Cellular Pathways

1. Cellular Pathways • Metabolic pathways occur in small steps, each catalyzed by a specific enzyme. • Metabolic pathways are often compartmentalized and are highly regulated.

2. Cellular Respiration Overview When glucose burns, energy is released as heat and light: C6H12O6 + 6 O2 6 CO2 + 6 H20 + energy The same equation applies to the metabolism of glucose by cells, but the reaction is accomplished in many separate steps so that the energy can be captured as ATP. Obtaining Energy and Electrons from Glucose

3. Energy Overview

4. C6H12O6 + 6O2 ---------> 6CO2 + 6H2O + energy via heat and ATP

5. O atoms draw shared electron pairs to themselves (oxidation of C). • Do the electrons get more/less stable? • Exergonic/endogonic?

6. C6H12O6 + 6CO2 6CO2 + 6H2O + ATP • Breaking the bonds between the six C atoms of glucose, results in 6 CO2 molecules. • Moving hydrogen atom electrons from glucose to oxygen, forms 6 H2O molecules. • As much of the free energy released in the process as possible is trapped as ATP.

7. Obtaining Energy and Electrons from Glucose • As a material is oxidized, the electrons it loses transfer to another material, which is thereby reduced. • Such redox reactions transfer a lot of energy. Much of the energy liberated by the oxidation of the reducing agent is captured in the reduction of the oxidizing agent.

8. Redox Reactions

9. The ultimate goal of cellular respiration is to capture as much of the available free energy in the form of ATP. This goal is accomplished through 2 different energy-transfer mechanisms: 1.substrate-level phosphorylation 2.oxidative phosphorylation

10. 1. Substrate-Level Phosphorylation (energy invested) • ATP is formed directly • a phosphate-containing compound transfers a PO4- directly to ADP, forming ATP • 30.5 kJ/mol of potential energy is transferred. (translation: sticking on a phosphate, which can later be used to make ATP from ADP)

11. 2. Oxidative Phosphorylation • ATP is formed indirectly • involves sequential redox reactions, with O being the final electron acceptor

12. Energy Transfer Oxidative Phosphorylation • Involves sequential redox reactions. • O is the final electron acceptor. • NAD+ removes 2H from glucose and is reduced to NADH • FAD is reduced to FADH2 • FADH2 and NADH move free energy from one place to another. Subtrate-level Phosphorylation • ATP is formed directly in an enzyme-catalyzed reaction. • Phosphate containing compound transfers a phosphate group directly to ADP forming ATP. • 31KJ/Mol is transferred • 50KJ/Mol in living cells

13. Energy Carriers • NAD+ and FAD+ are low energy, oxidized coenzymes that act as electron acceptors. • When an electron(s) is added to these molecules, they become reduced to NADH and FADH2. • In this case, reducing a molecule gives it more energy.

14. The coenzyme NAD is a key electron carrier in biological redox reactions. It exists in two forms, one oxidized (NAD+) and the other reduced (NADH + H+).

15. Highly folded Smooth Fluid-filled intermembrane space Folds of the inner membrane Protein-rich liquid Mitochondria • Specialize in the production of ATP. • Only in eukaryotic cells. • Double membrane – smooth outer layer and folded inner membrane (cristae). • Matrix is protein rich and fills the innermost space of the mitochondria. • Intermembrane space (between the 2 membranes) • Contain their own DNA (mtDNA) which lead to the endosymbiosis hypothesis.

16. The Big Picture • The entire process occurs in 4 stages and in 3 different places within the cell: • Stage 1: Glycolysis - a 10 steps occurring in the cytoplasm • Stage 2: Pyruvate Oxidation - 1 step occuring in the mito. matrix • Stage 3:Krebs cycle - 8 steps occuring in the mito. matrix • Stage 4:ETC and chemiosmosis - many steps occuring in the mito. cristae

17. Overview

18. Energy Pathways

19. Glycolysis • There are 2 main phases of glycolysis:Glycolysis I - activation phase, which uses ATP molecules • Glycolysis II - oxidative and phosphorylation reactions, which not only reduce glucose to pyruvate but also produce ATP molecules • C6H12O6 + 2ADP + 2Pi + 2 NAD+ -----> 2 pyruvate + 2ATP + 2(NADH + H+)

20. Stage 1: Glycolysis • Occurs in cytoplasm • Anerobic and does not require oxygen • Glucose is split into two 3-C molecules called pyruvate (pyruvic acid). • Transfers only 2.2% of free energy available in 1 mol of glucose to ATP.

22. Glycolysis: From Glucose to Pyruvate • O is not a strong enough oxidizer to strip electrons from C-H bonds in glucose at room or body temperature.Enzymes required. • Glycolysis is a pathway of ten enzyme-catalyzed reactions located in the cytoplasm. It provides starting materials for both cellular respiration and fermentation.

23. Glycolysis: From Glucose to Pyruvate • The energy-investing reactions of glycolysis use two ATPs per glucose molecule and eventually yield two glyceraldehyde 3-phosphate molecules. In the energy-harvesting reactions, two NADH molecules are produced, and four ATP molecules are generated by substrate-level phosphorylation. Two pyruvates are produced for each glucose molecule. Review

24. Glycolysis - Energy In

25. Glycolysis - Energy Out

26. Glycolysis - Energy Out So… How much in? 2 ATP, a glucose and 2 NAD+ How much out? 4ATP – 2ATP = 2ATP 2NADH 2 Pyruvate molecules (and a couple of water to boot!)

27. GLYCOLYSIS PRODUCTS • 2 ATPs are used in steps 1 & 3 to prepare glucose for splitting. • F 1,6-BP splits into DHAP and G3P. • DHAP converts to G3P. • 2 NADH are formed in step 6. • 2 ATP are formed by substrate-level phosphorylation in both steps 7 and 10. • 2 pyruvates are produced in step 10.

28. Pyruvate Oxidation When O2 is available (aerobic respiration)

29. A multi-enzyme complex catalyzes the following three changes: • 1.the carboxyl group of pyruvate is removed as a CO2 molecule. This is a decarboxylation reaction catalyzed by the enzyme pyruvate decarboxylase. • 2.The 2-C fragment is oxidized to form an acetate ion. Electrons from this reaction are picked up by NAD+ which is reduced to form NADH + H+ • 3.The acetyl group of the acetate ion is transferred to coenzyme A, forming acetyl CoA

30. Pyruvate Oxidation • Occurs in matrix of mitochondria. • 2 pyruvate + 2NAD+ + 2 CoA  2 acetyl-CoA + 2NADH + 2H+ + 2CO2

31. 2 pyruvate + 2 NAD+ + 2 CoA ------> 2 acetyl- CoA + 2 NADH + 2 H+ + 2 CO2

32. Krebs Cycle • 8 steps that oxidize acetyl-CoA to CO2 and H2O, forming a molecule of ATP. In addition, the cycle removes electrons, which are carried by 3 NADH and 1 FADH2 molecules to the ETC. The following is the overall chemical equation: oxaloacetate + acetyl-CoA + ADP + Pi + 3NAD+ + FAD ---> CoA + ATP + 3 NADH + 3H+ + FADH2 + 2CO2+ oxaloacetate

34. Production of Citrate • The energy in acetyl CoA drives the reaction of acetate with oxaloacetate to produce citrate. The citric acid cycle is a series of reactions in which citrate is oxidized and oxaloacetate regenerated. It produces two CO2 , one FADH2, three NADH, and one ATP for each acetyl CoA.

35. Occurs twice for each molecule of glucose, 1 for each acetyl-CoA. Animation: How the Krebs Cycle Works (Quiz 1)

36. What’s going on?? • In step 1, acetyl-CoA combines with oxaloacetate to form citrate. • In step 2, citrate is rearranged to isocitrate. • NAD+ is reduced to NADH in steps 3, 4 and 8. • FAD is reduced to FADH2 in step 6. • ATP if formed in step 5 by substrate-level phosphorylation. The phosphate group from succinyl-CoA is transferred to GDP, forming GTP, which then forms ATP. • In step 8, oxaloacetate is formed from malate, which is used as a reactant in step 1. • CO2 is released in steps 3 and 4.

37. Complete Overview respiration

38. The Electron Transport Chain • NADH and FADH2 molecules derived from glycolysis and the Kreb’s Cycle each contain electrons they gained from their formation • NADH (or FADH2) molecules carry these electrons to the innermembrane of the mitochondrion and electron transfer begins*NAD+ and FAD = electron carriers – takes H’s (and their e-) to electron transport chain!

39. REDOX • ÙThese electrons are passed through a series of electron carriers, one step at a time. As the electrons are passed along, one substance is oxidized, the other is reduced. (REDOX rxns) *What is the “driving force”? • ÙAt each step some energy is released. • ÙThis energy is used in certain places in the chain to pumpprotons (H+) from the matrix out into the inner membrane space.

40. ETC

41. Electron Transport Chain (ETC) • A series of electron acceptors (proteins) are embedded in the cristae. • These proteins are arranged in order of increasing electronegativity. • The weakest attractor of electrons (NADH dehydrogenase) is at the start of the chain and the strongest (cytochrome oxidase) is at the end.

42. These proteins pass electrons from NADH and FADH2 to one another through a series of redox reactions. • ETC protein complexes are alternately reduced and oxidized as they accept and donate electrons.

43. Energy Pumping • As the electrons pass from one molecule to the next, it occupies a more stable position. • The free energy released is used to pump protons (H+) to the intermembrane space. • 3 for every NADH and 2 for every FADH2. • This creates an electrochemical gradient, creating potential difference (voltage) similar to a battery.

44. ETC

45. NADH and FADH2 transfer the electrons they got from glucose to the ETC. • The electrons move through a series of redox reactions. Energy is released and used to pump protons (H+ ions) from the matrix into the intermembrane space. • At the end of the chain, the electrons are so stable that only a oxygen is strong enough to oxidize the last protein complex. • Oxygen strips 2 electrons from the final protein complex and forms water with 2 protons from the matrix.

46. Electron Transport Chain

48. ATP Synthase • Protons enter the matrix through proton channels associated with ATP synthase (ATPase). • For every H+ that passes through, enough free energy is released to create 1 ATP from the phosphorylation of ADP. • Conditions must be aerobic because oxygen acts as the final electron and H+ acceptor (water is formed as a byproduct).

49. Oxidative phosphorylation

50. Chemiosmosis