Free radical biology 自由基生物學

1. Free radical biology 自由基生物學 耿全福 Chuian-Fu Ken

2. Oxygen is a toxic gas: an introduction to oxygen toxicity and reactive oxygen species • 1. The history of oxygen. • 2. Oxygen and anaerobes. • 3. Oxygen and aerobes: mitochondria electron transport. • 4. Oxidases and oxygenases: Cytochromes P450, lipoxygenase. • 5. Oxygen toxicity in aerobes.

3. 6. What is a free radical? • 7. What is ‘reactive oxygen species’? • 8. Source of superoxide in aerobes.

4. 1. The history of oxygen.

8. 2. Oxygen and anaerobes • Strict anaerobes: Treponema denticola and several Clostridium spp. • Gangrene: Clostridium infection, treated patient with high pressure O2, which called hyperbaric oxygen therapy. • Moderate anaerobes: grow up to 10% O2; Bacteroides fragilis. • Microaerophiles: require low O2, but can’t tolerate 21% O2. ex: • Diarrhoea:Campylobacterjejuni. • Syphilis: Treponema pallidum . • Human body contains many anaerobes: gum, colon…etc.

9. Why does oxygen injure anaerobes? • Oxidation produce oxygen free radicals. • Oxygen inhibit some key enzymes: nitrogenase, N2----NH3 • All known nitrogenase enzymes are inactivated by O2 to some extent. • Some photosynthetic N2-fixing blue-green algae located their nitrogenase in thick-walled, O2 resistent cells known as heterocysts. • In the root nodules of leguminous plants that engage protein, leghaemoglobin is present, prevent O2 damage N2-fixing system.

10. 3. Oxygen and aerobes: Oxygen transport in mammals: haemoglobin, contains 2 α,2βsubunit, each carries a haem group with Fe2+. Myoglobin contains only 1 polypeptide with 1 haem occurred in heart and red muscle, binding O2 more tightly than haemoglobin. Leghaemoglobin perform a similar function. Haem can be synthesis in mammalian cells and by many bacteria, two steps in this pathway, catalysed by the enzymes coproporphyrinogen oxidase and protoporphyrinogen oxidase, mammalian use O2, bacteria use NADP+ as electron acceptor.

12. Mitochondria electron transport • 85-90% O2 is consumed by mitochondria. • Why only 0.5mmHg O2? • Glycolytic pathway • The citric acid cycle • The electron transport chain

14. glycolytic Transport into mitochondria Energy metabolism

15. The glycolytic pathway

17. Pyruvate transport into mitochondria matrix • Pyruvate → acetyl CoA Pyruvate dehydrogenase

18. The Krebs cycle

20. NADH

22. Electron transport chain

23. Electron transport chain Complex IV Complex I Complex III Complex II

25. Complex V

26. F1 F0 Inner membrane

27. Raha & Robinson, 2000 mitochondria

29. Free radical

30. Summary

31. ATP-ADP antiporter

32. 4. Oxidases and oxygenases: • 10-15% O2 is consumed by oxidase and oxygenase. • D-amino acid oxidase uses O2 to oxidize unwanted D-amino acid. • Xanthine oxidase uses O2 to oxidize xanthine and hypoxanthine into uric acid. • When collagen is being synthesized, proline and lysine hydroxylase use O2 to help put essential hydroxyl groups on to the proline and lysine.

33. ROS are generated in the cell Mates et al., 1999 In cell

34. Cytochromes P450 • Over 150 genes encoding the P450 superfamily have been described. • The reduced form of the cytochromes bind carbon monoxide to produce a complex that absorbs light strongly at 450 nm.

37. Common and Uncommon Cytochrome P450 Reactions Related to Metabolism and Chemical Toxicity F. Peter Guengerich • Cytochrome P450 (P450) enzymes catalyze a variety of reactions and convert chemicals to potentially reactive products as well as make compounds less toxic. • Most of the P450 reactions are oxidations. • The majority of these can be rationalized in the context of an FeO3+ intermediate and odd electron abstraction/rebound mechanisms. P450s also catalyze less generally discussed reactions including reduction, desaturation, ester cleavage, ring expansion, ring formation, aldehyde scission, dehydration, ipso attack, one-electron oxidation, coupling reactions, rearrangement of fatty acid and prostaglandin hydroperoxides, and phospholipase activity. • Most of these reactions are rationalized in the context of high-valent iron-oxygen intermediates and Fe2+ reductions, but others are not and may involve acid-base catalysis. Some of these transformations are involved in the bioactivation and detoxication of xenobiotic chemicals.

38. Contents • 1. Introduction 612 • 2. General Information about Cytochrome P450 Enzymes 612 • 2.1. Nomenclature and Distribution 613 • 2.2. Structures of P450s 613 • 2.3. Roles of P450s in Drug Metabolism 614 • 2.4. Roles of P450s in Chemical Toxicity 614 • 3. General Features of Catalysis 615 • 3.1. General Aspects of the Catalytic Cycle 615 • 3.2. Rationalization of Major Reactions with a General Mechanism 616 • 3.2.1. Carbon Hydroxylation 616 • 3.2.2. Heteroatom Oxygenation 617 • 3.2.3. Heteroatom Release (Dealkylation) 617 • 3.2.4. Epoxidation 618 • 3.2.5. 1,2-Group Migrations and Heme Inactivation in the Oxidation of Olefins and Acetylenes 618 • 3.3. Alternate Oxidation Mechanisms 620 • 3.4. Rate-Limiting Steps in P450 Reactions 622

39. 4. Unusual P450 Reactions: General Issues Regarding Reaction Diversity 624 • 4.1. Reductions 625 • 4.2. Desaturation 627 • 4.3. Oxidative Ester Cleavage 629 • 4.4. Ring Expansions 630 • 4.5. Ring Formation 630 • 4.6. Aldehyde Scissions 631 • 4.7. Dehydration 632 • 4.8. Ipso Attack and Related Reactions of Aromatic Rings 632 • 4.9. One-Electron Oxidation 633 • 4.10. Coupling Reactions 636 • 4.11. Rearrangement of Fatty Acid and Prostaglandin Hydroperoxides 637 • 4.12. Isomerization 639 • 4.13. Phospholipase D Activity 639 • 5. Conclusions 640

42. Lipoxygenase (LOX) • 動物 LOX種類 • 動物組織之脂氧合酶主要有三種異構 5-、12-及15-脂氧合酶，乃根據其氧化 arachidonic acid (20:4)之位置而命名

43. 哺乳類 LOX結構 • Composed two domains • C-terminal : catalytic domain, substrate binding site is a hydrophobic pocket , two long central helices contains four ligands (His-361, 366, 541 and 545). • N-terminal : -barrel domain, 115 a.a., 8-strands, 23% 類似mammalian lipase C-terminal ( Phe 70 and Leu 71). The structure of rabbit 15-LOX with a bound inhibitor (Sarah A. Gillmor, 1997)

44. (Sarah A. Gillmor, 1997) 470Å3 420Å3 390Å3

45. 15-LOX 的活性中心 These five ligands ( His-361, 366, 541, 545 and Ile 663 ) coordinate the iron with excellent octahedral gemotry. inhibitor RS75091 The structure of the inhibitor RS75091 bound ate the active site

46. 哺乳類動物的分子歸納 Hartmut Kuhn , 1999

47. LOX 應用性相關研究 • 食品加工方面 • 改善魚類製品風味 • 養殖魚罹病探針 • 延長食品貯藏期限 • 醫學研究方面 • 抑制5-LOX 活性以治療氣喘 (Israel, 1996) • 在發炎及牛皮癬組織中發現12-LOX被過量表現 (Shannon, 1993) • 表現15-LOX可降低 LDL 進而促進動脈硬化(Steinberg, 1989)

48. 5. Oxygen toxicity in aerobes