Biological Oxidation

1. Biological Oxidation Dr.S.Chakravarty, MD

2. Oxidation is defined as the removal of electrons and reduction as the gain of electrons. Oxidation is always accompanied by reduction of an electron acceptor.

3. Oxidation and reduction: Oxidation : Reduction : Loss of electrons Loss of hydrogen Gain of oxygen Gain of electrons Gain of hydrogen Loss of oxygen

4. REDOX POTENTIAL • When a substance exists both in the reduced state and the oxidised state , the pair is called a REDOX COUPLE. • The redox potential of this couple is estimated by measuring the EMF of a sample half cell connected to a standard half-cell.

5. Salt bridge 1M H + ~ H gas @ 1 Atmpspheric pressure E0’ =0 meV

6. WHEN A SUBSTANCE HAS LOWER AFFINITY FOR ELECTRONS THAN HYDROGEN IT HAS A NEGATIVE REDOX POTENTIAL • LOWER AFFINITY FOR ELECTRONS = NEG. REDOX POTENTIAL = STRONG REDUCING AGENT AND VICE VERSA. • ELECTRONS MOVE ALWAYS FROM MORE ELECTRONEGATIVE TO ELECTROPOSITIVE LOSS OF FREE ENERGY (THIS ALWAYS ENSURES THAT FREE ENERGY DECREASES)

8. ½ O2 + 2H+ H2O (E0’ = +0.82 ) • NAD+ + H +  NADH (E0’ = -0.32) •  E0’ = +0.82 – (-0.32 ) = 1.14 • COMBINE BOTH OF THESE :- ½ O2 + NADH + H+ H2O + NAD+ Δ G0 = -nFΔE0 = - 2 x 23.06 x 1.14 = -52.6 kcal/mol

9. FOOD (REDUCED ) BIOLOGIOCAL OXIDATION ENERGY SMALLER MOLECULES e- REDUCED COENZYMES e.g NAD ELECTRON TRANSPORT CHAIN 02  H2O MITOCHONDRION

10. Harper’s Illustrated Biochemistry

11. With the help of successive reductions in the electron transport chain assembly , this energy change is released in small increments so that the body can utilize it .

12. HIGH ENERGY COMPOUNDS • THESE COMPOUNDS WHEN HYDROLYSED RELEASE A LARGE AMOUNT OF ENERGY • INDICATED BY SQUIGGLE (~) • FREE ENERGY VARIES FROM -7 TO -15 kcal/mol

14. Substrate level Phosphorylation • Defn :- When the energy of high energy compound is directly transferred to nucleoside diphosphate to form a triphosphate without the help from electron transport chain. • Examples :- 1.Bisphosphoglycerate kinase ( Glycolysis) (1,3 bisphosphoglycerate 3-phosphoglycerate) 2. Pyruvatekinase (Glycolysis) (Phosphoenolpyruvate Pyruvate) 3. Succinatethiokinase (TCA cycle) (SuccinylCoA Succinate )

15. Biological Oxidation and Oxidative Phosphorylation • Biological Oxidation :- The transfer of electrons from the reduced co-enzymes though the respiratory chain to oxygen is known as biological oxidation. • Energy released during this process is trapped as ATP. This coupling of oxidation with phosphorylation is called as OXIDATIVE PHOSPHORYLATION.

16. MITOCHONDRION The mitochondrion contained the enzymes responsible for electron transport and oxidative phosphorylation Impermeable to ions and most other compounds USMLE concept!

17. The MITOCHONDRION – powerhouse of the cell Harper’s Illustrated Biochemistry

18. IMPORTANT MITOCHONDRIAL TRANSPORTERS PyruvatePyruvate Hydrogen Hydrogen MalateMalate Citrate Citrate ADP ADP ATP ATP INNER MITOCHONDRIAL MEMBRANE

19. The mitochondrial membrane is impermeable to NADH….. So, how does cytosolic NADH reach there ??

20. Malate –Aspartate shuttle Operates in Liver , Kidney and Heart

21. Glycerol -3 –phosphate shuttle Operates mainly in muscle and Brain.

22. Electron transport chain • The flow of electrons occurs through successive dehydrogenase enzymes in mitochondria , together known as the electron transport chain (ETC). (the electrons are transferred from lower to higher redox potential)

23. Electron Carriers- The transfer of electrons is not directly to oxygen but through coenzymes

24. Protein complexes: • NADH-CoQ Dehydrogenase (Complex I) • Succinate-CoQ Dehydrogenase (Complex II)  • CoQ-cytochrome cReductase (Complex III) • CytochromecOxidase (Complex IV) • Mobile complexes: • Co-enzyme Q or ubiquinone • Cyt C

25. SITE 1 4 PROTONS PUMPED OUT SITE 2 4 PROTONS PUMPED OUT SITE 3 2 PROTONS PUMPED OUT Complex IV Cyt a-a3 (Fe+3,Cu+2) Complex III FeS-Cytb-Cyt c1 COMPLEX I FMN-FeS Cyt C Co Q 2H + COMPLEX II FeS Inner mitochondrial membrane + 8.Succinate DH(TCA CYCLE) 9. AcylCoa A DH(fatty acid oxidn.) 10. Glycerol 3-P DH(mitochondrial) NAD ½ O2 FAD 1.Glyceraldehyde -3P 2.Isocitrate 3.Malate 4.Glutamate 5.β-OH-acylCoA H2O Fp(FAD) lipoate 6.Pyruvate 7.α-ketoglurarate Mitochondrial Matrix

26. Pathways for flow of electrons: There are 2 sites of entry for electrons into the electron transport chain: Using either • For NADH: Complex 1 -> complex 3 -> complex 4 • For FADH2: (more positive redox) Complex 2 -> complex 3 -> complex 4 NAD+ or FAD Both are coenzymes for dehydrogenase enzymes

27. 2 3 4 1

28. COENZYME Q • The ubiquinone is reduced successively to semiquinone (QH) and finally to quinol (QH2) • It accepts a pair of electrons from NADH or FADH2 through complex I or complex II respectively. • Co-enzyme Q is a quinone derivative having long isoprenoid tail. • 2 molecules of cytochrome c are reduced. • The Q cycle thus facilitates the switching from the 2 electron carrier ubiquinol to the single electron carrier cytochrome c. • This is a mobile carrier.

29. CHEMIOSMOTIC THEORY(Peter Mitchell. N.P 1978) • The transport of electrons from inside to outside of inner mitochondrial membrane is accompanied by the generation of a proton gradient across the membrane. • Protons accumulate outside the membrane creating an electrochemical potential. • This drives the synthesis of ATP by ATP synthase .

30. Harper’s Illustrated Biochemistry

31. Harper’s Illustrated Biochemistry

32. The pH outside is 1.4 units lower than inside . • The outside is positive 0.14V relative to inside. • The proton motive force (PMF ) IS 0.224 v corresponding to a free energy change of 5.2 kcal/mol of protons.

33. ENERGETICS OF ATP SYNTHESIS • ENERGY RELEASED = 52kcal/mol • Synthesis of 1ATP and Pi requires 7.3 kcal/Molmolecules • Chemical energy trapped = 7.3 x 3 = 21.9kcal = 40% • Rest 60% energy is dissipated as HEAT !!

34. The resting human body consumes approx. 420 Kjoules of energy / hour . This is only slightly greater than that of a 100 w LIGHT BULB. • BODY’S Energy Requirements are met by merely a 0.2 VOLTS difference in b/w the membranes of the mitochondrion .( St.Kitts – 110 V ) • The Trans membrane current is about 500amps ( approx 3 x 10 21 protons / sec.) • A laptop uses approximately 4.5 amps.

35. F0 – F1 complex : Fo complex: – O STANDS FOR OLIGOMYCIN Made of 12 subunits . H+ passes through each subunit from membrane space to inner space rotating the Fo complex (turbines).

36. F1 complex:Has 9 polypeptide chains ,(3 alpha , 3 beta , 1 gamma , 1 sigma , 1 epsilon) the α chains have binding sites for ATP and ADP and beta chains have catalytic activity. ATP SYNTHESIS NEEDS Mg +2 IONS • ADP and Pi bind the alpha subunit • Binding change mechanism - conformation change of beta subunits causes release of ATPs from the complex. • ATPs formed in the mitochondrial matrix are translocated to cytosol by ATP/ADP translocase

37. 1 ) ADENINE NUCLEOTIDE TRANSPORTER 2)H+/Pi SYMPORT

38. EARLIER ESTIMATES WERE WRONG!! • According to recent estimates , NADH may generate only 2.5 ATPs while FADH2 may generate only 1.5 ATP. • So , instead of 38 ATP , only 32 ATPs are generated from glucose .

39. ATP IS THE ENERGY CURRENCY

40. Thank you