Download MBBS Biochemistry PPT 44 Final Biologcal Oxidation 18 Lecture Notes

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Induction To Todays Topic
Any Guesses Of Todays Topic???

Energy Metabolism

Bioenergetics

BIOLOGICAL OXIDATION
Specific Learning Objectives

Questions Which Wil be Answered

What is system of Bioenergetics ?

How is chemical form of energy ATP formed (Generation)
and utilized (Operation) in human body ?

What Factors are associated to bioenergetics system?

Metabolites
Enzymes
Coenzymes
Cofactors
Hormones

Which disorders suffered due to defective system ?
Synopsis

vWhat is Bioenergetics?
vHigh Energy Compounds
vSubstrate Level Phosphorylation
vWhat is Biological Oxidation?
vEnzymes and Coenzymes of Biological

Oxidation Reactions

vElectron Transport Chain (ETC)

Continued---------

vOxidative Phosphorylation Mechanism
vInhibitors of ETC and Oxidative Phosphorylation

vUncouplers- Mode of Action

vShuttle System

vFactors Involved in Oxidative Phosphorylation

mechanism


Lets Get Introduced To
What Is Bioenergetics?

Bioenergetics or biochemical

thermodynamics is:

Study of energy changes during

biochemical reactions.

Biological Systems Conform to General

Laws of Thermodynamics

Energy Is Never Destructed

(Soul is energy never destructed and it is Immortal)

Total energy of a system, including its surroundings,

remains constant

Energy is neither lost nor gained during any change

May be transformed into another form of energy

May be transferred from one part of system to another

or


Conditions Of Bioenergetics

Isothermic (mostly)

Endothermic/

Endergonic/Anabolic

Exothermic/Exergonic

/Catabolic

ENDERGONIC ( Anabolic) PROCESSES

PROCEED BY COUPLING

OF EXERGONIC(Catabolic)

PROCESSES
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

OR

Fates Of High Energy Compound

In Catabolic And Anabolic

Pathways
During Catabolic pathways/reaction



High energy compounds follow

substrate level phosphorylation

reaction.

High energy compounds cleave high

energy bond to generate high energy

used for phosphorylation of ADP with

pi at reaction level.

Generate ATP at substrate/reaction

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 at

reaction level without involvement of ETC
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
During 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.

HIGH-ENERGY PHOSPHATES

PLAY A CENTRAL ROLE IN ENERGY

CAPTURE AND TRANSFER


High Energy Compounds Generated In

Catabolic Pathways Are Utilized In

Anabolic Reactions

HIGH-ENERGY PHOSPHATES

ACT AS

"ENERGY CURRENCY" OF CELL


Free Energy of hydrolysis Of High

Energy Phosphate Bonds has

Important Bioenergetics

Significance

Adenylate Kinase (Myokinase)

Interconverts Adenine Nucleotides
Important Features Of ATP

Contains three high energy phosphate bonds

Drive endergonic reactions

It is chemical energy currency of body

Functions in body as a complex with Mg2+

Biosynthesized by ATP synthase

Couples thermodynamically Unfavorable reactions to Favorable Ones

ATP synthesis is inhibited by Uncouplers

What Is Biological Oxidation?
Biological oxidations :

Oxidation

reactions/Process

Occurring in living

cells.

Importance/Features Of

Biological Oxidation
Biological Oxidation Reactions/Process :

Involves Oxygen

Associated with metabolism

Generates ATP

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)

Feature Of Biological Oxidation

Oxidation of a molecule (electron

donor) is always accompanied by

reduction of a second molecule

(electron acceptor)
Most predominant type of

Oxidation reaction in body

is:

Dehydrogenation Reaction

Catalyzed by Dehydrogenases

Dehydrogenases catalyzes

to remove Hydrogen from

substrates.

Which are temporarily

accepted by Coenzymes.
Coenzymes

and

Enzymes

of

Biological Oxidation

Reactions

Coenzymes

and

Inorganic Cofactors

Of

Biological Oxidation

Reactions
FMN
FAD
NAD+
NADP+
THBP (Tetra Hydro Biopterin)
Cu++
Fe+++

Oxidized Coenzymes involved in

Oxidation/Dehydrogenation

reactions.

NAD+

NADP+

FAD

FMN
Oxidized Coenzymes temporarily accept

the hydrogen from substrates and get

transformed to reduced coenzymes.

NADH+H+

FADH2

NADPH+H+

FMNH2

The reduced and oxidized forms of NAD


The reduced and oxidized forms of FAD

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
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.

DEHYDROGENASES CANNOT

USE OXYGEN AS A HYDROGEN

ACCEPTOR

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

Has to enter ETC for its

reoxidation.

Oxygen is involved

indirectly at an end of

ETC as electron and

proton acceptor .

Metabolic water is an

end product of ETC.
Remember

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

Enzymes

Pathway /Reaction

Glyceraldehyde -3-PO4

Glycolysis

Dehydrogenase

Pyruvate Dehydrogenase

PDH Complex

Isocitrate Dehydrogenase

TCA cycle

Ketoglutarate Dehydrogenase

TCA cycle

Malate Dehydrogenase

TCA cycle

Lactate Dehydrogenase

Pyruvate/Lactate metabolism

Glutamate Dehydrogenase

Glutamate metabolism

Hydroxy Acyl Dehydrogenase

Beta Oxidation of Fatty acids
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) into substrate.

Form Oxidized Products
OXYGENASES CATALYZE

DIRECT TRANSFER AND

INCORPORATION OF OXYGEN

INTO A SUBSTRATE MOLECULE

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)




Cytochromes P450

Are Monooxygenases

Important in Steroid Metabolism

& for

Detoxification of Many Drugs
Oxidases

Oxidases involve activated

molecular Oxygen as

Hydrogen (electron and proton )

acceptor.

Oxidases Reduce Oxygen to

form Water (H2O)

OXIDASES USE OXYGEN

AS A HYDROGEN ACCEPTOR

AH2 + ? O2 Oxidase A+ H2O

Tyrosine+ O2 Tyrosinase -Cu++ DOPA +

H2O
Examples Of Oxidases

Cytochrome Oxidase-Classic Example

(Hemoprotein ETC enzyme)

Ascorbate Oxidase
Mono Amine Oxidase
Catechol Oxidase

HYDROPEROXIDASES USE HYDROGEN PEROXIDE

OR AN ORGANIC PEROXIDE AS SUBSTRATE

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 Reduce Peroxides Using Various

Electron Acceptors

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)

Oxidative Phosphorylation

Synonyms Of ETC
1. Electron Transport Chain (ETC)
2. Oxidative Phosphorylation
3. Electron Transport System (ETS)
4. Fate of Reduced Coenzymes of

FADH2 and NADH+H+

5. Respiratory Chain
6. Internal/Cellular Respiration
7. Tertiary metabolism
8. Final Oxidative Pathway

What is Electron Transport Chain?
Electron Transport chain

Biological oxidation process very vital for human

being survival

Truly Aerobic in nature(indispensable on O2)

Located and operated at inner membrane of

Mitochondria

Alternate Oxidation and Reduction Reactions

carried out in process

What is Oxidative Phosphorylation?


Oxidation process (ETC) is tightly

coupled with Phosphorylation of

ADP with pi to generate ATP.

Illustrated as Sun and Day Light

Oxidative

Phosphorylation is a

major mode of ATP

generation in

human body
What is Fate of ETC/

Oxidative Phosphorylation ?

REOXIDIZES

REDUCING EQUIVALENTS

(NADH+ H+ and FADH2)

GENERATED DURING ANAEROBIC

DEHYDROGENASE REACTION
Electron Transport Chain

On Operation

Transports Electrons and Protons

Through series of ETC components

Finally H2 is received by activated

molecular Oxygen (1/2 O2)

Generates significant byproduct ATP

and metabolic water at end of process

Condition In which ETC Operates

ETC operates in truly aerobic

condition.

Oxygen unloaded at cellular

level by HbO2

Gets utilized at an end of ETC

process. (Respiratory Chain)
Site Of

Electron Transport Chain

OR

Oxidative Phosphorylation

ETC is located and operated

in all cells which contain

Mitochondria (Power house

of Cell)

(Except mature Erythrocytes which are

devoid of mitochondria)


Location of Mitochondrial ETC Complexes

? Inner membrane of Mitochondria

? Rich In Cardiolipin

Components and Enzymes of ETC

are arranged towards inner

surface of inner membrane of

mitochondria as:

Vectorial conformation

Increased order of positive redox

potential
Number of Mitochondria Vary in

Different cells

Number of Mitochondria changes

from cell to cell , tissue to tissue,

organ to organ, organism to

organism

Factors Responsible For Number of

Mitochondria in Cell
Type of cell, organ and its

function

Metabolic status of an individual

Physical activity of an individual

How much energy cell needs to

produce?

High number of Mitochondria present in

Heart, Rod cells, Sperm,

ciliated cells

Muscle cells for example, contain more

number of mitochondria compared to

Kidney cells.

Marathon runners have more number of

mitochondria in their leg muscle cells than

people with desk jobs
Components Of ETC

Series Of Protein Complexes

Flavoproteins & Iron-Sulfur

Proteins (Fe-S),Cytochromes

are Components

of Respiratory Chain Complexes


1.Flavo Protein- (First Component)
NADH Dehydrogenase-FMN and FeS

centers(Warburg's Yellow Enzyme)

2. Coenzyme Q/ Ubiquinone

3. Series of Cytochromes-
Cytochrome b-Cytochrome c1-

Cytochrome c- Cytochrome aa3

Coenzyme Q / Ubiquinone

Coenzyme Q (CoQ)/ Ubiquinone)

is located in lipid core of

mitochondrial membrane.

It is a Quinone derivative
Lipophilic dissolves in

hydrocarbon core of a membrane.
Coenzyme Q is very

hydrophobic.
Coenzyme Q has a long Poly

isoprenoid tail, with multiple

units of isoprene.

In human cells, most often n = 10

Q10 isoprenoid tail is longer than

width of a bilayer.

Coenzyme Q functions as a

mobile e- carrier within

mitochondrial inner

membrane.

Its role in trans-membrane H+

transport coupled to e-

transfer (Q Cycle).
Coenzyme Q

Accepts

Both Protons and Electrons

Quinone ring of

coenzyme Q can

be reduced to

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




Cytochrome Heme

Cytochrome Heme Iron is in

transitional state

Carries only electrons
Fe (III) + e- Fe (II)
Only one electron is transferred at

a time.
Cytochrome heme iron

can undergo 1 e-

transition between ferric

and ferrous states:

Fe+++ + e- Fe++
(oxidized) (reduced)

Cytochromes May Also Be

Regarded as Dehydrogenases

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

Dehydrogenase

activity.

Cytochromes absorb light at

characteristic wavelengths.

Absorbance changes upon

oxidation/reduction of

Heme Iron
Components of Respiratory Chain

are Contained in

Protein Complexes Embedded in

Inner Mitochondrial Membrane

Five Complexes of

Oxidative Phosphorylation
Complexes of Oxidative

Phosphorylation

There exists 5 complexes

Processing Oxidative

Phosphorylation to generate

ATP

Complexes are combination of one

or two components

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 Oxidative Phosphorylation Complexes

No. of

Prosthetic Groups

Complex

Name

Proteins

Complex I

NADH ?CoQ

46

FMN,

Reductase

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

ETC Components Associated With

Multiple Iron Sulfur Centers

Iron exists in Transitional State

Responsible for Oxidation and

Reduction Reactions
Complex I,I and II contains Iron

Sulfur Centers

Complex IV and V do not Contain

Iron Sulfur Centers

ETC Components

With Iron Sulfur Centers

NADH Dehydrogenase

Coenzyme Q-Cytochrome Reductase

Succinate ?Coenzyme Q Reductase
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 close

proximity of iron atoms.

Fe+++ (oxidized) + 1 e- Fe++ (reduced)


COMPLEX IV

Cytochrome a-a3/ Cytochrome Oxidase
large protein
Both a and a3 contain heme and Cu
Does not contain Fe ?S clusters
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

following irreversible reaction:
O + 4

O

2

H+ + 4 e- 2 H2

Four electrons are transferred into 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)

? Proton channel is ? between c

ring and a subunit.


Complex V ATP Synthase

How do ATPase and ATP Synthase

Differ?

ATPase is an enzyme that

hydrolyze ATP to form ADP

ATP synthase synthesize ATP

Both enzyme found in mitochondria


F- ATPase/ATP Synthase

F-ATPase belong to

superfamily of related ATP

Synthases

F-ATPase is a Rotating Motor
Mechanism Of Oxidative

Phosphorylation

Salient Features/Required Criteria's

Of

ETC/Oxidative Phosphorylation

OR

Criteria's Required For Oxidative

Phosphorylation
1

Arrangement Of

Electron Transport Chain

Components In Increased Order

Of Positive Redox Potential

Redox Potentials & Redox Couples
FREE ENERGY

CHANGES CAN BE

EXPRESSED IN TERMS

OF REDOX POTENTIAL

Redox Potential is a

measure of tendency of a

redox couple to accept or

donate electrons under

standard condition.
Components that have most negative

redox potentials

Have weakest affinity for electrons

Hence has capacity to donate its

electrons.

Redox couple with most positive

redox potentials have


Strongest affinity for electrons

therefore

Possess strongest tendency to

accept electrons.
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


Higher redox potential

(high tendency to accept

electrons)

Redox Couple

Components of ETC has capacity

to exist in oxidant and reduced

forms.

This pair is known as redox

couple

CoQ/CoQH2
Cyt b Fe+++/Cyt b Fe++


Sequence of

Respiratory

Electron

Carriers

Inhibitors

in green

2

Development Of

Proton Gradient And Proton Motive Force

In Intermembrane Space


Complex I,I I and IV

Pumps Protons From Matrix side

to Intermembrane Space

and generates

Proton Motive Force


Complex I,I I and IV

Serve as Proton Channels

Complex I ,III and IV act as a

Proton Pump.

Pump out 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.


A Large Drop in Redox

Potential across each of the

three Respiratory Enzyme

Complexes (I,III,IV).

Provides the Energy for H+

Pumping


3

Free Energy Change Occurs

Due To Transport OF Proton

Pumping and Electron Exchange

During Oxidative Phosphorylation
4

Heat Energy Generated At Certain

Specific Sites During Oxidation

Is Transformed By Chemical

Phosphorylation Reaction of ADP

and pi to form ATP

5

ATP Synthase /Complex V

Activation for Phosphorylation

Reaction
Proton gradient runs

downhill through ATP

Synthase to drive

synthesis of ATP
qF F of ATP Synthase catalyzes

1 o

phosphorylation reaction for ATP

synthesis

q Transport of H+ from intermembrane

space to into the mitochondrial matrix

through ATP Synthase is mandatory.

qTransport of at least 3 H+ per ATP is

required through ATP Synthase for its

activation and catalysis.

Thus heat energy is

transformed to

chemical form of

energy (ATP) in

Oxidative

Phosphorylation.
6

Oxygen is Terminal Acceptor of

Protons and electrons During

Oxidative Phosphorylation To

Generate Metabolic Water

Oxygen has 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

At end of E.T.C by 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.


Cytochrome oxidase

controls rate of O2

uptake which

Means this enzyme

determines how

rapidly we breathe.

Respired Oxygen

transported by Hb

unloaded at tissue/

cellular level is

utilized during E.T.C.
7

Coenzyme Q Accepts Electrons

Via Complexes I & II
Point To Note

In ETC electrons flow from
Most electro negative potential

NADH+H+ (-0.32) to most

electro positive potential

(+0.82) ? O2 .

HOW

ETC /Oxidative

Phosphorylation Operates ?


Most Oxidative

Metabolic Pathways

(TCA and Beta Oxidation Of Fatty acids)

Located In Mitochondrial Matrix

Generate Reduced Coenzymes
Reduced coenzymes

NADH+H+/FADH2

Generated during Anaerobic

Dehydrogenase reactions of

Carbohydrates, Lipids metabolic

pathways.

Get reoxidized on entering E.T.C

Reduced coenzymes NADH+H+ and

FADH2 are formed in Mitochondrial

matrix:

Oxidative Decarboxylation of

Pyruvate to Acetyl CoA by PDH

complex.

Oxidation of Acetyl CoA by TCA cycle

Beta Oxidation of fatty acids
NADH+H+ and FADH2 are

energy rich molecules


Contains a pair of

electrons having a high

transfer potential.

Entry Of NADH+H+ in ETC

When NADH+H+ enters ETC reducing

equivalents Protons and Electrons are

taken up by first component

/Complex I (Flavoproteins)

Then from complex I the reducing

equivalents are transferred to CoQ.
Entry Of FADH2 in ETC

FADH2 is generated at Succinate

Dehydrogenase reaction(Complex II)

FADH2 enters ETC process and its

reducing equivalents are taken up by

CoQ .

CoQH2 then here onwards transfers

only electrons to series of arranged

Cytochromes and Protons are

released in matrix.


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).

Sequence of Electron Carriers in ETC

NADH FMN-Fe-S Co-Q Fe cyt b

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.

ETC Process
A PROTON GRADIENT POWERS THE

SYNTHESIS OF ATP

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
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.

Flow of electrons through ETC

complexes leads to pumping of

protons out of the mitochondrial

matrix in intermembrane space.

This accumulation 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

Electron Transport (Oxidative

Process) is coupled to Phosphorylation
ATP is synthesized when 3

protons flow back from

intermembrane space of

mitochondria to

mitochondrial matrix through

an enzyme complex ATP

synthase.

Oxidation of fuels and

phosphorylation of ADP

are coupled by a proton

gradient across an inner

mitochondrial membrane.
Thus Oxidative phosphorylation

is process in which ATP is

formed

As a result of transfer of

electrons from NADH or

FADH2 to O2 by a series of

electron carriers.

Mechanism

Of

Oxidative Phosphorylation
Oxidative Phosphorylation

Oxidation tightly coupled

with Phosphorylation

E.T.C (Oxidation)Process

coupled with phosphorylation

of ADP+pi to generate ATP.

Hypothesis And Theories Mechanism

Of

Oxidative Phosphorylation

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 is

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

complexes I,III and IV.

Generates a Proton gradient and

in intermembrane space of

mitochondria.




Proton pumps are Complexes I,
III and IV.

Protons return through ATP synthase

Oxidative Phosphorylation
Proton gradient in inter

membrane space creates Proton

Motive Force due to:

Proton gradient have a

thermodynamic tendency

Proton gradient creates

Electrochemical potential

difference

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 through it.

Translocation of protons through

ATP Synthase

Stimulates and activates ATP

Synthase

For catalytic action of

phosphorylation- ADP with pi to

form ATP.

Supports mechanism of Oxidative

Phosphorylation.


Flow of three H+ through an ATP

Synthase complex

Brings a conformational change n

domains of ATP Synthase

Which causes the ATP synthase

activate and catalyze

phosphorylation reaction

To synthesize ATP from ADP + Pi.

ATP Synthase, a Molecular Mill.



ATP synthesis at F1 results from

repetitive conformational changes
as rotates

rotates 1/3 turn-
energy for ATP release

This process of producing ATP

is known as oxidative

phosphorylation.


Entire process of using Proton

gradient and proton motive

force to make ATP is called

Chemiosmosis.
During oxidative

phosphorylation 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.


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 Translocation From

Mitochondria

Through ATP/ADP Translocases


ATP molecules produced in Oxidative

Phosphorylation mechanism are

Transported out of mitochondrial

matrix through specific transporters

Operation Of ETC



Glycolysis, Fatty

acid oxidation

TCA cycle

supplies NADH

and FADH2 to

the Electron

Fatty Acids

Acetyl Co A

Transport Chain

Pyruvate

Amino Acids

Glucose
WHY ETC OPERATES ?

During E.T.C operation 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.

At this site free energy in the form of

heat released which is more than 7.3

Kcal.

This free heat energy is conserved to

undergo Phosphorylation reaction

and generate chemical form of

energy-ATP.

Sites in E.T.C at which

energy liberated is less

than 7.3 Kcal is simply

dissipiated in the

form of heat.
Three sites in E.T.C (Complex

I,III and IV) where heat energy

liberated more than 7.3 Kcal

Utilized for phosphorylation

reaction of ADP with pi to

form ATP.

Electrons are transferred from

NADH+H+ 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 initial e- acceptor.

FAD is reduced to FADH2 during oxidation of

Succinate to Fumarate.

FADH2 generated by Succinate

Dehydrogenase reaction gets reoxidized

by transfer of electrons through a series

of 3 iron-sulfur centers to CoQ, yielding

CoQH2.

QH2 product may be reoxidized via

complex III.

Providing a pathway for transfer of

electrons from Succinate into respiratory

chain.
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

intermembrane space.

It alternately binds to

complex III or IV during e-

transfer.
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

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

matrix to intermembrane Space

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

10 protons translocates

per NADH+H+

6 protons translocates

per FADH2
Proton gradient and Proton

motive Force created as

electrons transferred to

Oxygen forming water

10 H+ / NADH+H+
6 H+ / FADH2

F F couples ATP synthesis to H+

1 o

transport into the mitochondrial matrix.
Transport of least 3 H+ per ATP is

required.
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.

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

Oxidative Phosphorylation
ATP is a high energy phosphate

compound

Biologically important free

nucleotide

ATP has Two High Energy Phosphate

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
Thus ATP Generation

Is Due To Transformation Of

Heat Energy Into Chemical Form Of Bond Energy

Which Satisfy Law Of Thermodynamics

Energy Is never Destructed

Energy Is Transformed From One Form To Another

From One System To Another

One Body To Another

Significance OF ATP

ATP allows coupling of

thermodynamically

unfavorable reactions

to favorable reactions.
Uses of ATP generated in

Oxidative Phosphorylation

?Synthetic/Anabolic reactions
?Active transport mechanism.
?Muscular contraction
?Nerve impulse conduction.

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)

Remember

CoQ accepts electrons and

Protons by complexes I and II

Acceptance of Protons and

Electrons from Complex II by

CoQ does not generate ATP
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/ROS)


What is a Free Radical ?

Any chemical species with one of more

unpaired electrons

Unstable/Highly Reactive to get

stabilized

Powerful Oxidant
Short half life (nanoseconds)
Can exist freely in environment

Main Factors Responsible for

ETC
Factors For Universal Metabolism

Nutrition
Environment
Life Style Habits

Factors Associated To ETC

Metabolites- Carbs ,Proteins , Lipids

Vitamins , Minerals and Antioxidants

Oxygen Concentration

Respiration Process

Hemoglobin Structure and Function

Mitochondrial DNA

Metabolic Status

Oxidative Stress
REGULATORS OF OXIDATIVE

PHOSPHORYLATION

Important Direct Substrates

Regulators Of Oxidative Phosphorylation

and ATP Generation

NADH/FADH2

O2

ADP and pi
Indirect Substances Involved

Glucose
Fatty acids
Insulin
Amino acids and Proteins
Iron
Vitamin C
Vitamin B Complex members- Niacin,

Riboflavin

ATP/ADP Ratio

Regulates Mechanism Of

Oxidative Phosphorylation


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

ADP and pi is required for ETC

process.

Intramitochondrial ratio

ATP/ADP is a control

mechanism
At high ATP/ADP ratio
ATP acts as an allosteric

inhibitor for Complex IV

(Cytochrome Oxidase)

Inhibition is reversed by

increasing ADP levels.

ADP levels reflect rate of ATP

consumption and energy state

of the cell.

At low ADP levels ? Low

oxidative phosphorylation
Electron transport is tightly coupled to

phosphorylation.

ATP cannot be synthesized by

oxidative phosphorylation unless there

is heat energy released from electron

transport.

Electrons do not flow through the

electron-transport chain to O2 unless

ADP is phosphorylated to ATP.

Inhibitors OF

ETC Complexes

OR

Inhibitors Of

Oxidative Phosphorylation
ETC Complexes Inhibitors

OR

Interruptors of Oxidative Phosphorylation

Mechanism

Enemies/Distractors of ETC components

who stop its normal operation.

Block ETC operation and stop ATP

generation.

ETC Complexes Inhibitors

Chemical compounds having affinity for ETC

components/complexes

Chemically interact with ETC complexes, bind and

inactivate them

Affects normal functional operation of ETC

Low/No ATP production

Cessation of cellular activities
Complex I/Site I - E.T.C Inhibitors

vAmobarbital /Amytal
vRotenone (Fish/Rat Poison)
vMercurials
vPiercidin ?A


(Volatile Anesthetics)
vHalothane (Malignant Hyperthermia)
vFluothane
vIsoflurane
vSevoflurane

Complex III/ Site II -E.T.C Inhibitors

vBritish Anti Lewisite ( BAL)
vAntimycin ?A
vDimercaprol


Complex IV/Site III /Cytochrome

Oxidase Inhibitors :



vCyanide
vCarbon Monoxide
vH2S
vAzide
Complex V Inhibitors

ATP Synthase Inhibitors

Oligomycin

Fo particle of ATP Synthase serve as

proton channel

An antibiotic Oligomycin binds with

Fo particle of ATP Synthase

Do not translocate Protons through

it.

Inhibits activation of ATP Synthase

phosphorylation of ADP to ATP.
Atractyloside

A Glycoside prevents

translocation of ADP across

mitochondrial membrane.

Make it unavailable for

phosphorylation reaction

Bongregate

Pseudomonas toxin has

inhibitory action similar

like Atractyloside.
Artificial Electron Acceptors/

Distractors Of ETC

These chemicals

arrest respiration

by inhibition of

ETC complexes


Specific inhibitors of

Electron Transport Chain

and

ATP-Synthase



Uncouplers Of

Oxidative

Phosphorylation

What are Uncouplers?


Uncouplers are chemical agents

Uncouplers are mostly lipid soluble

aromatic weak acids

They Uncouple/Delink two tightly

coupled natural processes

E.T.C (Oxidation) uncoupled from

Phosphorylation (ATP generation)

They just carry out Oxidation

without 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 Action Il ustrates

As Total Solar Eclipse
Uncouplers just bring oxidation

(E.T.C/Sun Rise) without

phosphorylation(Interrupted Sun Light)

Uncoupler (Moon In between) inhibits

generation of ATP ( Dark/No Day)

Types Of Uncouplers
Physiological Uncouplers

Thermogenin /Uncoupling Protein-1
Excess of Thyroxine
Long Chain Fatty acids
Unconjugated Hyperbilirubinemia

Chemical Uncouplers

2,4 Di Nitro Phenol
Di Nitro Cresol
Dicumarol
Aspirin
p-Triflouromethoxy Carbonyl Cyanide

Phenylhydrazone (FCCP)

Valinomycin
Pentachlorophenol
Snake Venom-Phospholipases


Mode Of Action Of Uncouplers

Certain Uncouplers are ionophores,

lipophilic substances.

They carry protons from

intermembrane space across

mitochondrial membrane to matrix

From site other than specific site.

(i.e not through F0 and F1 particles of

ATP Synthase).
Certain Uncouplers changes

permeability of mitochondrial

membrane to protons.

Translocate protons easily

through mitochondrial

membrane.


2,4 DNP dissolve in membrane and function as

carriers for H+.



Uncouplers block oxidative phosphorylation by

dissipating H+ electrochemical gradient.
Protons pumped out leak back into mitochondrial

matrix,
preventing development of proton gradient and

proton motive force.

ATP Synthase reaction runs backward

in presence of an uncoupler.
w Hydrolysis of ATP is spontaneous.
Thus Uncouplers by their action

deplete proton gradient of

intermembrane space during

ETC operation.

Uncouplers Dissipate More Heat

Uncouplers Do not allow to develop

required proton gradient and

Do not form proton motive force in the

intermembrane space of mitochondria

No translocation of Protons through ATP

Synthase

Causes no stimulation or activation of ATP

Synthase

No catalysis of Phosphorylation of ADP with

pi to generate ATP.
During uncoupling phenomena



Free energy released as Heat energy

more than 7.3 Kcal is not conserved for

Phosphorylation reaction dissipiated as

it is in form of heat

A very high heat energy released then

causes swelling of Mitochondria and

exhibit malignant hyperthermia.

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 UCP-1 protein of an inner

mitochondrial membrane functions as a

H+carrier.



Uncoupling by UCP-1 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)


Significance Of Physiological

Uncouplers

In extreme cold conditions and in

hibernating animals

Physiological Uncouplers bring

uncoupling phenomena

The heat liberated inside body helps

to restore and maintain body

temperature.

Brown adipose
(fat) cells
contain natural
Uncouplers to
warm animals
cold adaptation
and hibernation.


As per the Required condition Of

Body

This "non-shivering

thermogenesis" is costly in

terms of respiratory energy

Heat energy unavailable for

ATP synthesis

But provides valuable warming

to an organism.

Effect Of Poor Antioxidant Activity


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
Shuttle Systems

Shuttling Reducing Equivalents

OF NADH+H+

from Cytosol into the

Mitochondrion


Shuttle

A vehicle or aircraft that travels regularly

between two places

Biochemical shuttle is a biochemical

system for translocating Protons and

electrons produced during Glycolysis

Across a semipermeable inner membrane

of mitochondrion

For oxidative phosphorylation mechanism

NADH+H+ is generated in the cytosol during

Glycolysis
Cytosolic NADH+H+ itself is not

carried across the mitochondrial

membrane.

Instead its Protons and

Electrons of NADH+H+ are

carried through shuttle

systems.

Since NAD+ and NADH +H+ are

impermeable to an inner

mitochondrial membrane

This reducing equivalents must be

shuttled into mitochondrial matrix

before they can enter the ETC.
Cytosolic NADH+H+

Enter Mitochondria

via

2 Shuttle Systems

Two shuttles Involved:

Malate-Aspartate Shuttle

Glycerol 3-phosphate Shuttle


Malate-Aspartate Shuttle

Malate/Aspartate Shuttle System
Malate Aspartate Shuttle

Active in Heart and Liver.
2.5 molecules of ATP are produced

Glycerol-3-Phosphate Shuttle


Glycerol-3-Phosphate Shuttle

Glycerol 3 Phosphate Shuttle
Glycerol Phosphate Shuttle

Active in Skeletal muscles and

Brain

FADH2 formed in this enter the

electron-transport chain through

CoQ

Generates only 1.5 molecules of

ATP

Summary of Shuttle Systems

Total ATPs Generated / 1 Glucose Oxidation

Heart and Liver

32.0 ATP

Uses Malate Aspartate Shuttle

Muscle and Brain

30.0 ATP

Uses Glycerol phosphate Shuttle
Factors Affecting

Oxidative Phosphorylation

Mechanism

Oxygen supply to cells
Hemoglobin structure and function
Respiratory system and its function
Mitochondrial structure and ETC

components.
Presence of Nutrients
Enzyme function and

Coenzymes availability

Adequate amount of ADP

and pi.

Presence of ETC inhibitors

Pathological Conditions Affecting

Oxidation Phosphorylation

Mechanism

Which

Lower Down ATP Production
1. Hypoxia

2. Anemia

3. Ischemia



4. Hemoglobinopathies

5. Emphysema

6. Respiratory Distress Syndrome

7. Asthma

8. Prolonged Starvation

9. Malnutrition

10. Diabetes mellitus

11. ETC inhibition by chemicals/drugs

12. Inherited Disorders of Mitochondria

Inherited /Genetic Disorders

Related To Mitochondrial

Oxidative Phosphorylation

Mechanism
Mitochondrial DNA

Mitochondrial genes encode for ETC complexes

Complex I
Complex III
Complex IV
Complex V

Mutations in any one or more genes of

mitochondrial DNA controlling mechanism of Oxidative

phosphorylation lead to its inherited disorders

1. MELAS

An inherited disorder caused due to defect

of complex I or IV of E.T.C

Associated with

Mitochondrial Myopathy
Encephalopathy
Lactate accumulation
Acidosis
Stroke
2. Fatal Infantile Mitochondrial Myopathy

Defect in E.T.C components

located in mitochondria

Cytochrome c Oxidase defect
Associated with renal

dysfunction.

Mostly fatal in early age

3. Leber's Hereditary

Optic Neuropathy (LHON)

Caused due to mutations in

mitochondrial DNA

Affects oxidative

phosphorylation mechanism

Loss of bilateral vision due to

neuroretinal degeneration.


Mutant Genes Of LHON



4. Mitochondrial DNA Deletion

Syndrome







5. Luft's Disease

Luft's Disease is a mitochondrial disease

First patient who was diagnosed with this

disease was a 30 year old Swedish woman by

Dr Rolf Luft

Caused by abnormal mitochondria
Biochemical Cause

Mitochondria Respire Wildly

Respiratory control is lost
Partial Uncoupling is caused by an abnormality in

mitochondrial membrane

Electron transport is only loosely coupled to ATP

production

Oxidation process proceed independent of ADP

phosphorylation to generate ATP

An extra energy evolves in form of heat
This elevates body temperature up to 38.4 ?C which

raises BMR

Luft's Disease Is Characterized By

Abnormal excessive production of heat

Characterized by hypermetabolism and

abnormal transpiration.

Patient experiences excessive sweating

during winter

Make them to change their clothes 10 times a

day.
Onset is in childhood

Thyroid function is normal

Since there is less ATP production

and an extra energy is lost in the

form of heat

Metabolic processes are stimulated

Luft's Disease

Non Thyroidal Hypermetabolism

Due to high BMR and low ATP production
High caloric intake
There is failure to put on weight despite a good diet
There is progressive weight loss despite increased food

intake

Excessive perspiration
Excessive thirst indicate a state of severe hyper

metabolism of non thyroid origin (since thyroid hormones

-T3 and T4 are normal)


Manifestations of Luft's Disease

Heat intolerance
Profuse perspiration
Polydipsia without polyuria
Severe hyper metabolism
Polyphagia
Muscular wasting and weaken
Absent deep reflexes, and Resting

tachycardia.

Multiorgan Dysfunction Risk In

Luft's Disease
Case Study

An elderly couple was brought by ambulance to an emergency

department after their daughter noticed that they were both

acting "strangely." The couple had been in good health prior

to the weekend. Their daughter had gone out to spend the

week-end with her friends. The couple had been snowed in at

their house until the snowplows cleared the roads. They had

plenty of food and were kept warm by a furnace and blankets.

On reaching home after two days, their daughter noticed that

they both were complaining of bad headaches, confusion,

fatigue, and some nausea. On arrival to an emergency

department, both patients were afebrile with normal vital

signs and O2 saturation of 99 percent on 2 L of O2 by nasal

cannula. Their lips appeared to be very red. Both patients were

slightly confused but otherwise oriented. The physical

examinations were within normal limits.

Carboxyhemoglobin levels were drawn and were elevated.

What is most likely cause of these patients' symptoms?


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.

Inherited Disorders of

Mitochondrial Dysfunction
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

Laboratory data showed lactic acidosis,
Proteinuria
Glycosuria and
Generalized aminoaciduria
Muscle biopsy showed large clumps of

granules positive with oxidative enzyme

stains and increased lipid droplets.

Ultrastructural studies showed large

aggregates of mitochondria, many of which

were greatly enlarged and contained

disoriented or concentric whorls of cristae

and paracrystalline inclusions.
A 1-month-old boy was admitted because of failure to thrive.

He was floppy and had bilateral ptosis, diminished reflexes, and

poor suck. He had aspiration pneumonia, developed seizures,

and died at age 3 1/2 months.

He was an only child, and family history was negative.
Cytochrome c oxidase was absent in fresh frozen sections

by histochemical staining.

By biochemical assay, cytochrome c oxidase (cytochrome aa3)

was 6% of normal in muscle biopsy and undetectable in autopsy

muscle; spectra and content of cytochromes showed lack of

cytochrome aa3, decreased cytochrome b and normal

cytochrome cc1.

In kidney, cytochrome-c-oxidase activity was 38% of normal

and spectra showed decreased cytochromes aa3 and b.

The association of fatal infantile mitochondrial myopathy,

lactic acidosis and renal dysfunction was previously reported

by Van Biervliet et al and appears to be a distinct nosologic

entity, one of the few biochemically defined mitochondrial

myopathies.

A case of cytochrome c oxidase deficiency primarily affecting

skeletal muscle is described. The child was admitted at 4

weeks due to failure to thrive and examination at that time

revealed weakness and hypotonia. His condition deteriorated

until at 11 weeks respiratory arrest necessitated artificial

ventilation and death occurred at 14 weeks. Biochemical

investigation showed lactic acidemia and generalized

aminoaciduria. Histochemical examination of muscle obtained

at biopsy showed strong reactions for some oxidative enzymes,

but by contrast cytochrome c oxidase could not be detected.

Cytochrome c oxidase activity was less than 5% of control

values in an extract of fresh muscle. The reduced-minus

oxidized absorption spectra of muscle mitochondrial fractions

prepared from post-mortem tissue showed an absence of

cytochrome aa3 and a partial deficiency of cytochrome b. Ultra

-structural examination showed abnormal mitochondria with

loss of cristae and an abnormal granular matrix. The family

history suggests autosomal recessive inheritance.

This post was last modified on 05 April 2022