Mitochondrial-Produced

Reactive Oxygen Species

Matthew Zimmerman, PhD

Assistant Professor

Cellular & Integrative Physiology

University of Nebraska Medical Center

mczimmerman@unmc.edu

Summer 2012 BIOC 998-590 UNL

Karolinska Institutet

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)

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 of removing O2

- from mitochondria MnSOD knock-out mice are perinatal lethal

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

Mitochondrial-Produced Superoxide One-electron reduction of oxygen is thermodynamically favorable for many mitochondrial

oxidoreductases 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-

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 (Dp) 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/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.

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.

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 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 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

6. a-Ketoglutarate dehydrogenase complex:

• Located on the matrix side of inner membrane

• Uses NAD+ as electron acceptor and catalyzes oxidation of a-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

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-

Nox4, NADPH oxidase catalytic subunit,

in mitochondria

PNAS, 2009

106(34):14385-14390

0

Nox4 in neuron mitochondria

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

Silencing Nox4 with siRNA attenuates AngII-

induced increase in superoxide levels

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

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)

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

of reducing equivalents

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

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

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

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. Collection of mitochondrial-localized antioxidants also play a significant role in the levels of ROS

in mitochondria

4. Mitochondrial superoxide can be elevated experimentally with rotenone (Complex I inhibitor) or

antimycin A (Complex III) inhibitor

5. 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

• 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

Mutant CuZnSOD increases superoxide

levels in mitochondria

Overexpressing MnSOD attenuates mutant

CuZnSOD-mediated increase in

mitochondrial superoxide

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

Intramuscular injection of AdSOD2 delays

motor dysfunction in ALS transgenic mice