Biochemistry - 30 - Oxygen Tocicity and Free Radical Injury study guide verison.pdf

Reactive Nitrogen Species: RNS Objective D • Formed by the reaction of nitric oxide, NO• with either molecular oxygen or the superoxide ion. • Most reactive RNS is peroxynitrite, ONOO‒, which is very destructive to cells. • RNS eventually break down to nitrite or nitronium ion and can be detected...

Reactive Nitrogen Species: RNS Objective D • Formed by the reaction of nitric oxide, NO• with either molecular oxygen or the superoxide ion. • Most reactive RNS is peroxynitrite, ONOO‒, which is very destructive to cells. • RNS eventually break down to nitrite or nitronium ion and can be detected in the urine as a biomarker for RNS. 9 Mitochondrial Electron Transport Chain: The Electron Leak Objective F • Normally CoQ is bound to COMPLEX I and receives both electrons there. Leaves COMPLEX I in a fully reduced form. • Statistical possibility that CoQ will dissociate after receiving only 1 electron: semi-quinone radical form. • With high O2 levels in mitochondria, likelihood of superoxide formation is high. 11 Objective G Enzymes that Use Oxygen as a Substrate Molecular oxygen has two unpaired electrons, and four electrons are shared between the two oxygen atoms. When O2 is split, each oxygen atom will be “short” two electrons to fill the valence shell. 4 electrons will fill the shells, and two protons per oxygen can bind to form H2O. If only two electrons are available, O2 won’t split. Rather the two electrons will pair the two unpaired electrons and then two protons will bind to form H2O2. Watch for reactions that form water versus H2O2 from O2 For Monooxygenases, O2 is split into two oxygen atoms. One oxygen atom will get two electrons from the donor to form water. The other oxygen will be “short” by two electrons, and so will form a covalent bond with the substrate to share the needed electrons. For Dioxygenases, both oxygen atoms end up on the substrate with shared electrons in two covalent bonds. Potential Problem: Most oxidases, peroxidases and oxygenases can transfer single e- to O2 via a metal à ROS 13 Cytochrome P450 Objective G Superfamily of monooxygenase enzymes that use a heme group and O2 to catalyze the oxidation of molecules. Substrates include unreactive hydrophobic molecules (cholesterol, fats), polar molecules (alcohols), and xenobiotics (drugs, toxins). [Fe3+O2-] + RH à ROH Cytochrome: Fe2+ in heme binds O2 and forms Fe2+-O2‒ tightly bound. Substrate (RH) binds to active site and superoxide attacks it. One oxygen atom binds to the substrate forming ROH. Reductase: two electrons donated by NADPH are delivered to the heme group and transferred to the second oxygen atom, allowing it to form water with 2 H+ 14 Objective A Oxidative Stress: Free Radical-Mediated Cellular Injury Oxidative stress occurs when ROS are produced faster than our cell defenses can destroy them. The results in damage to cells: 18 Lipid Peroxidation Unsaturated fatty acid chains are a rich source of electrons for free radical reactions. • Hydroxyl radical can extract a hydrogen (H+ + 1e) from a fatty acid carbon, reading a carbon radical on the chain. • O2 can react with the unpaired electron creating a covalently bound peroxyl radical. • The lipid peroxyl radical can take a hydrogen from another unsaturated fatty acid, etc, etc. • The lipid peroxide is not stable, and the chain will fragment and rearrange. • Malondialdehyde is very reactive toward other cell components. • Malondialdehyde in the blood and urine is evidence of ROS-mediated lipid damage in cells. To terminate the reaction, to radicals must react which stops their propagation, or an antioxidant must “quench” the radical. Objective I 20 Objective H Mitochondrial DNA Damage by ROS • Mitochondrial DNA is 10x more susceptible to ROS damage than nuclear DNA. • Nuclear DNA is protected by histones and DNA packaging. Mitochondria lack protective histones and higher-level structural organization. • There is a high rate of ROS generation from the mitochondrial ETC at CoQ (electron leak). • Mitochondrial DNA is also located near the inner membrane of mitochondria where the ETC is located, • ROS generated from ETC does not have to travel very far to attack the DNA. 21 Objective M Cellular Defense Mechanisms • Ferritin binds free iron to keep it away from H2O2 • SOD and Catalase destroy superoxide and H2O2 • Three isoforms (below left) • Glutathione (GSH) provides electrons for glutathione peroxidase to destroy H2O2 and glutathione reductase replenishes reduced GSH for more protection. Note 3 isoforms of Superoxide Dismuatase: Cu-Zn SOD (cytosolic) Mn SOD (mitochondrial) EC-SOD (extracellular) • Vitamin E and β-carotene reside in lipid bilayers to destroy lipid radicals 24 Ferritin as an Antioxidative Defense 25 Antioxidant Enzymes Objective M 26 Objective M Glutathione: GSH • GSH is synthesized by a specific enzyme pathway in the cytosol of nucleated cells • Present in all mammalian cells (many non-mammalian cells too) except in neurons • Deficiency of GSH causes death in newborn (rats) and cataract & mitochondrial swelling in adults • Glutathione Synthase deficiency very rare disorder • Glutathione levels can be low due to dietary or environmental factors (chronic free radical stress) 27 Objective M Vitamin E • Fatty acid peroxyl radicals are very reactive and perpetuate free-radical mediated damage within the membrane bilayer. • α-Tocopherol (Vitamin E) is very hydrophobic and partitions into the membrane bilayer. • Physical contact between Vitamin E and lipid peroxyl radicals “quenches” (destroys) the radicals. Two step process with 2 radicals. • Tocopheryl quinone is metabolized and removed from the body. • Note a peroxide is still “reactive” but far less reactive than a peroxyl radical. 28