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Sources of the instigators A. Exogenous sources of radicals B. Endogenous NADPH oxidase NADPH P450 oxidor

Redox Biology: Biochemistry David A. Wink and Murali C. Krishna National Institutes of Health National Cancer Institute Radiation Biology Branch Bldg. 10, Room B3-B69 Bethesda, Maryland 20892.

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Sources of the instigators A. Exogenous sources of radicals B. Endogenous NADPH oxidase NADPH P450 oxidor

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  1. Redox Biology: BiochemistryDavid A. Wink and Murali C. KrishnaNational Institutes of HealthNational Cancer InstituteRadiation Biology BranchBldg. 10, Room B3-B69Bethesda, Maryland 20892

  2. Biochemical Lecture I. Sources of the instigators A. Exogenous sources of radicals B. Endogenous NADPH oxidase NADPH P450 oxidoreductases Xanthine oxidase Mitochondria Nitric oxide Synthase Heme oxygenase II. Detoxification: A. Enzymatic SOD, CAT, GPx DT-Diaphorase (2-e transfer) B. Scavenging GSH Ascorbate Tocopherol Biochemical Lecture Sources of the instigators A. Exogenous sources of radicals B. Endogenous NADPH oxidase NADPH P450 oxidoreductases Xanthine oxidase Mitochondria Nitric oxide Synthase Heme oxygenase II. Detoxification: A. Enzymatic SOD, CAT, GPx DT-Diaphorase (2-e transfer) B. Scavenging GSH Ascorbate Tocopherol

  3. ● Formation and Abatement of Oxidants in Redox Biology -Source of Instigators: ●PAO, XO, NOX, Xenobiotic, Catalase/GPX, Prx, LOOH, TrxProtein, GSSG(GR)GSH, NOSNOHbO2 and Cellular Consumption ● NOO2- H2O2 and ●oxidationLOOH, Protein, GSSG - Antioxidant systems Formation and Abatement of Oxidants in Redox Biology NOS uncoupled NO Catalase/GPX HbO2 Cellular Consumption Prx GR GSH Trx Source of Instigators NOX XO PAO Xenobiotic H2O2 O2- SOD oxidation LOOH Proteinox GSSG Antioxidant systems

  4. Reduction of oxygen to O2- and H2O2 ● Outer-sphere electron transfer Thus, the determining factor is E and access ● O2 + e- O2- - 0.33 V O2 + e- + H+ HO2 - 0.46 V ●O2- + e- H2O2 +0.95 V pKa = 4.7● Substance with more negative reduction potential than -0.33 V can spontaneously reduce oxygen to superoxide.● Paraquat/methyl viologen MV+ + O2 MV2+ + O2-ΔE = (+0.45) + (-0.33) = +0.12 MV2+ + O2- Paraquat/methyl viologen MV+ + O2 DE = (+0.45) + (-0.33) = +0.12 Reduction of oxygen to O2- and H2O2 Outer-sphere electron transfer Thus, the determining factor is E and access O2 + e- O2- - 0.33 V O2 + e- + H+ HO2 - 0.46 V pKa = 4.7 O2- + e- H2O2 +0.95 V Substance with more negative reduction potential than -0.33 V can spontaneously reduce oxygen to superoxide.

  5. Redox Cycling to form Reactive Oxygen Species ● Types of Compounds● Ubiquinone (CoQ)● Menidione● Adriamycin (Doxorubicin)● Tocopherol● Quinone● Semi-Quinone (GSH) GS-Q  Prot-SQ● Diphenol● Dopamine derivatives, Catecholic Estrogens● Adrenaline● Quinone● Semi-Quinone● Diphenol OH OH Dopamine derivatives, Catecholic Estrogens Adrenaline Ortho O O O - e- O- + e- Quinone Semi-Quinone Diphenol Redox Cycling to form Reactive Oxygen Species Para Types of Compounds O O2 O O2- OH - e- Ubiquinone (CoQ) Menidione Adriamycin (Doxorubicin) Tocopherol + e- O2- H2O2 O O- OH Quinone Semi-Quinone Diphenol GSH GS-Q Prot-SQ

  6. ●Menadione (Vitamin K3) and dopamine produce ROS. ● dopamine hydroxylase● 6-hydroxydopamine O CH3 Thiol Met Hb + SQ- HbO2 O2- O Menadione (Vitamin K3) CH2CH2NH3+ CH2CH2NH3+ HO O2 OH OH hydroxylase OH OH dopamine 6-hydroxydopamine

  7. Pro-oxidant versus Antioxidant Quinone Pro-oxidant Pro-oxidant: SQ to QuinoneAnti-oxidant: reduce the SQ to QH2. Location is essential and pharmacokinetic stability water versus fat soluble Pro-oxidant versus Antioxidant +e +e SQ QH2 Quinone Fe(III) reduction Pro-oxidant O2- Pro-oxidant: SQ to Quinone Anti-oxidant: reduce the SQ to QH2. Location is essential and pharmacokinetic stability water versus fat soluble

  8. Vitamins Working in Concert Vitamin E produces ROS in the lipid. Caretinoid produces ROS in the cytosol Caretinoid CarOH or CarOOL R R Asc Car + OH; LOO CarOH or CarOOL NO2 Asc Car Vitamins Working in Concert Vitamin E = TcOH Pro-oxidant TcOH + OH; LOO or NO2 TcO Lipid Asc Cytosol Asc DHA and Asc

  9. Xenobiotic Metabolism ●Activation of Quinones, Paraquat etc. NADPH Cytochrome P450 Oxidoreductases x________________________x + 1 electron Detoxification of Quinones, Paraquat etc. DT-Diaphorase or Quinone Reductase ( NQ1, P450 reductase super family)x_________________________xH2 + 2 electrons ● Broccoli extracts induce DT-Diaphorase (NQ1) expression Xenobiotic Metabolism Activation of Quinones, Paraquat etc. X X- NADPH Cytochrome P450 Oxidoreductases + 1 electron Detoxification of Quinones, Paraquat etc. DT-Diaphorase or Quinone Reductase ( NQ1, P450 reductase super family) X XH2 + 2 electrons Broccoli extracts induce DT-Diaphorase (NQ1) expression

  10. ●Mitochondrial Sources of ROS ● Polarization/depolarization of the mitochondria can effect reductionwhere the proton gradient can be important to reduction of O2. H2O2 O2- -0.04 V Ubiquinone (Q) Ubisemiquinone (SQ) Ubiphenol (H2Q) -0.25 V HO2 O2- O2 pKa = 4.7 Mitochondrial Sources of ROS O2 Site I Site II Site III Site IV H2O Cytochrome c oxidase O2 O2- NADH Polarization/depolarization of the mitochondria can effect reduction where the proton gradient can be important to reduction of O2.

  11. ● Energetics of ROS formation from Site III ● Over expression of MnSOD leads to increase DCHF oxidation Energetics of ROS formation from Site III Eo = -0.33 Eo = 0.04 DE = -0.29 DG ~6 kcal K =0.01 SQ +O2 e- Water reduction at Site IV K = 0.01 Aconitase +NO Q +O2- NO3- ONOO- MnSOD e- H2O2 Spin trap Over expression of MnSOD leads to increase DCHF oxidation DHR DCHF

  12. Examples of Inner-sphere reduction of Oxygen Prolyl Hydroxylase +e- O2 Fe(III) Fe(II) Fe(II)O2 Hydroxylation of proline +e- Fe(O2-) Fe(III)O2- Examples of Inner-sphere reduction of Oxygen Bleomycin BLMFe(II) e- O2- BLMFe(III) Fe(II)O2 or Fe(III)O2- Substrate oxidation H2O2 e- Fe(V) or Fe(III)O2H+ OH and Fe(IV) 2 electron, oxygen transfer hydroxylation 1 electron oxidants

  13. Different reduction mechanism of O2 by P450 system ●Cytochrome P450 Different reduction mechanism of O2 by P450 system Cytochrome P450 Fe2+ + O2 Fe2+O2 +e- +e- Resting state Peroxide shunt Fe3+ -O22- Fe3+ +H2O2 Substrate oxidation H2O Fe5+=O 1.4 -1.6 V P450 reductase NADPH FAD FMN P450 O2 Q SQ

  14. Enzymatic generation of Reactive Oxygen Species Purpose: ●As an antimicrobial agent ● as a by product of metabolism ● Part of signal transduction mechanisms Enzymatic generation of Reactive Oxygen Species • Purpose: • As an antimicrobial agent • as a by product of metabolism • Part of signal transduction mechanisms

  15. Examples of Hydrogen peroxide formation ● Glucose Oxidase (Glc oxidase: Bacterial) ● O2 + Glucose --> gluconolactone + H2O2 ● Polyamine oxidase (PAO) O2 + spermine ---> acrolein + putrescine + H2O2●Xanthine Oxidase (XO) ● O2 + Xanthine --> Urate and O2-/H2O2 ● O2 + Hypoxanthine --> Xanthine and O2-/H2O2 ● NADPH oxidase (NOX) ● NADPH + 2 O2 --> 2 O2- + NADP+ Examples of Hydrogen peroxide formation Glucose Oxidase (Glc oxidase: Bacterial) O2 + Glucose --> gluconolactone + H2O2 Polyamine oxidase (PAO) O2 + spermine ---> acrolein + putrescine + H2O2 Xanthine Oxidase (XO) O2 + Xanthine --> Urate and O2-/H2O2 O2 + Hypoxanthine --> Xanthine and O2-/H2O2 NADPH oxidase (NOX) NADPH + 2 O2 --> 2 O2- + NADP+

  16. Xanthine Oxidase ●Purpose: Purine metabolism Secretion of milk dropsDetoxification of AldehydesGeneration of ROS Xanthine Oxidase Purpose: Purine metabolism Secretion of milk drops Detoxification of Aldehydes Generation of ROS XD Normal (XD) Uric acid + NADH Xanthine Uric acid + O2-/H2O2 Disease and injury (XO) XO XO HX --> X + ROS XD to XO by proteolysis or thiol oxidation

  17. Xanthine Oxidase ●Inhibited by allopurinol (abundant in goats milk)●Mouse utilizes XO more than humans Xanthine Oxidase Homodimer Inhibited by allopurinol (abundant in goats milk) HX or X Mo O2 FAD FADH2 Urate Fe2S2 O2-/H2O2 S O Mo Mouse utilizes XO more than humans S O H2O H S H N N 3-O3PO O N Moco H

  18. NADPH oxidase (NOX) ●Purpose:AntimicrobialRegulation of cell-surface signalingregulation of physiological function ● NOX-1 colon VSMC, prostate● NOX-2 innate immune system● NOX-3`inner ear● NOX-4 kidney● NOX-5 spleen (human only)● Vascular NOX● Phagocytic (Phox) ● Membrane bound ● gp91phox p21phox● Cytosolic regulators ● p47phox ● p67phox● p40phox ● Small GTPase/rac1/Rac2 NADPH oxidase (NOX) Purpose: Antimicrobial Regulation of cell-surface signaling regulation of physiological function Phagocytic (Phox) Membrane bound gp91phox NOX-1 colon VSMC, prostate NOX-2 innate immune system NOX-3`inner ear NOX-4 kidney NOX-5 spleen (human only) p21phox Cytosolic regulators p47phox p67phox p40phox Small GTPase/rac1/Rac2 Vascular NOX

  19. ● NADPH oxidase ●cytochrome b558 O2- NADPH oxidase O2 cytochrome b558 -Fe- -Fe- gp91phox FAD gp91 NADPH p22 P rac p47 gp67 P p40 P P

  20. Nitric Oxide Synthase ● Homodimer ● NOS-1 nNOS or neuronal NOS ● NOS-3 eNOS or endothelial NOS ● NOS-2 iNOS or inducible NOS ● P450 reductase domain ● Heme oxidase domain ● Constitutive and calcium sensitive ● Induced and calcium insensitive ● NADPH ● FMN ● FAD ● Calmodulin binding O2 ● Arginine/N-hydroxyarginine (NOHA) Nitric Oxide Synthase Homodimer NOS-1 nNOS or neuronal NOS NOS-3 eNOS or endothelial NOS NOS-2 iNOS or inducible NOS Constitutive and calcium sensitive Induced and calcium insensitive P450 reductase domain Heme oxidase domain Calmodulin binding NADPH FMN FAD O2 Arginine/N-hydroxyarginine (NOHA)

  21. Nitric Oxide Synthase ●In cells can exist as monomers which then are converted to dimers Nitric Oxide Synthase Tetrahydrobiopterin (BH4) Calmodulin (Cm) 130kd Arginine Reductase domain Heme oxygenase Cm BH4 BH4 Heme oxygenase BH4 BH4 Citrulline + NO Reductase domain Cm FMN FAD NOSox NADPH NOSred NADPH-dependent P450 reductase In cells can exist as monomers which then are converted to dimers

  22. NOS Biochemistry NOS Biochemistry NO Arginine Oxygen NADPH NOS NOHA O2- Control Factors Calcium Glycosylation BH4 HSP90 PZT Phosphorylation H2O2 HNO Primary Products Threshold of NO Fe(NO) Fe(II) + NO NO iNOS>> nNOS or eNOS Ki O2 Km eNOS, 25 µM; iNOS, 120 µM; nNOS, 400 µM

  23. Heme Oxygenase ● HO-1● HO-2● P450 superfamily● Numerous factors induce HO-2 ● Heme ● Biliverdin ● Bilirubin Guanylyl cyclase● p38 activation● Antioxidant N N N N N N N N Heme Oxygenase HO-1 HO-2 P450 superfamily Numerous factors induce HO-2 Heme HO M M V M V M V V + CO ? O O Guanylyl cyclase p38 activation H H H NADPH Biliverdin NADP+ M M V M V M V V Antioxidant O O H H H H Bilirubin

  24. ●Biochemistry of Prevention of Oxidative or Nitrosative stress ● Different classes ● Preventive -Inhibition of production of instigators ● Interception and Quenching -Scavenging of reactive intermediates formed 1) Want the product to be innocuous2) The product could be biologically recycled3) Prevent chain reactions from occurring (chain breaking) Biochemistry of Prevention of Oxidative or Nitrosative stress Different classes Preventive Inhibition of production of instigators Interception and Quenching Scavenging of reactive intermediates formed Want the product to be innocuous The product could be biologically recycled 3) Prevent chain reactions from occurring (chain breaking)

  25. Biochemistry Reactive Oxygen Species. XO, NOX and NOS produce O2.. XO produces H2O2. H202 can produce LOOH via the Fenton reaction. H202 can inhibit Aconitase, COX and MAPK. Peroxidases can metabolize H202 to HOCl, HOBr or NO2 leading to antimicrobial activity. Biochemistry Reactive Oxygen Species XO NOX Peroxidases O2- NOSunc NO2- Cl- Br- NO2 HOCl HOBr Fenton H2O2 Antimicrobial LOOH MAPK NFkB p53 Aconitase inhibition COX LipOX

  26. Chemical Biology of Nitric Oxide ●NO Directly inhibits respiration and P450 but stimulates guanylyl cyclase and scavenges radicals. ● NO Indirectly forms N203 leading to nitration. NO forms ONOO- and NO2 leading to oxidation and nitration. Chemical Biology of Nitric Oxide Direct NOS Radical scavenging Antioxidant properties Inhibition of Respiration Guanylyl cyclase Inhibition of P450 NO O2- Indirect +NO N2O3 NO2 ONOO- Antitumor Antipathogen Nitrosation Oxidation Nitration

  27. Preventive Mechanisms. 02. is metabolized to H202 by SOD. Catalase in the peroxisome metabolizes H202. Peroxiredoxin metabolize H202 in the cytosol, mitochondria, peroxisome and plasma. Glutathione peroxidase is a selenium containing protein in the cytosol and mitochondria. Catalase Heme protein peroxisome Peroxiredoxin (Prx) Isoforms: Prx 1-6 Contains reactive thiol 1-4 two cys one cys one cys Location Cytosol Mitochondria Peroxisome plasma Glutathione peroxidase (GPX) Selenium containing protein Cytosol and mitochondria LOOH Preventive Mechanisms O2- SOD H2O2

  28. ● Scavenging of NO (Prevention of RNOS formation). NOS produces NO which HbO2 or MbO2 converts into nitrite. Fe=O can metabolize NO into N02-. NO is consumed by the cells. ● Critical to compartmentalizing NO effects Scavenging of NO (Prevention of RNOS formation) NOS General Cellular Consumption Mechanism to be determined HbO2 MbO2 NO3- NO Fe=O Mitochondria? k > 107 M-1 s-1 NO2- Critical to compartmentalizing NO effects

  29. Biochemistry of Glutathione Peroxidase. H202, LOOH and ONOO- are metabolized by GPX to Prot-Se-OH plus H2O, LOH, or NO2-. GSH are metabolized by GPX to Prot-SeSG and H2O. ●Selenium compound Eblselen Biochemistry of Glutathione Peroxidase NADPH Glutathione reductase (GR) GPX H2O2 LOOH ONOO- GSSG Prot-Se- GSH H2O LOH NO2- Prot-Se-OH + Prot-SeSG + H2O GSH Selenium compound Eblselen

  30. Peroxiredoxin (Prx)is overexpressed in breast carcinoma, hepatocellular carcinoma, prostate, oral and lung vancer. Prx-(SH)2 is oxidized by H202 to Prx-disulfide. Thioredoxin (Trx) reduces Prx disulfide. ● Both GR and Trxred use hydride transfer to reduce the disulfides to sulfides Peroxiredoxin (Prx) SH Prx Prx (1-4) Over expressed in cancer cells Breast carcinoma Hepatocellular carcinoma Prostate Oral Lung SH H2O2 Trx(S-S) S Prx NADPH Trxred S Trx(SH)2 Trx = Thioredoxin (ER) Trxred = thioredoxin reductase (FAD) FADH2 Reduced S-S NADPH Both GR and Trxred use hydride transfer to reduce the disulfides to sulfides

  31. Reduction of a seleno-cysteine ●A selenylsulfide in a protein is reduced by a cysteine-exchange reaction and the resulting disulfide is then reduced by electron transfer. This example shows the reduction of thioredoxin (Trx) by thioredoxin reductase (TrxR).Sulfur and Selenium: The Role of Oxidation State in Protein Structure and Function.Angewandte Chemie International Edition. 42: 4742-4758. Reduction of a seleno-cysteine A selenylsulfide in a protein is reduced by a cysteine-exchange reaction and the resulting disulfide is then reduced by electron transfer. This example shows the reduction of thioredoxin (Trx) by thioredoxin reductase (TrxR).

  32. Glutathione Metabolism. GSH form GS. Which can react with O2 to form GSOO. Which can be oxidized to GSOH which reacts with GSH to form GSSG. GS. Can react with GSH to for GSSG.. GSSG. Can be oxidized to GSSG. GSSG is reduced by GR. O2 reacts with SOD to form H202 which is metabolized by GPX to H2O. Glutathione Metabolism SOD GPX H2O H2O2 O2- GSSG O2 NADPH GR GSH GSSG GSH GSH ---> GS ONOO- Oxidant OH/Fe=O NO2 GSH O2 GSSG GSOO GSOH SOD GPX H2O O2 H2O2 O2-

  33. Some two-electron reduction potentials Some two-electron reduction potentials

  34. ●Abatement of Nitrosative Stress ● Both NO and HNO toxicity is abated by GSH Abatement of Nitrosative Stress NO Both NO and HNO toxicity is abated by GSH O2 N2O3 GSH Cu(II)ZnSOD Cu(I)ZnSOD GSH + NO GSNO GSH ProteinS-SG GSSG +HNO Protein SNO +GSH GSH GSH GR GS(O)NH2 GSH GSNOH GSSG NH2OH

  35. ●Ascorbate metabolism. ● Human Plasma 0.2 mM Ascorbate Human Plasma 0.2 mM

  36. Glutathione and Ascorbate Pools Communicate. Protein disulfide isomerase (PDI) metabolizes GSH and DHA to form reduced Asc. Glutathione and Ascorbate Pools Communicate 2 GSH GSSG Protein Disulfide Isomerase (PDI) Glutaredoxin (cytosol) Trxred NADPH Y Asc AscH- DHA -0.28 +0.17 Reduce metals Increase Metal-mediated Oxidation

  37. Xanthine is metabolized to Urate and Uric Acid. ●Human Plasma 0.2-0.4 mM ●Urate and Uric Acid Urate and Uric Acid Human Plasma 0.2-0.4 mM Xanthine XD or XO OH NO2 107 Urea and HOC-CHO >109 Glyoxylic acid Oxalic acid

  38. ●Lipid Peroxidation ●Tocopherol reacts with LOO. To form TcO. and LOOH. Ascorbate or CoQH2 can react with TcO. To form TcOH. Lipid Peroxidation Tocopherol Prx Gpx LOO + TcOH TcO + LOOH DE = 0.9-0.5 = +0.4 Asc + TcOH Asc + TcO DE = 0.5-0.28 = +0.22 CoQH2 + TcO SQ + TcOH DE = 0.5-0.3 = +0.2

  39. ●Interaction of NO and ROS. NO binds to heme and inhibits Ferrochelatase and NOX. O2. interacts with RNOS resulting in GSH metabolism. O2. is metabolized to H202 which can reslut in lipid peroxidation. ● Heme and iron regulation is critical to the redox balance Interaction of NO and ROS ? CO and Bilirubin HO-1 Heme NOX Lipid Peroxidation NOS NO O2- SOD LOOH H2O2 RNOS Ferrochelatase Ox GPX Prx GSSG GPX Prx GR ROH GSH Heme and iron regulation is critical to the redox balance

  40. ●All enzymes require Oxygen and NADPH ● NADPH versus NADH ● Metabolism of iron, oxygen and glucose will be critical factors in the cellular redox state All enzymes require Oxygen and NADPH O2 HO XO NOS NOX NADPH versus NADH Metabolism of iron, oxygen and glucose will be critical factors in the cellular redox state Now the adventure begins in biology

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