Mitochondrial-Produced Reactive Oxygen Species

Matthew Zimmerman, PhDAssistant Professor

Cellular & Integrative PhysiologyUniversity of Nebraska Medical Center

mczimmerman@unmc.edu

Lecture Outline

1. Sources of mitochondrial-produced reactive oxygen species (ROS)

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

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)

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

O2

O2-HO2

H2O2

OH

H2O

e-

e-

e-

e-

superoxide

hydrogen peroxide

hydroxyl radical

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

Sources of Mitochondrial-Produced Reactive Oxygen Species

One-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)

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

Sources of Mitochondrial-Produced Reactive Oxygen Species

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

dihydroorotate 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

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

Sources of Mitochondrial-Produced Reactive Oxygen Species

Modified from Turrens JF, 2003

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

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)

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

Complex III (aka ubiquinone:cytochrome c reductase)

Modified from Turrens JF, 2003

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.

Mitochondrial-localized antioxidants1. 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)

Mitochondrial-localized antioxidants3. 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 antioxidants6. 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• 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

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-hydroxyethium 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

• 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. Control2. Wild-type CuZnSOD3. Mutant CuZnSOD #14. Mutant CuZnSOD #25. Mutant CuZnSOD #36. 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 neuronsControl AdSOD2

Intramuscular injection of AdSOD2 delaysmotor dysfunction in ALS transgenic mice

Summary1. Numerous sources of ROS production in mitochondria2. Complex I and III have been the most studied and best characterized, and, to date, are

generally considered the primary sources of mitochondrial-produced ROS3. Collection of mitochondrial-localized antioxidants also play a significant role in the levels of

ROS in mitochondria4. Mitochondrial superoxide can be elevated experimentally with rotenone (Complex I inhibitor) or

antimycin A (Complex III) inhibitor5. Mitochondrial ROS can be reduced experimentally by using antioxidant compounds linked to a

triphenylphosphonium group or by increasing expression of endogenous antioxidant proteins

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

• 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

Zhang and Gutterman

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

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

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.

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