MITOCHONDRIA

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MITOCHONDRIA

• STRUCTURE
• THE ELECTRON TRANSPORT CHAIN
• MITOCHONDRIAL ATP PRODUCTION
• MITOCHONDRIA AND THE ENVIRONMENT
• MITOCHONDRIAL ION CHANNELS
• MITOCHONDRIA AND APOPTOSIS
• MITOKONDRIAL DISEASES

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MITOKONDRIUMOK
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1) The membrane-located ATPase systems of
mitochondria and chloroplasts are
hydro-dehydration systems with terminal
specificities for water and ATP and their normal
function is to couple reversibly the
translocation of protons across the membrane to
the flow of anhydro-bond equivalents between
water and the couple ATP/(ADP Pi). 2) The
membrane-located oxido-reduction chain systems of
mitochondria and chloroplasts catalyse the flow
of reducing equivalents, such as hydrogen groups
and electron pairs, between substrates of
different oxido-reduction potential and their
normal function is to couple reversibly the
translocation of protons across the membrane to
the flow of reducing equivalents during
oxido-reduction. 3) There are present in the
membrane of mitochondria and chloroplasts
substrate-specific exchange- diffusion carrier
systems that permit the effective reversible
transmembrane exchange of anions against OH- and
of cations against H and the normal normal
function of these systems is to regulate the pH
and osmotic differential across the membrane, and
to permit entry and exit of essential metabolites
(e.g., substrates and phosphate acceptor) without
collapse of the membrane potential. 4) The
systems of postulates 1, 2, and 3 are located in
a specialized coupling membrane which has a low
permeability to protons and to anions and cations
generally.
MITCHELL, P. Coupling of phosphorylation to
electron and hydrogen transfer by a chemiosmotic
type of mechanism. Nature 191144148, 1961.
Nobel Prize in Chemistry 1978
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control

• Condensed state
• Matrix contracted
• Cristae expanded

ADP

• Ortodox state
• Matrix expanded
• Cristae contracted

ATP
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ENZYMES IN MITOCHONDRIAL COMPARTMENTS
OUTER MEMBRANE INTERMEMBRANE SPACE MAO
(monoamine oxidase) Adenylate kinase, Kinurenin
hidroxilase Nukleozid diphospho kinase NADH cyt
c reduktáz Carnitine acyl transferase
I Porin INNER MEMBRANE MATRIX Elektron
transport chain Citric acid cycle enzymes ATP
synthase Enzymes of beta oxidation Transporters
Pyruvate dehydrogenase Glutamate
dehydrogenase OTC
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Partially condensed
ADP
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orthodox
ATP
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Cell, Vol. 112, 481490, February 21, 2003
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Redox potenciálok a komplex I-ben
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A model of the F1 and F0 components of the ATP
synthase, a rotating molecular motor. The a, b,
a, b, and d subunits constitute the stator of the
motor, and the c, g, and e subunits form the
rotor. Flow of protons through the structure
turns the rotor and drives the cycle of
conformational changes in a and b that synthesize
ATP.
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The binding change mechanism for ATP synthesis by
ATP synthase. This model assumes that F1 has
three interacting and conformationally distinct
active sites. The open (O) conformation is
inactive and has a low affinity for ligands the
L conformation (with loose affinity for
ligands) is also inactive the tight (T)
conformation is active and has a high affinity
for ligands. Synthesis of ATP is initiated (step
1) by binding of ADP and Pi to an L site. In the
second step, an energy-driven conformational
change converts the L site to a T conformation
and also converts T to O and O to L. In the third
step, ATP is synthesized at the T site and
released from the O site. Two additional passes
through this cycle produce two more ATPs and
return the enzyme to its original state.
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Energetics Energy derived from NADH
oxidation NADH H ½ O2 ?Go-NF?Eo
2(96.5 kJ/Vmol)(1.14 V) 220
kJ/mol Proton motive force (pmf) ??H??m-60?pH
(mV) How much energy is released when one proton
moves from the intermembrane space to the
matrix?l ?pH0.75 ??150-200 mV Proton motive
force ?G2.3RT?pH F??5.70kJ/mol?pH
(96.5kJ/Vmol)?? Energy required to pump out one
proton cca 20 kJ/mol 10 protons ?G200 kJ
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How much energy is required to synthesize one mol
ATP?

• ?G ?Go RTlnADPPi/ATP
• ?G-30.5kJ/mol 8.315298
• -54.7 kJ/mol muscle cell
• ?G-51.8 kJ/mol RBC
• 2 protons are not enough
• 3 proton is more than enough but

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How much energy is required to synthesize one mol
ATP?

• ?G ?Go RTlnADPPi/ATP
• ?G-30.5kJ/mol 8.315298
• -54.7 kJ/mol muscle cell
• ?G-51.8 kJ/mol RBC
• 2 protons are not enough
• 3 proton is more than enough but
• Because of the ATP/ADP exchanger one extra proton
  is required
• Total cost 4 protons/ATP

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Lets play with mitochondria

• Uncouplers Oxidation yes, Phosphorylation NO
• Rotenon inhibition of complex I
• Atractyloside Inhibition of ANT
• Oligomycin Inhibition of ATP synthase

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rotenon
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ELEKTRON TRANSPORT and ATP SYNTHESIS
INNER MEMBRANE
NADHO2 NADH2O ADPPI ATP
MEMBRANE VESICLES ELEKCTRON TRANSPORT ATP
SYNTHÉZIS -
F1
Fo
ULTRASOUND
MECHANICAL SHAKING
RECONSTRUCTION
EL. TRPORT ATP SYNTHESIS -
F1 PARTICLES ELEKTRON TRANSPORT ATP SYNTHEZIS
ATP-ASE ACTIVITY
INSIDE OUT SUBMITOCHONDRIAL PARTICLES
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Mitochondria respire (oxygen consumption and pump
protons OXYGEN DECREASES, PROTON PUMPING
ACTIVITY DECREASES
pH elektrode
O2 injektor
O2 injection
?H
Time (sec)
mitochondria
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The role of proton motive force in ATP synthesis
II.
Liposomes
Artifitial vesicles From purified phospholipids
Bakteriorhodopsin
F0
H
H
ADPPi
H
H
H
H
H
H
H
H
H
H
ATP
H
H
H
F1
H
H
Protons pumped by bacteriorhodopsin Can be
utilized to synthesize ATP
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The role of proton motive force in ATP synthesis
1961 Peter Mitchell
ATP synthesis Artifial constructed pH gradient
Equilibration pH 4.0 pufferrel
Buffer pH 8.0 ADP Pi
pH 4.0
pH7.5
pH 4.0
ATP
ADPPi
ATP
ADPPi
pH7.5
pH 4.0
pH 8.0
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The essence of oxidative phosphorylation
MEMBRANE
ADPPI
ATP
O2 H2O
- - - - - -

H
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NADH shuttles

• Malate- aspartate
• Glycerophosphate

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Mitochondrial Uptake of Ca2
? slower kinetics
ATP purinergic agonist Gq-gtPLC-gtIP3
? ??m dependent
FCCP uncoupler
? Na/Ca2-exchange is important
CGP-37157 Na-Ca exch inhib.
Cell Calcium 2001 30.5 311-321
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• Ca2 uniporter
• Ca2 elecrtophoretic uptake (charge carried 2)
• Voltage dept, Ca2 activated
• Km10?M
• Ruthenium red, Ru360 (-)
• Kompetetive inhibitors bound and transported in
  the channels Sr2,Mn2,Ba2
• Allosteric inhibitors Mg2,Mn2,
• Spermin ()
• ATPgtADPgtAMP (-)
• Unknown molecular identity

• Rapid uptake mode
• Ca2 uptake Ca2o400nM körül
• Transient uptake,
• Reset
• Ruthenium red, Ru360 (-, but at higher conc.)
• ATP activated
• Mg2 does not inhibit
• Unknown molecular identity

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• Na-indept Ca2-efflux (NICE)
• nH-Ca2 antiporter, ngt2 ??m required
• Ruthenium Red inszenzitív
• Slow, early saturation

• Na-dept Ca2-efflux (NCE)
• 3Na-Ca2 antiporter
• ??m required
• Mg2,Sr2,Ba2,Mn2 (-)
• Amilorid, trifluoperazin, diltiazem, CGP-37157
  (-)
• Mátrix pH dept (opt. pH 7.6)

• Ca2-cycling
• A Ca2 efflux alacsony vmax-a és korai telítodése
  ellene hat
• A Ca2matrix finom szabályzását teszi lehetové
  (pl. cardiomyocyta)
• Térbeli szegregáció ???

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Ca2 microdomain
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Mitochondria can be ATP consumers
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Mechanism of uncoupling
Allosteric interaction
Cofactor
Shuttling
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Mitochondria and apoptosis
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Mitochondrial permeablity transition pore (mPTP)
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Ca2 microdomain