Dr. Subodhini Abhang

Points to be covered Over view

Redox potential .Arrangement of components in ETC.

Coupled nature of respiration in mitochondria

Substrate Level Phosphorylation

Components of electron transport chain

P : O ratio and its calculation

Mechanism of oxidative phosphorylation

Inhibitors

An Overview

• Biological oxidations are catalyzed by intracellular enzymes, to obtain energy.

• Electron Transport: Electrons carried by reduced coenzymes (NADH or FADH2) are passed sequentially through a chain of proteins and coenzymes ( electron transport chain)to O2 .

• Oxidative Phosphorylation: Coupling e- transport, oxidation and ATP synthesis (Phosphorylation) .

• Site of oxidative phosphorylation : inner mitochondrial membrane (Eukaryotic cells)

Redox potential E₀

Redox potential or oxidation –reduction potential is a quantitative measure of the tendency of redox pair to loose or gain electrons.

Free energy changes can be expressed in terms of

Free energy change is proportionate to the tendency of reactants to donate or accept the electrons.

−ve redox potential

+ve redox potential

Arrangement of components in ETC

• Arranged in the order of increasing redox potential.

From electro − ve to electro + ve

Redox pair E₀

NAD⁺/NADH -0.32 FMN/FMNH₂ -0.22Pyruvate/Lactate -0.19Cytochrome c Fe³⁺/Fe²⁺ +0.07O₂/ H₂O + 0.82

NADH & FADH₂

C

Carbohydrates

LipidsProteins

TCA cycle

Oxidation is coupled to phosphorylation of ADP

Respiration (consumption of oxygen)proceeds only when ADP is present

•Amount of o₂ consumeddepends on amount of ADP added .

Coupled nature of respiration in mitochondria.

Energy rich

A pair of electrons

Having high transfer potential

O₂ H₂O

Donated to

Free energyliberated

Utilized for ATP generation

Substrate Level Phosphorylation

Glyceraldehyde -3-Phosphate+ NAD ⁺+ Pi 1~3 bis phosphoglycerate + NADH

1~3bis phosphoglycerate + ADP 3 Phosphoglycerate + ATP

2) 2~ Phosphoenol pyruvate Pyruvate + ATP

3) α-ketoglutarate + NAD⁺ + CoA Succinyl ~ CoA + NADH + H⁺

Succinyl~CoA + GDP + Pi Succinate + GTP

1~

ADP

Synthesis of ATP without involving electron transport chain.

Definition

• The process of synthesizing ATP from

ADP and Pi coupled with the electron

transport chain is known as oxidative

phosphorylation.

Oxidative Phosphorylation

• Energy is released when electrons are transported from higher energy

NADH/FADH2 to lower energy O2 .

• This energy is used to phosphorylate ADP.

• This coupling of ATP synthesis to NADH/FADH2 oxidation is called oxidative phosphorylation.

• Oxidative phosphorylation is responsible for 90 % of total ATP synthesis in the cell.

Site …................ Mitochondria

Mitochondria

Impermeable

to ions and

most other

compounds

In inner

membrane

knobs

MitochondrionThe enzymes responsible for electron transport and

oxidative phosphorylation present in mitochondria.

∙∙∙∙∙∙∙∙∙ ∙∙∙∙ ∙∙∙

Structure of Mitochondria

Β-oxi

TCA

Phosphorylating subunits

Matrix

Inner Membrane

Outer membrane

Cristae

(B)

(B)

Inner membrane

F1 subunit

Fo subunit

ATP synthase

Electron Carriers

NAD+

FMN

FeS

ubiquinoneFAD FeS

Cyt b

FeS Cyt c1 Cyt c Cyt a Cyt a3

1/2 O2

ubiquinone

NAD+ or FAD

There are 2 sites of entry

for electrons into the

electron transport chain:

Both are coenzymes for

dehydrogenase enzymes

The transfer of electrons is not directly to oxygen but

through coenzymes

Components of electron transport chain

• Components are arranged in order of increasing redox potential.

• From electro –ve to +ve

• NAD⁺/NADH to O₂/H₂O

• -0.32 to +0.82

Components of electron transport chain

Complex I

• NADH -ubiquinone oxidoreductase (NADH dehydrogenase )

• Embedded in mitochondrial membrane

• Transfers electrons from NADH to Q

• NADH transfers two electrons as a hydride ion (H: H:-) to FMN

• Electrons, one at a time , are passed through Complex I to Q via FMN and iron -sulfur proteins

• Succinate -ubiquinone oxidoreductase(or succinate dehydrogenase complex)• Accepts electrons from succinate and catalyzes the reduction of Q to QH 2• FAD of II is reduced in a 2 -electron transfer of a hydride ion fromsuccinate• Complex II does not contribute to proton gradient , but supplieselectrons from succinate

Complex II

Fe

Fe

S

S

S

Fe

Fe

S

S

S

SS

Cys

Cys

Cys

Cys

S

Fe

S

Fe

S

S

S

S

Cys

CysCys

Cys

Iron-Sulfur Centers

3. Iron sulfur proteins-: Exist in oxidized ( Fe³⁺) & reduced (Fe²⁺) state.

Transfers electrons FMNH₂ Q & Q b, c₁

Iron-sulfur centers (Fe-S) have prosthetic groups containing 1-4 iron atoms

Iron-sulfur centers transfer only one electron, even if they contain two or more iron atoms.

E.g., a 4-Fe center might cycle between redox states:

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

1, Fe++ (reduced)

Iron-sulfur Centers (clusters)

biquinone

QCoenzyme Q CoQ

Other names and abbreviations:

FAD FeS

FeS

FeS

FMN

NAD+

ubiquinone

Cyt b

ubiquinone

O

O

CH3O

CH3CH3O

(CH2 CH C CH2)nH

CH3

OH

OH

CH3O

CH3CH3O

(CH2 CH C CH2)nH

CH3

2 e- + 2 H

+

coenzyme Q

coenzyme QH2

Free CoQ can undergo a 2 e-

oxidation/reduction:

Q + 2 e- + 2 H+ QH2.

Ubiquinone or Coenzyme Q

Coenzyme Q

• Coenzyme Q (CoQ, Q or ubiquinone) is lipid-soluble. It dissolves in the hydrocarbon core of a membrane.

• The only electron carrier not bound to a protein. It is a mobile electron carrier.

• It has ability to accept electrons in pairs and pass them one at a time through a semiquinone intermediate to complex III.

• This is called Q cycle.

Cytochromes

NAD+

FMN

FeS

ubiquinoneFAD FeS

Cyt b

FeS Cyt c1 Cyt c Cyt a Cyt a3

1/2 O2

ubiquinone

proteins that accept

electrons from QH2 or

FeS

Ultimately transfers the

electrons to oxygen

Cytochromes are electron carriers containing heme . Heme in the 3 classes of cytochrome (a, b,c) differ in substituents on the porphyrin ring.

Some cytochromes(b,c1,a,a3) are part of large integral membrane protein complexes.

Cytochrome c is a small, water-soluble protein.

Cytochrome c is also a mobile electron carrier.

Cytochromes

Oxidative Phosphorylation

Oxidation and phosphorylation are coupled processes by proton gradient across the inner mitochondrial membrane.

Mechanism of oxidative phosphorylation :

1. Chemical hypothesis.

2. Chemiosmotic

Chemical Hypothesis

• A series of phosphorylated high energy intermediates are formed and utilized for ATP synthesis.

• 1.Only substrate level phosphorylation can be explained.

• 2.Lacks experimental evidence.

Chemiosmotic Theory

Proposed by Peter Mitchell in 1961

Most accepted.

It explains how transport of electrons through respiratory chain is utilized to produce ATP from ADP +Pi

Explains action of uncouplers.

NAD+

FMN

FeS

ubiquinoneFAD FeS

Cyt b

FeS Cyt c1 Cyt c Cyt a Cyt a3

1/2 O2

ubiquinone

I

II

III IV

Mitochondrial Complexes

NADH Dehydrogenase

Succinate

dehydrogenase

CoQ-cyt c Reductase

Cytochrome Oxidase

NADH

NADHα-Ketoglutarate

IsocitratePyruvate

β-HydroxybutyrateMalate

β-hydroxy acyl coA

FMN,Fe.SComplex I

ADP + Pi

ATP

QFADFe.S

Complex II

Fatty acyl coA

Glycerol -3-Phosphate

Succinate

CCcccCCyt b, Fe-S, cytC₁Complex III

ADP + Pi

ATP

Cyt. C

Heme a heme a₃Cu Cu

O₂

ADP + Pi

ATP

Complex IV

Entry of reducing equivalents and synthesis of ATP

Complex I

Complex I: NADH-CoQ oxidoreductase

*Entry site for NADH + H+

*Contains:Fe-S cluster (non-heme protein)

flavin mononucleotide phosphate (FMN)

Coenzyme Q (free in membrane)

*Net reaction: NADH + H+ + CoQ ---> NAD+ + CoQH2*ΔG°' = -81.0 kJ/mol•complex I pumps protons outside the mitochondria•ATP produced

Complex II

Complex II: Succinate-CoQ reductase

*Entry site for FADH2

*Contains:Fe-S cluster (non-heme protein)Coenzyme Q (free in membrane)*Net reaction: Succinate + CoQ --> Fumarate +CoQH2*ΔG°' = -13.5 kJ/mol* Conversion of succinate to fumarate is reaction of TCAcycle and is catalyzed by succinate dehydrogenase

Not a proton pumpNo ATP produced

Complex III

Complex III: CoQH2-cytochrome c oxidoreductase

*Contains:cytochrome c (free in membrane)cytochrome bcytochrome c1Several Fe-S cluster (non-heme protein)*Net reaction: CoQH2 + 2 cyt c [Fe ³⁺] ---> CoQ + 2 cytc[Fe ²⁺ ] + 2 H+*ΔG°' = -34.2 kJ/mol•Complex III pumps protons outside the mitochondria•ATP produced.

Complex IV

Complex IV: cytochrome oxidase

*Contains:cytochrome acytochrome a3Copper*Net reaction: 2 cyt c [Fe ²⁺]+ 1/2 O2 + 2 H+ ---> 2 cyt c[Fe ³⁺] + H2O*ΔG°' = -110.0 kJ/mol* Complex IV pumps protons outside the mitochondria* ATP produced

III IVI

F1

FоQ

NADH+H⁺ NAD

II

Succinate Fumarate

4H⁺4H⁺2H⁺

Cyt c H⁺

H⁺

H⁺

H⁺

Uncouplers

H⁺

H⁺½O₂ + 2H⁺ H₂O

Inter membrane space +++++ +++++++ +++ +++

Inner mitochondrial membrane

Mitochondrial matrix

ADP + Pi ATP

The chemiosmotic theory 0f oxidative phosphorylation

−− −−−−− − −− −−−

Salient features of chemiosmotic theory

Inner mitochondrial membrane is impermeable to ions particularly to protons (H

Complex I, III, and IV acts as a proton pump.Pumping of electrons results in: a) Electrical gradient : as

protons are +vely charged , inter membrane space becomes more electro +ve as compare to mitochondrial matrix or in other words mitochondrial matrix becomes electro –ve. Thus potential difference is produced. b)Chemical gradient : accumulation of H+ causes lowering of pH in inter membrane space where as mitochondrial matrix become alkaline as compare to inter membrane space . Thus chemical gradient is produced.

Hence this is called electrochemical or proton gradient.

Salient features of chemiosmotic theory

The electrochemical potential difference across the membrane, once established as a result of proton translocation , inhibits further transport of reducing equivalents through the respiratory chain unless discharged by back translocation of protons across the membrane through ATP synthase .

This in turn depends on availability of ADP and Pi.

P : O Ratio

Refers to number phosphate group incorporated into ATP for every atom of O₂ consumed in oxidation.

OR

Represents number of ATP synthesized per pair electron carried through ETC.

P:o = 3 Mitochondrial oxidation of NADH

NADH + H⁺ +½ O₂ + 3ADP + 3Pi 3 NAD + 3ATP + 4 H₂O

P:o = 2 Mitochondrial oxidation of FADH₂

Calculation of the P:O ratio

molecules of ADP phosphorylated

P:O ratio = -----------------------------------------atoms of oxygen reduced

Complex I II III IV#H⁺ translocated/2e 4 0 4 2

Since 4 H⁺ are required for each ATP synthesized:

For NADH: 10 H⁺ translocated / O (2e⁻ ) P/O = (10 H ⁺/ 4 H⁺ ) = 2.5 ATP/O

For succinate substrate = 6 H⁺ / O (2e⁻ )P/O = (6 H⁺ / 4 H⁺ ) = 1.5 ATP/O

Energetics of oxidative phosphorylation

NAD⁺/NADH 1/2O₂/H₂O—O.32 +0.82

1/2O₂ + NADH + H⁺ H₂O + NAD⁺

Potential difference 1.14V = 52 cal/mol

3 ATP = 21.9cal

Efficiency of energy conservation

21.9 x 100 52

42%

Sites of ATP Synthesis• Site 1---Oxidation of FMNH₂ by Coenz Q

• Site2--- Oxidation of cyt.b by cyt.c₁

• Site3---cyt. Oxidase reaction (bet. a+a₃)

• When difference of redox potential betweentwo redox pairs >0.15 volts

• Or ∆ G > 7.3 Kcal

NADH

FMN,Fe.SComplex I

ADP + Pi

ATP

QFADFe.S

Complex II

Fatty acyl coA

Glycerol -3-Phosphate

Succinate

CCcccCCyt b, Fe-S, cytC₁Complex III

ADP + Pi

ATP

Cyt. C

Heme a heme a₃Cu Cu

O₂

ADP + Pi

ATP

Complex IV

PiericidineAmobarbitalRotenone

Oligomycin

BAL(dimercaprol)Antimycin A

Sites of ATP synthesis & Inhibitors

H₂S, CO, CN

Uncouplers

Uncouplers

Can uncouple or delink Allow oxidation without phosphorylation No ATP formation O₂ Consumption

Eg.1)2-4 dinitro phenol(DNP) - lipid soluble uncouple

2) Thermogenin- Physiological uncoupler

3) High doses of AspirinTri-fluorocarbamylcynide phenylhydrazone(FCCP) : 100 times more effective as an than dinitrophenol (DNP)

IONOPHORES

• Ionophores—Lipid soluble compounds

permiability of lipid bilayers

to certain ions.

Eg. Valinomycin & Nigercin

Permit potassium ion to penetrate through mitochondrial membrane discharging the membrane potential..

K⁺ ions exchanges with H⁺

Rate limiting factors

• Availabity of ADP & Substrate.

• Availabity of Substrate.

• Availibity of ADP only.

• Availibity of O₂ only.

• Capacity of respiratory chain.

The inner mitochondrial membrane is impermeable.

Therefore ,NADH produced in cytosol cannot directly enter mitochondria.

• Two shuttle systems for transport of reducing equivalents:

• Transport : Cytosol To Mitochondria but not vice versa

(1) Glycerol phosphate shuttle : insect flight muscles

(2) Malate Malate-aspartate shuttle : predominant in liver and other mammalian tissues

Aerobic Oxidation of Cytosolic NADH

Oxaloacetate Glutamate

Cytosolic malatedehydrogenase

NADH + H⁺

NAD⁺

MalateCytosol

Oxaloacetate Glutamate

Malate

Mitochondrial malatedehydrogenase

NADH + H⁺

NAD⁺

Electron transport chain

α-Ketoglutarate Aspartate

Aspartatetransaminase

α-Ketoglutarate Aspartate

Aspartatetransaminase

Mitochondrial Matrix

Malate – Aspartate shuttle

Glycerophosphate shuttle