Respiratory chain ~ Reactive oxygen species © Department of Biochemistry, MU Brno (J.D.) 2009 Transformation of energy in human body Energy transformations in the human body are accompanied with continuous production of heat Energetic data about nutrients Two ways of ATP formation in the body Substrate level phosphorylation • phosphorylation of ADP (GDP) is performed by the high-energy intermediates • succinyl-CoA (CAC) • 1,3-bisphosphoglycerate (glycolysis) • phosphoenolpyruvate (glycolysis) Phosphorylation of GDP in citrate cycle Phosphorylation of ADP by 1,3-bisphosphoglycerate Phosphorylation of ADP by phosphoenolpyruvate Aerobic phosphorylation follows the reoxidation of reduced cofactors in R.CH. NADH formation in matrix of mitochondria (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 • Dehydrogenation+deamination glutamate NADH formation in cytoplasm • Glycolysis (dehydrogenation of glyceraldehyde-3-P) • Gluconeogenesis (dehydrogenation of lactate to pyruvate) • Dehydrogenation of ethanol (to acetaldehyde and to acetic acid) FADH[2] formation in matrix of mitochondria • b-Oxidation of FA (dehydrogenation of saturated acyl-CoA) • Citrate cycle (dehydrogenation of succinate) Transport of NADH from cytoplasm to matrix • NADH produced in cytoplasm must be transported into matrix to be reoxidized in R.CH. • IMM is impermeable for NADH • two shuttle systems: • aspartate/malate shuttle (universal) • glycerol phosphate shuttle (brain, kidney) Aspartate/malate shuttle Glycerol phosphate shuttle Respiratory chain is the system of redox reactions in IMM which starts by the NADH oxidation and ends with the reduction of O[2][ ]to water Four types of cofactors in R.CH. • flavine cofactors (FMN, FAD) • non-heme iron with sulfur (Fe-S) • ubiquinone (Q) • heme (in cytochromes) Redox pairs in respiratory chain • 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 O[2] • the value of potential depends on protein molecule (compare cytochromes) R.CH. Entry points for reducing equivalents Enzyme complexes in R.CH. NADH+H^+ + Q + 4 H^+[matrix] ® NAD^+ + QH[2] + 4 H^+[ims] [] Complex I oxidizes NADH and translocates 4 H^+ into intermembrane space Complex II (independent entry) oxidizes FADH[2] from citrate cycle and reduces ubiquinone Complex III oxidizes QH[2], reduces cytochrome c and translocates 4 H^+ across IMM Complex IV oxidizes cyt c[red] and two electrons reduce monooxygen (½O[2]) Complex IV: real process is four-electrone reduction of dioxygen Utilization of proton motive force ATP-synthase is a molecular rotating motor: 3 ATP/turn Stoichiometry of ATP synthesis is not exactly recognized • transfer of 2 e^- from NADH to ½ O[2] .... 3 ATP • transfer of 2 e^- from FADH[2] to ½ O[2] .... 2 ATP 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. Reactive oxygen species (ROS) Reactive oxygen species in human body Superoxide anion-radical •O[2]^- • One-electrone reduction of dioxygen Superoxide formation ^• Respiratory burst (in neutrophils) 2 O[2] + NADPH ¾® 2 •O[2]^- + NADP^+ + H^+ • Spontaneous oxidation of heme-proteins heme-Fe^2+ + O[2] ¾® heme-Fe^3+ + •O[2]^- Radical •OH is the most reactive species; it is formed from superoxide and hydrogen peroxide Hydrogen peroxide H[2]O[2] is a side product in the deamination of certain amino acids Compare: reduction of dioxygen Singlet oxygen ^1O[2] • excited form of triplet dioxygen • formed after absorption of light by some compounds (porphyrins) Hypochlorous acid HClO • in some neutrophils • myeloperoxidase reaction • HClO has strong oxidative and bactericidal effects Nitric oxide NO· is released from arginine • exogenous sources: drugs - vasodilators • NO· activates guanylate cyclase Þ cGMP  relaxation of smooth muscles • NO· is a radical and affords other reactive metabolites: Compounds releasing NO 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 Bad effects of ROS 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) Elimination of superoxide • Superoxide dismutase • Catalyzes the dismutation of superoxide 2 •O[2]^- + 2 H^+ ¾® O[2] + H[2]O[2] • Oxidation numbers of oxygen -½ ¾® 0 -I • two forms: SOD1 (Cu, Zn, cytosol), SOD2 (Mn, mitochondria) Elimination of H[2]O[2 ] • catalase - in erythrocytes and other cells H[2]O[2 ]® ½ O[2] + H[2]O • glutathione peroxidase • contains selenocystein, reduces H[2]O[2] and hydroperoxides of phospholipids (ROOH) 2 G-SH + H-O-O-H  G-S-S-G + 2 H[2]O 2 G-SH + R-O-O-H  G-S-S-G + R-OH + H[2]O Lipophilic antioxidants Hydrophilic antioxidants 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 stabble radical PUFA-O-O· + Toc-OH ® PUFA-O-O-H + Toc-O· • Toc-O· is partially regenerated by ascorbate or GSH 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.) Lycopene does not have the β-ionone ring Ubiquinol (QH[2]) • occurs in all membranes • Endogenous synthesis by intestinal microflora from tyrosine and farnesyl diphosphate (biosyntheis of cholesterol, Harper p. 232) • Exogenous sources: liver, meat and other foods • Reduced form QH[2] regenerates tocopherol • Toc-O· + QH[2]  Toc-OH + ·QH L-Ascorbate (vitamin C) • cofactor of proline hydroxylation (maturation of collagen) • cofactor of dopamine hydroxylation (to noradrenaline) • potent reducing agent (Fe^3+® Fe^2+, Cu^2+  Cu^+) • supports iron absorption in GIT • Reduces many radicals: ·OH, ·O[2]^-, HO[2]·, ROO· .... • Regenerates tocopherol • It is catabolized to oxalate!! (high doses are not recomended) • excess of ascorbate has pro-oxidative effects!! L-Ascorbic acid is a weak diprotic acid L-Ascorbic acid has reducing properties (antioxidant) Flavonoids and other polyfenols • 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) • reduce free radicals, themselves are converted to unreactive phenoxyl radicals • they chelate free metal ions (Fe^2+, Cu^+) blocking them to catalyze Fenton reaction and lipoperoxidation Main sources of flavonoids • vegetable (onion) • fruits (apples, grapes) • green tea • cocoa, quality chocolate • olive oil (Extra Virgin) • red wine Glutathione (GSH) • tripeptide • γ-glutamylcysteinylglycine • made in all cells • reducing agent (-SH) • reduces H[2]O[2] and ROOH (glutathione peroxidase) • reduces many ROS • regenerates -SH groups of proteins and coenzyme A • regenerates tocopherol and ascorbate 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^+ Dihydrolipoate • cofactor of oxidative decarboxylation of pyruvate, 2-OG • reduces many radicals (mechanism not well understood) 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 Uric acid (lactim) is a weak diprotic acid Uric acid is the most abundant plasma antioxidant