‹#› 1 Respiratory chain ~ Reactive oxygen species © Department of Biochemistry, MU Brno (J.D.) 2013 ‹#› 2 Transformation of energy in human body chemical energy of nutrients = work + heat energy of nutrients = BM + phys. activity + reserves + heat energy input energy output BM = basal metabolism reserve = adipose tissue, glycogen any work requires ATP chemical: synthesis of proteins, urea ... osmotic: transport of ions against gradient ... mechanical: muscle contraction ... ‹#› 3 chemical energy of nutrients heat NADH+H+ FADH2 proton gradient across IMM heat heat ATP 1 2 3 1 ... metabolic dehydrogenations with NAD+ and FAD 2 ... respiratory chain (oxidation of reduced cofactors + reduction of O2 to H2O) 3 ... oxidative phosphorylation, IMM inner mitochondrial membrane 4 ... transformation of chemical energy of ATP into work + some heat ¢... high energy systems Energy transformations in the human body are accompanied with continuous production of heat work 4 ‹#› 4 Nutrient Energy (kJ/g) Thermogenesis Energy supply/day Lipids 38 4 % 30 % SAFA 5 %, MUFA 20 %, PUFA 5 % Saccharides 17 6 % 60 % Proteins 17 30 % 10 % Energetic data about nutrients ‹#› 5 glucose: 6.7 % H average ox. num. of C = 0.0 alanine: 7.9 % H average ox. num. of C = 0.0 stearic acid: 12.8 % H average ox. num. of C = -1.8 Þ C is the most reduced Oxidation numbers of carbon and the content of hydrogen in nutrients ‹#› 6 Two ways of ATP formation in body 1. Oxidative phosphorylation in the presence of O2 (~ 95 % ATP) ADP + Pi + energy of H+gradient ® ATP 2. Substrate-level phosphorylation (~ 5 % ATP) ADP + macroergic phosphate-P ® ATP + second product Compare: Phosphorylation substrate-OH + ATP ® substrate-O-P + ADP (e.g. phosphorylation of glucose, proteins, etc., catalyze kinases) higher energy content than ATP ‹#› 7 Distinguish Process ATP is Oxidative phosphorylation produced Substrate-level phosphorylation produced Phosphorylation of a substrate consumed ! ‹#› 8 Substrate level phosphorylation •phosphorylation of ADP (GDP) is performed by the high-energy intermediates •succinyl-CoA (CAC) •1,3-bisphosphoglycerate (glycolysis) •phosphoenolpyruvate (glycolysis) • ‹#› 9 Phosphorylation of GDP in citrate cycle guanosine diphosphate ATP succinate guanosine triphosphate succinyl-CoA succinyl phosphate is made from succinyl-CoA + Pi ‹#› 10 Phosphorylation of ADP by 1,3-bisphosphoglycerate 1,3-bisP-glycerate 3-P-glycerate ‹#› 11 Phosphorylation of ADP by phosphoenolpyruvate pyruvate enolpyruvate phosphoenolpyruvate ‹#› 12 Aerobic phosphorylation follows the reoxidation of reduced cofactors in R.CH. Nutrients (reduced forms of C) CO2 + reduced cofactors (NADH+H+, FADH2) reoxidation in R.CH. dehydrogenation O2 Proton gradient + H2O ADP + Pi ® ATP decarboxylation ‹#› 13 NADH formation in mitochondrial matrix (substrates of the important reactions) •Citrate cycle isocitrate 2-oxoglutarate malate •b-oxidation of FA b-hydroxyacyl-CoA • •Oxidative decarboxylation pyruvate 2-oxoglutarate 2-oxo acids from Val, Leu, Ile •Dehydrogenation of KB b-hydroxybutyrate •Oxidative deamination glutamate • ‹#› 14 •Glycolysis (dehydrogenation of glyceraldehyde-3-P) •Gluconeogenesis (dehydrogenation of lactate to pyruvate) •Dehydrogenation of ethanol (to acetaldehyde) • NADH formation in cytoplasm ‹#› 15 FADH2 formation in matrix of mitochondria •b-Oxidation of FA (dehydrogenation of saturated acyl-CoA) •Citrate cycle (dehydrogenation of succinate) ‹#› 16 Transport of NADH from cytoplasm to matrix •NADH produced in cytoplasm is transported into matrix to be reoxidized in R.CH. •inner mitochondrial membrane is impermeable for NADH •two shuttle systems: •aspartate/malate shuttle (universal) •glycerol phosphate shuttle (brain, kidney) • ‹#› 17 Aspartate/malate shuttle malate malate oxaloacetate Inner mitochondrial membrane oxaloacetate cytoplasm hydrogenation dehydrogenation transamination MD malate dehydrogenase AST aspartate aminotransferase ‹#› 18 Glycerol phosphate shuttle glycerol 3-P dihydroxyacetone phosphate Inner mitochondrial membrane GPD = glycerol 3-P dehydrogenase ubiquinol ubiquinone ‹#› 19 Respiratory chain is the system of redox reactions in IMM which starts by the NADH oxidation and ends with the reduction of O2 to water. The free energy of oxidation of NADH/FADH2 is utilized for pumping protons to the outside of the inner mitochondrial membrane. The proton gradient across the inner mitochondrial membrane represents the energy for ATP synthesis. H+ H+ H+ H+ – – – + + + ADP + Pi ATP NADH+H+ O2 2H2O NAD+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ e– proton gradient Matrix (negative) IMS (positive) ‹#› 20 Four types of cofactors in R.CH. •flavine cofactors (FMN, FAD) •non-heme iron with sulfur (Fe-S) •ubiquinone (Q) •heme (in cytochromes) Distinguish: heme (cyclic tetrapyrrol) × cytochrome (hemoprotein) ‹#› 21 Coenzyme FMN (as well as FAD) transfers two atoms of hydrogen. N N N NH O O H3C N N N N O O H H + 2 H – 2 H FMN oxidized form FMNH2 reduced form H3C H3C H3C CH2–O– P CH2 H–C–OH H–C–OH H–C–OH CH2–O– P CH2 H–C–OH H–C–OH H–C–OH Flavoproteins contain flavin prosthetic group as flavin mononucleotide (FMN, complex I) or flavin adenine dinucleotide (FAD, complex II): ‹#› 22 Iron-sulfur proteins (FeS-proteins, non-heme iron proteins) Despite the different number of iron atoms present, each cluster accepts or donates only one electron. Fe2S2 cluster Fe4S4 cluster ‹#› 23 R = –(CH2–CH=C–CH2)10-H CH3 It accepts stepwise two electrons (one from the complex I or II and the second from the cytochrome b) and two protons (from the mitochondrial matrix), so that it is fully reduced to ubiquinol: + e + H+ ubiquinone, Q semiquinone, •QH ubiquinol, QH2 + e + H+ Ubiquinone (coenzyme Q) The very lipophilic polyisoprenoid chain is anchored within phospholipid bilayer. The ring of ubiquinone or ubiquinol (not semiquinone) moves from the membrane matrix side to the cytosolic side and translocates electrons and protons. ‹#› 24 Cytochromes are heme proteins, which are one-electron carriers due to reversible oxidation of the iron atom: Mammalian cytochromes are of three types – a, b, c. They differ in the substituents attached to the porphin ring. All these types of cytochromes occur in the mitochondrial respiratory chain. Cytochromes type b (including cytochromes class P-450) occur also in membranes of endoplasmic reticulum and the outer mitochondrial membrane. N N N N F e 2+ N N N N F e 3+ + e– – e– ‹#› 25 Heme a of cytochrome aa3 Heme of cytochrome c Some differences in cytochrome structures Cytochrome c central Fe ion is attached by coordination to N-atom of His18 and to S-atom of Met80; two vinyl groups bind covalently S-atoms of Cys14+Cys17. The heme is dived deeply in the protein terciary structure so that it is unable to bind O2, CO. Cyt c is water-soluble, peripheral protein that moves on the outer side of the inner mitochondrial membrane. Cytochrome aa3 central Fe ion is attached by coordination to two His residues; one of substituents is a hydrophobic isoprenoid chain, another one is oxidized to formyl. The heme a is the accepts an electrons from the copper centre A (two atoms CuA). Its function is inhibited by carbon monoxide, CN–, HS–, and N3– anions. ‹#› 26 Redox pairs in the respiratory chain Oxidized / Reduced form E°´(V) NAD+ / NADH+H+ FAD / FADH2 Ubiquinone (Q) / Ubiquinol (QH2) Cytochrome c1 (Fe3+ / Fe2+) Cytochrome c (Fe3+ / Fe2+) Cytochrome a3 (Fe3+ / Fe2+) O2 / 2 H2O -0,32 0,00 0,10 0,22 0,24 0,39 0,82 ‹#› 27 •redox pairs are listed according to increasing E°´ •they are standard values (1 mol/l), real cell values are different •the strongest reducing agent in R.CH. is NADH •the strongest oxidizing agent in R.CH. is O2 •the value of potential depends on protein molecule (compare cytochromes) ‹#› 28 Entry points for reducing equivalents in R.Ch. I. acyl-CoA enoyl-CoA succinate fumarate cytoplasm shuttle beta-oxidation pyruvate, CAC, KB CAC ‹#› 29 Enzyme complexes in respiratory chain No. Name Cofactors Oxidation Reduction I. NADH-Q oxidoreductase* FMN, Fe-S NADH ® NAD+ Q ® QH2 II. succinate-Q reductase FAD,Fe-S,cyt b FADH2 ® FAD Q ® QH2 III. Q-cytochrome-c-reductase Fe-S, cyt b, c1 QH2 ® Q cyt cox® cyt cred IV. cytochrome-c-oxidase cyt a, a3, Cu cyt cred ® cyt cox O2 ® 2 H2O * also called NADH dehydrogenase ‹#› 30 NADH+H+ + Q + 4 H+matrix ® NAD+ + QH2 + 4 H+ims Complex I oxidizes NADH and reduces ubiquinone (Q) FMN, Fe-S The four H+ are translocated from matrix to intermembrane space (ims) ‹#› 31 Complex I oxidizes NADH and translocates 4 H+ into intermembrane space VMM 2 H+ I. 2 H+ 2 H+ intermembrane space IMM ‹#› 32 Complex II (independent entry) oxidizes FADH2 from citrate cycle and reduces ubiquinone matrix VMM II. succinate fumarate IMM Complex II, in contrast to complex I, does not transport H+ across IMM. Consequently, less proton gradient (and less ATP) is formed from the oxidation of FADH2 than from NADH. ‹#› 33 Complex III oxidizes QH2, reduces cytochrome c, and translocates 4 H+ across IMM matrix VMM mezimembránový prostor 2 H+ 2 H+ III. 4 H+ IMM intermembrane space Q-cycle ‹#› 34 Complex IV oxidizes cyt cred and two electrons reduce monooxygen (½O2) VMM ? IV. 2 H+ cyt cred IMM intermembrane space ‹#› 35 Complex IV: real process is four-electrone reduction of dioxygen complete reaction: 4 cyt-Fe2+ + O2 + 8 H+matrix ® 4 cyt-Fe3+ + 2 H2O + 4 H+ims partial reaction (redox pair): O2 + 4 e- + 4 H+ ® 2 H2O For every 2 electrons, 2 H+ are pumped into intermembrane space metabolic water ‹#› 36 Three times translocated protons create electrochemical H+ gradient across IMM It consists of two components: 1) difference in pH, ΔG = RT ln ([H+]out /[H+]in) = 2.3 RT(pHout – pHin) 2) difference in electric potential (ΔY, negative inside), depends not only on protons, but also on concentrations of other ions, ΔG = – nFΔY The proton motive force Δ p is the quantity expressed in the term of potential (milivolts per mole of H+ transferred): Δ p = – ΔG / nF = ΔY + 60 Δ pH . Utilization of proton motive force • synthesis of ATP = aerobic phosphorylation • heat production - especially in brown adipose tissue • active transport of ions and metabolites across IMM ‹#› 37 H+ H+ H+ H+ – – – + + + ADP + Pi ATP NADH+H+ O2 2H2O NAD+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ e– Terminal respiratory chain Electrochemical gradient matrix (negative) IMS (positive) Aerobic phosphorylation of ADP by ATP synthase The endergonic phosphorylation of ADP is driven by the flux of protons back into the matrix along the electrochemical gradient through ATP synthase. ‹#› 38 ATP synthase consists of three parts F1 head FO segment connecting section 1) F1 complex projects into the matrix, 5 subunit types (a3, b3, g, d, e) catalyze the ATP synthesis 2) connecting section 3) Fo inner membrane component, several c-units form a rotating proton channel ‹#› 39 ȱÀ ATP-synthase is a molecular rotating motor: 3 ATP/turn H+ H+ H+ H+ H+ H+ H+ H+ H+ Fo rotates F1 does not rotate ADP + Pi ® ATP a,b,δ subunits hinder rotation of F1 a α β ‹#› 40 Stoichiometry of ATP synthesis is not exactly recognized •transfer of 2 e- from NADH to ½ O2 .... 3 ATP •transfer of 2 e- from FADH2 to ½ O2 .... 2 ATP new research data indicate somewhat lower values (see Harper) •transfer of 2 e- from NADH to ½ O2 .... 2.5 ATP •transfer of 2 e- from FADH2 to ½ O2 .... 1.5 ATP ‹#› 41 Control of the oxidative phosphorylation Production of ATP is strictly coordinated so that ATP is never produced more rapidly than necessary. Synthesis of ATP depends on: – supply of substrates (mainly NADH+H+) – supply of dioxygen – the energy output of the cell; hydrolysis of ATP increases the concentration of ADP in the matrix, which activates ATP production. This mechanism is called respiratory control. ‹#› 42 Inhibitors of the terminal respiratory chain Complex I is blocked by an insecticide rotenone. A limited synthesis of ATP exists due to electrons donated to ubiquinone through complex II. Complex III is inhibited by antimycin A – complexes I and II become reduced, complexes III and IV remain oxidized. Ascorbate restores respiration, because it reduces cyt c. Complex IV is blocked by carbon monoxide, cyanide ion, HS– (sulfane intoxication), azide ion N3–. Respiration is disabled. CN–, CO HS–, N3– rotenone amobarbital antimycin A ascorbate ‹#› 43 occurs after ingestion of alkali cyanides or inhalation of hydrogen cyanide. Bitter almonds or apricot kernels contain amygdalin, which can release HCN. Cyanide ion, besides inhibition of cytochrome c oxidase, binds with high affinity onto methemoglobin (Fe3+). The lethal dose LD50 of alkali cyanide is about 250 mg. Symptoms - dizziness, gasping for breath, cramps, and unconsciousness follow rapidly. Antidotes may be effective, when applied without any delay: Hydroxycobalamin (a semisynthetic compound) exhibits high affinity to CN– ions, binds them in the form of harmless cyanocobalamin (B12). Sodium nitrite NaNO2 or amyl nitrite oxidize hemoglobin (FeII) to methemoglobin (FeIII), which is not able to transport oxygen, but binds CN– and may so prevent inhibition of cytochrome c oxidase. Sodium thiosulfate Na2S2O3, administered intravenously, can convert cyanide to the relatively harmless thiocyanate ion: CN– + S2O32– ® SCN– + SO32– . Cyanide poisoning Carbon monoxide poisoning CO binds primarily to hemoglobin (FeII) and inhibits oxygen transport, but it also blocks the respiratory chain by inhibiting cytochrome oxidase (complex IV). Oxygenotherapy improves blood oxygen transport, administered methylene blue serves as acceptor of electrons from complex III so that limited ATP synthesis can continue. ‹#› 44 Uncoupling of the respiratory chain and phosphorylation is the wasteful oxidation of substrates without concomitant ATP synthesis: protons are pumped across the membrane, but they re-enter the matrix using some other way than that represented by ATP synthase. The free energy derived from oxidation of substrates appears as heat.. DNP is very toxic, the lethal dose is about 1 g. More than 80 years ago, the long-term application of small doses (2.5 mg/kg) was recommended as a "reliable“ drug in patients seeking to lose weight. Its use has been banned, because hyperthermia and toxic side effect (with fatal results) were excessive. 2 Ionophors that do not disturb the chemical potential of protons, but diminish the electric potential ΔY by enabling free re-entry of K+ (e.g. valinomycin) or both K+ and Na+ (e.g. gramicidin A). 3 Inhibitors of ATP synthase – oligomycin. 4 Inhibitors of ATP/ADP translocase like unusual plant and mould toxins bongkrekic acid (irreversibly binds ADP onto the translocase) and atractylate (inhibits binding of ATP to the translocase). ATP synthase then lacks its substrate. There are four types of artificial or natural uncouplers: 1 "True“ uncouplers – compounds that transfer protons through the membrane. A typical uncoupler is 2,4-dinitrophenol (DNP): ‹#› 45 •is a inner mitochondrial membrane protein that transports protons back into the matrix, bypassing so ATP synthase. •It occurs in brown adipose tissue of newborn children and hibernating animals, which spend the winter in a dormant state. Thermogenin is a natural uncoupler H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ NADH+H+ 2H2O H+ H+ H+ H+ H+ H+ H+ H+ H+ O2 NAD+ e– ‹#› 46 The outer membrane is quite permeable for small molecules and ions – it contains many copies of mitochondrial porin (voltage-dependent anion channel, VDAC). The inner membrane is intrinsically impermeable to nearly all ions and polar molecules, but there are many specific transporters which shuttles metabolites (e.g. pyruvate, malate, citrate, ATP) and protons (terminal respiratory chain and ATP synthase) across the membrane. (the C side and the opposite M side) Mitochondrial metabolite transport ‹#› 47 Transport through the inner mitochondrial membrane Primary active H+ transport forms the proton motive force (the primary gradient) Free diffusion of O2, CO2, H2O, NH3 cytosolic side positive matrix side negative Ca2+ pyruvate OH– ADP3– ATP4– OH– malate succinate citrate isocitrate aspartate H2PO4– HPO42– malate malate Secondary active transports driven by a H+ gradient and dissipating it: ATP/ADP translocase pyruvate transporter phosphate permease – forms a (secondary) phosphate gradient dicarboxylate carrier tricarboxylate carrier the malate shuttle for NADH + H+ ‹#› 48 Mitochondria and apoptosis •apoptosis is a controlled process of cell death with minimal effect on surrounding tissue •apoptosis is important for physiological tissue turnover •apoptosis is regulated by a number of cell signals •regulatory protein family Bcl-2 (B-cell lymphoma 2) •some proteins are anti-apoptotic (Bcl-xl), other pro-apoptotic (Bax, Bak) •Bax and Bak proteins oligomerize and make a pore in outer mitochondrial membrane •cytochrome c is released into cytosol, binds with inactive caspases and other pro-apoptotic factors - creates apoptosome - and triggers the executive phase of apoptosis (caspase cascade) ‹#› 49 •about 98 % of O2 is consumed in respiratory chain for the complete reduction to water (cytochrome-c-oxidase) •however, other partly reduced oxygen species are also produced •they are called reactive oxygen species (ROS) •mainly in compl. I, III, especially, if electron trasport is slowned down or reversed •mitochondria contains a number of antioxidants (GSH, QH2, superoxide dismutase) • Mitochondria and oxidative stress respiratory chain cyt c ROS mitochondrial dysfunctions apoptosis necrosis lipoperoxidation of OMM diseases, ageing mutation of mtDNA defective proteins release of cytochrome c ‹#› 50 Reactive oxygen species in human body Radicals Neutral / Anion / Cation Superoxide ·O2- Hydroxyl radical ·OH Peroxyl radical* ROO· Alkoxyl radical RO· Hydroperoxyl radical HOO· Nitric oxide NO· Hydrogen peroxide H-O-O-H Hydroperoxide* R-O-O-H Hypochlorous acid HClO Singlet oxygen 1O2 Peroxynitrite ONOO- Nitronium NO2+ * Typically phospholipid-PUFA derivatives during lipoperoxidation: PUFA-OO·, PUFA-OOH ‹#› 51 Superoxide anion-radical •O2- •One-electrone reduction of dioxygen • • O2 + e- ® •O2- [one redox pair] ‹#› 52 Superoxide formation •Respiratory burst (in neutrophils) 2 O2 + NADPH ¾® 2 •O2- + NADP+ + H+ •Spontaneous oxidation of heme proteins heme-Fe2+ + O2 ¾® heme-Fe3+ + •O2- [complete redox reactions, combinations of two redox pairs] ‹#› 53 Radical •OH is the most reactive species; it is formed from superoxide and hydrogen peroxide •O2- + H2O2 ® O2 + OH- + •OH Catalyzed by reduced metal ions (Fe2+, Cu+) (Fenton reaction) ‹#› 54 Singlet oxygen 1O2 •excited form of triplet dioxygen •formed after absorption of light by some compounds (porphyrins) 3O2 ® 1O2 for electron configuration see Medical Chemistry I, p. 18 ‹#› 55 H2O + ½ O2 Hydrogen peroxide H2O2 is a side product in the deamination of certain amino acids imino acid catalase oxo acid two-electron reduction ‹#› 56 Xanthin oxidase reaction produces hydrogen peroxide •hypoxanthin + O2 + H2O ® xanthin + H2O2 • •xanthin + O2 + H2O ® uric acid + H2O2 • most tissues, mainly liver ‹#› 57 Compare: reduction of dioxygen Type of reduction Redox pair Four-electron O2 + 4 e- + 4 H+ ® 2 H2O One-electron O2 + e- ® ·O2- Two-electron O2 + 2 e- + 2 H+ ® H2O2 ! ‹#› 58 Hypochlorous acid HClO •in some neutrophils •myeloperoxidase reaction •HClO has strong oxidative and bactericidal effects H2O2 + Cl- + H+ ® HClO + H2O ‹#› 59 Nitric oxide NO· is released from arginine •exogenous sources: drugs - vasodilators •NO· activates guanylate cyclase Þ cGMP Þ relaxation of smooth muscles •NO· is a radical and affords other reactive metabolites: • NO· + ·O2- ® O=N-O-O- ® O=N-O-O-H (peroxonitrous acid) H+ NO2+ + OH- ·NO2 + ·OH nitration of tyrosine NO3- (plasma, urine) peroxonitrite nitrosylation ‹#› 60 Compounds releasing NO Na2[Fe(CN)5NO] sodium nitroprusside (natrii nitroprussias) sodium pentacyanonitrosylferrate(III) glycerol trinitrate (glyceroli trinitras) isosorbid dinitrate (isosorbidi dinitras) amyl nitrite isobutyl nitrite ‹#› 61 Good effects of ROS •intermediates of oxidase and oxygenase reactions (cyt P-450), during reactions the radicals are trapped in enzyme molecule so that they are not harmful •bactericidal effect – fagocytes, respiratory burst (NADPH-oxidase) •signal molecules – clearly proved in NO·, perhaps other radical species can have similar action • ‹#› 62 Bad effects of ROS Substrate Damage Consequences PUFA formation of aldehydes (MDA) and peroxides changes in membrane permeability, damage of membrane enzymes Proteins aggregation, cross-linkage fragmentation oxidation of –SH, phenyl changes in ion transport influx of Ca2+ into cytosol altered enzyme activity DNA deoxyribose decomposition modification of bases chain breaks mutations translations errors inhibition of proteosynthesis ‹#› 63 Antioxidant systems in the body •Enzymes •superoxide dismutase, catalase, glutathione peroxidase •Low molecular antioxidants = reducing compounds with •phenolic -OH (tocopherol, flavonoids, urates) •enolic -OH (ascorbate) •-SH (glutathione GSH, dihydrolipoate) •or compounds with extended system of conjugated double bonds (carotenoids) ‹#› 64 Elimination of superoxide •Superoxide dismutase •Catalyzes the dismutation of superoxide 2 •O2- + 2 H+ ¾® O2 + H2O2 •Oxidation numbers of oxygen • -½ ¾® 0 -I •two forms: SOD1 (Cu, Zn, cytosol), SOD2 (Mn, mitochondria) Dismutation is a special type of redox reaction in which an element is simultaneously reduced and oxidized so as to form two different products. ‹#› 65 Elimination of H2O2 •catalase - in erythrocytes and other cells H2O2 ® ½ O2 + H2O •glutathione peroxidase •contains selenocystein, reduces H2O2 and hydroperoxides of phospholipids (ROOH) • 2 G-SH + H-O-O-H ® G-S-S-G + 2 H2O • 2 G-SH + R-O-O-H ® G-S-S-G + R-OH + H2O 3% H2O2 aplied to a wound releases bubbles ‹#› 66 Lipophilic antioxidants Antioxidant Sources Tocopherol Carotenoids Ubiquinol Plant oils, nuts, seeds, germs Fruits, vegetables (most effective is lycopene) Formed in the body from tyrosine ‹#› 67 Hydrophilic antioxidants Antioxidant Sources L-ascorbate Flavonoids Dihydrolipoate Uric acid Glutathione Fruits, vegetables, potatoes Fruits, vegetables, tea, wine Made in the body from cysteine Catabolite of purine bases Made in the body from cysteine ‹#› 68 Tocopherol (Toc-OH) •Lipophilic antioxidant of cell membranes and lipoproteins •Reduces peroxyl radicals of phospholipids to hydroperoxides which are further reduced by GSH, tocopherol is oxidized to stable radical Toc-O· PUFA-O-O· + Toc-OH ® PUFA-O-O-H + Toc-O· •Toc-O· is partially reduced to Toc-OH by ascorbate or GSH • • • ‹#› 69 Carotenoids •polyisoprenoid hydrocarbons (tetraterpens) •eliminate peroxyl radicals •they can quench singlet oxygen •food sources: green leafy vegetables, yellow, orange, red vegetables and fruits •very potent antioxidant is lycopene (tomatoes, more available from cooked tomatoes, ketchup etc.) • ‹#› 70 Lycopene does not have the β-ionone ring lycopene beta-carotene ‹#› 71 Lycopene in food (mg/100 g) Tomato purée Ketchup Tomato juice/sauce Watermelon Papaya Tomatoes fresh Apricots canned Apricots fresh 10-150 10-14 5-12 2-7 2-5 1-4 ~ 0.06 ~ 0.01 In order to effectively absorb lycopene, tomatoes should be • chopped and mashed • stewed slowly • combined with oil ! ‹#› 72 Zeaxanthin and lutein zeaxanthin (two chiral centers) lutein (three chiral centers) • belong to xanthophylls – oxygen derivatives of carotenoids • they differ in the position of double bond and in the number of C* • occur mainly in green leafy vegetables (spinach, cabbage, kale) • contained in macula lutea, prevents it against degeneration • many pharmaceutical preparations available ‹#› 73 Ubiquinol (QH2) •occurs in all membranes •Endogenous synthesis by intestinal microflora from tyrosine and farnesyl diphosphate (biosyntheis of cholesterol) •Exogenous sources: liver, meat and other foods •Reduced form QH2 regenerates tocopherol •Toc-O· + QH2 ® Toc-OH + ·QH • • • ‹#› 74 L-Ascorbate (vitamin C) •cofactor of proline hydroxylation (maturation of collagen) •cofactor of dopamine hydroxylation (to noradrenaline) •potent reducing agent (Fe3+® Fe2+, Cu2+ ® Cu+) •supports intestinal absorption of iron •Reduces many radicals: ·OH, ·O2-, HO2·, ROO· .... •Regenerates tocopherol •It is catabolized to oxalate!! (high doses are not recomended) •excess of ascorbate has pro-oxidative effects: • Fe2+ and Cu+ catalyze the formation of hydroxyl radical • ascorbate + O2 ® ·O2- + ·monodehydroascorbate • • ‹#› 75 L-Ascorbic acid is a weak diprotic acid Two conjugate pairs: Ascorbic acid / hydrogen ascorbate Hydrogen ascorbate / ascorbate two enol hydroxyls pKA1 = 4.2 pKA2 = 11.6 ‹#› 76 L-Ascorbic acid has reducing properties (antioxidant) ascorbic acid dehydroascorbic acid (reduced form) (oxidized form) ‹#› 77 Flavonoids and other polyphenols •commonly spread in plant food •total intake about 1 g (higher than in vitamins) •derivatives of chromane (benzopyrane), many phenolic hydroxyls •a main example: quercitin (see also Med. Chem. II, p. 76) •they reduce free radicals, themselves are converted to unreactive phenoxyl radicals •they chelate free metal ions (Fe2+, Cu+) blocking them to catalyze Fenton reaction and lipoperoxidation ‹#› 78 Main sources of flavonoids •vegetable (onion) •fruits (apples, grapes) •green tea •cocoa, quality chocolate •olive oil (Extra Virgin) •red wine • quercitin ‹#› 79 Glutathione (GSH) •tripeptide •γ-glutamylcysteinylglycine •made in all cells •reducing agent (-SH) •reduces H2O2 and ROOH (glutathione peroxidase) •reduces many ROS •regenerates -SH groups of proteins and coenzyme A •regenerates tocopherol and ascorbate ‹#› 80 Regeneration of reduced form of GSH •continuous regeneration of GSH proceeds in many cells •glutathione reductase, esp. in erythrocytes •GSSG + NADPH + H+ ® 2 GSH + NADP+ • pentose cycle ‹#› 81 Dihydrolipoate •cofactor of oxidative decarboxylation of pyruvate, 2-OG •reduces many radicals (mechanism not well understood) • dihydrolipoate (reduced form) lipoate (oxidized form) ‹#› 82 Uric acid •final catabolite of purine bases •in kidney, tubular cells, 90 % of urates are resorbed •the most abundant antioxidant of blood plasma •reducing properties, reduces various radicals •has ability to chelate iron and copper ions ‹#› 83 Uric acid (lactim) is a weak diprotic acid uric acid hydrogen urate urate pKA1 = 5.4 pKA2 = 10.3 2,6,8-trihydroxypurine ‹#› 84 Uric acid is the most abundant plasma antioxidant stable radical (oxidized form) R· is ·OH, superoxide hydrogen urate (reduced form) Compare plasma concentrations Ascorbic acid: 10 - 100 μmol/l Uric acid: 200 - 420 μmol/l