Matthew Zimmerman PhD Assistant Professor Cellular amp Integrative Physiology University of Nebraska Medical Center mczimmermanunmcedu Lecture Outline Sources of mitochondrialproduced reactive oxygen species ROS ID: 908724
Download Presentation The PPT/PDF document "Mitochondrial-Produced Reactive Oxygen S..." is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.
Slide1
Mitochondrial-Produced Reactive Oxygen Species
Matthew Zimmerman, PhD
Assistant Professor
Cellular & Integrative Physiology
University of Nebraska Medical Center
mczimmerman@unmc.edu
Slide2Lecture 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)
Slide3What 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
Slide4Sources 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
Slide5Sources 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
Slide6Sources 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
Slide76. 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
Slide8Complex 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-
Slide9Complex 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)
Slide10Complex 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.
Slide11Complex 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
Slide12Complex 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.
Slide13Mitochondrial-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)
Slide14Mitochondrial-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
Slide15Mitochondrial-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
Slide16Smith 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)
Slide17Methods 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
Slide18Methods 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.
Slide19Methods 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
Slide20Fatal 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
Slide2120-25% 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)
Slide22Expression of different CuZnSOD mutants in cultured neurons decreases cell survival
Slide23Mutant CuZnSOD increases
superoxide levels in
mitochondria
Slide24Overexpressing
MnSOD attenuates
mutant CuZnSOD-mediated increase in
mitochondrial superoxide
Slide25Overexpression of MnSOD (SOD2) inhibits mutant CuZnSOD-mediated neuronal toxicity
Slide26Intramuscular injection of AdSOD2 results in retrograde transport and SOD2 expression in
spinal
cord motor
neuronsControl AdSOD2
Slide27Intramuscular injection of AdSOD2 delays
motor dysfunction in ALS transgenic mice
Slide28Summary
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
Slide29Slide30Andreyev A.U., et al. (2005). Biochemistry (Moscow). 70:200-14.
Slide31superoxide 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
Slide32Zhang and
Gutterman
Slide33Mitochondrial 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
Slide34Mitochondrial 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
Slide35Mitochondrial 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.
Slide36Pathological 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