1. OXIDATIVE PHOSPHORYLATION

2. INTRODUCTION:
RESPIRATION is the oxidative breakdown of organic compound to release energy.
ORGANIC COMPOUND:
Lipids ) proteins ) carbohydrates
Main aim of these metabolic reactions is to produce ATP.
But how ATP is produced ?

3. RECALL: LOCATION Glycolysis occurs in cytoplasm of the cell.
Electron transport chain (ETC) starts in the MITOCHONDRIAL MATRIX.
The NADH produced during glycolysis and other metabolic processes must make its way to enter into the mitochondria.
NADH produced during glycolysis must transport from cytoplasm into mitochondria to enters ETC.
Mitochondria is not permeable to NADH produced in the cytoplasm.
Shuttling of NADH is INDIRECT.
TWO SHUTTLE MECHANISM:
1) Malate- aspartate shuttle
2) Glycerol Phosphate Shuttle

4. TWO METHODS OF ATP SYNTHESIS:
Oxidative Phosphorylation
Substrate - level phosphorylation

5. TWO METHODS OF ATP SYNTHESIS:
1) SUBSTRATE LEVEL PHOSPHORYLATION:
Direct ATP formation through phosphate transfer from substrate to ADP.
Occurs in glycolysis and kreb cycle.
2) OXIDATIVE PHOSPHORYLATION :
Indirect ATP formation through redox reactions involving O2 as a final electron acceptor.
Driven by electron transport chain.

6. SUBSTRATE - LEVEL PHOSPHORYLATION:
Enzyme transfers a phosphate group from a substrate molecule to ADP.
Rather than adding inorganic phosphate to ADP as in oxidative Phosphorylation.

7. OXIDATIVE PHOSPHORYLATION :
Involves two processes that occurs in the INNER MITOCHONDRIAL MEMBRANE (IMM).
ELECTRON TRANSPORT CHAIN :
Energy in electrons of NADH and FADH2 used to drive H+ against its concentration gradient.
Electrons falls to final electron acceptor oxygen.
CHEMIOSMOSIS:
Using facilitated diffusion of H+ to drive the synthesis of ATP.
The protons flow back down their electrochemical gradient through transmembrane protein complexes called ATP synthase, which bind ADP + Pi and cause them to combine to form ATP.

8. ELECTRON TRANSPORT CHAIN :
ETC removes the energy stored in the NADH and FADH2 molecule to :
Moves protons against its concentration gradient creating proton gradient across the inner mitochondrial membrane.
Final electron acceptor is oxygen which accepts electrons and form water H2O .
All reactions are redox reaction.

9. CHEMIOSMOTIC MODEL: Proposed by PETER MITCHELL.
Transfer of protons from matrix to intermembrane space is accompanied by:
Generation of PROTON GRADIENT across the membrane.
PROTON ( H+) accumulate intermembrane space creating an electrochemical potential difference, proton gradient or electrochemical gradient.
This PROTON MOTIVE FORCE drives the synthesis of ATP by ATP synthase complex.

10. DEFINATION:
According to this model , the electrochemical energy inherent in the differences in proton concentration and separation of charge across the inner mitochondrial membrane – the PROTON MOTIVE FORCE – drives the synthesis of ATP as protons flow passively back into the matrix through a proton pore associated with ATP synthase.

11. GENERATION OF ATP : ( ATP SYNTHESIS)
Proton dependent ATP synthase.
Uses Proton gradient to make ATP.
Proton pumps through the proton channel on enzyme
From intermembrane space into matrix.
~4H+/ATP
Called CHEMIOSMOTIC THEORY.

12. ATP SYNTHESIS :
ATP SYNTHASE is a protein assembly in the inner mitochondrial membrane.
ATP synthase has two sub-units.
F1 subunit – projects into the matrix.
it has 9 subunits.
3 alpha , 3 beta, epsilon, delta and gamma subunit.
Catalysis ATP SYNTHESIS.
Peripheral catalytic sites are present on B- subunit.
F0 SUBUNIT – embedded in the membrane.
It is composed of 3 different type of subunit A , B2 , C
Acts as channel for transport of H+.

13. MECHANISM OF ATP SYNTHESIS : (ROTATIONAL CATALYSIS )
PAUL BOYER proposed a rotational catalysis mechanism.
Synthesis of ATP occurs on the surface of F1 unit.
BOYER’ s binding change hypothesis :
Binding change mechanism states that 3 beta subunits change CONFIRMATIONS during catalysis with only one beta subunit acting as a catalytic site.

14. β - SUBUNIT OCCURS IN THREE FORMS :
β – EMPTY – It has low affinity for substrate ADP + Pi.
β – ADP – can bind substrate ADP and Pi but it is catalytically it is inactive.
β – ATP – Binds substrate ADP and Pi tightly and catalytically active i.e. catalyses ATP synthesis.

16. ROTATIONAL CATALYSIS :
A given β – subunit starts in the β- ADP confirmation ,which binds ADP and Pi from the surrounding medium.
The subunit now changing confirmation, assuming the β- ATP form that tightly binds and stabilizes ATP.
Finally, the β- ATP subunit changes to the β- empty confirmation , which has very low affinity for ATP ,
Newly synthesized ATP leaves the enzyme surface.
NOTE :
One complete rotation of gamma subunit causes each β- subunit to cycle through all three of its possible confirmations.

17. CALCULATING FORMULA OF ATP :
P:O = No. of protons goes inside
Proton backflow
FOR NADH :
P : O = 10/4 = 2.5 ATP
FOR FADH2 :
P : O = 6/4 = 1.5 ATP

18. REGULATION OF OXIDATIVE PHOSPHORYLATION :
Regulation of rate of oxidative phosphorylation by ADP level is called RESPIRATORY CONTROL.
ADP level increases when ATP is consumed and so oxidation is coupled to utilization of ATP.
Electron transport is tightly regulated to oxidative phosphorylation.
Controlled by ADP.
Oxidation cannot be proceed via ETC without simultaneous phosphorylation of ADP.

19. MASS ACTION RATIO : [ATP] = MASS ACTION RATIO [ADP] / [Pi]
With more ADP available for oxidative phosphorylation , rate of respiration increases, causes regeneration of ATP.
This process continues until mass action ratio returns to normal high level.
HIGH – energy sufficient , signifies high ATP.
LOW- energy debt , signifies high ADP or low ATP.

20. INHIBITORS OF OXIDATIVE PHOSPHORYLATION :
Oligomycin blocks the synthesis of ATP by preventing movements of protons through ATP synthase.
Oligomycin – acts through one of the proteins present in F0 – F1 stalk.

21. THANK YOU 