1. Electron Transport Chain/Respiratory Chain Proton gradient formed Four large protein complexes Mitochondria localized Energetically favorable electron flow

2. Mitochondrion Inner Membrane Respiration site Surface area for humans ca. 3 football fields Highly impermeable (no mitochondrial porins) Matrix and cytoplasmic sides

3. Standard Reduction Potentials

4. ΔG˚΄ = -n F Δ E˚΄ F = 96,480 J mol -1 V -1 Favorable Electron Flow: NADH to O 2 Net electron flow through electron transport chain: ½O 2 + 2H + + 2e - H 2 O ΔE˚΄ = + 0.82V NAD + + H + + 2e - NADH ΔE˚΄ = - 0.32V Subtracting reaction B from A: ½O 2 + NADH + H + H 2 O + NAD + ΔE˚΄ = + 1.14V ΔG˚΄ = -220 kJ mol -1

5. Electron Transport Energetic’s

6. Electron Transport Chain Components Protein complexes: I.NADH-Q reductase II.Succinate dehydrogenase III.Cytochrome C reductase IV.Cytochrome C oxidase Bridging components: Coenzyme Q and Cytochrome C What is the driving force for this electron flow?

7. Coupled Electron- Proton Transfer Through NADH-Q Oxidoreductase FMN bridges: NADH 2 e - donor with FeS 1 e - acceptor L-shaped Complex I Overall reaction: NADH + Q + 5H + NAD + + QH 2 + 4H +

8. Coupled Electron- Proton Transfer Through NADH-Q Oxidoreductase H + movement with 1 NADH Iron-sulfur clusters (a.k.a. nonheme-iron proteins) 2Fe – 2S or 4Fe – 4S complexes

9. NADH-Q Oxidoreductase (Complex I) Structure Largest of respiratory complexes Mammalian system contains 45 polypeptide subunits; 8 Fe-S complexes; 60 transmembrane helices

10. Different Quinone (Q) Oxidation States QH 2 generated by complex I & II Membrane-bound bridging molecule Overall reaction: QH 2 + 2Cyt C ox + 2H + Q + 2Cyt C red + 4H + X

11. Oxaloacetate Enzyme Regeneration from Succinate Succinate Dehydrogenase Fumerase Malate Dehydrogenase

12. Pathways that Contribute to the Ubiquinol Pool Without Utilizing Complex I

13. Alternative Q-Cycle Entry Points Complex I Complex II (citric acid cycle) Glycerol 3-phosphate shuttle Fatty acid oxidation (electron-transferring- flavoprotein dehydrogenase)

14. Electron-Transport Chain Reactions in the Mitochondria

15. The Q Cycle Electron transfer to Cytochrome c Reductase via 3 hemes and a Rieske iron-sulfur center Overall reaction: QH 2 + 2Cyt C ox + 2H + Q + 2Cyt C red + 4H + ISP – iron sulfur protein

16. The Q Cycle

17. Cytochrome c Oxidoreductase Structure Intermembrane side Heme-containing homodimer with 11 subunit monomers Functions to: Transfer e - to Cyt c Pump protons into the intermembrane space Matrix side

18. Cytochrome c Oxidase: Proton Pumping and O 2 Reduction

19. Cytochrome c Oxidase: O 2 Reduction to H 2 O Reaction shown: 2Cyt C red + 2H + + ½ O 2 2Cyt C ox + H 2 O Overall reaction: 2Cyt C red + 4H + + ½ O 2 2Cyt C ox + H 2 O + 2H +

20. Cytochrome c Oxidase O 2 to H 2 O reduction site Intermembrane space Matrix Oxygen requiring step 13 subunits; 10 encoded by nuclear DNA Cu A /Cu A prosthetic group positioned near intermembrane space

21. Cytochrome c Oxidase

22. Electron-Transport Chain Reactions in the Mitochondria

24. Mitochondrial Electron-Transport Chain Components

25. ATP Synthesis via a Proton Gradient The two major 20 th century biological discoveries: DNA structure and ATP synthesis

26. ATP-Driven Rotation in ATP-Synthase: Direct Observation γ rotation with ATP present With low ATP 120-degree Incremental rotation Glass microscope slide

27. ATP Synthase with a Proton-Conducting (F 0 ) and Catalytic (F 1 ) Unit Matrix side Intermembrane side F 1 matrix unit contains 5 polypeptide chain types (α 3, β 3, γ, δ, ε) Proton flow from intermembrane space to matrix

28. Matrix side ATP-Synthase with Non-Equivalent Nucleotide Binding Sites Side view F 1 contains: α 3, β 3 heximeric ring and γ, ε central stalk Central stalk and C-ring form the rotor and remaining molecule is the stator Top view

29. γ-Rotation Induces a Conformational Shift in the β Subunits Each β subunit interacts differently with the γ subunit ATP hydrolysis can rotate the γ subunit

30. Proton Flow Around C-Ring Powers ATP Synthesis Subunit C Asp protonation favors movement out of hydrophylic Subunit a to membrane region Deprotonation favors Subunit a movement back in contact with Subunit a

31. Proton Motion Across the Membrane Drives C-Ring Rotation

32. C-Ring Tightly Linked to γ and ε Subunits C-ring rotation causes the γ and ε subunits to turn inside the α 3 β 3 hexamer unit of F 1 Columnar subunits (2 b) with δ prevent rotation of the α 3 β 3 hexamer unit What is the proton to ATP generation ratio?

33. Mitochondrial ATP-ADP Translocase Net movement down the concentration gradient for ATP (out of matrix) and ADP (into matrix) No energy cost

34. Mitochondrial Transporters for ATP Synthesis Net movement against the concentration gradient for P i (into matrix) and charge balance - OH (out of matrix) Proton gradient energy cost

35. ATP Yield With Complete Glucose Oxidation

36. Heat Generation by an Uncoupling Protein UCP-1 Brown adipose tissue rich in mitochondria for heat generation Pigs nest, shiver, and have large litters to compensate for lack of brown fat

37. ATP Synthesis Chemical Uncoupling What physiological effect might DNP have in humans?

38. Electron Transport Chain Inhibitors Toxins (e.g. fish and rodent poison rotenone) Site specific inhibition for biochemical studies What impact will rotenone have on respiration (O 2 consumption)?

39. Proton Gradient Central to Biological Power Transmission

40. Problems: 13, 21, 23, 31, 33