20. Electron Transport and Oxidative Phosphorylation

Transcription

1 20. Electron Transport and Oxidative Phosphorylation

2 20.1 What Role Does Electron Transport Play in Metabolism? Electron transport - Role of oxygen in metabolism as final acceptor of electrons - In inner mitochondrial membrane Oxidative phosphorylation - Production of ATP by oxidation of nutrients - In mitochondria Definition Fe(Ⅲ) + e- Fe(Ⅱ) (reduction) Fe(Ⅱ) Fe(Ⅲ) + e- (oxidation)

3 20.1 What Role Does Electron Transport Play in Metabolism? Reactions of electron transport chain - Coupled to synthesis of ATP 1 Pumping of protons across inner mitochondrial membrane 2 Creating a ph gradient 3 Storing potential energy 3/44

4 20.1 What Role Does Electron Transport Play in Metabolism? Oxygen, ultimate electron acceptor, is reduced to water - Complete series of oxidation-reduction reactions

5 20.2 What Are the Reduction Potentials for the Electron Transport Chain? Energy changes in electron transport - Movement of electrons from one carrier to another - Reduction potential: whether electrons would be more likely to be transferred from NADH to coenzyme Q or the other way around? - Hydrogen ion is in aqueous solution in equilibrium with hydrogen gas. 2H + + 2e- H 2

6 20.2 What Are the Reduction Potentials for the Electron Transport Chain? - Hydrogen/H+ pair has a higher reduction potential than ethanol/ acetaldehyde pair.

7 20.2 What Are the Reduction Potentials for the Electron Transport Chain? - Electron flow in opposite direction (H2/H+ pair has a lower reduction potential.)

8 20.2 What Are the Reduction Potentials for the Electron Transport Chain? 8/44

9 20.2 What Are the Reduction Potentials for the Electron Transport Chain? Example - Reduction of oxygen : volts - If this reaction were paired directly with NAD + /NADH, what would happen? NADH + H + NAD + + 2H + + 2e /2O 2 + 2H + + 2e- H 2 O Sum NADH + 1/2O 2 + H + NAD + + H 2 O ΔG of redox reaction ΔG = -nfδe ΔG = -(2)( kj V -1 mol -1 )(1.136V) = -219 kj mol -1 Very large number if NADH reduced oxygen directly

10 20.3 How Are the Electron Transport Complexes Organized? Respiratory complexes - Four mutienzyme systems - Can be isolated from inner mitochondrial membrane - Integral parts of inner mitochondrial membrane Complex Ⅰ NADH-CoQ oxidoreductase - Transfer of electrons from NADH to coenzyme Q (CoQ)

11 20.3 How Are the Electron Transport Complexes Organized? Complex Ⅰ - Flavoprotein has a flavin coenzyme or FMN (flavin mononucleotide). 11/44

12 20.3 How Are the Electron Transport Complexes Organized? Complex Ⅰ Reaction in several steps - Successive oxidation and reduction of flavoprotein and iron-sulfur moiety 1 NADH + H + + E-FMN NAD + + E-FMNH 2 2 E-FMNH 2 + 2Fe-S oxidized E-FMN + 2Fe-S reduced + 2H + 3 2Fe-S reduced + CoQ + 2H + 2Fe-S oxidized + CoQH 2 - Coenzyme Q as ubiquinone

13 20.3 How Are the Electron Transport Complexes Organized? Complex Ⅰ - Overall equation NADH + H + + CoQ NAD + + CoQH 2 - Proton pumping create ph (proton) gradient

14 20.3 How Are the Electron Transport Complexes Organized? Complex Ⅰ - Phosphorylation of ADP to ATP : ΔG = ~ kj mol -1.

15 20.3 How Are the Electron Transport Complexes Organized? Complex Ⅰ Some of carriers - Carry electrons and hydrogens in reduced forms: NADH - Carry only electrons: iron-sulfur protein

16 20.3 How Are the Electron Transport Complexes Organized? Complex Ⅱ Succinate-CoQ oxidoreductase - Transfer of electrons to coenzyme Q - Substrate is succinate from citric acid cycle. Succinate + E-FAD Fumarate + E-FADH 2 E-FADH 2 + Fe-S oxidized E-FAD + Fe-S reduced Fe-S reduced + CoQ + 2H + Fe-S oxidized + CoQH 2 - Overall reaction is Succinate + CoQ Fumarate + CoQH 2 - ΔG = kj mol -1 - No ATP production, and no hydrogen ions are pumped out.

17 20.3 How Are the Electron Transport Complexes Organized? Complex Ⅲ Cytochromes - Electrons are passed from CoQ to first of a series of very similar proteins. - Successive oxidation-reduction reactions of cytochromes, Fe(Ⅲ) + e- Fe(Ⅱ) (reduction) Fe(Ⅱ) Fe(Ⅲ) + e- (oxidation)

18 20.3 How Are the Electron Transport Complexes Organized? Complex Ⅲ CoQH 2 -cytochrome c oxidoreductase - Oxidation of reduced coenzyme Q (CoQH 2 ). - Electrons are passed along to cytochrome c in a multistep process. - Overall reaction is CoQH 2 + 2Cyt c[fe(Ⅲ)] CoQ + 2Cyt c[fe(Ⅱ)] + 2H + 18/44

19 20.3 How Are the Electron Transport Complexes Organized? Complex Ⅲ - CoQ can exist in three forms.

20 20.3 How Are the Electron Transport Complexes Organized? Complex Ⅲ Q cycle - One electron is passed from reduced coenzyme Q to iron-sulfur clusters to cytochrome c 1. CoQH2 Fe-S Cyt c1 - By omitting iron-sulfur proteins, CoQH 2 + Cyt c 1 (oxidized) Cyt c 1 (reduced) + CoQ - (semiquinone anion) + 2H + - Proton pumping and ATP production is coupled. - ΔG = kj mol -1 for each mole of NADH.

21 20.3 How Are the Electron Transport Complexes Organized? Complex Ⅳ Cytochrome c oxidase - Transfer of electrons from cytochrome c to oxygen - Overall reaction is, 2Cyt c[fe(Ⅱ)] + 2H + + 1/2O 2 2Cyt c[fe(Ⅲ)] + H 2 O - Proton pumping as a result - Contain cytochrome a and a 3, as well as two Cu 2+ ions Cyt c Cyt a Cu 2+ Cyt a 3 O 2

22 20.3 How Are the Electron Transport Complexes Organized? Complex Ⅳ - Reactions of cytochromes more explicitly, Cyt c [reduced, Fe(Ⅱ)] + Cyt aa 3 [oxidized, Fe(Ⅲ)] Cyt aa 3 [reduced, Fe(Ⅱ)] + Cyt c [oxidized, Fe(Ⅲ)] - Half-reaction for reduction of oxygen is, 1/2 O 2 + 2H + + 2e- H 2 O - Overall reaction is, 2Cyt aa 3 [reduced, Fe(Ⅱ)] + 1/2 O 2 + 2H + 2Cyt aa 3 [oxidized, Fe(Ⅲ)] + H 2 O - In this final reaction we have finally seen the link to molecular oxygen in aerobic metabolism. - ΔG = -220 kj mol -1 for each mole of NADH

23 20.3 How Are the Electron Transport Complexes Organized? Complex Ⅳ Energetics of electron transport reactions.

24 20.3 How Are the Electron Transport Complexes Organized? Cytochromes and Other Iron-Containing Proteins of Electron Transport All cytochromes contain heme group - Differences in side chains of heme group: various stages of electron transport. 24/44

25 20.3 How Are the Electron Transport Complexes Organized? Cytochromes and Other Iron-Containing Proteins of Electron Transport Nonheme iron proteins - Contain sulfur, as case with iron-sulfur proteins

26 20.4 What Is the Connection between Electron Transport and Phosphorylation? Some of energy released by electron transport is used for phosphorylation of ADP - How each of reactions catalyzed by three of four respiratory complexes 1 Energy-releasing oxidation reaction 2 Proton pumping 3 ph gradient across inner mitochondrial membrane 4 Voltage difference across membrane by concentration difference of ions

27 20.4 What Is the Connection between Electron Transport and Phosphorylation? Coupling factor (ATP synthase) - To link oxidation and phosphorylation - Complex protein oligomer - F0: portion of protein that spans membrane, three polypeptide chains (a, b, and c) - F1: portion that projects into matrix, five chains (α 3 β 3 γδε) 27/44

28 20.4 What Is the Connection between Electron Transport and Phosphorylation? ATP synthase - The whole protein complex - Mitochondrial ATPase

29 20.4 What Is the Connection between Electron Transport and Phosphorylation? Uncouplers - Inhibit phosphorylation of ADP without affecting electron transport - Ex) 2,4-dinitrophenol, various antibiotics (valinomycin, gramicidin A)

30 20.4 What Is the Connection between Electron Transport and Phosphorylation? P/O ratio - To indicate coupling of ATP production to electron transport - Number of moles of Pi consumed in the reaction ADP + Pi ATP for each mole of oxygen atoms consumed in the reaction 1/2O 2 + 2H + + 2e- H 2 O - P/O ratio is 2.5 when NADH is substrate oxidized. - P/O ratio is 1.5 when FADH 2 is substrate oxidized.

31 20.5 What Is the Mechanism of Coupling in Oxidative Phosphorylation? Chemiosmotic Coupling Chemiosmotic coupling mechanism - Difference in proton concentration between intermembrane space and matrix. - By British scientist Peter Mitchell in 1961.

32 20.5 What Is the Mechanism of Coupling in Oxidative Phosphorylation? Chemiosmotic Coupling Experimental evidence 1) A system with definite inside and outside compartments is essential for oxidative phosphorylation. 2) Submitochondrial preparations that contain closed vesicles can be prepared.

33 20.5 What Is the Mechanism of Coupling in Oxidative Phosphorylation? Chemiosmotic Coupling 3) A model system for oxidative phosphorylation can be constructed with proton pumping in the absence of electron transport. 4) The existence of ph gradient has been demonstrated and confirmed experimentally.

34 20.5 What Is the Mechanism of Coupling in Oxidative Phosphorylation? Chemiosmotic Coupling How does proton gradient lead to production of ATP? - Ion channels through inner mitochondrial membrane

35 20.5 What Is the Mechanism of Coupling in Oxidative Phosphorylation? Chemiosmotic Coupling Reasonable mode of action for uncouplers - In light of the existence of a proton gradient - Dinitrophenol (acid): Conjugate base is uncoupler. - Ionophores: antibiotic uncouplers, such as gramicidin A and valinomycin

36 20.5 What Is the Mechanism of Coupling in Oxidative Phosphorylation? Conformational Aspects of Coupling Proton gradient is indirectly related to ATP production - Proton gradient leads to conformational changes in proteins.

37 20.5 What Is the Mechanism of Coupling in Oxidative Phosphorylation? Conformational Aspects of Coupling - Three sites for substrate on synthase and three possible conformational sites 1 Open (O), with low affinity for substrate 2 Loose-binding (L), not catalytically active 3 Tight-binding (T), catalytically active - F 1 portion of ATP synthase as a rotary motor - c, γ, and ε subunits constitute rotor. - γ and ε subunits constitute rotating shaft.

38 20.6 How Are Respiratory Inhibitors Used to Study Electron Transport? If a pipeline is blocked, there will be a backup - In electron transport, this analogy is useful. - When a flow of electrons is blocked in a series of redox reactions, reduced compounds will accumulate before blockage point in pathway.

39 20.6 How Are Respiratory Inhibitors Used to Study Electron Transport? Respiratory inhibitors - To determine order of electron transport chain - Determining relative amounts of oxidized and reduced forms of various electron carriers in intact mitochondria - On the spectroscopic properties

40 20.6 How Are Respiratory Inhibitors Used to Study Electron Transport? Three sites at which inhibitors have an effect 1) Barbiturates block transfer of electrons from flavoprotein NADH reductase to coenzyme Q. 2) Blockage when electron transfer involving b cytochromes, coenzyme Q, and cytochrome c 1 3) Transfer of electrons from cytochrome aa 3 complex to oxygen

41 20.7 What Are Shuttle Mechanisms? Electrons can be transferred to a carrier that can cross membrane. - NADH from glycolysis to mitochondria Glycerol-phosphate shuttle - Carrier system - In insect flight muscle ATP

42 20.7 What Are Shuttle Mechanisms? Malate-aspartate shuttle - More complex and more efficient shuttle mechanism - In mammalian kidney, liver, and heart ATP

43 20.8 What Is the ATP Yield from Complete Oxidation of Glucose? How many molecules of ATP are produced for each molecule of glucose oxidized? - ATP production and reoxidation of NADH and FADH 2.

44 THE END!! 44/44