The Paradox of Aerobic Life

1. The Paradox of Aerobic Life • All life on earth is based on redox reactions (reduction; gain of ê, oxidation; loss of ê), using reductive processes to store energy and oxidative processes to release it. The unusual chemistry of O2 makes it possible to integrate highly reactive oxygen in life-giving redox metabolism. • Oxygen is essential, but toxic • Aerobic cells face constant danger from reactive oxygen species (ROS). • ROS can act as mutagens, cause lipid peroxidation and denature proteins.

2. The role of oxygen in plant growth and responses to environment Oxygen as the regulator of environmental responses • We will talk about • What are ROS • ROS chemistry • ROS generation & decomposition (during Environmental stress) • ROS importance in plants • ROS signaling • - ROS perception and signal transduction; • - the downstream physiological effects of ROS • ( ROS in plant disease) • - induction of Programmed cell death (Apoptosis) • - induction of defense reactions • The role of ROS in adaptation to stress(es) • - the role of mitochondria and of intracellular repair systems • - ROS in stress cross-talk

3. Free radicals • a radical is any chemical species that has unpaired electrons, i.e. contains at least one electron that occupies an atomic or molecular orbital by itself. • free radicals are capable of independent existence, while bound radicals are part of a larger molecular structure. • Radicals can have positive, negative, or neutral charge • For example, O2- (superoxide anion radical) and OH- (hydroxyl ion) are negatively charged radicals, while H. (hydrogen radical) and OH. (hydroxyl radical) are uncharged. • A) Ionization: H-O-H  H+ + OH- • B) Radiolysis: H-O-H  H. + OH. • In A), 2ê are transferred to oxygen, with the resultant production of charged products; • in B), 1 ê goes to oxygen and the other to hydrogen, with the consequence that the reaction products are uncharged

4. The Earth was originally anoxic • Metabolism was anaerobic • O2 started appearing ~2.5 x 109 years ago Anaerobic metabolism-glycolysis Glucose + 2ADP + 2PiLactate+2ATP+ 2H2O O2 an electron acceptor in aerobic metabolism Glucose +6O2+ 36ADP + 36Pi 6CO2 +36ATP+ 6H2O

5. There are just enough electrons to make the whole atom electrically neutral

6. Basics of Redox Chemistry Term Definition Oxidation Gain in oxygen 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

7. Oxidation-reduction (redox) reactions comprisea major class of biochemical reactions • BioEnergetics, the reactions that lead to the generation of > 95% of the energy utilized by aerobic organisms. • 2) Chemical transformations e.g. alcohol dehydrogenase, fatty acid desaturase (introduces double bonds into fatty acids). • 3) Detoxification-the conversion of the predominantly lipid-soluble toxic compounds present in our environment • (e.g. DDT, many drugs) into water-soluble derivatives that can then be excreted. • Electron transfers --> the oxidation of intermediary metabolites by O2 in the mitochondria . It often requires the successive transfer of H atoms or electrons, first to NAD+, then from NADH to an ubiquinone (Q), next from QH2 to ferricytochrome c and finally from ferrocytochrome c to O2. These reactions are catalysed, e.g., by an oxidoreductase using NAD+ or NADP+ as acceptor, NADH:Q oxidoreductase Good info source: http://www.plantstress.com/Articles/Oxidative%20Stress.htm

8. The Paradox of Aerobiosis • Oxygen is essential, but toxic. • Aerobic cells face constant danger from reactive oxygen species (ROS). • ROS can act as mutagens, they can cause lipid peroxidation and denature proteins.

9. Environmental factors that induce oxidative stress Root growth Good study source: http://cropsoil.psu.edu/Courses/AGRO518/Oxygen.htm

10. 2 billion years of REDOX regulation • ALL LIVING ORGANISMS are oxidation–reduction (redox) systems. They use anabolic, reductive processes to store energy and catabolic, oxidative processes to release it. • Plants have perfected the art of redox control. Indeed, redox signals are key regulators of plant metabolism, morphology, and development. These signals exert control on nearly every aspect of plant biology from chemistry to development, growth, and eventual death.

11. Atomic and molecular oxygen Molecular oxygen can accept a total of 4 electrons atomic oxygen: 1s22s22px22py12pz1 molecular oxygen: s1s2 s*1s2 s2s2 s*2s2 s2pz2 p2px2 p2py2 p*2px1 p*2py1

12. Molecular oxygen is a di- or biradicalit has two unpaired electrons and is paramagnetic

13. Superoxide The addition of one electron to O2 gives the electron configuration s1s2s*1s2s2s2s*2s2s2pz2p2px2p2py2p*2px2p*2py1 - superoxide, O2- .

14. Peroxide (O-O2-) And another gives the electron configuration s1s2s*1s2s2s2s*2s2s2pz2p2px2p2py2p*2px2p*2py2 - peroxide, O22-/H2O2 Bond order = (10-8)/2 = 1 4 anti-bonding p* electrons, rapidly stabilised by accepting 2 protons → H202

15. Hydroxyl radical and ion • HO• HO-  O2- (H2O) and O -· (oxyl and/or hydroxyl radical), Bond order = (10-9)/2 = ½; Highly unstable

16. Oxygen-summary

17. : : . . O:O : : • Ground-state oxygen has 2-unpaired electrons • The unpaired electrons have parallel spins • Oxygen molecule is minimally reactive due to spin restrictions

18. Free radicals have one or more unpaired electrons in their outer orbital, indicated in formulas as []. As a consequence they increased reactivity to other molecules. This reactivity is determined by the ease with which a species can accept or donate electrons. • The prevalence of oxygen in biological systems means that oxygen centered radicals are the most common type found • O2 is central to metabolism in aerobic life, as a terminal electron acceptor, being reduced to water. Transfer of electron to oxygen yields the reactive intermediates.

19. The beginnings • 1775 - Priestley: • discovery of O2 • observation of toxic effect of O2 • 1900 – Moses Gomberg: • discovery of triphenylmethyl radical • Until 1950/60: • minimal attention was given to biological • actions of free radicals and reactive • oxygen species (ROS)

20. Evidence on the existence of ROS • 1954 - Gerschman et al. :Recognition of similarities between radiation and oxygen toxicity • 1969 - McKord and Fridovich:Discovery of superoxide dismutase; suggested the existence of endogenous superoxide • 1973 - Babior et al.:Recognition of the relationship between superoxide production and bactericidal activity of neutrophils • 1981 - Granger et al.:recognition of the relationship between ROS production and ischemia/reperfusion induced gut injury

21. “Longevity” of reactive species Reactive Species Half-life Hydrogen peroxide Organic hydroperoxides ~ minutes Hypohalous acids Peroxyl radicals ~ seconds Nitric oxide Peroxynitrite ~ milliseconds Superoxide anion Singlet oxygen ~ microsecond Alcoxyl radicals Hydroxyl radical ~ nanosecond

22. Half-life of some reactive species Reactive species Half-life (s) Physiol conc. (mol/l) Hydroxyl radical (OH) Alcoxyl radical (RO) Singlet oxygen (1O2) Peroxynitrite anion (ONOO-) Peroxyl radical (ROO) Nitric oxide (NO) Semiquinone radical Hydrogen peroxide (H2O2) Superoxide anion (O2-) Hypochlorous acid (HOCl) 10-9 10-6 10-5 0.05 – 1.0 7 1 - 10 minutes/hours spontan. hours/days (accelerated by enzymes) spontan. hours/days (by SOD accel.to 10-6) dep. on substrate 10-9 10-9- 10-7 10-12 - 10-11

23. Oxidation reactions Oxidation loss of H2 or gain of O, O2, or X2 Reduction  gain of H2 or loss of O, O2, or X2 The loss or gain of H2O or HX are not considered oxidation-reduction reactions. X=halogen

24. 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 Radical-mediated reactions

25. Fenton reaction (1894) Cu1+  Cu2+ Haber and Weiss extension (1934) Oxidizing molec Reducung molec

26. Hydroxyl radical reactions addition of OH to the organic molecule Stable oxidised products abstraction reaction of the .OH radical: oxidation of organic substrates Chain reactions

27. Enzymatic sources of ROS Xanthine oxidase Hypoxanthine + 2O2 --> Xanthine + O2.-+ H2O2 NADPH oxidase NADPH + O2 --> NADP+ + O2.- Amine oxidases R-CH2-NH2 + H2O+ O2 --> R-CHO + NH3 + H2O2 Myeloperoxidase Hypohalous acid formation H2O2+ X- + H+ --> HOX + H2O NADH oxidase reaction Hb(Mb)-Fe3+ + ROOH --> Compound I + ROH Compound I + NADPH --> NAD·+ Compound II Compound II + NADH --> NAD·+ E-Fe3+ NAD·+ O2 --> NAD+ + O2.- Aldehyde oxidase 2R-CHO + 2O2--> 2R-COOH + O2.- Dihydroorotate dehydrogenase Dihydroorotate + NAD·+ O2 --> NADH + O2.- + Orotic acid

28. Nonenzymatic sources of ROS and autooxidation reactions Fe2+ + O2 --> Fe3++ O2.- Hb(Mb)-Fe2+ + O2 --> Hb(Mb)-Fe3++O2.- Catecholamines + O2 --> Melanin + O2.- Reduced flavin Leukoflavin + O2 --> Flavin semiquinone + O2.- Coenzyme Q-hydroquinone + O2 --> Coenzyme Q (ubiquinone) + O2 .- Tetrahydropterin + 2 O2 --> Dihydropterin + 2 O2.-

29. Lipid peroxidation 1.1 - Initiation Peroxidation sequence starts with the attack of a ROS (with sufficient reactivity) able to abstract a hydrogen atom from a methylene group (- CH2-), these hydrogen having very high mobility. This attack generates easily free radicals from polyunsaturated fatty acids. .OH is the most efficient ROS to do that attack, whereas O2.- is much less reactive Under aerobic conditions conjugated dienes are able to combine with O2 to give a peroxyl (or peroxy) radical, ROO.. peroxyl radical is able to abstract H from another lipid molecule (adjacent fatty acid), especially in the presence of Fe/Cu, causing a chain reaction.

30. The peroxidation of linoleic acid initiation, propagation and termination Peroxidation is initiated when a reactive oxygen species abstracts a methylene hydrogen from an unsaturated fatty acid found in the lipid membrane forming a lipid radical (L·).  This lipid radical then reacts with molecular oxygen forming a lipid hydroperoxyl radical (LOO·) which can then react abstract a methylene hydrogen from a neighboring unsaturated fatty acid forming a lipid hydroperoxide (LOOH)

31. Mitochondrion Post-transcriptional Effects Chloroplast Post-transcriptional Effects Paraquat High Light + Chilling Sulfur Dioxide ROS Arise Throughout the Cell Wounding Pathogens Chilling Ozone Cell Wall Drought Salinity Cytosol Antioxidant genes Nucleus Gene Expression ROS subcellular sites unclear

32. The electron transport system in the thylakoid membrane showing 3 possible sites of activated oxygen production auto-oxidizable Mehler reaction a) Singlet oxygen may be produced from triplet chlorophyll in the light harvesting complex. b) Superoxide and hydrogen peroxide may "leak" from the oxidizing (water-splitting) side of PSII. c) Triplet oxygen may be reduced to superoxide by ferredoxin on the reducing side of PSI, especially when NADP is limiting (NADPH oxidation by Calvin cycle low).

33. The water–water cycle. • The ascorbate–glutathione cycle. • The glutathione peroxidase (GPX) cycle. • CAT. SOD acts as the first line of defense converting O2− into H2O2. Ascorbate peroxidases (APX), GPX and CAT then detoxify H2O2. In contrast to CAT (d), APX and GPX require an ascorbate (AsA) and/or a glutathione (GSH) regenerating cycle (a–c). This cycle uses electrons directly from the photosynthetic apparatus (a) or NAD(P)H (b,c) as reducing power. ROIs are indicated in red, antioxidants in blue and ROI-scavenging enzymes in green. • Abbreviations: DHA, dehydroascorbate; DHAR, DHA reductase; Fd, ferredoxin; GR, glutathione reductase; GSSG, oxidized glutathione; MDA, monodehydroascorbate; MDAR, MDA reductase; PSI, photosystem I; tAPX, thylakoid-bound APX.

34. The redox cycling of ascorbate in the chloroplast often referred to as the Halliwell-Asada pathw

35. ROS production in Mitochondria Electron transfers oxidation of intermediary metabolites by O2 require the successive transfer of H+ or ê, first to NAD+, then from NADH to an ubiquinone (Q), next from QH2 to ferricytochrome c and finally from ferrocytochrome c to O2. These reactions are catalysed, e.g., by an oxidoreductase using NAD+ or NADP+ as acceptor, NADH:Q oxidoreductase ETC in the inner plant mitochondria membrane H+-pumping of CI, III, and IV. ROS production at the two main sites, CI and III. Since UQ• is bound to the inner and outer membranes in CIII, ROS can be formed on either side of the membrane. CI, NADH dehydrogenase; CII, succinate dehydrogenase; CIII, ubiquinol-cytochrome bc1 reductase; CIV, cytochrome c oxidase The more you eat the more mitochondria respiration and more ROS you getMol Cel Biol, 2000, p. 7311-7318, Vol. 20,

36. Mitochondria as a source of ROS The source of mitochondrial ROS involves a non-heme Fe protein that transfersê to O2. This occurs primarily at Complex I (NADH-coenzyme Q) and, to a lesser extent, following the auto-oxidation of coenzyme Q from the Complex II (succinate-coenzyme Q) and/or Complex III (coenzyme QH2-cytochrome c reductases) sites. The precise contribution of each site to total mitochondrial ROS production is probably determined by local conditions including chemical or physical damage to the mitochondria, oxygen availability and the presence of xenobiotics. Kehrer JP (2000) Toxicology 149: 43-50

37. Functions of the alternative oxidase Option for envir stress regulation In the electron-transport chains of mitochondrial (a) and chloroplast (b), AOX diverts electrons that can be used to reduce O2 into O2- and uses these electrons to reduce O2 to H2O. In addition, AOX reduces the overall level of O2, the substrate for ROI production, in the organelle. AOX is indicated in yellow and the different components of the electron-transport chain are indicated in red, green or gray. AOX may also work as a bypass to oxidize NADH and FADH2 under ADP-limiting conditions under which the cytochrome oxidase pathway is restricted

38. plant mitochondria in stress response In mammalian mitochondria, 1-5% of the oxygen consumed in vitro goes to ROS production. Antimycin, a complex III inhibitor that does not block O2.- formation, increased both O2.- generation and membrane damage (BBA1268,249) The major sites of ROS production are complex I and the ubisemiquinone in complex III. The latter activity is completely inhibited by the complex IV inhibitor KCN, which interrupts the Q cycle and prevents the formation of ubisemiquinone. KCN can thus be used to distinguish between complex I and III contributions to ROS Annu. Rev. Plant Physiol. Plant Molec. Biol. 52, 561-591

39.  Extra- and intracellular sources of ROS in plants. XOD, xanthine oxidase

40. Prooxidants R3C.Carbon-centered R3N.Nitrogen-centered R-O.Oxygen-centered R-S.Sulfur-centered • Free 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 H2O2Hydrogen peroxide HOCl- Hypochlorous acid O3Ozone 1O2 Singlet oxygen ONOO- Peroxynitrite Men+ Transition metals • Non-Radicals: • Species that have strong oxidizing potential • Species that favor the formation of strong oxidants (e.g., transition metals)

41. Reactive Oxygen Species (ROS) Radicals: O2.- Superoxide .OH Hydroxyl RO2. Peroxyl RO. Alkoxyl HO2.Hydroperoxyl Non-Radicals: H2O2Hydrogen peroxide HOCl- Hypochlorous acid O3Ozone 1O2 Singlet oxygen ONOO- Peroxynitrite

42. Oxidative Protection Oxidative Stress Antioxidants Oxidants Oxidative Stress Oxidative Protection • Oxidants: • Superoxide, Hydrogen peroxide, hydroxyl, nitric oxide, peroxynitrite • Auto-oxidation, Enzymes, Ischaemia-Reperfusion, Respiratory burst, organelles • Damage to lipids, protein, DNA • Consequences  Repair, adaptation or death • Antioxidants ??? Oxidative stress occurs when the ROS generation exceeds the ROS removal

43. Flavonoids ROS scavenging molecules plant antioxidants Ascorbate Glutathione Polyphenols Flavonoids Lipoic acid Ponce de León Enzymes: SOD Catalase Glutathione peroxidase Ascorbate peroxidase Thioredoxins Glutaredoxins Nature425, 132-133

44. Reactive Nitrogen Species (RNS) Non-Radicals: ONOO-Peroxynitrite ROONO Alkyl peroxynitrites N2O3 Dinitrogen trioxide N2O4 Dinitrogen tetroxide HNO2 Nitrous acid NO2+Nitronium anion NO- Nitroxyl anion NO+ Nitrosyl cation NO2Cl Nitryl chloride Radicals: NO. Nitric Oxide NO2.Nitrogen dioxide

45. Nitric Oxide • NO refers to nitrosyl radical (•NO) and its nitroxyl (NO–) and nitrosonium (NO+) ions • Freely diffusible, gaseous free radical. • First described in 1979 as a potent relaxant of peripheral vasculature. • Used by the body as a signaling molecule. • Used as neurotransmitter, bactericide. • Environmental Pollutant • First gas known to act as a biological messenger N O

46. Nitric Oxide in plants • Affects aspects of plant growth and development. • Affects the responses to: light, gravity, oxidative stress, pathogens. • Can be a maturation and senescence factor • Has a concentration dependent cytotoxic or protective (antioxidant) effects.

47. NO-induced cell death in Arabidopsis occurs independently of ROS Cells were treated with methyl viologen (MV) to generate O2 · , NO donor (RBS), and/or the peroxynitrite scavenger and SOD-mimetic MnTBAP

48. cGMP in NO-induced cell death The effects of the caspase-1 inhibitor Ac-YVAD-CMK on NO- and H2O2-induced cell death Cells were pre-treated with ODQ (guanylate cyclase inhibitor) and/or 8Br-cGMP prior to RBS.

49. NO and Cell Death +PBITU Psm (avrRpm 1) NO + H2O2 cause cell death NO + O2- react to form peroxynitrite % Cell Death Peroxynitrite (ONOO -) does not cause cell death Too much O2- ‘mops up NO’ – no death Delladonne et al. (2001) PNAS 98:13454

50. Microsomal Oxidation, Flavoproteins, CYP enzymes Myeloperoxidase (phagocytes) Xanthine Oxidase, NOS isoforms Endoplasmic Reticulum Transition metals Lysosomes Cytoplasm Fe Cu Oxidases, Flavoproteins Peroxisomes Mitochondria Plasma Membrane Electron transport Lipoxygenases, Prostaglandin synthase NADPH oxidase Endogenous sources of ROS and RNS (in animals)