The Role of Electron Transport in Metabolism

2. The Role of Electron Transport in Metabolism • Electron transport is carried out by four closely related multisubunit membrane-bound complexes and two electron carriers, coenzyme ___ and cytochrome ___ • In a series of oxidation-reduction reactions, electrons from FADH2 and NADH are transferred from one complex to the next until they reach _____ • O2 is reduced to H2O • As a result of electron transport, ____________ are pumped across the inner membrane to the intermembrane space, creating a pH gradient

3. ATP Production in the Mitochondrion • The production of ATP in the mitochondria is the result of oxidative phosphorylation • The proton gradient establishes a voltage gradient • The proton and voltage gradients together provide the mechanism to couple electron transport with _______________________________________

4. Establishment of the Proton Gradient

5. Summary • Electron transport from one carrier to another creates a proton gradient across the inner mitochondrial membrane • The proton gradient is coupled to the production of ATP in aerobic metabolism

6. Reduction Potentials • A useful way to look at electron transport is to consider the change in free energy associated with the movement of electrons from one carrier to another • If we have two electron carriers, for example NADH and coenzyme Q, are electrons more likely to be transferred from NADH to coenzyme Q, or vice versa? • What we need to know is the _____________ _____________ for each carrier • A carrier of high reduction potential will tend to be _____________ if it is paired with a carrier of _____________ reduction potential

7. Reduction Potentials

8. Reduction Potentials

9. Summary • Standard reduction potentials provide a basis for comparison among redox reactions • The sequence of reactions in the electron transport chain can be predicted by using reduction potentials.

10. Electron Transport Complexes • Complex I: NADH-CoQoxidoreductase • Electrons are passed from NADH to FMN NADH + H+ E-FMN NAD+ + E-FMNH2

11. Electron Transport Complexes • Electrons are then passed to the iron-sulfur clusters • The last step of Complex I involves electrons being passed to coenzyme Q (also called ubiquinone)

12. Energetics of Electron Transport • The transfer of electrons is strongly ____________ and sufficient to drive the phosphorylation of ADP

13. Electron Transport Complexes Complex II:Succinate-coenzyme Q oxidoreductase Succinate + E-FAD Fumarate + E-FADH2 • The overall reaction is exergonic (-13.5 kJ/mol), but not enough to drive ATP production • No H+ is pumped out of the matrix during this step

14. Electron Transport Complexes Complex III: CoQH2-cytochrome c oxidoreductase CoQH2 + 2Cyt c[Fe(III)] CoQ + 2Cyt c[Fe(II)] + 2H+ • This reactions of this complex results in a decrease in free energy that is sufficient to drive the phosphorylation of ADP to ATP • The flow of electrons from reduced CoQ, a quinone that can exist in 3 forms, is known as the _____________

15. Oxidized and Reduced Forms of CoQ

16. Electron Transport Complexes Complex IV: Cytochrome c oxidase • Catalyzes the final step in electron transport 2Cyt c[Fe(II)] + 2H+ + ½O2 2 Cyt c[Fe(III)] + H2O • Complex IV contains cytochrome a, cytochrome a3, and Cu(II), which are also involved in the electron transport • Complex IV is the link to __________ __________

17. Electron Flow

18. The Energetics of Electron Transport Reactions

19. The Heme Group of Cytochromes • All cytochromes contain a _____________ group • Side chain differences depending on the heme

20. Connection between Electron Transport & Phosphorylation • The energy-releasing oxidations give rise to proton pumping and a pH gradient across the _____________ _____________ membrane • Differences in the concentration of ions across the membrane generates a _____________ _____________ • A coupling process converts the electrochemical potential to the chemical energy of ATP • The coupling factor is ATP _____________, a complex protein oligomer, separate from the electron transport complexes • Uncouplers inhibit the phosphorylation of ADP without affecting electron transport; examples are ________________, _____________, & _____________

21. Uncouplers

22. P/O Ratio • P/O ratio: the number of moles of Pi consumed in _____________ to the number of moles of oxygen atoms consumed in _____________ • Phosphorylation: ADP + Pi ATP + H2O • Oxidation: 1/2O2 + 2H+ + 2e- H2O • P/O = ______ when NADH is oxidized • P/O = ______ when FADH2 is oxidized

23. Summary • The coupling of electron transport to oxidative phosphorylation requires a multisubunit membrane-bound enzyme, ATP synthase. • This enzyme has a channel for protons to flow from the intermembrane space into the mitochondrial matrix. • The proton flow is coupled to ATP production in a process that appears to involve a conformational change of the enzyme.

24. Chemiosmotic Coupling • Chemiosmotic coupling • based on a ________ concentration gradient between the intermembrane space and the matrix • a proton gradient exists because the various proteins that serve as electron carriers are not symmetrically oriented with respect to the two sides of the inner mitochondrial membrane • these proteins take up protons from the matrix when they are reduced and release them to the intermembrane space when they are reoxidized • the reactions of NADH, CoQ, and O2 all require _______________________

25. Chemiosmotic Coupling

26. Chemiosmotic Coupling Evidence for chemiosmotic coupling suggested (Mitchell 1961): • A system with definite inside and outside _____________ (closed vesicles) is essential. • Submitochondrial vesicles can be prepared, which carry out oxidative phosphorylation and have an asymmetric orientation of respiratory complexes. • A model system for oxidative phosphorylation can be constructed with proton pumping in the absence of electron transport; the model system consists of reconstituted membrane vesicles, mitochondrial ATP synthase, and a proton pump. • The existence of the pH gradient has been demonstrated and _____________ _____________

27. Chemiosmotic Coupling • The mechanism by which the proton gradient leads to the production of ATP depends on ion channels through the inner mitochondrial membrane • Protons flow back into the matrix through channels in the F0 unit of ATP _____________ • The flow of protons is accompanied by formation of ATP in the F1 unit of ATP _____________ • The details of how phosphorylation takes place as a result of the linkage to the proton gradient are not explicitly specified by this mechanism

28. Chemiosmotic Coupling

29. Conformational Coupling • The proton gradient leads to changes in conformation in a number of proteins, including ATP _______________ • There are 3 sites for substrate on ATP _____________, and 3 possible conformations: - Open (O); a low affinity for substrate - Loose-binding (L); not catalytically active, binds ADP & Pi - Tight-binding (T); catalytically active, binds ATP • These sites interconvert as a result of proton flux through ATP synthase • Proton flux converts L to T, which produces ATP • Proton flux converts T to O, releasing ATP

30. Release of ATP from ATP Synthase

31. Summary • In chemiosmotic coupling, the proton gradient is the crux of the matter. The flow of protons through pores in the synthase drives ATP production. • In conformational coupling, a change in the shape of the synthase releases bound ATP that has already been formed.

32. Respirator Inhibitors & Electron Transport • Respiratory inhibitors can be used to determine the order of reactions in the electron transport chain • Isolate intact _____________ from cells • Provide an oxidizable substrate so that electron transport can occur • Add a respiratory inhibitor • Measure relative amounts of oxidized and reduced forms of various electron carriers • Inhibitors have an effect on three sites in the electron transport chain

33. The Effect of Respiratory Inhibitors

34. Sites of Action of Some Respiratory Inhibitors

35. Shuttle Mechanisms • Shuttle mechanisms: transport metabolites between _____________ and _____________ • Glycerol phosphate shuttle: • We know glycolysis in the _____________ produces NADH • NADH does not cross the _____________________ membrane, but glycerol phosphate and dihydroxyacetone phosphate do • Through the glycerol phosphate shuttle, ____ ATP are produced in the mitochondria for each cytosolic NADH

36. The Glycerol-Phosphate Shuttle

37. The Malate-Aspartate Shuttle • The Malate-Aspartate Shuttle: • Has been found in mammalian kidney, liver, and heart • Malate crosses the mitochondrial membrane, while oxaloacetate cannot • The transfer of electrons from NADH in the cytosol produces NADH in the mitochondria • In the malate-aspartate shuttle, _____ mitochondrial ATP are produced for each cytosolic NADH

38. The Malate-Aspartate Shuttle

39. Summary • Shuttle mechanisms transfer ___________, but not _____________, from the cytosol across the mitochondrial membrane • In the malate-aspartate shuttle, 2.5 molecules of ATP are produced for each molecule of cytosolic NADH, rather than 1.5 ATP in the glycerol-phosphate shuttle… • This affects the overall yield of ATP in these tissues

40. ATP Yield from Complete Oxidation of Glucose • In the complete oxidation of glucose, a total of ______ or ______ molecules of ATP are produced for each molecule of glucose, depending on the shuttle mechanism

41. The ATP Yield from Complete Oxidation of Glucose