18.oxidative phosphorylation

OXIDATIVE PHOSPHORYLATION

DEFINITION

Oxidative phosphorylation is the name given to the synthesis of ATP from ADP ( phosphorylation), that occurs when NADH and FADH2 are oxidized by electron transport throughout the respiratory chain (hence called oxidative)

Mitochondria are the site of oxidative phosphorylation in eukaryotes ,

There is two ways to synthesize ATP

Oxidative phosphorylation : the phosphorylation of ADP to ATP coupled

to electron transfer

Substrate level phosphorylation : direct transfer the phosphate from chemical intermediate (also called substrate ) to ADP or GDP forming ATP or GTP , independent of electron transfer chain

Example of Substrate level phosphorylation

3-phosphoglycerate

1) 1,3-bisphosphoglycerate

ADP

ATP

Phosphoglycerate kinase

Glycolysis

2) Phosphoenolpyruvate

ADP

ATP

Pyruvate

Pyruvate kinase

3) succinyl CoA

GDP

GTP

succinate

succinyl CoA synthetase

Glycolysis

TCA cycle

CHEMIOSMOTIC HYPOTHESIS

Is the mechanism of oxidative phosphorylation ,

proposed by Peter Mitchell in 1961

The chemiosmotic hypothesis : it proposed that energy liberated by electron

transport is used to create a proton gradient across the mitochondrial inner membrane

and it is this proton gradient is used to drive ATP synthesis

Thus the proton gradient couples electron transport and ATP synthesis

ATP SYNTHASE Also called complex Ⅴ

It is the enzyme that actually synthesize ATP

It located in the inner mitochondrial membrane

It utilizes energy from the proton gradient to promote phosphorylation of ADP forming ATP

THE STRUCTURE OF ATP SYNTHASE The ATP synthase can be seen as spherical projections

from the inner membrane .

Is composed of two major components part ,

F1 unit or called F1 ATPase :

is the spheres of the ATP synthase

and point outward

F0 unit :

spans the inner mitochondria membrane

So the ATP synthase is also called F0F1 ATPase

The stalk between F0 and F1 contains several additional polypeptide

F1 unit

F0 unit

Proton channel

Matrix

Intermembran space

The structure of ATP synthase

F1 unit : contains 5 polypeptides in the form α3β3γδε.

only F1 components can hydrolyze ATP, have ATPase activity , so also called ATPase ,

F1 with F0 together can synthesize ATP

F0 unit: made of abc polypeptides, is proton channel, can be seen as the proton transport ,

The complete complex harnesses the energy released by electron transport to drive ATP synthesis

The function of ATP synthase

In summary : the oxidative phosphorylation process is as follow

Electron transport down the respiratory chain from NADH or FADH2

Complex , , Cause protons be pumped out Ⅰ Ⅲ Ⅳof the mitochondrial matrix into the intermembrane space

The pumping out of H+ generates a higher concentration of H+ and an electrical potential , thus an electrochemical proton gradient is formed .

The H+ flow back into the mitochondrial matrix through ATP synthase and the electrochemical proton gradient drives ATP synthesis

ELECTROCHEMICAL PROTON GRADIENT

Is the sum of chemical gradient : a higher concentration of H+ ions form the chemical

gradient of H+ outer : high concentration H+ inner : low concentration H+

Electrical potential : electrical charge potential across the inner mitochondrial

membrane outer: positive charge inner: negative charge

matrix

+ + + +

- - - -

H+

O2

H2O

H+

e-

ADP+

Pi ATP

The chemiosmotic hypothesis :

High H+Positive charge

Low H+Negitive charge

ⅢⅠ Ⅱ Ⅳ

F0

F1

Cyt c

Q

NADH+H+

NAD+

Fumarate

Succinate

H+

1/2O2+2H+

H2O

ADP+Pi ATP

H+

H+

H+

Intermembrane space

mattix

+ + + + + + + + + +

- - - - - - - - -

The chemiosmotic hypothesis :

GENERATE ATP SUMS

3 ATP are synthesized per NADH oxidized through the NADH

respiratory chain

2 ATP are synthesized per FADH2 oxidized through the FADH2

respiratory chain

WHY ATP SYNTHESIZE FROM FADH2 RESPIRATORY CHAIN IS LESS THAN NADH RESPIRATORY CHAIN ?

NADH respiratory have 3 H+ pump, complex , ,Ⅰ Ⅲ Ⅳ

FADH2 respiratory only have 2 H+ pump, complex ,Ⅲ Ⅳ

So the ATP made from FADH2 is less than from NADH

The process of biological oxidation

COUPLING OF ELECTRON TRANSPORT AND PHOSPHORYLATION

Electron transport is normally tightly coupled to ATP synthesis

electrons do not flow through the electron transport chain to oxygen unless ADP is simultaneously phosphorylated to ATP

Also , ATP is not synthesized unless electron transport is occurring to provide the proton gradient

Thus we can know that oxidative phosphorylation needs NADH or FADH2 , oxygen , ADP , and inorganic phosphate

COUPLING SITE OF ELECTRON TRANSPORT AND PHOSPHORYLATION

complex , ,Ⅰ Ⅲ Ⅳ

How to know this ?

P:O ratio: in oxidative phophorylation the P:O ratio is the number of ATP formed per oxygen atom is consumed

NADH respiratory chain : P:O ratio=3

forming 3 ATPs per oxygen atom consumed ,

NADH respiratory chain have 3 coupling site

FADH2 respiratory chain : P:O ratio=2

forming 2 ATPs per oxygen atom consumed ,

FADH2 respiratory chain have 2 coupling site

UNCOUPLERS

WHAT IS UNCOUPLERS? Some chemicals act as uncoupling agents,

that is, when added to cell, they stop ATP synthesis but electron transport still continues and so oxygen is still consumed

The chemicals for example : • 2,4-dinitrophenol (DNP) • uncoupling protein

DNP DNP are lipid-soluble small molecule

that can bind H+ ion and transport the H+ across the mitochondria membrane ,

So it is H+ ionophores

HOW DNP UNCOUPLING ? electron transport occurs and pump out H+ ions across

the inner mitochondrial membrane to build the H+ gradient .

But DNP in the same membrane carriers the H+ ions back into the mitochondrion , preventing formation of a proton gradient.

Since no proton gradient forms, so no ATP can be made by oxidative phosphorylation

the energy derived from electron transport is released as heat

UNCOUPLING PROTEIN There is brown adipose tissue in the body ,

this tissue is rich in mitochondria, the inner mitochondrial membranes of which contains a protein called uncoupling protein or thermogenin

HOW UNCOUPLING PROTEIN WORK ? Uncoupling protein can be seen as a H+ passageway

, allows H+ to flow back into mitochondria without having to enter the ATP synthase ,

thus preventing formation of a proton gradient , so uncouples electron transport and oxidative phosphorylation ,

And also energy derived from electron transport is released as heat

mechanism of uncoupling protein （ brown adipose tissue mitochondrial ）

Ⅲ ⅢⅠ Ⅰ Ⅱ Ⅱ

FF0 0

FF1 1

Ⅳ Ⅳ

Cyt c

Q

Intermembrane space

Matrix

Uncoupling protein

Heat energy HH+ +

HH+ + ADP+Pi ATP

THERMOGENESIS

The production of heat by uncoupling is called nonshivering thermogenesis .

It is important in certain biological situation ,

For example ,the brown adipose tissue is found in sensitive body areas of some new brown animals (including human ), where the heat production provides protection from cold condition

In addition, thermogenesis by brown adipose tissue plays a important role in maintaining body temperature in hibernating animals

RESPIRATORY CONTROL

ADP: The actual rate of oxidative phosphorylation is set by

the availability of ADP

If ADP is added to mitochondrial, the rate of oxygen consumption rises as electrons flow down the chain

Then when all the ADP has been phosphorylated to ATP, the rate of oxygen consumption falls

This process called respiratory control

this mechanism ensures that electrons flow down the chain only when ATP synthesis is needed.

If the level of ATP is high ,ADP is low,

no electron transport occurs

NADH and FADH2 build up,

so does excess citrate

then the citric acid cycle and glycolysis are all inhibited

OVER ALL ADP high

Oxidative phosphorylation rises Oxygen consumption rises

ADP low

Oxidative phosphorylation falls Oxygen consumption falls

ATP

Adenosine triphosphate: ATP

Glycosidic bond

NO

CH2O

OHOH

N

NN

NH2

P

O

OH

OP

O

OH

OP

O

OH

OH

ATPATP

Ester bond

αβr

Ribose

Adenine

Production and application of ATP

ATP

ADP

oxidative Phosphorylation

~P ~P

~P ~P

Mechnism energyOsmotic energyChemical energyElectric energyHot energy

substrate level Phosphorylation

RIOXIDATION OF CYTOSOLIC NADH

The inner membrane of mitochondria impermeable to some molecule and ions

Permeable to : pyruvate 、 succinate 、 citrate α-ketoglutarate 、 malate 、 Glu ect

Impermeable to : H+ 、 NADH 、 NADPH 、 oxaloacetate ect

The inner mitochondrial membrane is impermeable to NADH.

Therefore NADH produced in the cytoplasm during glycolysis go into the mitochondria through the membrane shuttle , then in the mitochondria go into the respiratory chain .

The membrane shuttle is a combination of enzyme reaction that bypass this impermeability barrier

WHICH REACTION OF GLYCOLYSIS PRODUCE NADH

Glyceraldehyde 3-phosphate

1,3-bisphosphoglycerate

Glyceraldehyde 3-phosphate

Dehydrogenase

NAD+

NADH

The reaction take place in the cytosol

THERE IS TWO SHUTTLE SYSTEM IN THE MITOCHONDRIAL MEMBRANE

• glycerol 3-phosphate shuttle

• Malate-asparate shuttle

NADH+H+

Glycerol 3-phosphate

Dehydrogenase

FADH2

NAD+ FAD

innerMembrane

electron chain

Dihydroxyacetone Phosphate




Dehydrogenase


Cytosol


NADH+H+


Dehydrogenase

FADH2

NAD+

FAD

innerMembrane outer

Membrane

IntermembraneSpace

matrix

electron chain


PiCH2O-

CH2OH

C=O

PiCH2O-

CH2OH

C=O

Glecerol 3-phosphate

PiCH2O-

CH2OH

CHOH

PiCH2O-

CH2OH

CHOH



Dehydrogenase

Dihydroxyacetone phosphate in the cytosol is

reduced to glycerol -3-phosphate ,

and NADH reoxidized to NAD+,

by cytosolic glycerol 3- phosphate dehydrogenase

The glycerol -3-phosphate diffuse across the inner mitochondrial membrane

In the inner membrane the glycerol 3-phosphate is converted back to dihydroxyacetone phosphate by

mitochondrial glycerol 3-phosphate dehydrogenase

the mitochondrial glycerol 3-phosphate dehydrogenase does not use NAD+ but instead uses FAD.

The FADH2 is then reoxidized by FADH2 respiratory chain.So 2 ATPs are synthesized )

The dihydroxyacetone phosphate then diffuse back to the cytosol .

Note :

The shuttle does not allow cytoplasm NADH to enter the mitochondrion,

but transports the two electrons from NADH into the mitochondria

and feed the electron into the FADH2 electron transport chain .

So synthesize 3ATPs

NADH +H+

NAD+ malate

α-ketoglutaratecarrier

malate dehydrogenase

inner memebrane malateMalate

Oxaloacetate Oxaloacetate

NAD+

NADH +H+


glutamate –aspartate

carrier

Aspartate

Aspartate Aminotransferase

Aspartate Aminotransferase

Aspartate

glutamate

α-ketoglutarate

α-ketoglutarate

glutamate

Cytosol

NADH +H+

NAD+

-OOC-CH2-C-COO-

O

-OOC-CH2-C-COO-

OH

H

NADH +H+

NAD+

glutamate –aspartate

carrier

malateα-ketoglutarate

carrier

-OOC-CH2-C-COO-

OH

H

malate

-OOC-CH2-C-COO-

O

oxaloacetate

-OOC-CH2-CH2-C-COO-

O

-OOC-CH2-CH2-C-COO-

O

-OOC-CH

2-CH

2-C-COO

-

H3N

+

Hglutamate


aspartate aminotransferase

intermembrane space

inner memebrane

matrix

-OOC-CH

2-C-COO

-

H3N

+

Hasparate

-OOC-CH

2-C-COO

-

H3N

+

H

-OOC-CH

2-CH

2-C-COO

-

H3N

+

H

α-ketoglutarate

malate

Oxaloacetate in the cytosol is converted to malate by cytosolic malate dehydrogenase.

NADH oxidized to NAD+

The malate enters the mitochondrion by amalate-α-ketoglutarate carrier in the inner

mitochondrial membrane

In the matrix the malate is reoxidized to oxaloacetate , NAD+ form NADH.

Then NADH go into the NADH respiratory chain And 3ATPs are synthesized

Oxaloacetate does not easily cross the inner mitochondrial membrane and so is transaminated to form aspartate

And then the aspartate exits from the mitochondrion and is reconverted to oxaloacetate in cytosol , again by transamination

this cycle of reactions is to transfer the electrons from NADH in the cytosol to NADH in the mitochondrial matrix ,

The NADH in the mitochondria is then reoxidized by the NADH electron transport chain

So synthesize 3ATPs

Note

In summary : Cytosol NADH go into the mitochondria by this two shuttele


in the mitochondria go into the FADH2

respiratory chain .

so produce 2 ATPs

Malate-asparate shuttle:

in the mitochondria go into the NADH respiratory chain. so produce 3ATPs

Complex : NADH dehydrogenaseⅠ or called NADH-CoQ reductase

complex : Succinate-coenzyme Q reductase Ⅱcomplex : Cytochrome bc1 complexⅢ or called cytochrome reductase

complex : Cytochrome oxidase Ⅳ

1. Description Composition of respiratory chain complex

Practice exercises

NADH

CoQ

succinateFAD(Fe-S)

FMN(Fe-S)

complexⅠ

complexⅡ

O2Cyt

c

Cytaa3

CuCuB

Cytb 、 Fe-S 、 Cytc1

Complex Ⅲ

Complex Ⅳ

2. Write down the two respiratory chain

FADH2 respiratory chain

NADH respiratory chain

3. Filling the blank

There is two respiratory chain in the body : that is ( ) and ( )

NADH respiratory chain

FADH2 respiratory chain

4. Explain : electron transport chain

Electron transport chain:

The electrons are transferred from NADH to oxygen along a chain of electron carriers collectively called electron transport chain , also called respiratory chain.

5. Choice the sequence of Cytochoreme in respiratory chain A . c c1 b aa3 O2

B . c1 c b aa3 O2

C. b c1 c aa3 O2

D. b c c1 aa3 O2

E. c b c1 aa3 O2

6. ROTENONE INHIBIT ELECTRON TRANSPORT AT ( )

A. NADH dehydrogenaseB. cytochrome bc1 complexC. cytochrome oxidase D. Succinate –Q dehydrogenase E. cytochrome c

( A )

7. ANTIMYCIN A INHIBIT ELECTRON TRANSPORT AT ( )

• A. NADH dehydrogenase

• B. cytochrome bc1 complex

• C. cytochrome oxidase

• D. Succinate –Q dehydrogenase

• E. cytochrome c

( B )

8. CARBON MONOXIDE (CO) INHIBIT ELECTRON TRANSPORT AT ( )

• A. NADH dehydrogenase

• B. cytochrome bc1 complex

• C. cytochrome oxidase

• D. Succinate –Q dehydrogenase

• E. cytochrome c

( C )

9. COUPLING SITE OF ELECTRON TRANSPORT AND PHOSPHORYLATION ARE( )

A. complex Ⅰ B. complex Ⅱ C. complex Ⅲ D. complex Ⅳ E. complex Ⅴ

( A,C,D )

10. THE TWO WAYS TO SYNTHESIZE ATP ARE ( ) AND ( )

-Oxidative phosphorylation -substrate level phosphorylation

11. THE MECHANISM OF OXIDATIVE PHOSPHORYLATION IS ( )

-Chemiosmotic hypothesis

12. THE ENZYME THAT ACTUALLY SYNTHESIS ATP IS ( ), IT IS MADE UP OF ( )UNIT AND ( ) UNIT

ATP synthase F0 , F1

13. NADH PRODUCED IN THE CYTOPLASM MUST BE REOXIDIZED VIA A MEMBRANE SHUTTLE . THE TWO SHUTTLE SYSTEM ARE ( ) AND ( )

-glycerol 3-phosphate shuttle

-Malate-asparate shuttle

14 . If ADP is high, The Oxidative phosphorylation and Oxygen consumption will ( ) .

if ADP is low , The Oxidative phosphorylation and Oxygen consumption will ( )

rises , falls

15 . the chemicals that can uncouple the electron transport with the ATP synthesis are ( ) and ( )

DNP, Uncoupling protein

Technology

18.oxidative phosphorylation