Mitochondria and the Tricarboxylic Acid Cycle: Energy Production in Cells

1. Mitochondria and the TriCarboxylic Acid Cycle (TCA or Citric acid cycle) are central to energy production in eukaryotic cells! 10/15 and 17 • Overview of intracellular metabolism • What are the 4 Stages of aerobic energy production? • How are mitochondrial organized inside? • Steps in TCA: structures, enzymes, and energy compounds, and carbon dioxide. • The energy Balance sheet for oxidation of a glucose to carbon dioxide. 1 glucose  38(sometimes36)ATP + 6 CO2 • Fatty acids are oxidized by Beta Oxidation • Amino acids “can” become glucose, though the process is VERY expensive.

2. Glycolysis: goal create a net of 2 ATP + 2 pyruvate from glucose during hyper or euglycemia (use LDH only durign short term hypoxia).Gluconeogenesis: Goal reverse reactions if needed with pyruvate carboxylase, PEP kinase, F-1,6-biphosphatase ,and G-6-phosphatase and regenerate glucose for glucose dependent tissues during periods of hypoglycemia.

3. There are 4 steps in aerobic respiration: • Step 1: Glycolysis in cytosol • Glucose 2 Pyruvate+2 ATP+2 NADH+ 2 H+ • Step 2: Tricarboxylic Acid Cycle/Acetyl-CO: Occurs in mitochondrial matrix • Pyruvate oxidized to carbon dioxide • Priming: 2Pyruvate2Acetly-CoA + 2 CO2 • Cycle yields CO2, ATP, FADH and NADH • Step 3: Electron Transport and Proton Pump • Occurs on inner mitochondrial membrane • Energy in electrons pumpsH+ out of matrix! • Last Step: 4e- and 4H+ bind O2  2 H2O • Step 4: ATP synthesis on inner membrane • Let H+ run back though a ATP-synthase enzyme and phosphorylate ADP to ATP!

4. Mitochondria are central to the huge ATP yield that the tricarboxylic acid cycle makes possible for eukaryotic cells. • Organelle with its own DNA: • Location inside cell: • Mitochondrial density relative to tissue type: • Aerobic tissues: • Anaerobic tissues: • Why Light and Dark Turkey meat? • Liver and kidney? • Maternal Inheritance of mitochondria: • You got it all from your mamma! • Mitochondrial Parts: • Matrix, Crista, Inner/Outer membranes, Porins • Unique double membrane system • Bacteria are sort of complex mitochondria

5. What do mitochondria look like? Classic View: discrete structures (sausage-like) New View: interconnected structures floating in the cytosol

6. The DNA found in mitochondria is entirely different from the DNA found in the nucleus and indicates that mitochondria probably evolved from a type of bacteria, amino acid sequence of the ATP synthase is well conserved across eukaryotes and prokaryotes.

7. Tricarboxylic Acid Cycle (TCA/Krebs Cycle) is the CENTRAL HUB for oxidation and energy production from sugars, fatty acids, and some amino acids! TCA is called a “cycle” because the last step creates the substrate for the first step! Acetyl-CoA is main entry molecule! • GlucoseAcetyl-CoA • Fatty Acids Acetyl-CoA • Amino Acids  Some make Acetyl-CoA Some aa turned into Glucose (Acetyl-CoA) Complete Oxidation of one Acetyl-CoA Acetyl-CoAGTP+3NADH+FADH2+2CO2 • GTPATP • NADH 3 ATP • FADH2 2 ATP Acetyl-CoA1ATP+9ATP+2ATP+2 CO2

8. What are the key steps in TCA? Please work at recognizing compound names, structures, enzymes and locations of energy generation? • Overview: CH3-CO-S-CoA  2 CO2 • Entry Point: • Oxaloacetate merges with Acetyl-CoA • Citrate is formed (3 carboxyl groups; TCA) • Last Step in Cycle: • Oxaloacetate(OAA) re-created from malate • Starting point is recycled! • VVIP: Remember: removal of any one intermediate in the cycle prevents OAA regeneration! @Intermediates back up into glycolysis too!@

9. TCA occurs in the mitochondrial matrix and produces CO2, GTP, FADH2 and NADH. TCA REQUIRES continual production of OAA All Reactions X2 for the oxidation of one glucose

10. Energy Balance Sheet for complete oxidation of a single glucose molecule to carbon dioxide. • Glycolysis in Cytosol: (8 ATP) • 1 glucose  2 pyruvate + 2 ATP + 2 NADH • System feeds forward only if NAD+ is available • Aerobic and sometimes anaerobic • Pyruvate transported into matrix • Pyruvate Decarboxylase in Matrix: (6 ATP) 2pyruvate2 Acetyl-CoA+2 NADH + 2H+ and2 CO2 • TCA in Matrix: (24 ATP) • 2Acetyl-CoA4CO2+2 GTP+2FADH2+6NADH • Conversion to ATP2 ATP + 4 ATP + 18ATP • Net Yield: 1 glucose8+6+24=38 ATP (assumes oxygen ) • Sometimes a less efficient system is used to transport cytosolic NADH into the mitochondrial matrix such that these two NADHs yield only 4 ATP, not 6.

11. Remember that a negative Delta G can help drive a reaction with a positive value if there is “No Membrane In Between”. This is why Delta G from the reactions in the mitochondria cannot help to drive the reactions of glycolysis in the cytosol. Although molecules such as pyruvate can “carry” the energy between different compartments.

12. Glycolysis Compared to Kreb CycleRemove products immediately after they are produced! ∆G= ∆G’o + 2.303 RT log(Keq) Keq= [Products]/[Reactants What intracellular substrate/product concentrations are typically maintained?

13. Beta-oxidation: is an enzyme pathway that oxidizes fatty acids into acetyl-CoA molecules that feed directly into TCA which produces 3 NADH, 1 FADH2 and 1 ATP for each ACETYL-CoA! Fatty Acids:9 kcal/g Carbohydrates:4 kcal/g AminoAcids:4 kcal/g Triglycerides + LipaseGlycerol + 3 fatty acids C-16 palmitate8 acetyl-CoA(7cuts)Each acetyl-CoATCAX8 (8X3 NADH)+(8X1FADH2)+(8X1GTP)  72 ATP + 16 ATP + 8 ATP = 96 ATP PLUS: each time acetyl-CoA cut off the FA chain: 1NADH+1FADH2 created Additional Energy: (7XNADH)+(7XFADH2)(7X3)+ (7X2)= 35 ATP Total oxidation of palmitic acid to CO2= 96+35=131 (130)ATP Compare to the 38 ATP from a single glucose molecule! Fats create tons of ATP! Fat utilization REQUIRES mitochondria and oxygen!

14. What does the Beta Oxidation pathway look like for an 8-carbon fat that gets cut three times to make four acetyl-CoA molecules? Fatty Acid Oxidation A 16 carbon fatty acid yields 16/2= 8 acetyl-CoA Remember: each acetyl-CoA (acetate) is sent to TCA and becomes oxidized to carbon dioxide plus 3 NADH, 1 FADH2 and 1 GTP(ATP)…assuming mitochondria are functioning and oxygen is available.

15. If fat yields SO MUCH energy why don’t cells burn fat all the time? Problems: • Fatty acid solubility in blood is a problem for transport! • Fatty acids not always completely oxidized to carbon dioxide! • Beta-oxidation requires plenty of oxygen and mitochondria! Beta-oxidation creates toxins during a diabetic crisis: • 1) Acetone • 2) Acetoacetone • 3) Ketones • 4) Beta-hydroxybutyric acid • Why do we see ketoacidosis during a diabetic crisis? • Blood acidity reduces ability of hemoglobin to carry oxygen! • Ketones react readily with DNA (mutations) and proteins (denaturation)

16. Why do amino acids make a poor fuel for making ATP?Answer: It is really expensive AND potentially toxic! • 1) Use results in protein breakdown!! expensive • 2) Not all A.A. can feed into glucose or the TCA!! expensive • 3) Ammonia and urea are created by degradation!! toxic • 4) Ketones are created by accident!! Toxic • During Starvation: Proteolysis occurs in liver cells so glucose can be produced for the other cells that MUST use glucose, like glucose dependent red blood cells. • These are “gluconeogenic” amino acids! • Some amino acids “can” feed into TCA following transamination! • Alanine………Glutamate…………..Aspartate: pull off ammonia • Pyruvate…….α-ketoglutarate……..oxaloacetate • Problem: ammonia accumulates!

17. Co-Enzyme A attaches to acetate with a thioester (-C-S-) bond. Pantothenic acid is a vitamin required in our diets!

18. FlavineAdenineDinucleotide has a triple-ringed flavin with alternating double bonds that temporarily hold (stabilize) electrons.

19. The Trick: the nicotinamide functional group either has a charge (NAD+; oxidized;low energy) or has an extra electron and proton (reduced; high energy; NADH). The Nic-ring structure stabilizes the extra electron so the energy in the electron can be transferred between molecules. NADH electrons mostly used in the mitochondria (not cytosol). Great T.Q. Recognize FADH2 and NADH structures and high/low energy states

20. Contrast TCA/Citric acid cycle in the mitochondria to what happens in the cytosol. Remember That: 1 Glucose yields 2 Pyruvates

21. Summary: What does the electron transport pathway look like? NADH and FADH2 feed e- into system via complex I OR II of the inner mitochondrial membrane depending on how much energy they contain.

22. The Electron Transport System describes how cells use electron energy to create a proton gradient used for ATP synthesis! • THE FOUR DISCRETE ENZYME COMPLEXES • NADH dehydrogenase: Complex I- 23 peptides • Accepts e- from highest energy NADH and pumps proton out! • Succinatedehydrogenase:Complex II- 4-5 peptides • Accepts e- from lower energy FADH2 • Complex I and II Feed e- into CoEnzyme Q (“Ubiquinone”) • Co-E Q feeds e- into Cytochromebc: Complex III- 9 peptides • Pumps proton out and feeds e- into Complex IV! • CytochromeOxidase: Complex IV- 9 more peptides • Pumps proton out and sends e- to final acceptor: oxygen • Final Reaction: ½ O2 + 2 H+ + e-  H2O (water created)

23. What happens to the potential energy of the electrons as they move down the electron transport pathway to water? Where does the energy go relative to the proton pumps?

24. The ATP-synthase is the protein complex that synthesizes ATP!But Only If ADP, Pi, and proton gradient are in the matrix!This is the ChemiOsmotic Model for ATP Synthesis! • Three Parts to ATP-synthase: Stalk + Fo + F1 • Part I: Proton Translocator (Fo)-- A pore that is selective for just protons • Part II: ATP-synthase (F1)-- Collects the energy harvested as protons go down the concentration gradient and uses this energy to attach Pi to ADP. • Part III: Stalk -- Links the F1 and Fo parts together

25. ATP is synthesized in a 3 step processes that is driven by the passage of protons through the Fo-translocator.

26. What are some metabolic toxins and inhibitors that are of major biological significance to eukaryotes? • Rotenone: Complex I/CoQ • Cyanide and Carbon Monoxide: Complex IV • Oligomycin antibiotic: ADP phosphorylation • Alcohol and membrane permeability to H+ • Brown fat cells: uncoupler proteins and heat generation • Why might some antibiotics given for bacterial infections affect mitochondrial function in human cells?

27. Outer Mitochondrial Membrane: relatively non-specific porins!How do cytosolic metabolites get inside the matrix?Inner Mitochondrial Membrane: specific transport enzymes!