Biological oxidation

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dr.tahira lecture on bilogical oxidation

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Biological oxidation

  1. 1. BIOLOGICAL OXIDATION
  2. 2. <ul><li>Oxidation is a reaction with oxygen directly or indirectly OR to lose hydrogen and / or electrons. This process is carried out by enzymes. </li></ul><ul><li>There can be the following three classes of biological oxidation :- </li></ul><ul><li>- loss of one or more electrons </li></ul><ul><li>- loss of one or more hydrogen atoms </li></ul><ul><li>- addition of one or more oxygen atoms </li></ul>
  3. 3. <ul><li>EXERGONIC and ENDERGONIC REACTIONS :- </li></ul><ul><li>In exergonic reactions, free energy is released while in endergonic reactions, free energy is absorbed. </li></ul><ul><li>This free energy moves between the two reactions through the common intermediate . This common intermediate is thus a carrier of chemical energy between the two reactions. </li></ul><ul><li>Two chemical reactions have a common intermediate when they occur in a sequence. For example </li></ul><ul><li>A + B -> C + D </li></ul><ul><li>D + X -> Y + Z </li></ul><ul><li>D is the common intermediate and can act as carrier of chemical energy. </li></ul>
  4. 4. ATP as an ENERGY CARRIER <ul><li>Many coupled reactions use ATP to generate common intermediate. These reactions may involve </li></ul><ul><li>ATP cleavage --- the transfer of a phosphate group from ATP to another molecule </li></ul><ul><li>ATP synthesis ---- the transfer of phosphate group from an energy rich intermediate to ADP, forming ATP. </li></ul>
  5. 5. Energy carried by ATP <ul><li>ATP ---- one molecule of adenosine ( adenine + ribose ) , and three phosphate groups. </li></ul><ul><li>The phosphate groups are attached to each other by high-energy phosphate bonds. </li></ul><ul><li>-Removal of one phosphate ->-> ADP </li></ul><ul><li>-Removal of two phosphates ->-> AMP </li></ul><ul><li>The standard free energy of hydrolysis of ATP , is approx. – 7.3 kcal/mol for each of the two terminal phosphate groups. </li></ul>
  6. 6. ELECTRON TRANSPORT CHAIN <ul><li>During the metabolism of energy rich molecules, the metabolic intermediates of these reactions donate electrons to specific co-enzymes– NAD and FAD to form energy rich reduced coenzymes, NADH and FADH 2 . These reduced coenzymes, in turn each donate a pair of electrons to special electron carriers, collectively called Electron Transport Chain. </li></ul><ul><li>Free energy is lost by these electrons as they pass down this chain. </li></ul><ul><li>Part of this energy is used to form ATP from ADP – OXIDATIVE PHOSPHORYLATION. </li></ul><ul><li>The remainder of this energy is used in processes like Ca transport and to form heat. </li></ul>
  7. 7. Site of ETC <ul><li>MITOCHONDRIA ---- inner mitochondrial membrane. It is impermeable to most small ions, small and large molecules --- requirement of specialized carriers. </li></ul><ul><li>The matrix of mitochondria contains </li></ul><ul><li>- TCA cycle enzymes </li></ul><ul><li>- fatty acid oxidation enzymes </li></ul><ul><li>- Mitochondrial DNA and RNA </li></ul><ul><li>NAD and FAD </li></ul><ul><li>ADP and Pi </li></ul>
  8. 8. Organization of the chain <ul><li>The inner mitochondrial membrane can be divided into five protein complexes :- </li></ul><ul><li>- complexes I, II, III, IV, and V </li></ul><ul><li>Complex I to IV, each contains a part of the ETC. Complex V is used for ATP synthesis. </li></ul><ul><li>Each complex accepts or donates electrons to electron carriers --- coenzyme Q and Cytochrome c. </li></ul><ul><li>Each carrier donates electrons to the next carrier in the chain. </li></ul><ul><li>The electrons ultimately combine with oxygen and protons to form water. </li></ul>
  9. 10. Reactions of the electron transport chain <ul><li>1- Formation of NADH:- </li></ul><ul><li>NAD is reduced to NADH </li></ul><ul><li>Enzyme --- dehydrogenase --- removal of two hydrogen atoms from the substrate ---- both electrons and a hydride ion are transferred to NAD , forming NADH and a hydrogen ion. </li></ul>
  10. 11. <ul><li>2 – NADH Dehydrogenase :- </li></ul><ul><li>NADH transfers the free proton and the hydride ion to NADH dehydrogenase --- complex I. </li></ul><ul><li>FMN is already present in this complex. It receives two hydrogen atoms and becomes FMNH 2. </li></ul>
  11. 12. <ul><li>3- Coenzyme Q :- </li></ul><ul><li>It is a quinone derivative. Also called ubiquinone. </li></ul><ul><li>It can accept hydrogen atoms from two sources --- FMNH 2 ( complex I ) and from FADH 2 , produced on succinate dehydrogenase ( complex II ). </li></ul><ul><li>CoQ links flavoporoteins to cytochromes. </li></ul>
  12. 13. <ul><li>4- Cytochromes :- </li></ul><ul><li>Each cytochrome contains a heme group made of a porphyrin ring with an iron atom. </li></ul><ul><li>The iron in cytochromes is converted from its ferric- Fe 3 to ferrous-Fe 2 form and functions as a reversible carrier of electrons. </li></ul><ul><li>The electrons are passed along the chain from coQ to cytochromes b and c1 ( complex III ), c and a + a 3 ( complex IV ). </li></ul>
  13. 14. <ul><li>5- Cytochrome a + a 3 :- </li></ul><ul><li>Also called cytochrome oxidase. </li></ul><ul><li>At this site, transported electrons, molecular oxygen and free protons come together to produce water. </li></ul>
  14. 15. Release of free energy during ETC:- <ul><li>Free energy is released during the transfer of electrons along the electron transport chain. </li></ul><ul><li>Coupled oxidation – reduction reactions --- Redox pairs e.g NADH -> NAD and FMN-> FMNH 2 . </li></ul><ul><li>Redox pairs differ in their tendency to lose electrons. </li></ul><ul><li>The transport of a pair of electrons from NADH to oxygen releases 52.58 calories--- used mainly for ATP formation. </li></ul>
  15. 17. <ul><li>Coenzyme Q (CoQ, Q or ubiquinone) is lipid-soluble. It dissolves in the hydrocarbon core of a membrane. </li></ul><ul><li>the only electron carrier not bound to a protein. </li></ul><ul><li>it can accept/donate 1 or 2 e - . </li></ul>
  16. 18. <ul><li>Cytochromes are electron carriers containing heme . Hemes in the 3 classes of cytochrome ( a , b , c ) differ in substituents on the porphyrin ring. </li></ul><ul><li>Some cytochromes(b,c1,a,a3) are part of large integral membrane protein complexes . </li></ul><ul><li>Cytochrome c is a small, water-soluble protein. </li></ul>Cytochromes
  17. 19. <ul><li>The heme iron can undergo 1 e - transition between ferric and ferrous states: Fe 3+ + e -  Fe 2+ </li></ul><ul><li>Copper ions besides two heme groups ( a and a 3 ) act as electron carriers in Cyt a,a3 </li></ul><ul><li>Cu 2+ +e -  Cu + </li></ul>Heme is a prosthetic group of cytochromes . Heme contains an iron atom in a porphyrin ring system.
  18. 20. <ul><li>NAD + , flavins and Q carry electrons and H + </li></ul><ul><li>Cytochromes and non-haem iron proteins carry only electrons </li></ul><ul><li>NAD +, FAD undergo only a 2 e - reaction; </li></ul><ul><li>cytochromes undergo only 1e - reactions </li></ul><ul><li>FMN, Q undergo 1e - and 2 e - reaction </li></ul>Electron carriers
  19. 21. ETC ---- REVIEW <ul><li>a series of highly organized oxidation-reduction enzymes </li></ul><ul><li>Final common pathway in aerobic cells to transfer electrons to oxygen. </li></ul><ul><li>Inner mitochondrial mem. </li></ul><ul><li>Four complexes/ components </li></ul>
  20. 22. <ul><li>Complex I – NAD + CoQ Reductase </li></ul><ul><li>Transfer of two electrons from NADH to CoQ via FMN, coverting it into FMNH 2. </li></ul><ul><li>- FMNH2 transfers electons to Fes proteins which transfer electrons to CoQ. </li></ul><ul><li>- CoQ accepts electrons from FMNH 2 </li></ul><ul><li>( complex I ) and from FADH 2 ( complex II ) . </li></ul><ul><li>CoQ is the link between flavoproteins and cytochromes. Pumping of protons. </li></ul>
  21. 23. <ul><li>Complex II – Succinate + CoQ Reductase </li></ul><ul><li>transfer of electrons from Succinate to CoQ via FADH 2 . Succinate is converted to Fumerate during this. </li></ul><ul><li>Complex III- CoQ + Cyto. C reductase </li></ul><ul><li>Transfer of electrons from CoQH 2 to Cyt.c via Cyt.b & Cyt. c1. Fe+++ of cyt c1accepts electrons and forms Fe++ . Complex is also a proton pump. </li></ul>
  22. 24. <ul><li>Complex IV ---- Cyt.c reductase </li></ul><ul><li>Transfer of electrons from cyt c to molecular oxygen via Cyt.a , Cu++ , and Cyt.a 3 . Water is formed as a result. </li></ul><ul><li>This complex also acts as proton pump. </li></ul>
  23. 26. Electron Carriers 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
  24. 27. Inhibitors of ETC <ul><li>Site I--- complex I – Rotenone and Amytal. Inhibit electron transfer from FMN to Fes. Rotenone is used as fish poison. </li></ul><ul><li>Site II – complex III – Antimycin A, blocks electron transfer from cyt b to Fes and then cyt c1. </li></ul><ul><li>Site III – complex IV- cyanide, CO, sodium azide. Block between cyt a+a3 and oxygen. </li></ul>
  25. 28. <ul><li>When the chain is blocked, electron carriers will be in a reduced state before the block point and in an oxidized state after it. </li></ul><ul><li>This blockage eventually causes inhibition of ATP synthesis. </li></ul>
  26. 29. H + Transport <ul><li>Complex I, III , IV drive H + transport from matrix to the cytosol When e - flow through , which creates p roton gradient ( electrochemical potential) across the inner membrane </li></ul><ul><li>Complex I and Complex IV : The mechanism of H + transport is still not known. </li></ul><ul><li>The mechanism of H + transport in Complex III is Q cycle. </li></ul>
  27. 30. <ul><li>4H + are pumped per 2e  passing through complex III. </li></ul><ul><li>The H + /e  ratio is less certain for the other complexes: probably 4H + /2e  for complex I; 2H + /2e  for complex IV. </li></ul>
  28. 31. <ul><li>Q Cycle :The mechanism of H + transport in Complex III </li></ul>
  29. 32. <ul><ul><li>Electrons are transported along the inner mitochondrial membrane, through a series of electron carriers </li></ul></ul><ul><ul><li>Protons (indicated by + charge) are translocated across the membrane, from the matrix to the intermembrane space </li></ul></ul><ul><ul><li>Oxygen is the terminal electron acceptor , combining with electrons and H + ions to produce water </li></ul></ul><ul><ul><li>4. As NADH delivers more H + and electrons into the ETS, the proton gradient increases , with H + building up outside the inner mitochondrial membrane, and OH - inside the membrane. </li></ul></ul>
  30. 33. Release of free energy during ETC <ul><li>Flow of electrons is accompanied by release of free energy . </li></ul><ul><li>The electrons can be transferred as hydride ions to NAD, as hydrogen atoms to FMN, CoQ, and FAD or as electrons to cytochromes. </li></ul><ul><li>Tendency to lose electrons can be quantitatively specified by a constant Eo– standard reduction potential. </li></ul>
  31. 34. Reduction Potentials Number of electrons transferred in the redox reaction Faraday’s constant (96485 J/volt/mole) Crucial equation:   G o ' = -n F  E o ' The relative tendency to accept e - s and become reduced.  = E o '(acceptor) - E o '(donor) E 0 ’=standard reduction potential. If  E o ' is positive, an electron transfer reaction is spontaneous (  G o ' <0)
  32. 35. OXIDATIVE PHOSPHORYLATION
  33. 36. <ul><li>In oxidative phosphorylation, ATP is produced by the combination of ADP and Pi. Energy is obtained from the flow of electrons from NADH to molecular Oxygen during . </li></ul><ul><li>FREE ENERGY CHANGES AND SITES OF ATP FORMATION : In inner mitochondrial membrane. Three sites:- </li></ul><ul><li>- complex I - complex III - complex IV </li></ul>
  34. 37. Chemiosmotic hypothesis of ATP synthesis <ul><li>Complex I,III and IV are proton pumps. Free energy of oxidation of components is coupled to the translocation of H+ from inside to outside of inner mitochondrial membrane. </li></ul><ul><li>Accumulation of protons at this site ----- electrochemical potential will drive the synthesis of ATP by activation of ATP synthase. </li></ul>
  35. 38. ATP synthase <ul><li>Complex V </li></ul><ul><li>Composed of two domains --- F1 and Fo </li></ul><ul><li>Fo domain ---- spans the inner mitochondrial membrane and serves as a channel through which protons reenter into mitochondrial matrix. </li></ul><ul><li>F1 ----- extra membranous part, projects into mitochondrial matrix. </li></ul>
  36. 39. <ul><li>The protons accumulated on the cytosolic side of inner mitochondrial membrane re-enter the mitochondrail matrix through Fo. This causes rotation of Fo. </li></ul><ul><li>Rotation of Fo results in confirmational changes in F1 and result is the activation of catalytic activity of ATP synthase. </li></ul><ul><li>ADP and Pi then combine to form ATP. </li></ul>
  37. 40. Inhibition of Oxidative Phosphorylation <ul><li>OLIGOMYCIN ------ binds with Fo domain, </li></ul><ul><li>- blocks H+ channels, no reentery of protons in mito. matrix. No oxid phospho. </li></ul><ul><li>ETC also stops due to accumulation of protons </li></ul><ul><li>Respiratory control ---- phosphorylation of ADP to ATP is essential for cellular respiration. Decreased level of ADP and Pi also decrease ATP synthesis. </li></ul>
  38. 41. Uncouplers of oxidative phosphorylation <ul><li>These compounds uncouple ETC and Oxidative phosphorylation. </li></ul><ul><li>They increase permebility of inner mito mem to protons. 2,4 dinitrophenol is a classic example. It causes ETC to proceed at a rapid rate without forming a proton gradient. Energy is released as heat, not used for ATP. High doses of Aspirin also act as uncoupler. </li></ul><ul><li>Natural uncoupler --- brown fat. </li></ul>
  39. 42. Transport of ADP and ATP <ul><li>Adenine nucleotide carrier --- transport of one molecule of ADP from cytosol into mito and exports one ATP from mito into cytosol. </li></ul><ul><li>Inhibited by plant toxin atractyloside. </li></ul>
  40. 43. Transport of reducing equivalents <ul><li>NADH produced in cytosol cannot enter mito. </li></ul><ul><li>Two electrons of NADH called reducing equivalents, enter mito using shuttle mechanism. </li></ul>
  41. 44. CYTOPLASM OUTER MEMBRANE MATRIX INNER MEMBRANE Figure 3. The malate-aspartate shuttle. OAA Malate (1) e - NAD + e - Glu 0 (6) Glu 0 Asp -1 (4) KG KG Malate (2) e - e - OAA NADH NAD + (3) e - Complex I e - NAD + Glucose Pyruvate GLYCOLYSIS NADH Asp -1 (5)
  42. 45. CYTOPLASM INNER MEMBRANE MATRIX FAD Glycerol-3-phosphate dehydrogenase (2) DHAP OUTER MEMBRANE Figure 4. Glycerol phosphate shuttle. Cytoplasmic glycerol 3-phosphate dehydrogenase (1) oxidizes NADH. Glycerol 3-phosphate dehydrogenase in the inner membrane (2) reduces FAD to FADH 2 . G3P Dihydroxyacetone phosphate (DHAP) NAD + 3-phosphate Glycerol e  (1) FADH 2 e  CoQ e  O 2 e  NADH Glucose Pyruvate GLYCOLYSIS NAD +
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