Free Radicals - Lecture 3 PDF

Endogenous Sources of ROS and RNS 1. Mitochondria (electron transport chain) 2. Cytochrome P450 3. Peroxisome 4. Phagocytosis (NADPH oxidase, myeloperoxidase) 5. Nitric oxide synthase 6. Xanthine oxidase 7. Non enzymatic (transition metals) 1- Electron transport chain ▪ Inner mitochondrial membr...

Endogenous Sources of ROS and RNS 1. Mitochondria (electron transport chain) 2. Cytochrome P450 3. Peroxisome 4. Phagocytosis (NADPH oxidase, myeloperoxidase) 5. Nitric oxide synthase 6. Xanthine oxidase 7. Non enzymatic (transition metals) 1- Electron transport chain ▪ Inner mitochondrial membrane ▪ The final stage of aerobic respiration is known as oxidative phosphorylation ▪ Electron transport chain is the transfer of electrons from NADH and FADH2 to oxygen via multiple carriers ▪ The reduction of O2 to water by the mitochondrial electron transport chain ▪ This process require - oxygen ( To accept the electrons and hydrogen at the end) - Reduced NAD+ and FAD which are carrying hydrogen - Electron carriers ▪ The electrons that transferred from NADH and FADH2 to the ETC involves 4 multi-subunit large enzymes complexes and 2 mobile electron carriers Components of ETC 1. Enzyme complex I, NADH dehydrogenase 2. Enzyme complex II, succinate dehydrogenase 3. Enzyme complex III, cytochrome bc1 reductase 4. Enzyme complex IV, cytochrome c oxidase - ATP synthase :- synthesis of ATP from ADP Two mobile carrier ▪ These are connected by two mobile carrier coenzyme Q and cytochrome c - Coenzyme Q (ubiquinone) connects between either Complex I or Complex II to Complex III - Cytochrome c connects complex III and IV ▪ Electron flow from more electronegative to electropositive components ▪ NADH passes its electron on to complex 1 – NADH dehydrogenase ▪ FADH2 passes its electron to complex 2- succinate dehydrogenase ▪ In complex IV, when a total of four electrons are transferred to oxygen, two water molecules are formed ▪ Some electrons can “escape” the electron transport chain and combine with oxygen to form a very unstable form of oxygen called a superoxide radical (O2 -) ▪ The electrons derived from NADH and FADH2 combine with O2 , resulting in the generation of reactive oxygen species. ▪ Mitochondrial electron transport chain complex I, II, III take part in O2-. ▪ Leakage of electrons at complex I and complex III from electron transport chains leads to partial reduction of oxygen to form superoxide. Subsequently, superoxide is quickly dismutated to hydrogen peroxide by SOD1 and SOD2 ▪ Products of partial reduction of oxygen are highly reactive and damage the living tissue - sometimes the levels of superoxide rise, for example after alcohol exposure (which generates a lot of NADH). - Thus, more hydrogen peroxide is formed and can’t be detoxified by the limited amount of catalase. - Instead hydrogen peroxide becomes reduced by iron (Fe2+), which donates an electron to produce the hydroxyl radical ( OH) - It is extremely reactive and a great oxidizing agent. 2- Cytochrome P450 ▪ Cytochrome P450 is a superfamily of heme enzyme can catalyze different reaction types, mainly hydroxylation. (P450 = absorbs a very characteristic wavelength (450 nm) of UV light when it is exposed to carbon monoxide) Located in the smooth endoplasmic reticulum of all major organs and tissues especially liver ▪ Use NADPH as a source of reducing equivalent ▪ Acting on both xenobiotics and endogenous compounds methene ▪ CYP enzymes catalyze the oxygenation of an organic substrate and the simultaneous reduction of molecular oxygen. ▪ If the transfer of oxygen to a substrate is not tightly controlled, uncoupling occurs and leads to the formation of reactive oxygen species ▪ In this oxidation – reduction process, two microsomal enzymes play a key role. ▪ The first enzyme:-A heme protein known as cytochrome p450, it is a terminal oxidase and plays the important role of transferring an oxygen atom to the substrate RH and convert it to ROH ▪ The second enzyme, the flavoprotein known as cytochrome p450 reductase which is Fe+3 NADPH dependent. It function as an electron carrier catalyzing the reduction of cytochrome p450 to the ferrous form by cytochrome p450 reductase transferring an electron from NADPH Cinnamate 4-hydroxylase It is a cytochrome P450 monooxygenase associated externally with the endoplasmic reticulum of plant cells. The enzyme uses NADPH-cytochrome P450 reductase as a donor of electrons and hydroxylates cinnamic acid to form 4-coumaric acid p-Coumaric acid serves as a precursor of organic compounds that are essential for plant metabolism including flavonoids and lignin cinnamic acid 4-coumaric acid ▪ Superoxide is produced by microsomal NAD(P)H dependent electron transport involving cytochrome P450 1. Cytochrome P450 reacts first with its organic substrate, RH 2. The complex is oxidized by a flavoprotein to form a radical intermediate 3. They can readily react with triplet oxygen because each has one unpaired electron. 4. This oxygenated complex may be reduced by cytochrome b or occasionally the complex may decompose releasing superoxide 3- Electron transport system with Microbodies ▪ A microbody is a type of organelles that is found in the cells of plants and animals ▪ They are two types of microbodies namely: 1. Peroxisome 2. Glyoxysome ▪ Peroxisomes and glyoxysomes are organelles with a single membrane that compartmentalizes enzymes involved in the ß-oxidation of fatty acids, and the glyoxylate acid cycle (TCA cycle) ▪ Glycolate oxidase produces H2O2 in a two-electron transfer from glycolate to oxygen ▪ Xanthine oxidase, urate oxidase and NADPH oxidase generate superoxide because of the oxidation of their substrates Peroxisome ▪ These also called microbodies are organelles found in all eukaryotic cells called peroxisome because of their ability to produce or utilize hydrogen peroxide ▪ They are small, oval or spherical in shape. They have a fine network of tubules in their matrix ▪ About 50 enzymes have been identified Glyoxysome ▪ These are found in plant ▪ They convert stored lipid into carbohydrates so they can be used for plant growth. ▪ In glyoxysome, the fatty acids are hydrolyzed to acetyl-coA by oxidation enzymes. ▪ One of the reactions catalyzed by using the coenzyme FMN involves the enzyme glycolate oxidase. ▪ Glycolate oxidase is a peroxisomal enzyme. ▪ It catalyzes the oxidation of alpha-hydroxy acids. It is one of the key enzymes in photorespiration, where it oxidizes glycolate to glyoxylate. ▪ This reaction can be divided into to main steps. First, glycolate is oxidized to glyoxylate by FMN in a two-electron transfer. Then, the reduced FMN is reoxidized by oxygen and peroxide is formed ▪ Xanthine oxidase (XO) is a form of xanthine oxidoreductase (XOR) catalyzes oxidative hydroxylation of hypoxanthine to xanthine to uric acid, accompanying the production of reactive oxygen species (ROS) x x ▪ NADPH oxidase catalyze the transfer of electron to O2 generating superoxide or H2O2 using NADPH as an electron donor. NADPH oxidase NADPH + 2O2 NADP+ + H+ + 2O2 − 4- During inflammation ▪ The tissue damage produced free radicals has to contribute to the injury process ▪ O2 − is generated by one-electron reduction of O2 through enzymatic catalysis by NADPH oxidase or xanthine oxidase (XO) ▪ H2O2 can change to highly reactive HOCl at the inflammatory sites by an enzyme known as myeloperoxidase (MPO), which is abundantly expressed in neutrophils ▪ H2O2 can also change to the highly toxic OH in presence of Fe2+ by Fenton's reaction Reaction ▪ In addition, metal containing protein (Hb) released from lysed erythrocyte at sites of inflammation. Metal containing in these proteins can react with free radical and ROS through a series of reactions called Haber – Weiss ▪ In the presence of the transition metal ion, O2 − and H2O2, in turn, generate the highly reactive OH− and OH (Haber–Weiss reaction) ▪ In the first step of this reaction, O2 − reacts with Fe3+ to form Fe2+ and O2. ▪ The second step of this reaction is also known as Fenton's reaction and occurs under the biological conditions in which Fe2+ reacts with H2O2 to form both OH and OH−