Mitochondrial-Produced Reactive Oxygen Species Matthew Zimmerman, - - PDF document

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Mitochondrial-Produced Reactive Oxygen Species

Matthew Zimmerman, PhD Associate Professor Cellular & Integrative Physiology University of Nebraska Medical Center mczimmerman@unmc.edu

Summer 2013 BIOC 998‐590 UNL

Lecture Outline

• 1. Complex I and Complex III – Primary

sources of ROS in mitochondria

• 2. Other sources of mitochondrial-

produced reactive oxygen species (ROS)  NADPH oxidase (Nox4)

• 3. Mitochondrial-localized antioxidants
• 4. Methods to measure mitochondrial-

produced ROS

• 5. Diseases associated with

mitochondrial-produced ROS

• Amyotrophic lateral sclerosis

(ALS; aka Lou Gehrig’s disease)

Murphy MP. (2009) Biochem J. 417:1-13)

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Sources of Reactive Oxygen Species

• Mitochondria
• NADPH oxidase
• Xanthine oxidase
• Lipoxygenase
• Nitric oxide synthases

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

NADPH oxidase

Mitochondrial‐Produced ROS

• Generally accepted that mitochondrial

energy metabolism is the most quantitatively important source of ROS is most cells

• Superoxide (O2

‐) is the primary (and

proximal) ROS generated by mitochondria

• ≈ 0.2 ‐ 2 % of oxygen consumed by

mitochondria is converted to superoxide

• As electrons flow down chain they can

“leak” off chain on to oxygen → superoxide

• Presence of SOD in both matrix and

intermembrane space indicates importance

• f removing O2

‐ from mitochondria

• MnSOD knock‐out mice are perinatal lethal

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

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Mitochondrial‐Produced Superoxide

• One-electron reduction of oxygen is thermodynamically favorable for many mitochondrial
• xidoreductases due to the moderate redox potential of the superoxide/dioxygen couple

Under what conditions can mitochondria electron transport chain (ETC) produced superoxide?

• 1. Mitochondria not making ATP

and electron carriers are fully reduced

• 2. High NADH/NAD+ ratio in

mitochondria matrix

• Can be caused by damage to ETC,

slow respiration, or ischemia

Complex I: A Primary Source of Mitochondrial- Produced Superoxide

Complex 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 O2

- from the reaction of oxygen

with the fully reduced FMN (dependent on NADH/NAD+ ratio)

• Electrons may also leak off FeS centers
• Inhibition of respiratory chain or increased

levels of NADH increases NADH/NAD+ ratio and, in turn produces O2

-

O2

-

6/4/2013 4 Complex I: A Primary Source of Mitochondrial- Produced Reactive Oxygen Species

Reverse 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 (p) and

reduced CoQ (succinate or -glycerophosphate supply electrons to reduce CoQ)

• Rate of RET-dependent superoxide production may be the highest that can occur in

mitochondria

RET: high p and high CoQH2/CoQ

Modified from Murphy MP. (2009)

Complex I: A Primary Source of Mitochondrial- Produced Reactive Oxygen Species

Increasing 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.

6/4/2013 5 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 superoxide

• In matrix and intermembrane space

Complex III (aka ubiquinone:cytochrome c reductase)

Modified from Turrens JF, 2003

O2

‐

O2

‐

Complex III: A Primary Source of Mitochondrial- Produced Reactive Oxygen Species

Increasing 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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Additional Sources of Mitochondrial- Produced Reactive Oxygen Species

• 1. Cytochrome b5 reductase:
• Outer mitochondrial membrane localization
• Oxidizes cytoplasmic NAD(P)H
• Reduces cytochrome b5 in outer membrane
• May produce O2

- (~ 300 nmol/min/mg protein)

• Upregulated in schizophrenic patients
• 2. Monoamine oxidase (MAO):
• Outer mitochondrial membrane localization
• Critical in turnover of monoamine

neurotransmitters

• Catalyze the oxidative deamination of biogenic

amines aldehyde and release of H2O2

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

Bortolato M et al. Adv Drug Deliv Rev. 2008

• 3. Dihyroorotate dehydrogenase (DHOH):
• Located at the outer surface of inner membrane
• In the process of pyrimidine nucleotide synthesis, DHOH converts

dihyroorotate to orotate

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

H2O2 (in vitro)

• Role in producing ROS in vivo remains unclear and controversial
• 4. Dehydrogenase of -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 H2O2
• 5. 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

Additional Sources of Mitochondrial- Produced Reactive Oxygen Species

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• 6. -Ketoglutarate dehydrogenase complex:
• Located on the matrix side of inner membrane
• Uses NAD+ as electron acceptor and catalyzes oxidation of -ketoglutarate

to succinyl-CoA

• Similar to other sources, limited supply of electron acceptor promotes

production of ROS

• 7. 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 receptor

• Isolated SDH can produce ROS

(again in absence of electron receptor)

• Mutations in SDH subunits results in an increase in mitochondrial-localized

ROS, particularly superoxide

Modified from Turrens JF, 2003

Additional Sources of Mitochondrial- Produced Reactive Oxygen Species Link between NADPH oxidase and mitochondria?

• Gp91phox (Nox2) is distributed in the cytoplasm of neurons

and is “particularly abundant near mitochondria”.

Modified from Wang G., et al. 2004 J Neurosci. 24(24):5516-24

NADPH oxidase

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NADPH Oxidase (NOX)‐Derived ROS

• Multi‐subunit, membrane bound complex that passes electrons through the membrane

from NADPH or NADH to oxygen → superoxide

• 100x more selective for NADPH over NADH
• Phagocyte NADPH oxidase first example of a system that produces ROS as the primary

function (not as a byproduct)

• Responsible for phagocyte respiratory burst
• Respiratory burst absent in Chronic Granulomatous Disease (CGD) – patients lacking

cytochrome b558 (gp91phox or Nox2 + p22phox)

• NADPH oxidase subunits include:
• 7 Nox isoforms (Nox1, Nox2, Nox3, Nox4, Nox5, Duox1, Duox2)
• Often referred to as the catalytic subunits
• 2 organizer subunits (p47phox, NOXO1)
• 2 activator subunits (p67phox, NOXA1)
• 2 Duox‐specific maturation subunits (DUOXA1, DUOXA2)
• 1 stabilizing subunit (p22phox)
• p40phox
• Active complexes made of a mixture of these subunits

NADPH Oxidase

Conserved Structural Properties Nox Enzymes

• 1. NADPH binding site in COOH terminus
• 2. Flavin adenine dinucleotide (FAD) binding

region in COOH terminus

• 3. Six conserved transmembrane domains
• 4. Four conserved heme‐binding histidines
• Electrons are passed from NADPH → FAD →

1st heme → 2nd heme → Oxygen = superoxide

Bedard and Krause, Physiol Rev 2007

O2

‐

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Nox4, NADPH oxidase catalytic subunit, in mitochondria

PNAS, 2009 106(34):14385-14390

Nox4 in neuron mitochondria

Case AJ, et al. Mitochondrial-localized NADPH Oxidase 4 is a Source of Superoxide in Angiotensin II-stimulated Neurons. Am J Physiol Heart Circ Physiol. 2013

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MitoProt Score*: Nox4 – 0.977; MnSOD – 0.985; Prx3 – 0.993; LDH – 0.023; Actin – 0.016

* M.G. Claros, P. Vincens. Computational method to predict mitochondrially imported proteins and their targeting sequences.1996.

• Eur. J. Biochem. 241, 770-786.

Nox4 in neuron mitochondria

Case AJ, et al. Mitochondrial-localized NADPH Oxidase 4 is a Source of Superoxide in Angiotensin II-stimulated Neurons. Am J Physiol Heart Circ Physiol. 2013

Silencing Nox4 with siRNA attenuates AngII- induced increase in superoxide levels

Case AJ, et al. Mitochondrial-localized NADPH Oxidase 4 is a Source of Superoxide in Angiotensin II-stimulated Neurons. Am J Physiol Heart Circ Physiol. 2013

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Mitochondrial-localized antioxidants

• 1. Manganese superoxide dismutase (MnSOD, SOD2):
• Catalyzes dismutation of superoxide producing hydrogen peroxide and oxygen
• Located exclusively in matrix of mitochondria
• Nuclear-encoded protein with a mitochondrial-target sequence
• Homozygous knockout mice only live for few days
• Large percentage of tumor cells have low MnSOD activity

O2

- + O2 - + 2H+ H2O2 + 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 unclear

• Mutant SOD1, associated with

amyotrophic lateral sclerosis (ALS), appears to accumulate in mitochondria

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

CuZnSOD is commonly thought of as the cytoplasmic localized SOD, but it is also expressed in mitochondria

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Mitochondrial CuZnSOD in neurons following adenovirus-mediated gene transfer

Control (Non-infected) AdCuZnSOD (50MOI, 96h) MitoTracker Red CuZnSOD Merged

Mitochondrial CuZnSOD protein and activity are increased following gene transfer

EM micrograph of Mito-fraction

1 2 3 1 2 3

• 1. Control (Non-infected)
• 2. AdEmpty
• 3. AdCuZnSOD

Whole cell lysate Mito-fraction

MnSOD LDH (Cytosolic marker) COXIV CuZnSOD MnSOD CuZnSOD (human) CuZnSOD Calnexin (ER marker)

(Mito markers)

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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-transporters
• Oxidized glutathione (GSSG) can be reduced back to GSH by glutathione

reductase localized in the matrix

• 4. Glutathione peroxidase (GPx1)
• Uses GSH for the reduction of hydrogen peroxide to water
• Found 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 C
• Present in intermembrane space
• Can scavenge superoxide
• The reduced cytochrome c is recycled by cytochrome c oxidase
• Biological significance of cytochrome c as a superoxide scavenger in vivo

remains to be fully elucidated

Mitochondrial-localized antioxidants

• 6. Peroxiredoxins (Prx)
• Reduce hydrogen peroxide and

lipid hydroperoxides

• Prx3 highly expressed in heart,

adrenal gland, liver and brain mitochondria

• Prx5 highly expressed testis
• 7. Thioredoxin (Trx) system
• Trx2 recycles Prx by reducing the

disulfide

• Oxidized Trx2 is then recycled by

thioredoxin reductase (TrxR), which uses NADPH as the source

• f 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)

Methods for measuring mitochondrial-produced ROS

• 1. 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-hydroxyethidium 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 probe
• Amount of spin trap/probe radical are detected using EPR

Important issues to consider:

• Purity and integrity of mitochondria preparation
• Depending 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.

Methods for measuring mitochondrial-produced ROS

• 3. Amplex Red to detect hydrogen peroxide efflux from isolated mitochondria
• Isolated mitochondria incubated with Amplex Red in the presence of HRP
• Measure levels of fluorescent product, resorufin

Important issues to consider:

• Purity and integrity of mitochondria preparation
• If 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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Summary

1. Numerous sources of ROS production in mitochondria 2. 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 3. Nox4 has also been reported to be a source of ROS in mitochondria 4. Collection of mitochondrial-localized antioxidants also play a significant role in the levels of ROS in mitochondria 5. Mitochondrial superoxide can be elevated experimentally with rotenone (Complex I inhibitor) or antimycin A (Complex III) inhibitor 6. Mitochondrial ROS can be reduced experimentally by using antioxidant compounds linked to a triphenylphosphonium group or by increasing expression of endogenous antioxidant proteins

• 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 disease

Amyotrophic 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 affected

From Valentine JS, 2005.

• Annu. Rev. Biochem
• 1. Control
• 2. Wild‐type CuZnSOD
• 3. Mutant CuZnSOD #1
• 4. Mutant CuZnSOD #2
• 5. Mutant CuZnSOD #3
• 6. Mutant CuZnSOD #4

Amyotrophic Lateral Sclerosis (ALS) Expression of different CuZnSOD mutants in cultured neurons decreases cell survival

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Mutant CuZnSOD increases superoxide levels in mitochondria Overexpressing MnSOD attenuates mutant CuZnSOD-mediated increase in mitochondrial superoxide

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Overexpression of MnSOD (SOD2) inhibits mutant CuZnSOD-mediated neuronal toxicity Intramuscular injection of AdSOD2 results in retrograde transport and SOD2 expression in spinal cord motor neurons

Control AdSOD2

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Intramuscular injection of AdSOD2 delays motor dysfunction in ALS transgenic mice