ELECTRON TRANSPORT CHAIN (oxidative phosphorylation)

1. ELECTRON TRANSPORT CHAIN(oxidative phosphorylation)

2. Oxidative Phosphorylation • Culmination of all aerobic-energy yielding metabolism • Energy from fuel oxidation is converted to the high energy bonds of ATP, via a chain of electron carriers Q: The high energy molecules that transfer their energy to ATP are _______ and ______ Q: The metabolic pathways where NADH and FADH2 are generated include __________ and ___________ NADH FADH2 Glycolysis Kreb Cycle

3. Carbohydrates, fatty acids, amino acids NAD+ FAD METABOLISM NADH + H+ FADH2 CO2 and H2O O2 NADH + H+ ADP + Pi FADH2 OXIDATIVE PHOSPHORYLATION NAD+ ATP FAD H2O

4. Location of the ETC • Q: Where in the mitochondria is the ETC located? What is the composition of the mitochondrial matrix • The inner membrane. 50% proteins/enzymes involved in oxidative metabolism (Krebcycle, oxidation of fatty acids, amino acids) , NAD+ and FAD, ADP and inorganic phosphate

5. Mitochondria

6. Organization of the ETC Q. Which one of the following is not protein in nature? • COMPLEX I (NADH dehydrogenase) • COMPLEX II (Succinate dehydrogenase) • COMPLEX III (cytochrome b-c1 complex) • COMPLEX IV (cytochrome a1 + a3 complex) • COMPLEX V (ATP synthase complex) And • Coenzyme Q • Cytochrome C

7. COMPLEX I NADH Dehydrogenase Q: The structural features of Complex I that allow it to accept 2H+ and 2e-s from NADH is _____________ and to pass the e-s on to CoQ is ____________ FMN Fe-S centers

8. COMPLEX I NADH Dehydrogenase • Protein complex that spans the cell membrane. It contains • a NADH binding site • a molecule of Flavin mononucleotide (FMN) • Iron–sulfur (Fe-S) centers • A binding site for CoQ • Transfers two H atoms (2e- and 2H+) from NADH (and H+) to FMN which become FMNH2. FMNH2 passes the electrons to the Fe-S centers which transfer electrons to CoQ. • Pumps four protons from the matrix into the intermembrane space

9. Coenzyme Q (ubiquinone) • Only component of the ETC that does not contain protein • Quinone derivative with a long hydrophobic isoprenoid tail • Transfers e-s from complex I and other flavoproteins to Complex III • Three other flavoproteins including complex II (succinate dehydrogenase), glycerol 3- phosphate- and fatty acyl CoA dehydrogenase shuttle e-s via FADH2

10. Coenzyme Q (ubiquinone) Q. What is the function of the isoprenoid tail found in CoQ? A. The isoprenoid tail makes CoQ lipophilic -allowing it to diffuse through the mitochondrial membrane

11. COMPLEX II Succinate Dehydrogenase Q. How does complex II differ from the other complexes of the ETC (at least 2 differences) A:It is a part of the Kreb cycle It is not a transmembrane protein and It does not pump protons

12. COMPLEX II Succinate Dehydrogenase • Part of the TCA cycle (Kreb cycle) • Oxidation of succinate to fumarate, reduction of FAD+ to FADH2 • Does not span the membrane, present towards the matrix • Transfers electrons from FADH2 to CoQ • No proton pumping action

13. Flavoproteins • FMN and FAD contain the water soluble vitamin Riboflavin (B2). • Although rare, a dietary deficiency of riboflavin can impair the function of these proteins and thereby the ETC

14. The Cytochromes Q: What structural feature of cytochromes allow it to accept/donate electrons: • Cytochromes are proteins that contain a bound heme group (an iron bound to a porphyrin ring similar to heme in hemoglobin) • The iron is in the form of Fe+++ rather than Fe++ (as in heme from hemogblobin) • Accept electrons (Fe+++ gets reduced to F++) and pass them on (Fe++ is oxidized back to F+++)

15. COMPLEX IIICytochrome bc1 • Accepts electrons from CoQ passes them on to cytochrome C • Three protons are pumped from the matrix during this reaction

16. Cytochrome c COM • Cytochrome c is loosely bound to the outer face of the inner membrane • Shuttles e-s from complex III to Complex IV

17. COMPLEX IVCytochrome a+a3 Q. How does cytochrome a+a3 differ from the other complexes of the ETC (at least 2 differences) • reacts directly with O2 • Contains Cu atoms

18. COMPLEX IVCytochrome a+a3 • Only component of the ETC that reacts directly with O2 • Reduces O2 to H2O by bringing together the transported e-s, O2 and protons from the matrix O2 + 4e- + 4H+ 2H2O • Bound Cu atoms facilitate this reaction. • Four protons are pumped out during this reaction

20. Redox pairs • Oxidation of a compound is always coupled with the reduction of another compound. • Q: Give an example of a redox pair: NADH + H+ FMN FMNH2 NAD+

21. Standard Reduction Potential The tendency of a redox pair to lose electrons can be specified by a constant Eo (the standard reduction potential) Q. More negative the Eo _______________ the potential to lose electrons • More positive the Eo greater the potential to accept electrons • Electrons therefore flow from the pair with the more negative Eo to the most positive one • The order of the complexes in the ETC is from the more negative to more positive greater

22. Organization of the ETC according to Eo Redox pair Eo NAD+/NADH -0.32 FMN/FMNH2 -0.22 Cytochrome c Fe+++/Fe++ +0.22 1/2O2/H2O +0.82 The order of the complexes in the ETC is from the more negative to more positive

24. Free Energy of ATP • Transport of e-s down the ETC from NADH to O2 produces 52.8 kcal of energy • Converting one ADP to ATP requires 7.3 Kcal/mol • __ ATPs are produced/molecule of NADH oxidized in the ETC • __ ATPS /mol of FADH2 oxidized • The remaining energy is lost as heat or used for ancillary reactions 3 2

25. No ATP generated so far!! The flow of electrons from NADH to oxygen does not directly result in ATP synthesis

26. Chemiosmotic Theory • Originally proposed by Peter Mitchell • was awarded the Nobel Prize in Chemistry 1978 • when proposed, chemiosmosis was a very radical idea and was not well received by other scientists!

27. Chemiosmotic Theory • The pumping of H+ from the mitochondrial matrix into the intermembrane space by the complexes I, III and IV results in an electrochemical gradient. • The H+ can go back to the matrix only through the ATP synthase molecules. • This exergonic flow of H+ is used by the enzyme to generate ATP. Q: What is the ChemiosmoticTheory

29. COMPLEX VATP Synthase

30. Binding-mechanism of ATP synthase It take at least 3 protons to produce 1 ATP

31. Vignette 6 Introduction: A 68-year-old female in a hypertensive crisis was being treated in the intensive care unit (ICU) with intravenous nitroprusside for 48 hours. The patient’s blood pressure was brought back down to normal levels. Presenting complaint: However, she started complaining of a burning sensation in her throat and mouth which was followed by nausea and vomiting, diaphoresis, agitation, and dyspnea. On Examination: The nurse noticed an almond-like smell in her breath. Lab investigations: An arterial blood gas revealed a significant metabolic acidosis. A serum test suggests a metabolite of nitroprusside, thiocyanate,is at toxic levels. Diagnosis: Cyanide poisoning from toxic dose of nitroprusside Treatment:Supportive therapy, gastrointestinal (GI) decontamination, oxygen, and antidotal therapy with amyl nitrite, sodium nitrite, andsodium thiosulfate.

32. Inhibitors of the ETC Q: Cyanide poisoning affects the ETC by inhibiting ________________ complex IV (a+a3)

33. Inhibitors of the ETC CO Cyanide Sodium Azide Rotenone Malonate Antimycin A Oligomycin INHIBITORS

34. Inhibitors of the ETC Q. What would be the oxidation/reduction status of complex I, II, III and IV incase of Antimycin A poisoning? • I, II, III would be reduced • IV would be oxidized

35. Uncouplers of Oxidative Phosphorylation Q: What is meant by uncoupling of Oxidative Phosphorylation? • Electron flow through the ETC without ATP synthesis.

36. Uncouplers of Oxidative Phosphorylation

37. Uncoupling Proteins • UCP1-5 • UCP1, also know as thermogenin is exclusively found in brown adipose tissue • Brown adipose tissue is abundant in newborns and some adult mammals • Provides body heat during cold stress in babies and to hibernating mammals • Uncouple the proton gradient, generating energy in the form of heat rather than ATP

38. Vignette 7 An unresponsive 25-year old woman was carried to the ER by her family. Her family members revealed that she had taken three doses of “weight loss” pills. She developed headache, fever, chest pain, profuse sweating and weakness soon afterwards. Initial findings were: temperature 105.5oF, pulse 151 beats per minute, blood pressure 40/10. She died within 15 minutes. After death, rigor mortis set in after 10 minutes and her temperature rose to 115oF after another 10 min. Among her personal effects a plastic bottle containing the weight loss pills were found, which on analysis proved to contain 2,4,dinitrophenol.

39. Q. Why does DNP cause weight loss? Decreased ATP and proton gradient result in increased electron flow by increasing fuel oxidation (i.e., more carbohydrates and fats (calories) will be consumed)

40. Inherited defects in the ETC • 13 of the ~120 polypeptides required for oxidative phosphorylation are encoded in the mitochondrial genome • rRNA and tRNAs required for protein synthesis are also encoded by the mitochondrial genome • Mutations in these any of these genes can cause OXPHOS diseases

41. THE END!