Reactive Oxygen Species I History Definitions Key reactions Sources of ROS Damage to proteins Damage to lipids Damage to DNA Antioxidants The Earth was originally anoxic Metabolism was anaerobic O2 started appearing 2 5 x 109 years ago Anaerobic metabolism glycolysis Glucose 2ADP 2Pi Lactate 2ATP 2H2O O2 an electron acceptor in aerobic metabolism Glucose 6O2 36ADP 36Pi 6CO2 36ATP 6H2O Ground state oxygen has 2 unpaired electrons O O The unpaired electrons have parallel spins Oxygen molecule is minimally reactive due to spin restrictions Before 1950s golden age of chemistry Many free radicals have been discovered and described Haber Weiss 1934 Superoxide Hydrogen peroxide Baeyer Villiger 1901 discovered peroxynitrite Hydroxyl radical Early 1950s presence of radicals in biological materials Commoner et al 1954 Nature 174 689 691 1956 Radicals may be formed as by products of enzymatic reactions in vivo Harman 1956 radicals are pandora s box of evils aging mutations cancer 1969 Discovery of superoxide dismutase SOD McCord Fridovich 1969 JBC 244 6049 6055 Free radicals must be important for biology if the body has a defense enzyme s Intensive investigations on the role of oxidants in disease begun Since late 70s Radicals are important for normal biology Mittal Murad 1977 superoxide anion stimulates formation of cGMP Ignarro Kadowitz 1985 and Moncada 1987 NO is important in circulation Basics of Redox Chemistry Term Definition Oxidation Gain in oxygen Loss of hydrogen Loss of electrons Reduction Loss of oxygen Gain of hydrogen Gain of electrons Oxidant Oxidizes another chemical by taking electrons hydrogen or by adding oxygen Reductant Reduces another chemical by supplying electrons hydrogen or by removing oxygen Prooxidants Free Radicals Radicals Any species capable of independent existence that contains one or more unpaired electrons A molecule with an unpaired electron in an outer valence shell Non Radicals Species that have strong oxidizing potential Species that favor the formation of strong oxidants e g transition metals R3C R3N R O R S Carbon centered Nitrogen centered Oxygen centered Sulfur centered H2O2 Hydrogen peroxide HOCl Hypochlorous acid O3 Ozone 1O Singlet oxygen 2 ONOO Peroxynitrite Men Transition metals Reactive Oxygen Species ROS Radicals Non Radicals O2 OH RO2 RO HO2 H2O2 HOClO3 1O 2 ONOO Superoxide Hydroxyl Peroxyl Alkoxyl Hydroperoxyl Hydrogen peroxide Hypochlorous acid Ozone Singlet oxygen Peroxynitrite Reactive Nitrogen Species RNS Radicals NO Nitric Oxide NO2 Nitrogen dioxide Non Radicals ONOOPeroxynitrite ROONO Alkyl peroxynitrites N2O3 Dinitrogen trioxide N2O4 Dinitrogen tetroxide HNO2 Nitrous acid NO2 Nitronium anion NONitroxyl anion NO Nitrosyl cation NO2Cl Nitryl chloride Longevity of reactive species Reactive Species Half life Hydrogen peroxide Organic hydroperoxides Hypohalous acids minutes Peroxyl radicals Nitric oxide seconds Peroxynitrite milliseconds Superoxide anion Singlet oxygen Alcoxyl radicals microsecond Hydroxyl radical nanosecond Oxidative Stress Antioxidants Prooxidants An imbalance favoring prooxidants and or disfavoring antioxidants potentially leading to damage H Sies Radical mediated reactions Addition R H2C CH2 R CH2 CH2 Hydrogen abstraction R LH RH L Electron abstraction R ArNH2 R ArNH2 Termination R Y R Y Disproportionation CH3CH2 CH3CH2 CH3CH3 CH2 CH2 Hydroxyl radical OH O2 Fe3 O2 Fe2 Fenton H2O2 Fe2 OH OH Fe3 Haber Weiss O2 H2O2 OH O2 OH Transition metal catalyzed Other reductants can make Fe2 e g GSH ascorbate hydroquinones Fe2 is an extremely reactive oxidant Important Enzyme Catalyzed Reactions From McMurry and Castellion Fundamentals of general organic and biological chemistry Biological Pathways for Oxygen Reduction Important physiological functions that involve free radicals or their derivates From Droge W 2002 Physiol Rev 82 47 95 Endogenous sources of ROS and RNS Microsomal Oxidation Flavoproteins CYP enzymes Xanthine Oxidase NOS isoforms Myeloperoxidase phagocytes Transition metals Endoplasmic Reticulum Cytoplasm Lysosomes Fe Cu Oxidases Flavoproteins Peroxisomes Mitochondria Plasma Membrane Lipoxygenases Prostaglandin synthase NADPH oxidase Electron transport Mitochondria as a source of ROS Turrens J Physiol 2003 Peroxisomes as a source of ROS and RNS Fatty Acid Fatty acyl CoA synthetase Acyl CoA H2O2 Acyl CoA oxidase Enoyl CoA Enoyl CoA hydrolase Hydroxyacyl CoA Hydroxyacyl CoA dehydrogenase Ketoacyl CoA Thiolase Acetyl CoA Acyl CoA shortened by two carbons Enzymes in mammalian peroxisomes that generate ROS Schader Fahimi Histochem Cell Biol 2004 NADPH oxidase as a source of ROS Present mainly in neutrophils oxidative burst but also in many other cell types Prostaglandin H Synthase PHS as a source of ROS Co oxidation of xenobiotics X during arachidonic acid metabolism to PGH2 PHS Cytoplasmic sources of ROS and RNS xanthine oxidase xanthine oxidase Nitric Oxide Synthases NOS neuronal nNOS I endothelial eNOS III inducible iNOS II NO Lysosome as a source of ROS and RNS Myeloperoxidase undergoes a complex array of redox transformations and produces HOCl degrades H2O2 to oxygen and water converts tyrosine and other phenols and anilines to free radicals and hydroxylates aromatic substrates via a cytochrome P450 like activity 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 is the protonation of the peroxycytochrome P450 e which forms of H2O2 In addition to these ROS producing branches another mechanism of electron leakage appears to be the fourelectron reduction of the oxygen molecule with the production of water Davydov