1. Oxidative Phosphorylation What is it? Process in which ATP is formed as a result of the transfer of electrons from NADH or FADH 2 to O 2 via a series of electron carriers Oxidation of glucose: Glycolysis: 2ATP2NADH (5ATP) Pyruvate dehyd: 2NADH (5ATP) Citric acid cycle: 2ATP6NADH (15ATP)2FADH 2 (3ATP) Oxidative phos: ~26-28ATP TOTAL:~30-32ATP K, lec18, p2 Occurs in mitochondria

2. Oxidative Phosphorylation What is mitochondria? 2 membranes: Inner - only permeable to O 2, H 2 O transporters req’d for ATP, P i, pyruvate, etc. folding increases surface area (site of ox. phos. machinery) Matrix contains: citric acid cycle enzymes Fatty acid oxidation enzymes (discuss later)

3. Oxidative Phosphorylation Summary F type transporter ATP synthase

4. Oxidative Phosphorylation History 1961 - Peter Mitchell proposed chemiosmotic hypothesis: energy from e - transport is stored in a proton gradient which is then used to make ATP Experimental support: 1. Uncouplers: dinitrophenol carries H + across membrane, dissipating the H + gradient DNP-treated mito endlessly consume O 2 with NO ATP synthesis 2. Artificial H+ gradients drive ATP synthesis K, lec18, p16 K, lec18, p17

5. Oxidative Phosphorylation What are the electron carriers? NADH, NADPH (cannot cross inner mito membrane, shuttle their e-) FMN, FAD (directly involved in Ox phos) NADH, NADPH and FADH 2 each carry 2e - FMN can carry 1 or 2e -

6. Membrane bound Hydrophobic quinone (coenzyme Q) Q can carry 1 or 2e - Q floats free in lipid bilayer and moves e - from complexes I and II to III Oxidative Phosphorylation

7. Iron-containing proteins (cytochromes and Fe-S proteins) Cyt carry 1 e -, heme, found in complexes III and IV and cytochrome c Oxidative Phosphorylation FeS carry 1 e -, found in complexes I, II, and III, Fe 2+ or Fe 3+

8. Cytochrome c Peripheral membrane protein that shuttles e - between complexes III and IV Fe is linked to His and Met side chains Oxidative Phosphorylation K, lec 18, p11

9. Oxidative Phosphorylation Electron transport chain (respiratory chain) Series of oxidation/reduction components that carry electrons Complex I (NADH-Q dehydrogenase) Complex II (Succinate-Q dehydrogenase) Coenzyme Q Complex III (Cytochrome reductase) Cytochrome c Complex IV (cytochrome oxidase) FMN, FeS FAD, FeS itself Cyt b H, Cyt b L, FeS, Cyt c1 itself Cyt a, Cyt a3, Cu A, Cu B Proteine- carrying components

10. Oxidative Phosphorylation Order of electron carriers determined by respiratory inhibitors NADH  FMN  FeS  Q  cyt b  FeS  cyt c1  cyt c  cyt a  cyt a3  O 2 rotenoneantimycin Acyanide, azide

11. Oxidative Phosphorylation Complex I: NADH:Ubiquinone oxidoreductase NADH to Q (Proton pump)

12. Oxidative Phosphorylation Complex II: Succinate dehydrogenase Succinate to Q QH 2

13. Oxidative Phosphorylation Complex III: Cytochrome bc 1 complex or ubiquinone:cytochrome c oxidoreductase Ubiquinol (QH 2 ) to cytocrome c

14. Oxidative Phosphorylation Complex IV: Cytochrome oxidase Cytocrome c to molecular O 2 (reducing it to H 2 O)

15. Oxidative Phosphorylation ATP synthase Multiprotein complex 3H + pass through for each ATP made K, lec18, p18 OUTER MEMBRANE INNER MEMBRANE

16. Oxidative Phosphorylation Energetics of Ox. Phos. 1/2 O 2 + NADH + H +  H 2 O + NAD +  G˚ = -220 kJ/mol An electrochemical gradient across the inner membrane is formed: electrical: outside is more positive chemical: proton concentration gradient (pH out is 1.4 units < pH in Energy of 3H + transported drives ATP synthesis ADP + P i + 3H +  ATP + H 2 O  G˚ = +30.5 kJ/mol

17. Oxidative Phosphorylation Control Ox. Phos cannot occur without: source of e - (NADH) sink for e - (O 2 ) substrates for ATP synthase (ADP and P i ) [ADP] is limiting factor Inhibit electron transfer

18. Oxidative Phosphorylation How does ATP made in mito get out?