Reactive Oxygen Species I:Reactive Oxygen Species I:• Definitions• Key reactions• Sources of ROS• Damage to proteins• Damage to lipids• Damage to DNA• Antioxidants• The Earth was originally anoxic• Metabolism was anaerobic• O2started appearing ~2.5 x 109years agoAnaerobic metabolism-glycolysisGlucose + 2ADP + 2PiLactate + 2ATP + 2H2OO2an electron acceptor in aerobic metabolismGlucose + 6O2+ 36ADP + 36Pi 6CO2+ 36ATP + 6H2O• Ground-state oxygen has 2-unpaired electronsO:O::::..• The unpaired electrons have parallel spins• Oxygen molecule is minimally reactive due to spin restrictionsTerm DefinitionOxidation Gain in oxygenLoss of hydrogenLoss of electronsReduction Loss of oxygenGain of hydrogenGain of electronsOxidant Oxidizes another chemical by takingelectrons, hydrogen, or by adding oxygenReductant Reduces another chemical by supplyingelectrons, hydrogen, or by removing oxygenBasics of Redox ChemistryBasics of Redox ChemistryFree RadicalsFree Radicals: Any species capable of independent existence that contains one or more unpaired electrons A molecule with an unpaired electron in an outer valence shellR3C.Carbon-centeredR3N.Nitrogen-centeredR-O.Oxygen-centeredR-S.Sulfur-centeredProoxidantsProoxidantsNon-Radicals: Species that have strong oxidizing potential Species that favor the formation of strong oxidants (e.g., transition metals)H2O2Hydrogen peroxideHOCl-Hypochlorous acidO3Ozone1O2Singlet oxygenONOO-PeroxynitriteMen+Transition metalsReactive Oxygen Species (ROS)Reactive Oxygen Species (ROS)Radicals:O2.-SuperoxideOH.HydroxylRO2.PeroxylRO.AlkoxylHO2.HydroperoxylNon-Radicals:H2O2Hydrogen peroxideHOCl-Hypochlorous acidO3Ozone1O2Singlet oxygenONOO-PeroxynitriteReactive Nitrogen Species (RNS)Reactive Nitrogen Species (RNS)Radicals:NO. Nitric OxideNO2.Nitrogen dioxideNon-Radicals:ONOO-PeroxynitriteROONO Alkyl peroxynitritesN2O3Dinitrogen trioxideN2O4Dinitrogen tetroxideHNO2Nitrous acidNO2+Nitronium anionNO-Nitroxyl anionNO+Nitrosyl cationNO2Cl Nitryl chloride““Longevity” of reactive speciesLongevity” of reactive speciesReactive Species Half-lifeHydrogen peroxideOrganic hydroperoxides ~ minutesHypohalous acidsPeroxyl radicals ~ secondsNitric oxidePeroxynitrite ~ millisecondsSuperoxide anionSinglet oxygen ~ microsecondAlcoxyl radicalsHydroxyl radical ~ nanosecond“An imbalance favoring prooxidants and/or disfavoring antioxidants, potentially leading to damage” -H. SiesAntioxidantsProoxidantsOxidative StressOxidative StressRadicalRadical--mediated reactionsmediated reactionsAdditionR.+H2C=CH2R-CH2-CH2.Hydrogen abstractionR.+LH RH+L.Electron abstractionR.+ArNH2R-+ArNH2.+TerminationR.+Y.R-YDisproportionationCH3CH2.+ CH3CH2.CH3CH3+ CH2=CH2Hydroxyl radical (Hydroxyl radical (..OH)OH)O2.-+ Fe3+O2+ Fe2+H2O2+ Fe2+OH-+ .OH + Fe3+O2.-+ H2O2OH-+ O2+ .OHHaber-WeissFenton•Transition metal catalyzed•Other reductants can make Fe2+ (e.g., GSH, ascorbate, hydroquinones)•Fe2+ is an extremely reactive oxidantImportant EnzymeImportant Enzyme--Catalyzed ReactionsCatalyzed ReactionsBiological Pathways for Oxygen ReductionBiological Pathways for Oxygen ReductionFrom: McMurry and Castellion “Fundamentals of general, organic and biological chemistry”Important physiological functions that Important physiological functions that involve free radicals or their derivatesinvolve free radicals or their derivatesFrom Droge W (2002) Physiol Rev 82: 47-95Endogenous sources of ROS and RNSEndogenous sources of ROS and RNSMitochondriaLysosomesPeroxisomesEndoplasmic ReticulumCytoplasmMicrosomal Oxidation, Flavoproteins, CYP enzymesMyeloperoxidase(phagocytes) Electron transportOxidases,FlavoproteinsPlasma MembraneLipoxygenases,Prostaglandin synthaseNADPH oxidaseXanthine Oxidase,NOS isoformsFeCuTransition metalsMitochondria as a source of ROSMitochondria as a source of ROSTurrens, J Physiol, 2003Localization of the main mitochondrial sources of superoxide anionMitochondrial electron chainQuinone cycleChandel & Budinger, Free Radical Biol Med, 2007Peroxisomes as a source of ROS and RNSPeroxisomes as a source of ROS and RNSFatty AcidAcyl-CoAEnoyl-CoAHydroxyacyl-CoAKetoacyl-CoAAcetyl-CoA Acyl-CoA shortened by two carbonsFatty acyl-CoA synthetaseAcyl-CoA oxidaseEnoyl-CoA hydrolaseHydroxyacyl-CoAdehydrogenaseThiolaseH2O2Fatty AcidAcyl-CoAEnoyl-CoAHydroxyacyl-CoAKetoacyl-CoAAcetyl-CoA Acyl-CoA shortened by two carbonsFatty acyl-CoA synthetaseAcyl-CoA oxidaseEnoyl-CoA hydrolaseHydroxyacyl-CoAdehydrogenaseThiolaseH2O2Enzymes in mammalian peroxisomes that generate ROSSchader & Fahimi, Histochem Cell Biol, 2004NADPH oxidase as a source of ROSNADPH oxidase as a source of ROSPresent mainly in neutrophils (oxidative burst), but also in many other cell typesPHSProstaglandin H Prostaglandin H SynthaseSynthase(PHS) as a source of ROS(PHS) as a source of ROSCo-oxidation of xenobiotics (X) during arachidonic acid metabolism to PGH2xanthine oxidase xanthine oxidaseCytoplasmicCytoplasmicsources of ROS and RNSsources of ROS and RNSNO•Nitric Oxide Synthases (NOS):neuronal nNOS (I)endothelial eNOS (III)inducible iNOS (II)LysosomeLysosomeas a source of ROS and RNSas a source of ROS and RNSMyeloperoxidase undergoes a complex array of redox transformations and produces HOCl, degrades H2O2to oxygen and water, converts tyrosine and other phenols and anilines to free radicals, and hydroxylates aromatic substrates via a cytochrome P450-like activityMicrosomes as a source of ROS (I)Microsomes as a source of ROS (I)A scheme of the catalytic cycle of cytochrome P450-containing monooxygenases. The binding of the substrate (RH) to ferric P450 (a) results in the formation of the substrate complex (b). The ferric P450 then accepts the first electron from CPR (cytochrome P450 reductase), thereby being reduced to the ferrous intermediate (c). This intermediate then binds an oxygen molecule to form oxycomplex (d), which is further reduced to give peroxycomplex (e). The input of protons to this intermediate can result in the heterolytic cleavage of the O–O bond, producing H2O and the ‘oxenoid’ complex (f), the latter of which then inserts the heme-bound activated oxygen atom into the substrate molecule to produce ROH. In eukaryotic monooxygenases, reactive oxygen species (ROS) are produced by ‘leaky’ branches (red arrows). In one such branch, a superoxide anion radical is released owing to the decay of the one-electron-reduced ternary complex (d). The second ROS-producing branch
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