrochemical
gradient, thereby stimulating
respiration.
Free energy of ETC is dissipated as
heat.
Uncoupling of ETC and
phosphorylation occurs in animals
as a means to produce heat
Non shivering thermogenesis.
Occurs in brown adipose tissues
(rich in mitochondria)
This "non-shivering
thermogenesis" is costly in
terms of respiratory energy,
Heat energy unavailable for ATP
synthesis, but provides valuable
warming of the organism.
Significance Of Physiological
Uncouplers
In extreme cold conditions and in
hibernating animals
Physiological Uncouplers bring
uncoupling phenomena
The heat liberated inside the body
helps to restore and maintain the
body temperature.
Brown adipose
(fat) cells
contain natural
Uncouplers to
warm animals
cold adaptation
and hibernation.
ETC Inhibitors and Uncouplers
Any compound that stops
electron transport will stop
respiration...this means you
stop breathing
Electron transport can be
stopped by inhibiting ATP
synthesis
An uncoupler breaks the
connection between ATP
synthesis and electron
transport
Inherited Disorders
Related To E.T.C
Infantile Mitochondrial Myopathy
Defect in E.T.C components
located in mitochondria.
Associated with renal
dysfunction.
Mostly fatal in early age.
MELAS
An inherited disorder caused due to
deficiency of complex I or IV of
E.T.C
Associated with
Mitochondrial Myopathy
Encephalopathy
Lactic Acidosis
Stroke
Inherited Disorders of
Oxidative Phosphorylation
Leber's Hereditary
Optic Neuropathy (LHON)
Caused due to mutations in
mitochondrial DNA
Affects oxidative
phosphorylation.
Loss of bilateral vision due to
neuroretinal degeneration.
Factors Affecting ETC Process
Oxygen supply to cells.
Hemoglobin structure and
function
Respiratory system and its function
Mitochondrial structure and ETC
components.
Availability of reduced
coenzymes.
Adequate amount of ADP
and pi.
Presence of ETC inhibitors.
Conditions Affecting ETC and ATP
Low/slow operation
of ETC and less
production of ATPs
is noted in
conditions of:
Hypoxia
Anemia
Ischemia
Hemoglobinopathies
Emphysema
Respiratory Distress Syndrome
Asthma
Inherited Disorders of Mitochondria
ETC inhibition by chemicals/drugs.
High Energy Compounds Of
Human Body.
High energy compounds are
energy rich compounds.
Possess high energy bonds in its
structures.
Cleavage of these high energy
bonds liberate more energy than
that of ATP hydrolysis.
S.No
Examples Of High
Free Energy
Energy Compounds
Released On
Hydrolysis.
Cal/mol
1
Phospho Enol Pyruvate
-14.8
2
Carbamoyl Phosphate
- 12.3
3
Cyclic AMP
-12.0
4
1,3 Bis Phospho Glycerate
-11.8
S.No
Examples Of High Energy
Free Energy
Compounds
Released On
Hydrolysis.
Cal/mol
5
Creatine Phosphate
-10.3
6
S Adenosine Methionine
-10.0
( SAM)
7
Succinyl CoA
-7.7
8
Acetyl CoA
-7.7
9
ATP
-7.3
Significance Of High Energy
Compounds.
In catabolic pathways/reaction
High energy compounds follow
substrate level phosphorylation
reaction.
High energy compounds cleave to
generate energy used for
phosphorylation of ADP with pi at
reaction level.
Generate ATP at substrate level.
Substrate Level Phosphorylation
Mode of generation of ATP at
substrate level.
Involves cleavage of high energy bond
present in high energy compound.
Bond energy released is used for
Phosphorylation reaction.
Generates ATP directly and instantly
without involvement of ETC process.
Examples Of High Energy
Compounds Undergoing Substrate
Level Phosphorylation.
S.No
High Energy
Enzyme
Product
High
Metabolic
Compound
Catalyzing
Obtained
energy
Pathway
Phosphate
Involved
Compound
Generated
1
1,3 Bis
Phospho
3
ATP
Glycolysis
Phospho
Glycerate Phospho
Glycerate
Kinase
Glycerate
2
Phospho
Pyruvate
Enol
ATP
Glycolysis
Enol
Kinase
Pyruvate
Pyruvate
3
Succinyl
Succinate Succinate
GTP
Krebs/TCA
CoA
Thio
Cycle
Kinase
In anabolic pathways/reaction
High energy compounds follow
condensation or bond building
reactions.
High energy compound cleave to
generate energy
Energy used for building C-C
bonds.
Questions
Long Essays.
Q.1 Define Biological oxidation.
Enumerate and Describe various
enzymes carrying out biological
oxidation reactions with suitable
examples.
Q.2 Describe Respiratory chain and
Give its significance.
OR
Explain the Electron. Transport chain
(E.T.C.) and its significance.
OR
How the reduced equivalents generated
in anaerobic dehydrogenase reactions
are reoxidized.
Q.3 What is oxidative
phosphorylation? Explain
the mechanism with
respect to various theories
and hypothesis.
Short Notes
Cytochromes
Inhibitors of E.T.C
Shuttle systems and its
significance
Inhibitors and Uncouplers of
oxidative phosphorylation
Complexes of E.T.C.
Redox potential and free energy
changes.
Inherited Disorders related to E.T.C.
abnormality.
ATP ? Mode of its formation and
it's role in the Body.
Short Answer Questions
Give the sites for ATP generation of
in E.T.C.
Enumerate the High energy
compounds of our body
Substrate level phosphorylation and
it's importance.
Enumerate the Enzymes
catalyzing Biological oxidation
reactions. Write the class to
which these enzymes classified.
Define P.O ratio. What is the P:O
ratio for reduced NADH+H+ &
FADH2 respectively.
List the components of E.T.C. and
their location.
Redox couple & Redox potential.
FlavoProteins
Product of Aerobic and
Anaerobic dehydrogenation
reactions.
Write enzymes catalyzing
Aerobic and Anaerobic
dehydrogenation reaction's
during metabolism.
THANK YOU
This post was last modified on 05 April 2022