2. 2.2 Cellular Respiration: The Details ► Goals;  Break the bonds b/w the 6 C atoms of glucose, resulting in 6 CO 2 molecules  Move H atom electrons from glucose to oxygen, forming 6 water molecules  Trap as much of the free energy released in the process as possible in the form of ATP

3. Overview of cellular respiration ► 4 metabolic stages  Anaerobic respiration 1. Glycolysis (substrate level phosphorylation)  respiration without O 2  in cytosol  Aerobic respiration  respiration using O 2  in mitochondria 2. Pyruvate oxidation 3. Krebs cycle 4. Electron transport chain and chemiosmosis (oxidative phosphorylation) C 6 H 12 O 6 6O 2 ATP6H 2 O6CO 2  +++ (+ heat )

4. Energy Transfer ► How is the chemical potential energy in glucose transformed into ATP? 1. Substrate-Level Phosphorylation 2. Oxidative Phosphorylation

5. Substrate-Level Phosphorylation ► A molecule containing a phosphate transfers it to ADP (with the aid of enzymes), forming ATP. 31kJ/mol of free energy is also transferred. ► For each glucose molecule, 4 ATP are made this way in glycolysis (stage 1) and 2 ATP in the Kreb’s cycle (stage 3)

7. Oxidative Phosphorylation ► ATP is formed indirectly through a series of enzyme-catalyzed redox reactions involving oxygen as the final electron acceptor ► It begins when the compound NAD+ (nicotinamide adenine dinucleotide), which is a coenzyme, removes 2 H atoms (2 protons and 2 electrons) from glucose. ► 2 electrons and 1 proton attach and reduce NAD+ to NADH, while the left over proton dissolves in surrounding solution H+ (aq)

8. Oxidative Phosphorylation ► NADH is formed during glycolysis, pyruvate oxidation and 3 times in the Kreb’s cycle ► A dehydrogenase enzyme catalyzes this rxn. ► FAD (flavin adenine dinucleotide) also acts like NAD+ and is reduced by 2 H atoms from glucose to form FADH 2 (occurs in Kreb’s cycle) ► These reductions are both energy harvesting rxns that will later transfer most of their free energy to ATP

9. Oxidative Phosphorylation ► These co-enzymes (reduced FADH 2 and NADH) function as energy carriers ► So, how does the free energy get transferred to ATP? It occurs in Stage 4 (electron transport and chemiosmosis) and requires oxygen…discussed later!

11. Aerobic respiration happens in 4 stages: Stage 1 – Glycolysis (10 step process occurring in cytoplasm) glyco lysis glucose splitting

12. ► Stage 2-Pyruvate oxidation-1 step process occurring in the mitochondrial matrix ► Stage 3-Kreb’s Cycle (citric acid cycle) 8 step cyclical process occurring in the mitochondrial matrix ► Stage 4 Electron Transport and chemiosmosis (oxidative phosphorylation) multi step process occurring in the inner mitochondrial membrane (cristae)

14. intermembrane space inner membrane outer membrane matrix cristae Mitochondria — Structure ► Double membrane energy harvesting organelle  smooth outer membrane  highly folded inner membrane ► cristae  intermembrane space ► fluid-filled space between membranes  matrix ► inner fluid-filled space  DNA, ribosomes  enzymes ► free in matrix & membrane-bound mitochondrial DNA What cells would have a lot of mitochondria?

16. In glycolysis, a glucose molecule (6 carbon) is broken down into two 3-carbon pyruvate (pyruvic acid) molecules. energy released to make small quantity of ATP (2 molecules) series of enzyme controlled reactions pyruvic acid glucose Glycolysis does not require oxygen

17. -ate or acid??? ► -ate replaces the word acid in organic acids to indicate the ionized form of the acid ► E.g. pyruvic acid-pyruvate ► E.g. aspartic acid-aspartate

18. Fig 11 (p.98) ► The overall chemical equation for glycolysis glucose + 2ADP + 2Pi + 2NAD+ 2 pyruvate + 2ATP + 2(NADH + H+) ► The energy yield for glycolysis 4 ATP produced 2 ATP used 2 ATP produced net (can be used immediately) 2 NADH produced (used later to obtain more ATP)

21. Glycolysis ► Alone, this process is not efficient in transferring energy from glucose (only 2.2%) ► Some is lost as heat but most of the energy is trapped in the pyruvate and NADH ► Glycolysis is thought to be the earliest form of energy metabolism. ► Video -http://www.youtube.com/watch?v=x- stLxqPt6E Video ► Song- http://www.youtube.com/watch?v=6JGXayUyNVw Song

22. Your turn… ► Read p.94-100 and note sheets ► Answer Q 1-10 on p.115 ► Fill out glycolytic Pathway ► Quiz on Wednesday, October 13th (all 10 steps with enzymes)

23. Stage 2 – Pyruvate oxidation ► The pyruvic acid made in glycolysis (stage1) still contains a lot of energy and are transported through the mitochondrial membranes into the matrix ► A multi-enzyme complex catalyzes 3 changes

24. Pyruvate oxidation 1. A carboxyl group is removed as CO 2 (a decarboxylation rxn using the enzyme pyruvate decarboxylase) 2. NAD + is reduced by 2 H atoms (food) to form NADH. The NAD + oxidizes the 2-C portion and becomes acetic acid. This is a redox rxn as pyruvate is oxidized and NAD + is reduced 3. Coenzyme A (contains S) is attached to the remaining acetic acid portion to form acetyl-CoA in an unstable bond (sets it up for stage 3)

25. Pyruvate oxidation equation 2 pyruvate + 2 NAD + + 2 CoA 2 acetyl-CoA + 2 NADH + 2 H + + 2 CO 2 Where do the products go? ► Acetyl-CoA move to stage 3-Krebs Cycle ► NADH move to stage 4-Electron Transport/Chemiosmosis (produce ATP by oxidative phosphorylation) ► CO 2 exits as waste ► H + remain dissolved in the matrix

26. What exactly is Acetyl CoA? ► It is multifunctional; If the body needs energy it moves into the Kreb’s cycle, if not it produces lipids (energy storing) ► Many nutrients catabolized for energy are converted to acetyl-CoA and then channeled toward fat or ATP production-depending on energy needs.

28. Anaerobic Respiration (in animals) anaerobic = in the absence of oxygen

29. In low oxygen conditions or during heavy exercise, when not enough oxygen can be supplied, muscle cells swap to anaerobic respiration

30. glycolysis still happens as it does not require oxygen in absence of oxygen pyruvic acid is turned into lactic acid. pyruvic acid lactic acid glucose 2 ADP + 2 Pi 2 ATP

31. A build up of lactic acid produces muscle fatigue. Muscle fatigue makes muscles ache and contract less powerfully. A recovery period is needed. During this time more oxygen is taken in to convert the lactic acid back into pyruvic acid again. The volume of oxygen needed is called the oxygen debt.

32. Summary oxygen debt e.g. during hard exercise oxygen debt repaid during recovery time glucose pyruvic acid lactic acid

33. Anaerobic Respiration in plants The same process occurs in plants and yeast in low oxygen conditions, e.g. muddy, flooded soils.

34. glycolysis still happens, producing 2 ATP molecules This time in absence of oxygen, pyruvic acid is turned into carbon dioxide and ethanol glucose pyruvic acid ethanol + carbon dioxide This is irreversible 2 ADP + 2 Pi 2 ATP

35. Comparison of aerobic and anaerobic respiration Aerobic respiration Anaerobic Respiration in animals in plants and yeast Oxygen required? yesnono Glycolysis occurs yesyesyes ATP yield 38ATP2ATP2ATP Glucose completely broke down? yesnono End products Carbon dioxide and water Lactic acid Ethanol and carbon dioxide

36. Fermentation (anaerobic) ► Bacteria, yeast 1C 3C2C pyruvate  ethanol + CO 2 ► Animals, some fungi pyruvate  lactic acid 3C ► beer, wine, bread ► cheese, anaerobic exercise (no O 2 ) NADHNAD + NADHNAD + back to glycolysis 

37. recycle NADH Alcohol Fermentation 1C 3C2C pyruvate  ethanol + CO 2 NADHNAD +  Dead end process  at ~12% ethanol, kills yeast  can’t reverse the reaction bacteria yeast back to glycolysis 

38. recycle NADH  Reversible process  once O 2 is available, lactate is converted back to pyruvate by the liver Lactic Acid Fermentation pyruvate  lactic acid 3C NADHNAD +  O2O2 animals some fungi back to glycolysis 

39. Pyruvate is a branching point Pyruvate O2O2 O2O2 mitochondria Krebs cycle aerobic respiration fermentation anaerobic respiration

40. Stage 3: Kreb’s Cycle ► 8-step process catalyzed by enzymes ► Considered cyclic b/c oxaloacetate (the product of step 8) is the reactant in step 1 ► Cycles through twice for every glucose molecule as there are 2 molecules of acetyl CoA. ► Equation; Oxaloacetate + acetyl-CoA +ADP +Pi +3 NAD + + FAD CoA + ATP + 3 NADH + 3H + + FADH 2 + 2 CO 2 + oxaloacetate

41. The Krebs Cycle ► Occurs in the matrix of the mitochondrion. Transfers energy from organic molecules to ATP, NADH, FADH 2 and removes C atoms as CO 2 ► Aerobic phase (requires oxygen). By the end of the Kreb’s Cylce the original glucose molecule is entirely consumed Steps 1. 2-carbon acetyl CoA joins with a 4-carbon compound (oxaloacetate) to form a 6- carbon compound called Citrate. CoA is released (recycled)

43. The Krebs Cycle Steps 2. Citrate (6-C) is re-arranged to isocitrate (6-C) 3. Isocitrate is converted to alpha- ketoglutarate (5-C) by losing a CO 2 and 2 H atoms that reduce NAD + to NADH

45. The Krebs Cycle Steps 4. Alpha-ketoglutarate (5-C) is converted into succinyl CoA (4-C). One CO 2 is removed, coenzyme A is added and 2 H atoms reduce NAD + to NADH. 5. Succinyl CoA (4-C) is converted to succinate (4-C). ATP is formed by substrate level phosphorylation and coenzyme A is released

47. The Krebs Cycle Steps 6. Succinate (4-C) is converted to fumarate (4-C). Two H reduce FAD to FADH 2 7. Fumarate (4-C) is converted to malate (4- C). This is a hydrolysis rxn 8. Malate (4-C) is converted to oxaloacetate (4-C). Two H reduce NAD + to NADH

49. Highlights ► Video- http://www.youtube.com/watch?v=XVWdeK oiEOc Video ► Energy is harvested in steps 3 (NADH), 4 (NADH), 5 (ATP-substrate-level phosphorylation), 6 (FADH 2 ), 8 (NADH) ► The last 4 C atoms of the original glucose leave as CO 2 (waste)

50. Grand Total so far… ► Glycolysis (2 ATP, 2 NADH) ► Pyruvate oxidation (2 NADH, 2 CO 2 ) ► Kreb’s Cycle (after 2 cycles)  6 NADH  2 FADH 2  2 ATP  4 CO 2 ► The 12 reduced coenzymes (energy carriers) will eventually be transferred to ATP in stage 4

51. Where did all the carbons go? ► 6-C (glucose) at the end of glycolysis is transformed into two 3-C pyruvate ► After pyruvate oxidation you are left with two CO 2 and two acetyl CoA (2 carbons each) ► Once the Kreb’s Cycle is completed you lose the last four original carbons as CO 2

52. Cellular Respiration Stage 4 ETC and Chemiosmosis ► Occurs in the inner mitochondrial membrane ► Requires oxygen (final acceptor of electrons in the ETC) without oxygen; Kreb’s, ETC and chemiosmosis stop. ► Energy carriers (NADH and FADH 2 ) transfer the H atom electrons to a series of compounds (mostly proteins) in the Electron Transport Chain (ETC)

53. ETC

54. Brief Overview ► Electrons move through a series of redox reactions that release the free energy used to pump protons into the intermembrane space (this creates an electrochemical gradient-source of free energy)

55. Brief Overview ► During chemiosmosis, protons move through ATPase complexes (within the membrane) releasing free energy (drives the synthesis of ATP)

56. Videos ► Video-ETC Video ► http://www.youtube.com/watch?v=xbJ0nbzt5Kw http://www.youtube.com/watch?v=xbJ0nbzt5Kw ► Video-Chemiosmosis Video ► http://www.youtube.com/watch?v=3y1dO4nNaKY http://www.youtube.com/watch?v=3y1dO4nNaKY ► Video-Oxidative Phosphorylation Video ► http://www.youtube.com/watch?v=Idy2XAlZIVA

57. ETC ► The ETC is arranged in increasing electronegativity (weakest attractor of electrons at start to strongest at the end.) ► Each component is reduced (gaining 2 electrons from the component before) and oxidized (losing 2 electrons from the component after.) ► Electrons are shuttled through like a baton from start to finish ► As they move they become more stable as they get closer to the nuclei of the atoms they associate with.

58. ETC ► Free energy is used to pump out H+ protons into the intermembrane space ► At the last component of the ETC, oxygen (highly electronegative) accepts (strips) the last 2 electrons and together with 2 protons from the matrix, forms water

59. Actual components of ETC ► NADH dehydrogenase, ubiquinone (Q), cytochrome b-c 1 complex, cytochrome c, cytochrome oxidase complex. ► The ETC is highly exergonic ► The free energy lost by the electron pair during transport is used to pump out H +.

62. Energy converted ► Chemical potential energy of electron position is converted to electrochemical potential energy of a proton gradient (accumulation of charged protons). ► FADH 2 passes on their electrons to complex Q (Ubiqinone) ► 2 ATP are formed for every FADH 2 ► 3 ATP are formed for every NADH

63. Chemiosmosis and Oxidative Synthesis ► The electrochemical gradient (of H+) stores free energy ► Creates a potential difference (voltage), like that of a battery ► B/c they cannot pass through the phospholipids bilayer, they must pass through proton channels associated with ATP-synthase.

64. Chemiosmosis and Oxidative Synthesis ► The free energy stored in the gradient produces a PMF (proton-motive force) that moves protons through ATPase complex. ► Free energy in gradient is reduced and is used to create ATP from (ADP and Pi) ► It is called chemiosmosis b/c the energy that drives the synthesis of ATP comes from the “osmosis” of protons through a membrane.

65. Chemiosmosis and Oxidative Synthesis ► Once created ATP move into cytoplasm through facilitated diffusion to do “work” (movement, cell division etc)

66. Cellular Respiration Summary ► Table 3 p.114

67. How much ATP is really made? ► Theoretically 36 is made but as the membrane is permeable (to protons) and some energy is used for other endergonic rxns, actual yield is about 30 ATP ► Efficiency of energy conversion is about 32%

68. Controlling Aerobic Respiration ► Phosphofructokinase (enzyme at step 3 of glycolysis) controls cellular respiration ► It is activated by ADP and citrate ► It is inhibited by ATP ► NADH inhibits pyruvate decarboxylase (enzyme that converts pyruvate to acetyl CoA).

69. Metabolic Rate ► Amount of energy consumed at a given time, and a measure of the overall rate at which the energy-yielding rxns of cellular respiration takes place. ► BMR (Basal Metabolic Rate) is the minimum amount of energy for survival (breathing, control temp.) Usually accounts for 60-70% of daily energy

70. Metabolic Rate ► BMR Calculator BMR Calculator BMR Calculator ► http://www.tlbc.ca/blog/index.php/bmr- calculator/ http://www.tlbc.ca/blog/index.php/bmr- calculator/ http://www.tlbc.ca/blog/index.php/bmr- calculator/ ► Varies with age, sex, health

71. Looking back… ► ATP is a high energy yielding nucleotide that biological systems need to power rxns (muscle contractions, cell division) ► Glucose is broken down during cellular respiration (covalent bonds are split) to provide energy for the synthesis of ATP. By-product is CO 2.

72. Looking back… ► The role of oxygen is to “grab” the excess H+ (don’t want the cell to be too acidic) forming water. ► Glucose is oxidized (into CO 2 ) and oxygen is reduced to H 2 O

73. Practice ► Worksheets ► P.115 Q12-18