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

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)

Oxidative Phosphorylation Summary F type transporter ATP synthase

Oxidative Phosphorylation History 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

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 -

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

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+

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

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

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

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

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

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

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

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

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˚ = kJ/mol

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

Oxidative Phosphorylation How does ATP made in mito get out?