Mitochondrial-Produced Reactive Oxygen Species - PowerPoint Presentation

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Mitochondrial-Produced Reactive Oxygen Species

Matthew Zimmerman, PhD

Assistant Professor

Cellular & Integrative Physiology

University of Nebraska Medical Center

mczimmerman@unmc.edu

Slide2

Lecture Outline

Sources of mitochondrial-produced reactive oxygen species (ROS)

Complex I and Complex III – Primary sources of ROS in mitochondria

Mitochondrial-localized antioxidantsMethods to measure mitochondrial-produced ROSDiseases associated with mitochondrial-produced ROSAmyotrophic lateral sclerosis (ALS; aka Lou Gehrig’s disease)

Murphy MP. (2009)

Biochem

J. 417:1-13)

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What are reactive oxygen species (ROS)

and free radicals?

ROS: species of oxygen, produced by all aerobic cells, that are in a more reactive state than molecular oxygen Free radicals: an atom or group of atoms possessing one or more unpaired electrons ROS best known for role in host defense mechanisms Often considered toxic byproducts of cellular metabolism More recently, ROS recognized as key signaling molecules

O

2

O

2

-

HO

2

H2O2

OH

H2O

e

-

e

-

e

-

e

-

superoxide

hydrogen peroxide

hydroxyl radical

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Sources of Reactive Oxygen Species

Mitochondria

NADPH

oxidase Hypoxanthine/xanthine oxidase Lipoxygenase Nitric oxide synthases

Turrens

JF. (2003) J Physiol. 552.2:335-344

NADPH oxidase

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Sources of

Mitochondrial-Produced

Reactive

Oxygen SpeciesOne-electron reduction of oxygen is thermodynamically favorable for many mitochondrial oxidoreductases due to the moderate redox potential of the superoxide/dioxygen couple (E1/2 = -0.16 V)Cytochrome b

5 reductase:

Outer mitochondrial membrane localization

Oxidizes cytoplasmic NAD(P)H

Reduces cytochrome

b

5 in outer membraneMay produce O2

- (~ 300 nmol/min/mg protein)Upregulated in schizophrenic patients

Monoamine oxidase (MAO):Outer mitochondrial membrane localizationCritical in turnover of monoamine neurotransmitters

Catalyze the oxidative deamination of biogenic amines aldehyde and release of H

2O2

May be involved in ischemia, aging, Parkinson’s disease

Bortolato

M et al. Adv Drug

Deliv

Rev. 2008

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Sources of

Mitochondrial-Produced

Reactive

Oxygen Species3. Dihyroorotate dehydrogenase (DHOH):Located at the outer surface of inner membraneIn the process of pyrimidine nucleotide synthesis, DHOH converts dihydroorotate

to oro

tate

Electron receptor is coenzyme Q and in absence of coenzyme Q produces H

2

O

2 (in vitro

)Role in producing ROS in vivo remains unclear and controversial

4. Dehydrogenase of a-glycerophosphate:Located at the outer surface of inner membrane

Uses coenzyme Q as electron receptor and catalyzes oxidation of glycerol-3-phosphate to dihydroxyacetone Studies in mice and drosophila suggest it produces H

2O2

Aconitase:

Localized in matrix

Catalyzes conversion of citrate to isocitrate (

tricarboxylic acid (TCA) cycle)

Inactivated by O2-

and, in turn, produces OH

most likely via Fe2+ release

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6. a

-

Ketoglutarate

dehydrogenase complex:Located on the matrix side of inner membraneUses NAD+ as electron acceptor and catalyzes oxidation of a-ketoglutarate to succinyl-CoASimilar to other sources, limited supply of electron acceptor promotes production of ROS7. Succinate

dehydrogenase (SDH; aka Complex II):Located at the inner surface of

inner membrane

Flavoprotein

that oxidizes

succinate to furmarate

using coenzyme Q as electron receptorIsolated SDH can produce ROS (again in absence of electron receptor)

Mutations in SDH subunits results in an increase in mitochondrial-localized ROS, particularly superoxideSources of Mitochondrial-Produced

Reactive Oxygen Species

Modified from Turrens JF, 2003

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Complex I: A Primary Source

of

Mitochondrial-Produced Reactive

Oxygen SpeciesComplex I (aka NADH-ubiquinone oxidoreductase; NADH dehydrogenase) Major entry point for electrons into the electron transport chain (ETC)

Flavin mononucleotide (FMN) accepts electrons from NADH

FMN passes electrons to chain of

FeS

centers (n=7) and finally to

CoQ

Produces O

2- from the reaction of oxygen with the fully reduced FMN (dependent on NADH/NAD+

ratio) Inhibition of respiratory chain or increased levels of NADH increases NADH/NAD+ ratio and, in turn produces

O2-

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Complex I: A Primary Source

of

Mitochondrial-Produced Reactive

Oxygen SpeciesReverse Electron Transfer (RET) production of superoxide Electrons are transferred against redox potential gradient (reduced CoQ NAD+) Occurs during low ATP production resulting in a high protonmotive

force (D

p

) and reduced

CoQ

(

succinate or a-glycerophosphate

supply electrons to reduce CoQ) Rate of RET-dependent superoxide production may be the highest that can occur in mitochondria

RET: high

Dp and high CoQH2

/CoQModified from Murphy MP. (2009)

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Complex I: A Primary Source

of

Mitochondrial-Produced Reactive

Oxygen SpeciesIncreasing Complex I-produced superoxide experimentally: Rotenone- induced inhibition of Complex I Rotenone binds to the CoQ-binding site Electrons in Complex I “leak” from either FMN or

FeS centers to oxygen producing superoxide

Modified from Liu Y. et al. (2002). J

Neurochem

. 780-7.

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Complex III: A Primary Source

of

Mitochondrial-Produced Reactive

Oxygen Species Oxidizes CoQ using cytochrome c as electron acceptor Reduced CoQ (QH2) transfers one electron to FeS

protein (ISP, aka Rieske

protein) and eventually cytochrome c

The resulting

semiquinone

(Q

-) transfers electrons to cytochrome b, then to the

Qi site which results in the reduction of another CoQ molecule (Q-cycle)

The semiquinone (Q-

) is unstable and can donate electron to oxygen forming superoxideComplex III (aka ubiquinone:cytochrome

c reductase)

Modified from

Turrens JF, 2003

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Complex III: A Primary Source

of

Mitochondrial-Produced Reactive

Oxygen SpeciesIncreasing Complex III-produced superoxide experimentally: Antimycin-induced inhibition of Complex III Antimycin blocks the transfer of electrons to the Qi-site, which results in the accumulation of the unstable semiquinone

The unstable semiquinone

can transfer electrons to oxygen producing superoxide

Andreyev A.U., et al. (2005). Biochemistry (Moscow). 70:200-14.

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Mitochondrial-localized antioxidants

Manganese superoxide dismutase (MnSOD, SOD2):

Catalyzes

dismutation of superoxide producing hydrogen peroxide and oxygenLocated exclusively in matrix of mitochondriaNuclear-encoded protein with a mitochondrial-target sequenceHomozygous knockout mice only live for few daysLarge percentage of tumor cells have low MnSOD activity

O

2

-

+

O

2

- + 2H+ H2O

2 + O2

MnSOD

2. Copper/Zinc superoxide dismutase (CuZnSOD, SOD1)

Catalyzes same reaction as MnSOD

Primarily found in cytoplasm, but also present in mitochondria

Precise mitochondrial localization is unclear – most evidence indicates intermembrane space

Mechanism of transport into mitochondria is also unclearMutant SOD1, associated with amyotrophic lateral sclerosis (ALS), appears to accumulate in mitochondria

Zhang DX. (2006). Am J Physiol. 292:H2023-31)

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Mitochondrial-localized antioxidants

3. Glutathione

~ 10% glutathione levels in cells is in mitochondria

Can be transported into mitochondria via specialized GSH-transportersOxidized glutathione (GSSG) can be reduced back to GSH by glutathione reductase localized in the matrix4. Glutathione peroxidase (GPx1)Uses GSH for the reduction of hydrogen peroxide to waterFound in mitochondrial matrix and intermembrane space

5. Phospholipid glutathione peroxidase (

PhGPx

; GPx4)

Reduces lipid hydroperoxides and hydrogen peroxide

GPx4 long form expressed in mitochondria

Knockout mice are embryonic lethal

6. Cytochrome CPresent in intermembrane spaceCan scavenge superoxide

The reduced cytochrome c is recycled by cytochrome c oxidaseBiological significance of cytochrome c as a superoxide scavenger in vivo remains to be fully elucidated

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Mitochondrial-localized antioxidants

Peroxiredoxins

(

Prx)Reduce hydrogen peroxide and lipid hydroperoxidesPrx3 highly expressed in heart, adrenal gland, liver and brain mitochondriaPrx5 highly expressed testisThioredoxin (Trx) systemTrx2 recycles

Prx by reducing the disulfide

Oxidazed

Trx2 is then recycled by thioredoxin reductase (

TrxR

), which uses NADPH as the source of reducing equivalents

Echtay

KS. (2007) Free Rad Biol Med. 43:1351-71

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Smith R.A.J., et al. (2008) Ann NY

Acad

Sci. 1147:105-111

Exogenous mitochondrial-targeted antioxidants Antioxidant compounds covalently attached to a lipophilic triphenylphosphonium cation target mitochondria

Such compounds include: SOD mimetic M40403 (

MitoSOD

) and tempol (

MitoTempol

); peroxidase mimetic

ebselen (MitoPrx); coenzyme Q (

MitoQ); tocopherol (MitoE)

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Methods for measuring mitochondrial-produced ROS

MitoSOX Red fluorescence

Mitochondrial-targeted superoxide sensitive fluorogenic probe (

Invitrogen/Molecular Probes)MitoSOX is dihydroethidum (DHE; aka hydroethidine) linked to a triphenylphosphonium group

Like DHE, fluorescence can be detected using 405, 488, 510 nm excitation

However, only the 405 nm excitation detects the 2-hydroxyethium fluorescent product which is specifically dependent on superoxide

Dikalov

S. et al. (2007). Hypertension

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Methods for measuring mitochondrial-produced ROS

2. Electron paramagnetic resonance (EPR) on isolated mitochondria

Mitochondria can be isolated from tissue or cultured cells

Isolated mitochondria incubated with EPR spin trap or spin probeAmount of spin trap/probe radical are detected using EPRImportant issues to consider:Purity and integrity of mitochondria preparationDepending on spin trap/probe selected use specific antioxidants (e.g. SOD) to selectively measure a particular ROS

Mariappan

N. et al. Free

Rad

Biol

Med. (2009). 46:462-70.

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Methods for measuring mitochondrial-produced ROS

3.

Amplex

Red to detect hydrogen peroxide efflux from isolated mitochondriaIsolated mitochondria incubated with Amplex Red in the presence of HRPMeasure levels of fluorescent product, resorufinImportant issues to consider:Purity and integrity of mitochondria preparationIf using this method to indirectly measure superoxide, remember not all superoxide is converted to hydrogen peroxide (nitric oxide in mitochondria)

Numerous matrix

peroxidases

will consume hydrogen peroxide

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Fatal neurodegenerative disease that specifically targets motor neurons in the spinal cord, brain stem, and cortex

Most common adult motor neuron disease; 5,600 cases diagnosed each year in U.S.

Disease onset usually begins with weakness in arms and legs and quickly progresses to total paralysis

Patients generally die of respiratory failure 2-5 years after the first symptoms appear ALS often referred to as Lou Gehrig’s diseaseAmyotrophic Lateral Sclerosis (ALS)From alsa.org

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20-25% of familial ALS cases

are associated with mutations in a cellular antioxidant enzyme called

CuZnSOD (SOD1

) Mutant CuZnSOD-induced neuronal toxicity is believed to involve a toxic gain of function; not a loss of SOD activity Many familial ALS mutant CuZnSOD proteins retain SOD activity CuZnSOD knockout mice do not develop motor neuron disease Overexpressing wild-type CuZnSOD in animal or cell culture models of ALS does not provide protection Mutant CuZnSOD expression is ubiquitous, although only motor neurons appear to be affectedFrom Valentine JS, 2005. Annu

. Rev. Biochem

1. Control

2. Wild-type CuZnSOD

3. Mutant CuZnSOD #1

4. Mutant CuZnSOD #2

5. Mutant CuZnSOD #36. Mutant CuZnSOD #4

Amyotrophic Lateral Sclerosis (ALS)

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Expression of different CuZnSOD mutants in cultured neurons decreases cell survival

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Mutant CuZnSOD increases

superoxide levels in

mitochondria

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Overexpressing

MnSOD attenuates

mutant CuZnSOD-mediated increase in

mitochondrial superoxide

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Overexpression of MnSOD (SOD2) inhibits mutant CuZnSOD-mediated neuronal toxicity

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Intramuscular injection of AdSOD2 results in retrograde transport and SOD2 expression in

spinal

cord motor

neuronsControl AdSOD2

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Intramuscular injection of AdSOD2 delays

motor dysfunction in ALS transgenic mice

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Summary

Numerous sources of ROS production in mitochondria

Complex I and III have been the most studied and best characterized, and, to date, are generally considered the primary sources of mitochondrial-produced ROS

Collection of mitochondrial-localized antioxidants also play a significant role in the levels of ROS in mitochondriaMitochondrial superoxide can be elevated experimentally with rotenone (Complex I inhibitor) or antimycin A (Complex III) inhibitorMitochondrial ROS can be reduced experimentally by using antioxidant compounds linked to a triphenylphosphonium group or by increasing expression of endogenous antioxidant proteins

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Andreyev A.U., et al. (2005). Biochemistry (Moscow). 70:200-14.

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superoxide not a strong oxidant, but precursor to other ROS and involved in propagation of oxidative chain

rxns

mitos are main cellular source of ROS other cellular sources include NADPH oxidase, cytochrome P450-dependent oxygenases, xanthine oxidase non-enzymatic production of superoxide occurs when 1 electron is transferred to molecular oxygen by reduced coenzymes (i.e. flavins

, iron sulfur clusters)

mitos

contain numerous redox centers that “leak” electrons to oxygen

1-4% of all oxygen consume is incompletely reduced to superoxide, thus increase oxygen consumption (hyperoxic conditions) can increase superoxide

however, hypoxic conditions can also increase superoxide ALS

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Zhang and

Gutterman

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Mitochondrial sources of superoxide

high reducing environment in mitochondria allows respiratory components, such as

flavoproteins

, iron-sulfur clusters, to transfer 1 electron to oxygen in fact most steps in ETC involve single-electron reductions steady state concentration of superoxide estimated to be 10-10 M and h2o2 estimated to be 5x10-10 M. superoxide can be produced on outer mito membrane, in matrix, and on both sides of inner membrane

matrix superoxide removed by

mnsod

superoxide in intermembrane space may be carried to cytoplasm via voltage-dependent anion channels (

han

et al. 2003) Complex III main source in heart and lung

Complex I main source in brain (important in aging and Parkinson’s) superoxide formation can increase both electron flow slows down and when concentration of oxygen increase

superoxide production increases as repiratory chain becomes more reduced

rotenone (cplx I) and antimycin (cplx III) may increase superoxide because upstream of site of inhibition carriers are fully reduced

however not all inhibitors have this effect

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Mitochondrial sources of superoxide

high reducing environment in mitochondria allows respiratory components, such as

flavoproteins

, iron-sulfur clusters, to transfer 1 electron to oxygen in fact most steps in ETC involve single-electron reductions steady state concentration of superoxide estimated to be 10-10 M and h2o2 estimated to be 5x10-10 M. superoxide can be produced on outer mito membrane, in matrix, and on both sides of inner membrane

matrix superoxide removed by

mnsod

superoxide in intermembrane space may be carried to cytoplasm via voltage-dependent anion channels (

han

et al. 2003) Complex III main source in heart and lung

Complex I main source in brain (important in aging and Parkinson’s) superoxide formation can increase both electron flow slows down and when concentration of oxygen increase

rotenone (cplx I) and antimycin (cplx III) may increase superoxide because upstream of site of inhibition carriers are fully reduced

however not all inhibitors have this effect

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Mitochondrial sources of superoxide

calcium, nitric oxide and

mito

membrane potential all play a role in generating mito ROS UCP, mito uncoupling proteins (mito anion carriers) induce protein leak across inner membrane and suppress mito membrane potential, thus decreasing ROS NO may inhibit complex IV, thus modulating

mito respiration and increase

mito

ROS

reverse electron transport increases superoxide and may come from III or II and can be blocked by rotenone, thus indicating that it is due to electrons entering Complex I thought the

CoQ

binding site.

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Pathological conditions

increase

mito

ROS can induce apoptosis by increasing mito permeablity via increasing opening of transition pores cytochrome c is released and can activate caspase cascade cytochrome c release can further increase mito superoxide because

cytochome c can reduce superoxide and because the ETC become more reduced because transfer of electrons between II and III slows down