The Paradox of Aerobic Life

1

The Paradox of Aerobic Life• All life on earth is based on redox reactions All life on earth is based on redox reactions ((reduction; reduction; gain of ê,gain of ê, oxidation; oxidation;

loss of êloss of ê)), using reductive processes to store energy and oxidative , using reductive processes to store energy and oxidative

processes to release itprocesses to release it. . The unusual chemistry of OThe unusual chemistry of O22 makes it possible makes it possible

to integrate highly reactive oxygen in lifeto integrate highly reactive oxygen in life--giving redox metabolismgiving 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 environmentOxygen 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

Free radicalsFree radicals a radical is any chemical species that has unpaired electrons, i.e. a radical is any chemical species that has unpaired electrons, i.e.

contains at least one electron that occupies an atomic or molecular contains at least one electron that occupies an atomic or molecular

orbital orbital by itselfby itself..

free radicals are capable of independent existence, while free radicals are capable of independent existence, while boundbound

radicals are part of a larger molecular radicals are part of a larger molecular

structure.structure.

Radicals can have positive, negative, or neutral chargeRadicals can have positive, negative, or neutral charge

• For example, OFor example, O22-- (superoxide anion radical) and OH (superoxide anion radical) and OH-- (hydroxyl ion) are (hydroxyl ion) are

negatively charged radicals, while Hnegatively charged radicals, while H.. (hydrogen radical) and OH (hydrogen radical) and OH..

(hydroxyl (hydroxyl

radical) are uncharged. radical) are uncharged.

• A) Ionization: H-O-H A) Ionization: H-O-H H H++ + OH + OH--

• B) Radiolysis: H-O-H B) Radiolysis: H-O-H H H.. + OH + OH..

In A), 2In A), 2êê are transferred to oxygen, with the resultant production of are transferred to oxygen, with the resultant production of

chargedcharged products; products;

in B), 1 in B), 1 êê goes to oxygen and the other to hydrogen goes to oxygen and the other to hydrogen, with the consequence , with the consequence

that the reaction products are that the reaction products are unchargeduncharged

4

•The Earth was originally anoxic

•Metabolism was anaerobic

•O2 started appearing ~2.5 x 109 years ago

Anaerobic metabolism-glycolysis

Glucose + 2ADP + 2Pi Lactate + 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

Term Definition

Oxidation Gain in oxygenLoss of electrons

Reduction Loss of oxygenGain of hydrogenGain of electrons

Oxidant Oxidizes another chemical by takingelectrons, hydrogen, or by adding oxygen

Reductant Reduces another chemical by supplyingelectrons, hydrogen, or by removing oxygen

Basics of Redox ChemistryBasics of Redox Chemistry

7

Oxidation-reduction (redox) reactions comprise

a major class of biochemical reactions1) 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

http://www.plantstress.com/Articles/Oxidative%20Stress.htm Good info source:

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

http://cropsoil.psu.edu/Courses/AGRO518/Oxygen.htm

Good study source:

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

atomic oxygen:

1s22s22px22py

12pz1

molecular oxygen:1s

2 *1s22s

2*2s22pz

2 2px2 2py

2 *2px1 *2py

1

Molecular oxygen can accept a total of 4 electrons

Molecular oxygen is a di- or biradicalMolecular oxygen is a di- or biradicalit has two unpaired electrons and is paramagneticit has two unpaired electrons and is paramagnetic

SuperoxideSuperoxideThe addition of one electron to O2 gives the electron configuration

1s2 *1s

2 2s2 *2s

2 2pz2 2px

2 2py2 *2px

2 *2py1 - superoxide, O2-

.

Peroxide (O-OPeroxide (O-O2-2-))And another gives the electron configuration1s

2 *1s2 2s

2 *2s2 2pz

2 2px2 2py

2 *2px2 *2py

2 - peroxide, O22-/H2O2

Bond order = (10-8)/2 = 14 anti-bonding electrons, rapidly stabilised by accepting 2 protons → H202

Hydroxyl radical and ionHydroxyl radical and ion

•

Bond order = (10-9)/2 = ½; Highly unstable

HO• HO-

O2- (H2O) and O -· (oxyl and/or hydroxyl radical),

16

Oxygen-summary

17

• Ground-state oxygen has 2-unpaired electrons

O:O

: ::: ..

• 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 beginningsThe beginningsThe beginningsThe 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 Evidence on the existence of ROSROS

Evidence on the existence of Evidence on the existence of ROSROS

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 Longevity” of reactive speciesspecies

Reactive Species Half-life

Hydrogen peroxideOrganic hydroperoxides ~ minutesHypohalous acids

Peroxyl radicals ~ secondsNitric oxide

Peroxynitrite ~ milliseconds

Superoxide anionSinglet oxygen ~ microsecondAlcoxyl radicals

Hydroxyl radical ~ nanosecond

22

Half-life of Half-life of some rsome reactive eactive sspeciespeciesHalf-life of Half-life of some rsome reactive eactive sspeciespecies

RReactive specieseactive species Half-lifeHalf-life (s)(s) Half-lifeHalf-life (s)(s)

Hydroxyl radical (Hydroxyl radical (OH)OH)Alcoxyl radical (ROAlcoxyl radical (RO))Singlet oxygen (Singlet oxygen (11OO22))Peroxynitrite anion (ONOOPeroxynitrite anion (ONOO--))Peroxyl radical (ROOPeroxyl radical (ROO))Nitric oxide (Nitric oxide (NO)NO)Semiquinone radicalSemiquinone radicalHydrogen peroxide (HHydrogen peroxide (H22OO22))

Superoxide anion (OSuperoxide anion (O22--))

HypochloHypochlorous acidrous acid (HOCl) (HOCl)

Hydroxyl radical (Hydroxyl radical (OH)OH)Alcoxyl radical (ROAlcoxyl radical (RO))Singlet oxygen (Singlet oxygen (11OO22))Peroxynitrite anion (ONOOPeroxynitrite anion (ONOO--))Peroxyl radical (ROOPeroxyl radical (ROO))Nitric oxide (Nitric oxide (NO)NO)Semiquinone radicalSemiquinone radicalHydrogen peroxide (HHydrogen peroxide (H22OO22))

Superoxide anion (OSuperoxide anion (O22--))

HypochloHypochlorous acidrous acid (HOCl) (HOCl)

1010-9-9

1010-6-6

1010-5-5

00..05 – 105 – 1..0077

1 - 101 - 10minutes/hoursminutes/hours

sspontpontan. hours/an. hours/daysdays((accelerated by accelerated by enzymeenzymess))

sspontpontan. hours/an. hours/daysdays((by SOD accel.by SOD accel. to to 1010-6-6))dep. on substratedep. on substrate

1010-9-9

1010-6-6

1010-5-5

00..05 – 105 – 1..0077

1 - 101 - 10minutes/hoursminutes/hours

sspontpontan. hours/an. hours/daysdays((accelerated by accelerated by enzymeenzymess))

sspontpontan. hours/an. hours/daysdays((by SOD accel.by SOD accel. to to 1010-6-6))dep. on substratedep. on substrate

Physiol Physiol conc.conc. ((mol/lmol/l))

Physiol Physiol conc.conc. ((mol/lmol/l))

1010-9-9

1010-9-9 - 10- 10--77

1010--12 12 - 10- 10--1111

23

Oxidation reactions Oxidation loss of H2 or gain of O, O2, or X2

Reduction gain of H2 or loss of O, O2, or X2The loss or gain of H2O or HX are not considered oxidation-reduction reactions. X=halogen

24

Radical-mediated reactionsRadical-mediated reactionsAddition

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

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

Enzymatic sources of ROSXanthine 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 peroxidation1.1 - InitiationPeroxidation 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

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)

initiation, propagation and termination

31

ROS Arise Throughout the Cell

Mitochondrion

Chloroplast

Nucleus

Cytosol

Cell Wall

WoundingChilling Ozone

Drought,Salinity

ExpressionGene

Antioxidant genes

Post-transcriptionalEffects

ParaquatHigh Light + Chilling

Sulfur Dioxide

,,

subcellularROS

sitesunclear

(

)

,Pathogens

Post-transcriptionalEffects

Mitochondrion

Chloroplast

Nucleus

Cytosol

Cell Wall

Wounding

Chilling Ozone

ExpressionGene

Antioxidant genes

Post-transcriptionalEffects

ParaquatHigh Light + Chilling

Sulfur Dioxide

Pathogens

Post-transcriptionalEffects

ROS subcellular sites unclear

Drought Salinity

32

The electron transport system in the thylakoid membrane

showing 3 possible sites of activated oxygen production

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).

Mehler reaction

auto-oxidizable

(a) The water–water cycle.

(b) The ascorbate–glutathione cycle.

(c) The glutathione peroxidase (GPX) cycle.

(d) 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.

3434

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

35

ROS production in Mitochondria

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 get Mol Cel Biol, 2000, p. 7311-7318, Vol. 20,

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

Electron transfers oxidation of intermediary metabolites by O2

36

The source of mitochondrial ROS involves a non-heme Fe protein that transfers ê 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

Mitochondria as a source of ROSMitochondria as a source of ROS

37

Functions of the alternative oxidase

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

Option for envir stress regulation

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

Free RadicalsFree 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

R3C. Carbon-centered

R3N. Nitrogen-centered

R-O. Oxygen-centered

R-S. Sulfur-centered

ProoxidantsProoxidants

Non-Radicals: Species that have strong oxidizing

potential Species that favor the formation of

strong oxidants (e.g., transition metals)

H2O2 Hydrogen peroxide

HOCl- Hypochlorous acid

O3 Ozone

1O2 Singlet oxygen

ONOO- Peroxynitrite

Men+ Transition metals

41

Reactive Oxygen Species (ROS)Reactive Oxygen Species (ROS)

Radicals:

O2.- Superoxide

.OH Hydroxyl

RO2. Peroxyl

RO. Alkoxyl

HO2. Hydroperoxyl

Non-Radicals:

H2O2 Hydrogen peroxide

HOCl- Hypochlorous acid

O3 Ozone

1O2 Singlet oxygen

ONOO- Peroxynitrite

Oxidants

Oxidative Stress

Oxidative Protection Oxidative Stress

Oxidative Protection

Antioxidants

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

ROS scavenging moleculesROS scavenging molecules

plant antioxidantsAscorbateGlutathionePolyphenolsFlavonoidsLipoic acid

Enzymes:SODCatalaseGlutathione peroxidaseAscorbate peroxidaseThioredoxinsGlutaredoxins

Flavonoids

Ponce de León

Nature 425, 132-133

44

Reactive Nitrogen Species (RNS)Reactive Nitrogen Species (RNS)

Radicals:

NO. Nitric Oxide

NO2. Nitrogen dioxide

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

Nitric OxideNitric Oxide

NO refers to nitrosyl radical (•NO) and its nitroxyl NO refers to nitrosyl radical (•NO) and its nitroxyl (NO–) and nitrosonium (NO+) ions(NO–) and nitrosonium (NO+) ions

Freely diffusible, gaseous free radical.Freely diffusible, gaseous free radical. First described in 1979 as a potent First described in 1979 as a potent

relaxant of peripheral vasculature. relaxant of peripheral vasculature. Used by the body as a signaling molecule.Used by the body as a signaling molecule. Used as neurotransmitter, bactericide. Used as neurotransmitter, bactericide. Environmental PollutantEnvironmental Pollutant First gas known to act as a biological First gas known to act as a biological

messengermessenger

N O

Nitric Oxide in plantsNitric Oxide in plants

Affects aspects of plant Affects aspects of plant growth and growth and development.development.

Affects the responses to:Affects the responses to:

light, gravity, oxidative stress, light, gravity, oxidative stress, pathogenspathogens..

Can be a Can be a maturationmaturation and and senescencesenescence factorfactor

Has a concentration dependent Has a concentration dependent cytotoxiccytotoxic oror

protective protective (antioxidant)(antioxidant) e effects. ffects.

NONO--induced cell death in induced cell death in ArabidopsisArabidopsis occurs independently of ROSoccurs 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

cGMP in NOcGMP in NO--induced cell deathinduced cell death

Cells were pre-treated with ODQ (guanylate cyclase inhibitor) and/or 8Br-cGMP prior to RBS.

The effects of the caspase-1 inhibitor Ac-YVAD-CMK on NO- and H2O2-induced cell death

49

NO and Cell DeathNO and Cell Death

NO + H2O2 cause cell death

NO + O2- react to form peroxynitrite

Peroxynitrite (ONOO -) does not cause cell death

Too much O2- ‘mops up NO’ – no death

Delladonne et al. (2001) PNAS 98:13454

+PBITUPsm (avrRpm 1)

0

10

20

30

40

50

60

70

80

90

100

1mM H2O2 10mM H2O2 NO 1mMH2O2+NO

% C

ell D

eath

50

Endogenous sources of ROS and RNSEndogenous sources of ROS and RNS (in animals) (in animals)

Mitochondria

Lysosomes

Peroxisomes

Endoplasmic Reticulum

Cytoplasm

Microsomal Oxidation, Flavoproteins, CYP enzymes Myeloperoxidase

(phagocytes)

Electron transport

Oxidases,Flavoproteins

Plasma Membrane

Lipoxygenases,Prostaglandin synthase

NADPH oxidase

Xanthine Oxidase,NOS isoforms

FeCu

Transition metals

51

PEROXISOMEPEROXISOME

Fatty Acid

Acyl-CoA

Enoyl-CoA

Hydroxyacyl-CoA

Ketoacyl-CoA

Acetyl-CoA Acyl-CoA shortened by two carbons

Fatty acyl-CoA synthetase

Acyl-CoA oxidase

Enoyl-CoA hydrolase

Hydroxyacyl-CoAdehydrogenase

Thiolase

H2O2

• -oxidation of fatty acids• bile acid synthesis• purine and polyamine

catabolism• amino acid catabolism• oxygen metabolism

52

NADH + H+ NAD+

FADH2 FAD

bIIbIII

cII cIII

aII aIII

H2O O2

-0.32 V

-0.06 V

0.04 V

0.25 V

0.29 V

0.82 V

Cytochromes

IncreasingReducingPower

e-

e-

e-

e-

e-

O2 + e- => O2.- -0.45 V

O2 + 2H+ + 2e- => H2O2 -0.11 V

O2 + 4H+ + 4e- => H2O 0.82 V

Oxidative Phosphorylation & ROS

53

Many key oxidoreductases such as dehydrogenases, hydrogenases, nitrogenases, and the many oxygen enzymes of synthesis, drug detoxification, respiration photosynthesis, include a chain of single electron transferring redox

cofactors. Porphyrins, chlorins, iron sulfur clusters, flavins or quinones are common members of the chains.

The chains, which can comprise 2 to 8 cofactors, serve to ferry single ê between one site of substrate oxidation/reduction and another, or to a place close to the surface of the enzyme where they are exchanged with other single ê transferring redox protein partners, such as cytochrome c or flavodoxin. The distance covered by these linear chains can be rather long.

54

Intracellular ROS abundance in WT and Aox1 transgenic cultured tobacco cells.

antisense sense

• Plant Mitochondria also Contain an Uncoupling Protein • Mammalian mitochondria do not contain the AOX. Instead they have

an uncoupling protein that increases the proton permeability of the inner mitochondrial membrane and in that way dissipates the proton gradient. This is another mechanism for reducing the ATP production and increasing heat production. Surprisingly, plant mitochondria also contain a protein resembling the uncoupling protein

55

Oxygen consumption in oxidatively stressed mitochondria.

C

G/GO

0.30

0.59

rot

mal+glutADP

A

O2 c

onsu

mp

tion

/µg

pro

t/m

in

Time (min)

C0.14

H2O2

0.67

B

mal+glut

ADP

O2 c

onsu

mp

tion

/µg

pro

t/m

in

C

G/GO

0.20

0.59

KCN

suc + ADP

O2 c

onsu

mp

tion

/ µg

pro

t/m

in C

0123

Time (min)

01

Time (min)

0123

A) Arabidopsis cells were treated with G/GO. Electron transport was initiated by addition of complex I substrates, malate plus glutamate and NAD+. Coupling between the electron transport and ATP production was estimated by the addition of ADP. The role of complex I on oxygen consumption was examined by addition of rotenone. Numbers indicate the rate of oxygen consumption.B) Cells were spiked with 5 mM H2O2 and mitochondria were isolated 3 h later. Electron transport across complex I was measured as described in (A).

C) Electron transport across complex III was measured with 10 mM succinate plus 100 mM ADP. The dependence of oxygen consumption on the cytochrome c pathway was examined by addition of 50 mM KCN

56

ROS production in isolated mitochondria

Control G/GO

A

A) Mitochondria isolated from control or cells treated for 3 h with G/GO and stained with DHDR123

0

0.1

0.2

0.3

0.4

0.5

0 1 5

H2O2 (mM)

B

mol

H2O

2/µ

gPro

t/m

in

0

0.1

0.2

0.3

0.4

0.5

succinate

mol

H2O

2/µ

gPro

t/m

in

C

malatecontrol

H2O2 pretreated

Amplificatio

n of the Oxidativ

e Stress

Amplificatio

n of the Oxidativ

e Stress

57

Mitochondrial Aconitase Is a Source of Hydroxyl Radical

-H2O

   + H2O

+H2O

   - H2O

)1(

citrate cis-Aconitate Isocitrate

[Fe4S4(]S   Cys)3(H2O)n [Fe3S4(]S

  Cys)3

Iron-sulphur clusters

Aconitase (aconitate hydratase; EC 4.2.1.3) catalyses the stereo specific isomerisation of citrate to isocitrate via cis aconitate in the tricarboxylic acid cycle, a non redox active process

58

Recently it has been proposed that the reaction between mitochondrial aconitase and superoxide plays a major role in mitochondrial oxidative damage. During this reaction, the iron is released from m-aconitase as iron(II) with the concomitant

generation of H2O2. This facilitates the formation of "free" hydroxyl radical in mitochondria. In the presence of intracellular reducing agents (e.g. glutathione, ascorbate, and NADPH), iron(II) is reincorporated into the inactive form of m-aconitase to regenerate the active form. According to this proposal, hydroxyl radical is continuously generated in mitochondria as a result of the reaction between superoxide and aconitase.

J Biol Chem, Vol. 275, 14064-14069, 2000

(Because of the Aconitase role in cellular energy production, this enzyme function is well positioned as an important marker relative to biological decline)

59

The plant mitochondria may integrate stress signals for programmed cell death (PCD). There are many different situations that lead to cytochrome c release. These include oxidative stresses that induce permeability transition (PT) pore formation, stresses on electron transport and a rise in Ca2+ levels. It is proposed that when cells are unable to maintain metabolic homeostasis and the stresses overwhelm the cell, that mitochondria release cytochrome c triggering death. These stresses are normal components of PCD in plants.

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Models for the release of cytochrome c from mitochondria

In models a and b, the outer mitochondrial membrane ruptures as a result of swelling of the mitochondrial matrix, allowing cytochrome c to escape from mitochondria. Model a involves opening of the PTP whereas model b involves closure of the VDAC and hyperpolarization of the inner mitochondrial membrane as the causes of matrix swelling. In models c–e, a large channel forms in the outer membrane (via VDAC), allowing cytochrome c release, but mitochondria are not damaged

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(a) In all cases Cytochrome c release into the cytosol requires calcium flux at low cellular ATP levels. In the first (b), the permeability transition pore (PT pore) forms as a complex with the voltage-dependent anion channel (VDAC), the adenine nucleotide translocator (ANT), cyclophilin D (not shown) and the benzodiazepine receptor (not shown). The PT pore permits water to move into the matrix; outer membrane rupturing occurs when the inner membrane swells. (c) Cytochrome c can also be released directly via the VDAC.

Integration of stress signals by Mitochondria

62

Increases in cytosolic Ca2+ due to activation of ion channel-linked receptors, can induce permeability transition (PT) of the mitochondrial membrane. PT constitutes the first rate-limiting event of the common pathway of apoptosis. Upon PT, apoptogenic factors leak into the cytoplasm from the mitochondrial intermembrane space. Two such factors, cytochrome c and apoptosis inducing factor (AIF), begin a cascade of proteolytic activity that ultimately leads to nuclear damage (DNA fragmentation) and cell death. Cytochrome c, a key protein in electron transport, appears to act by forming a multimeric complex with Apaf-1, a protease, which in turn activates procaspase 9, and begins a cascade of activation of downstream caspases. Smac/Diablo is released from the mitochondria and inhibits IAP (inhibitor of apoptosis) from interacting with caspase 9 leading to apoptosis. Bcl-2 and Bcl-X can prevent pore formation and block the release of cytochrome c from the mito

Mitochondria in Apoptosis

Bax

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Nitric oxide (NO) is a pleiotropic signalling molecule that binds to cytochrome c oxidase (complex IV) reversibly and in competition with oxygen. Endogenously generated NO disrupts the respiratory chain and causes changes in mitochondrial Ca2+ flux.

64

65

Oxidative Burst in the Plasma Membrane

apoplastic peroxidase

NADPH oxidase

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Activation of NADPH oxidase by pathogens (elicitors)

ArabidopsisRiceHumangp91phox

rbohA

EF hands – Ca2+-binding sites.

Resistance responses

Exogenous H2O2 rescues both Ca2+ channel activation and stomatal closing in atrbohD/F placing it upstream of Ca2+

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Activation of NADPH Oxidase Occurs within Intracellular Compartments

Molec. Cell 11, 35-47 (2003)

animals plants

6868

Oxidants

Oxidative Stress

Oxidative Protection Oxidative Stress

Oxidative Protection

Antioxidants

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 ???

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Antioxidants

Oxidants Oxidative Damage

Enzymatic Defences – catalytically remove ROS

Metal Sequestration Proteins

Low MW Antioxidants

Other Protective Compounds e.g. HSPs

(Repair Processes)

Amounts Variable - cell types & tissues

Effectiveness Variable - (production site, radical species)

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ROS Detoxification

Catalases: 2 H2O2 ---> 2 H2O + O2

Peroxidases: AH2 + H2O2 ---> A + 2 H2O A is an electron donor

Catalytic Activity:Mn3+ + O2

- (Mn3+-O2-) Mn2+ + O2

Mn2+ + O2- (Mn2+-O2

-) + 2H+ Mn3+ + H2O2

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Cellular localization of SODs

72

Halliwell-Asada pathway redox cycling of ascorbate in the chloroplast

Antioxidant concentration in plant cells ascorbate (10-100 mM), glutathione (1-10 mM)

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Light-induced necrosis in Cat1AS plants and protection by elevated CO2

Changes in ascorbate and glutathione contents in leaves of Cat1AS and wild-type tobacco during light stress

(A) Effect of a shift from LL to HL on the levels of reduced (L-AA) and oxidized (DHAA) ascorbate. (6 h and 48 h exposure to HL).

(B) Effect on reduced (GSH) and oxidized (GSSG) glutathione

Complementation by catalase

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Flavonoids are Chemo-preventive Agents

75

Flavonoid Structure

• 200-300 Related Polyphenols

• Substitution on the C ring distinguishes the classes flavonoids

• Substitution on the A and B rings distinguish structures within a class

• Three potential metal binding sites exist

5

6

7

8 O1 2

3

4

6'

2'

3'

4'

5'

A

B

C

OH O

OH

O

OH

OH

OH

1

23

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Phenotypes associated with Bax expression in transgenic plants

ROS Production in Plants Expressing Bax

PNAS | 2001 | vol. 98 | 12295PNAS 1999; 96: 7956-7961 .

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Ascorbate reaction with superoxide can serve a physiologically similar role to SOD:

2 O

2 + 2H+ + ascorbate --> 2H2O2 + dehydroascorbate

The reaction with hydrogen peroxide is catalysed by ascorbate peroxidase :

H2O2+ 2 ascorbate --> 2H2O + 2 monodehydroascorbate

The indirect role of ascorbate as an antioxidant is to regenerate membrane-bound antioxidants, like -tocopherol, that scavenge peroxyl radicals and singlet O2,

respectively:

tocopheroxyl radical + ascorbate tocopherol + monodehydroascorbate

The above reactions indicate that there are two different products of ascorbate oxidation, monodehydroascorbate and dehydroascorbate, representing 1e and

2e transfers, respectively.

The monodehydroascorbate can either spontaneously dismutate (below) or is reduced to ascorbate by NAD(P)H monodehydroascorbate reductase (below):

2 monodehydroascorbate ascorbate + dehydroascorbate

monodehydroascorbate + NAD(P)H ascorbate + NAD(P)

The dehydroascorbate is unstable above pH6, decomposing into tartrate and oxalate. To prevent this, dehydroascorbate is rapidly reduced to ascorbate by

dehydroascorbate reductase using reducing equivalents from glutathione (GSH):

2 GSH + dehydroascorbate GSSG + ascorbate

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interactions that lead to recruitment of IP3 receptors during apoptosis

The positive feedback between IP3 receptor-mediated Ca2+ release and mitochondria underlies the generation of Ca2+ signals that accelerate the rate of cell death.

The apoptosis-inducing cycle of Ca2+ between IP3 receptors and mitochondria can be initiated by a variety of mechanisms, including non-specific entry of Ca2+ following membrane damage.

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The role of Aquaporins and membrane damage in chilling and hydrogen peroxide induced changes

in the hydraulic conductance of maize roots

Scheme summarizing the interpretation of the results. Chilling causes an initial decrease of Lo in both genotypes. After 3 d at 5°C, the tolerant genotype recovers its Lo thanks to the increase in aquaporin abundance and phosphorylation and to the maintenance of membrane integrity. On the contrary, the sensitive genotype does not recover its Lo because of membrane damage caused by oxidative stress. The tolerant genotype can cope with the oxidative stress, but the sensitive genotype cannot.

Systemic Signaling and Acclimation in response to excess light

Photodamage & APX2 induction

Leaves grown in LL (control) exposed to EL. (A) Chlorosis on detached leaves after 2 hours in EL. (B) relative luciferase activity

Systemic induction of APX2-LUC expression.

Image of luciferase activity. A part of the whole rosette (as shown) was exposed to EL for 40 min (arrow -> the apical rosette region). A typical primary (1°) EL-exposed leaf and a secondary (2°) LL-exposed leaf are shown

H2O2 is a local and systemic signal involved in the adaptation of leaves to high light

(the arrow indicates the apical region of the rosette)

catalase but not SOD diminished APX2 expr.

Systemic induction of H2O2 by wounding