1. Chapter 7 Biological Oxidation

2. Biological oxidation is the cellular process in which the organic substances release energy (ATP), produce CO2 and H2O through oxidative-reductive reactions. organic substances: carbohydrate, fat and protein

3. 7.1 Principal of Redox Reaction The electron-donating molecule in a oxidation-reduction reaction is called the reducing agent or reductant; the electron-accepting molecule is the oxidizing agent or oxidant: for example: Fe2+ (ferrous) lose -e Fe3+ (ferric) gain +e

4. Redox reaction = reduction-oxidation reaction Several forms of Biological Reduction 1. Gain of electrons 2. Hydrogenation 3. Deoxygenation Several forms of Biological Oxidation 1. Loss of electrons 2. Dehydrogenation 3. Oxygenation

5. oxidation-reduction potential ( or redox potential), E : it is a measure of the affinity of a substance for electrons. It decide the loss (or the gain) of electrons. A positive E: the substance has a higher affinity for electrons , accept electrons easily. A negative E: the substance has a lower affinity for electrons , donate electrons easily.

6. E0`, the standard redox potential for a substance :is measured under stander condition(25, 1mmol/L reaction substance),at pH7, and is expressed in volts.

7. Section 7.2 Respiration Chain and Oxidative Phosphorylation

8. 7.2.1 Respiratory Chain Term: A chain in the mitochondria consists of a number of redox carriers for transferring electrons from the substrate to molecular oxygen to form oxygen ion, which combines with protons to form water.

9. Redox carriers including 4 protein complexes 1.Complex I: NADH:ubiquinone oxidoreductase NADH:CoQ oxidoreductase 2.Complex II: Succinate dehydrogenase 3.Complex III: cytochrome bc1 (ubiquinone Cyt c oxidoreductase) 4.Complex IV: cytochrome oxidase

10. Complex I ( NADH:ubiquinone oxidoreductase) Function: transfer electrons from NADH to CoQ Components: NADH dehydrogenase (FMN) Iron-sulfur proteins (Fe-S) complex NADH CoQ FMN; Fe-SN-1a,b; Fe-SN-4; Fe-SN-3; Fe-SN-2

11. R=H: NAD+; R=H2PO3:NADP+ 1. NAD(P)+: Nicotinamide Adenine Dinucleotide Phosphate)

12. Oxidation of NADH is a 2-electron(2e), 2-proton(2H) reaction NAD+ or NADP+ NADH or NADPH

13. 2. FMN can transfer 1 or 2 hydride ions ach time FMN: flavin mononucleotide Accepts 1 H+ and 1 e- Accepts 2 H + and 2 e to form semiquinone = stable free radical to give fully reduced form

14. 3. Iron-sulfur clusters (Fe-S) transfers 1electron at a time, without proton involved Fe3++e- Fe2+

15. 4.Ubiquinone (CoQ) is lipid-soluble, not a component of complex , can transfer 1 or 2 hydride ions each time. Function: transfer electrons and protons from complex , to complex .

16. NADH+H+ NAD+ FMN Reduced Fe-S Q FMNH2 Oxidized Fe-S QH2 Matrix Intermembrane space

17. Complex II : Succinate dehydrogenase (Succinate: CoQ oxidoreductase ) Function: transfer electrons from succinate to CoQ Components: Succinate dehydrogenase (FAD, Fe-S) Cytochrome b560 Complex Succinate Fe-S1; b560; FAD; Fe-S2 ; Fe-S3 CoQ

18. Cytochromes a, b, c are heme proteins, their heme irons participate redox reactions of e- transport. Fe3++e- Fe2+

19. Intermembrane space Matrix Succinate

20. Complex III: cytochrome bc1 (ubiquinone Cyt c oxidoreductase) Function: transfer electrons from CoQ to cytochrome c Components: iron-sulfur protein cytochrome b(b562, b566) cytochrome c1 complex QH2 b562; b566; Fe-S; c1 Cyt c

21. Cytochrome c is soluble, which will transfer electrons to complex Intermembrane space Matrix

22. Complex IV: cytochrome oxidase Function: transfer electrons from Cyt c to molecule oxygen, the final electron acceptor. Components: cytochrome aa3 copper ion (Cu2+) Cu2+ + e- Cu+ Complex IV Cyt c CuAaa3CuB O2

23. Cytochrome c Coenzyme Q ubiquinone/ol

24. Sequence of respiratory chain Principles: e- tend to flow from a redox pair with a lower Eto one with a higher E In the e--transport chain, e--carriers are arranged in order of increasing redox potential, making possible the gradual release of energy stored in NADH, FADH2

25. Redox potential redox pair NAD+/NADH+H+ FMN/ FMNH2 FAD/ FADH2 Cyt b Fe3+/Fe2+ Q10/Q10H2 Cyt c1 Fe3+/ Fe2+ Cyt c Fe3+/Fe2+ Cyt a Fe3+ / Fe2+ Cyt a3 Fe3+ / Fe2+ 1/2 O2/ H2O E'0(V) E -0.32 -0.30 -0.06 0.040.10 0.07 0.22 0.25 0.29 0.55 0.82

26. There are two respiratory chains NADH respiratory chain NADH Complex CoQ Complex cytochrome c Complex O2 Succinate (FADH2) respiratory chain Succinate Complex CoQ Complex cytochrome c Complex O2

27. NADH respiration chain FADH2 respiration chain

28. 7.2.2 Oxidative Phosphorylation The oxidation of organic nutritions produces the energy-rich molecules, NADH and FADH2. The oxidation of NADH or FADH2 in mitochondrial is the electron transferring through respiration chain. The free energy produced in electron transferring supports the phosphorylation of ADP to form ATP. The oxidation of NADH or FADH2 and the formation of ATP are coupled process, called Oxidation Phosphorylation.

29. The Chemiosmotic Theory The free energy of electron transport is conserved by pumping protons from the mitochondrial matrix to the intermembrane space so as to create an electrochemical H+ gradient across the inner mitochondrial membrane. The electrochemical potential of this gradient is harnessed to synthesize ATP. Peter Mitchell

30. Electrochemical H+ gradient (Protonmotive force) 2 components involved 1. Chemical potential energy due to difference in [H+] in two regions separated by a membrane 2. Electrical potential energy that results from the separation of charge when a proton moves across the membrane without a electron.

31. Complex I: 4 H+ expelled per e--pair transferred to Q Complex III: 4 H+ expelled per e--pair transferred to Cyt c Complex IV: 2e- + 2 H+ from matrix convert O2 to H2O; 2 further H+ expelled from

32. Proton pumping: Reductiondependent conformational switch of an e--transport complex Conformation 1 (high affinity for H+) Conformation 2 (low affinity for H+).

33. ATP Synthase Intermembrane space Inner (ab2c9-12) Membrane Matrix C ring (33 )

34. -subunit take up ADP and Pi to form ATP ADP + Pi ATP Each of 3 subunits contains an active site F1: multisubunit complex that catalyzes ATP synthesis F 0 = proton-conducting transmembrane unit

35. When protons flow back through F0 channel, -subunit is rotated by the rotation of c ring, then the conformations of -subunits are changed, this lead to the synthesis and release of ATP. To form a ATP need 3 protons flow into matrix. H+ flow -subunit has three conformations:T (tight), L (loose), O (open)

36. Translocation of ATP , ADP and Pi. ADP3- ATP4- H+ H2PO4- H+ Intermembrane space F0 Matrix F1 ATP4ADP3H+ H2PO4- H+

37. When protons flow back through F0 channel, -subunit is rotated by the rotation of c ring, then the conformations of -subunits are changed, this lead to the synthesis and release of ATP. To form a ATP need 3 protons flow into matrix. H+ flow -subunit has three conformations:T (tight), L (loose), O (open)

38. P/O ratios P/O ratio is the rate of phosphate incorporated into ATP to atoms of O2 utilized. It measure the number of ATP molecules formed per two electrons transfer through the respiratory chain. NADH respiratory chain : 2.5, FADH2 respiratory chain: 1.5

39. During two electrons transfer through NADH respiratory chain, ten protons are pumped out of the matrix. To synthesis and translocation an ATP, four protons are needed. So, two electrons transport can result in 2.5 ATP. To succinate respiratory chain , two electrons transport can result in 1.5 ATP.

40. Regulation of Oxidative Phosphorylation 1.PMF (proton motive force) regulate the electron transport. higher PMF lower rate of transport 2.ADP concentration resting condition: energy demanded is low, ADP concentration is low, the speed of Oxidative Phosphorytion is low. active condition: the speed is high.

41. Inhibitor of Oxidative Phosphorylation 1.Inhibitor of electron transport Succinate Antimycin A Cyanide, Azide Carbon Monoxide Retonone Amytal

42. 2.Uncoupling agents uncoupling protein (in brown adipose tissue), 2,4-dinitrophenol, Pentachlorophenol heat H+ Intermenbran space H Cyt c uncoupling protein F0 Q F1 Matrix + H+ H+ ADP+Pi ATP 2,4-dinitrophnol

43. 3.Oligomycin bonds at the connection of F 0 and F1, inhibit the function of ATP synthase. Intermembrane space Matrix Oligomycin C ring

44. Succinate Retonone Amytal Antimycin A Uncoupling agent Oligomycin

45. ATP and other Energy-rich compounts ATP has two energy-rich phosphoric acid anhydride bonds, the hydrolysis of each bond release more energy than simple phosphate esters. NH 2 N N OH OH OH N OH OH N p O p OCH2 O O= P O OH H H H H OH OH AMP ADP ATP

46. Some Energy-rich compounds Structure Exemple creatine phosphate phosphoenolpyruvate acetyl phosphate Acetyl CoA G

47. The hydrolysis of energy-rich bond: G = -5 -15kcal/mol The compounds with energy-rich bond are high-energy compounds. The hydrolysis of low-energy bond: G = -1 -3kcal/mol The compounds with low energy bond are compounds. low-energy

48. Transport of high-energy bond energies 1.Substrate level phosphorylation Glycerate 1,3-biphosphate + ADP Glycerate 3-phosphate +ATP G = -4.5kcal/mol Phosphoenolpyruvate +ADP Pyruvate + ATP G = -7.5kcal/mol

49. 2.ATP is the center of energy producing and utilizing. ATP Oxidative Phosphorylation Energy utilization Substrate level phosphorylation ~P ~P ADP

50. 3.Other nucleoside triphosphates are involved in energy transport. GTP: gluconeogenesis protein synthesis UTP: glycogen CTP: lipid synthesis

51. 4.Transport of the terminal phosphate bond of ATP to the other nucleoside Function of nucleoside diphosphate kinase ATP + UDP ATP + CDP ATP + GDP ADP + UTP ADP + CTP ADP + GTP Function of adenylate kinase ADP + ADP ATP + AMP

52. 7.3 Energy from cytosolic NADH A mitochondrial NADH produce 2.5 ATP A cytosolic NADH must be transported into mitochondrial for oxidation by two methods. Glycerol phosphate shuttle 1.5 ATP Malate aspartate shuttle 2.5 ATP

53. Glycerol phosphate shuttle CH2OH CH2OH Electron chain Glycerol phosphate dehydrogenase NAD+ C=O C=O CH2O- Pi NADH+H + CH2O- Pi dihydroxyacetone phosphate dihydroxyacetone phosphate CH2OH CH2OH CHOH CHOH CH2O- Pi CH2O- Pi Glycerol phosphate FADH2 FAD Glycerol phosphate Intermembran space Glycerol phosphate dehydrogenase Inner menbran

54. Malate aspartate shuttle O Aspartate -OOC-CH2-C-COO- H 3N Malate -ketoglutarate carrier + - - OOC-CH2-C-COO Aspartate H oxaloacetate oxaloacetate Glutamate NADH +H+ Glutamate Electron chain NADH +H+ NAD+ -ketoglutarate -ketoglutarate OH NAD+ -OOC-CH2-C-COOH Malate cytosol Glutamate-aspartate carrier inner mitochondrial membran Malate matrix

55. 7.4 Other Biological Oxidations Monoxygenases dioxygenase --add 2 atoms of O2 oxygenase to organic compounds. monoxygenase (mixed-function oxidase, hydroxylase) --adds 1 oxygen atom to organic compounds as a hydroxyl group. RH + NADPH + H+ + O2 ROH + NADP+ + H2O

56. The chief compounds of monoxygenase: Cyt b5, Cyt P450, Cyt P450 reductase(FAD,FMN)

57. Free Radical Scavenging Enzymes Free Radical: the groups with an unpaired electron. (such as O2 H2O2 OH) 1.Superoxide dismutases(SODs) 2O2 + 2H+ SOD H2O2 + O2 peroxidase H2O + O2

58. 2.Glutathione peroxidase H2O2 (ROOH) 2G SH NADP+ Glutathione Glutathione reductase peroxidase H2O (ROH+H2O) G S S G NADPH+H+

59. 3.Catalase (in peroxisomes) 2H2O2 catalase 2H2O + O2

60. summary