M ITO C H O N D R IA L D N A

A N D O X ID ATIV E
P H O S P H O R Y LATIO N
D IS EA S ES (O X P H O S
D
ProfIS EABagiada
Agus S ESSpBiok
)
What is a Mitochondrion?

A cellular organelle probably of endosymbiotic

origin that resides in the cytosol of most
nucleated (eurkaryotic) cells.
This organelle produces energy by oxidising
organic acids and fats with oxygen by the
process of oxidative phosphorylation and
generates oxygen radicals (reactive oxygen
species ROS )as a toxic by-product
Mitochondrial Genetics

Each cell contains many mitochondria,

each of which contains multiple copies of
16.5-k-b circular DNA molecule
The mitochondrial genome is subject to a
number of peculiarities of inheritance
Mitochondrial Genetics

Interest in mitochondrial genetics comes

mostly from:
interest in diseases caused by mutations
in mDNA
interest in human history
mitochondrial enthusiast)
The nuclear and Mitochondrial genetic codes
are similar but not identical
M t-D N A genetic code

A minimum 32 t-RNA are required to

read a nucclear genetic code
In the mt DNA the codon anticodon
pairing is simplified to only 22 t-
RNA . One mito-chondrial t-RNA can
recognize an entire 4 member codon
family rather than 2 by nuclear t-
RNA.
 H strands : 12 oxphos subunit genes
(12S and 16S rRNA, 14 tRNA genes.
L strands : one oxphos subunit gene
(ND6 of complex I), 8 tRNA genes
D loop (Displacement loop) are 1122-
bp contain genetic information
necessary for replication and
transcription.
The human nuclear and mitochondrial genomes

Nuclear Genome Mitochondrial Genome

Size 3200 Mb 16.6 kb

No. of different DNA 23 (in XX cells) or 24 (in One circular DNA

molecules XY cells); all linear molecule
Total no. of DNA 46 in diploid cells, but Often several thousands
molecules per cell varies according to (but variable
ploidy
Associated protein Several classes of Largely free of protein
histone & nonhistone
protein
No. of genes ~ 30 000 ~35-000 37

Gene density ~ 1/100 kb 1/0.45 kb

 Table continued..
Repetitive DNA Over 50% of genome Very little

Transcription The great bulk of genes are Co-transcription of multiple

transcribed individually genes from both the heavy
and light strands

Introns Found in most genes Absent

% of coding DNA ~ 1.5% ~ 93%

Codon usage Slightly different see slide

Recombination At least once for each pair of No evidence for this

homologs at meiosis occurring naturally

Inheritance Mendelian for sequence on X Exclusively maternal

and autosomes; paternal for
sequence on Y
 Maternal genetic
transmission

An affected woman transmits the trait to

all her children. Affected men
(represented by squares do not pass
the trait to any of their offspring
Mitochondrial
inheritance. Sperm
mitochondria are shed
before entry of the
sperm nucleus. All
mitochondrial in the
zygote are contributed
by the egg cell
 OXPH O S D ISEASES

OXPHOS (respiration chain) is

responsible for producing much of
the ATP that is required by the cells.
The OXPHOS pathway incorporate
over 100 polypeptides, whose genes
are located in either n-DNA and mt-
DNA
The expression of these genes and
the assembly of the five enzymes
complex is highly ordered and
 It is now clear that human disease
can result from mutations in either
nuclear and mitochondrial DNA
genes or in the system that
coordinate their interaction.
Consequently : OXPHOS disease CAN
HAVE a complex array of inheritance
patterns and a wide spectrum of
clinical manifestation.
Biochem istry and genetic

OXPHOS is composed of five protein-

lipid enzymes complex which contain
flavins (FMN and FAD), quinoids
compounds (CoEQ10), transition
metals compounds (iron sulfur
cluster, hems, protein bond copper)
and cytochrom.
Located in the mitochondrial inner
membrane and are designated as:
Complex I until complex V
Five protein lipid enzym es
com plex
Complex I (NADH:ubiquinon
oxidoreductase EC 1.6.5.3)
Complex II (succinate:ubiquinone
oxidoreductase. EC 1.3.5.1
Complex III (Ubiquinon:ferrocytochrome
oxidoreductase EC 1.10.2.21
Complex IV (ferrocytochromc:oxygen
oxidoreductase/cytochrom c oxidase EC
1.9.3.1
Complex V (ATP syntase EC 3.6.1.34)
ELECTRO N E TRAN SPO RT

Complex I and II collect electron from

various sources and transfer them to
Ubiquinone.
The electron then move sequentially
through complex III, cytocrome c,
complex IV, and finally react with
oxygen, the terminal electron acceptor.
As electrons move through its pathway, proton
are pumped across the inner mit membrane to
intermembrane space at complex I,III and IV
producing a proton gradient
ATP Synthesis

Complex V utilizes the potential

energy stored in proton gradient to
condensed ADP and inorganic
phosphate (P) into ATP. The resulting
ATP is exchanged across the inner
membrane with ADP by adenin
nucleotide translocase (ANT).
Because ADP is the rate-limiting
substrate of OXPHOS, ANT may play
a major role in regulating
mitochondrial energetic.
 Electron Transport Chain
inner membrane space of mitochondria

Cyt c1 Cyt c
CoQ Cyt a,a3
FP Fe-S Fe-S
Cyt b
inner membrane
4 H+ + 4 e- + O2
NADH + H+ FADH2

FPflavoprotein
Fe-S iron-sulfur protein 2 H 2O
CoQ coenzyme Q
Cyt cytochromes. Structurally related proteins
that each contain iron
Com plex I
In the cytosol the most important
NAD-link dehydrogenase are the
glycolytic enzymes, glyceraldehide
phosphate dehydrogenase, and
lactate dehydrogenase.
Because NADH cannot penetrate the
inner membrane of mit, the malate
aspartate shuttle need to carries the
reducing equivalent across the
membrane, donating the electron to
NAD+ in the mit matrix to form
Com plex I(cont)
Comp I transfer e to CoQ10 through a
long series of redox groups include FMN
and six iron-sulfur cluster. According to
Ohnishi nomenclatur are designated as
N-1a, N-1b, N-2, N-3, N-4 and N-5.
Purification of complex I (from bovine
heart) and analysis of the polypeptide
composition revealed about 41 different
sub-units. Seven of these polypeptides
are encoded by mt-DNA genes,
designated as ND1, ND2, ND3, ND4,
ND4i, ND5 and ND6. The remaining
about 34 polypeptides are encoded by
n-DNA
Com plex I(cont ..)

These complex I polypeptide can be

isolated in 3 major groups: 1. the
flavo protein fragment, 2.the iron
protein fragment, 3. the hydrophobic
protein fragment.
Flavoprotein fragment consist of :
FMN, six of iron , and 3 poly peptides
(51,24 and 10 kDa).
The NADH and FMN binding site is in
the 51 kDa polypeptide. A 15 kDa
binding to ubiquinon. ND6 gene
 The hydrophobic protein fragment
contains the iron-sulfur centers are
electron donor to Ubiquinone.
Of the seven mitochondrial DNA
complex I genes, the gene product
for ND1, ND3, and ND4L have been
localized to the hydrophobic protein
fragment (maybe also ND2,ND4 and
ND5). ND1 gene product bind to
rotenon and therefore also transfer
Com plex II

E.C 1.3.5.1. (succinate:ubiquinon oxido

reductase)
Perform a key step in the TCA cycle, in
which succinate is dehydrogenated to
fumarate and the electron are donated to
ubiquinone in the mitochondrial inner
membrane.
Localized to the matrix side of the
mitochondrial inner membrane and is the
only OXPHOS enzyme at which all the
subunits are code by n-DNA
Succinate acts as both a substrate and
positive modifier of complex II.
Com plex III

EC 1.10.2.2 (Ubiquinol:ferrocytochrome c
oxidoreductase)
Catalyzes electorn transfer between two
mobile electron carriers, ubiquinol and cyt.c
and also translocates protons across the
mitochondrial inner membrane
Using sodium dedocyl sulfat gel
electrophoresis can be resolved into 11
polypeptide subunits (subunit I XII).
These subunit play an important role in
maintaining the proper orientation of the
redox group.
Cyt b is the only complex III polypeptide
encoded by the mt-DNA . It is very
hydrophobic. Is the major trans-membrane
Com plex IV
EC 1.9.3.1 (ferrocytochrom c:oxygen oxido reductase
or cyt c oxidase)
Is the terminal enzyme complex for electron transport.
(e + O2 H2O)
Proton are pumped to inter-membrane space
Mammalian Complex IV composed of 13 polypeptides
subunit.
According to Kadenbach nomenclature, subunit I,II and
III are encoded by mt-DNA and the remaining are
encoded by n-DNA
(IV,Va,Vb,Via,Vib,Vic,VIIa,VIIb,VIIc,VIII)
Subunit I and II incorporate Cu and heme a and a3,
redox center for electron transfer.
Com plex V
EC 3.6.1.34 (ATP syntase). Composed of12-13
subunits, two coded by mt-DNA (ATPase 6 and 8
genes).
Use the electro-chemical gradient created by
complex I,III,IV as a source of energy for
synthesizing the ATP from ADP + Pi
The enzyme composed of two parts, the F1
segment and which catalyze ATP synthesis and
Fo segment which translocate proton into
mitochondrial matrix.
When F1 is purified from Fo segment it catalyze
the breakdown of ATP into ADP and Pi
Reduction in electron transport caused by
OXPHOS disease mutation would reduce proton
translocation thus impairing ATP production.
Com plex V (cont)

Couple reaction is a reaction between oxidation

and phosphorylation of complex V
In coupled mitochondria electron transport and
oxygen consumption increase when ADP is
available.
Compound that dissipate the electrochemical
gradient, such as 2,4 dinitro phenol(DNP), and
carbamyl cyanide m-chloro phenilhydrazine
(mCP) uncouple ATP synthesis.
A physiological uncoupler called uncoupler
protein (UCP), is found in mitochondria of brown
fat. His protein is a proton carrier that permit the
protons outside the mitochondrion to return to
the matrix. This causes the electron transport
system to run at its maximum rate, producing
heat rather than ATP thermogenic function of
 U CP = uncoupling protein

The UCP genes are maps to chromosome

4q31
In contrast to the thermogenic function of
UCP, two distinct inhibitor proteins, the
Pullman Monroy inhibitor (PMI), and
calcium binding protein (CBP), can bind to
the F1 segment of complex V and regulate
energy production by preventing ATP
hydrolysis and promoting ATP synthesis .
CBP found in higher concentration in heart
and skeletal muscle.
M itochondrialgenetic

Mt DNA has a unique genetic :

1. Maternal inheritance
2. Replicative segregation
3. Threshold expression of phenotype
4. Developmental stage and tissue
specific OXPHOS gene expression
5. High mutation rate
6. An accumulation of somatic mtDNA
mutation with aging and degenerative
disease.
M aternalinheritance
Is the rule of all vertebrates
The mtDNA of the mother is
transmitted to all of her children
through the egg cytopasma (200-
300.000 mtDNA)
Paternal transmission has not been
found to be a significant features of
mt DNA inheritance.
It occurs because of mtDNA location
of sperm is on the tail which are not
enter the ovum
H IG H M U TATIO N RATE

The mt-DNA has a much higher

mutation rate than the nuclear
DNA , including substitution mutation
and deletion.
Its cause by production of free
radical in respiratory chain.
The phenotype effect of mutation
depend on the severity of the
damage
Nucleotide substitution fall into 2
classes: synonymous and
Som atic m t-D N A m utations and aging

Aging is associated with variety of

degenerative changes in the protein,
lipid n-DNA and mt-DNA of the cell.
The accumulation of these changes
can then result in cellular dysfunction
and eventually cell death.
Cell with low replicate potential i.e
neuron cells, can damage/die rapidly
than cells with high replicate potential
(hematopoetic lineages) compensate
for cell damage by their rapid turn
over.
M itochondrialtheory ofaging

This hypothesis has 3 essential elements:

1. Free radical are continuously produced in
the mitochondria.
2. Free radical production is stimulated by
inhibition of electron transport chain
oxidative stress oxidative damage to cell
membrane, DNA and protein.
3. Accumulation of mt-DNA damage blocks
mitochondrial biogenesis permanent
organell dysfunction ultimately to cell
death
 Oxidatioand phosphorylation
(oxphos)

oxphos diseases
Paradigm for OXPH O S dysfunction and
O xygen radicaldam age
nDNA mtDNA Enviromental
Mutation mutation Toxin

OXPHOS DYSFUNCTION
DAMAGE TO oxphos cumulative impairment of oxphos function

INCREASE FREE RADICAL PRODUCTION

SOMATIC MtDNA MUTATION ACCUMULATION

OXPH O S D ISEASES

Devided into 4 genetic groups:

1. Class I mutation (n-DNA mutation)
2. Class II mutation (mt-DNA point
mutation)
3. Class III mutation (mt-DNA deletion
and duplication)
4. Class IV mutation (Undefined
genetic defect)
Class Im utation
Disorder of the nuclear OXPHOS gene
Large number of nuclear oxphos
gene many respiratory deficiency
diseases, are the result of nuclear
OXPHOS gene mutation.
To identify this mutation is detailed
pedigree analysis
Cybrid transfer methode
Class Im utation

1. BIMM (Benign Infantile

Mitochondrial Myopathy)
* weakness at birth, hypotonia,
difficulty feeding, respiratory
difficulties, myopathy, lactic
acidosis.
* only skeletal muscle appear to be
affected and improves
spontaneously at 6 to 9 months of
age.
Class Im utation
a. BIMC (Benign Infantile Mitochondrial
Myopathy and Cardiomyopathy.)
* More severe variant of BIMM and involve
both skeletal and cardiac muscle.
b. Lethal Infantile Cardiomyopathy.
c. CPEO (Chronic Progressive External
Opthalmoplegia) and KSS (Kearn-Sayre
Syndrome)
d. Leigh disease (Sub Acute Necrotizing
Encephalopathy)
e. Mitochondrial Myopathy
Class Im utation

2. LIMD (Lethal Infantile Mitochondrial

Disease)
* Can be relatively normal at birth.
However OXPHOS function rapidly
decline, resulting in death during
the first year of life.
* failure to thrive, weakness,
hypotonia, and severe lactic
acidosis
Class IIm utation
Point mutation of mtDNA can be
either
homoplasmic (single population of
mutant mtDNAs)
heteroplasmic (mixed population of
mutant and normal mtDNA)
Both types of mutations lead to
variable phenotype expression
Class IIm utation
1. LHON (Leber Hereditary Optic Neuropathy)
* the first human disease associated with
mtDNA point mutation.
* painless loss of central visual acuity
which occur between 12 and 30 years of
age.
2. MERRF (Myoclonic Epilepsy and ragged
Red Fiber)
3. Alzheimer disease(AD) and Parkinson
Disease (PD)
Class IIIm utation
Mt DNA deletion and duplication.
mtDNA deletion such as K-S
Syndrome and CPEO
Pearson syndrome
DM and deafnes
Migraines and stroke (malignant
maigrain)
Class IV m utation
Alpers disease (Progressive Infantile
Poiliodystrophy)
LIC (Lethal Infantile Cardiomyopathy)
Idiophatic Dystonia
Luft disease
D iagnostic approach

Pedegree analysis
(Mendelian or maternal Inheritance)

Clinical exam
(History and ph ysical exam)

Metabolic evaluation

Muscle biopsy Blood

 Metab evaluation:
1.Blood quantitative organic acids,
amino acids, and carnitine analysis
2.Urine 24 hours collection
3. CSF
 Muscle biopsy:
1. Histochemistry and electrone
microscopy
2. OXPHOS biochemistry (isolation of
mitochondria immediately from
fresh sample)
3. mtDNA analysis
 Blood: Direct mtDNA analysis for
pathogenic mutation
OXPH O S disease m anagem ent
Attempts to increase ATP production
Therapi : CoQ, Menadione and
philoquinon, succinate, Thiamine and
riboflavin, Corticosteroid, Idebenone,
dichlor acetate, Chloramphenicol
 R.O .S

Approximately 98-99 % of oxygen

delivered to the cell reduced to H2O by
complex IV
However 1-2% of the oxygen can be
converted to oxygen radical
In mammals superoxide radical (O2.-)
are generated by OXPHOS at the rate
above 107 of oxygen per mitochondrion per day.
Inhibition of the electron transport chain increase the
electro negativity of the early stages of OXPHOS,
greatly stimulating oxygen radical generation
 Hence mutation in mt-DNA genes will increase
radical oxygen production and increase damage to
mitochondrial inner membrane and the mt-DNAs.
Cytosolic enzyme xanthine oxidase and
mitochondrial monoamine oxidase A and B also
contribute in free radical accumulation within the
cell.
Alloxan, menadione, rotenon (complex I inhibitor),
and methylphenyl-pyridinium, tetrachloro-p-
dibenzyl, increase Ca levels, tumor necrosis factor
alpha, ferrous and cuppri ion and ischemic
reperfusion dramatically increase radical-oxygen
production.
Characeristic offree radical

FR : atom/mol with one or more

unpairing electrone
Difficult to detect (short half life)
High reactivity
Can produce new radical chain
reaction cell membrane damage
(lipid peroxidase) MBA, 9 hydroxy
nonenal, isoprostan etc.
D am age to D N A

Damage to DNA by free radical

including:
1. Opening the purine and pyrimidine
ring
2. Damages of phospho diester bridge
3. Mutation oncogenic gene
carcinogenesis
4. Break down of one or both DNA
strand.
D am age to protein

Damage to protein by free radical

including:
1. Cross linking between proteins or
between protein and CH/lipid.
 Damaged to lipid bilayer of cell
membrane
Lipid peroxidation
Oxidative damage of cell membrane
will produce : MDA, f2-isoprostan, 8-
OH noneal, pentena, etc
Anti oxidant

Oxidant is electron acceptor. Anti

oxidant is electron donor
Or substances who can quenced the
bad effect of oxidant/free radical.
Internal antioxidant and external
antioxidant
Preventive anti oxidant and chain
breaking anti oxidant
Enzymatic anti oxidant and non
enzymatic AO
A-O

Internal AO : SOD, catalase,

peroxidase (GPx)
Xantine oxidase, monoamine
oxidase A and B
External AO : A,E,C, flavonoid,
lecopen
thankyou