Transcript Hücrede Enerji Metabolizması

Electron transport
Chemiosmotic Theory
• Electron Transport: Electrons carried by
reduced coenzymes are passed through a chain
of proteins and coenzymes to drive the
generation of a proton gradient across the inner
mitochondrial membrane
• Oxidative Phosphorylation: The proton gradient
runs downhill to drive the synthesis of ATP
• Electron transport is coupled with oxidative
phosphorylation
• It all happens in or at the inner mitochondrial
membrane
Outer Membrane – Freely
permeable to small molecules
and ions. Contains porins
with 10,000 dalton limit
Inner membrane – Protein
rich (4:1 protein:lipid).
Impermeable. Contains ETR,
ATP synthase, transporters.
Cristae – Highly folded inner membrane structure.
Increase surface area.
Matrix- “cytosol” of the mitochondria. Protein rich (500
mg/ml) Contains TCA cycle enzymes, pyruvate
dehydrogenase, fatty and amino acid oxidation pathway,
DNA, ribosomes
Intermembrane Space – composition similar to cytosol
Reduction Potentials
• High Eo' indicates a strong tendency to be reduced
• Crucial equation: Go' = -nF Eo'
•
Eo' = Eo'(acceptor) - Eo'(donor)
• NADH + ½ O2 + H+  NAD++ H+ + H2O
NAD++ H+ + 2e- NADH
Eo’ = -0.32
½ O2 + 2e- + 2H+  H2O
Eo’ = 0.816
Go‘= -nF(Eo'(O2) - Eo'(NADH))
Go‘= -nF(0.82 –(-0.32)) = -nF(1.14)
= -2(96.5 kJ mol-1V-1)(1.136) = -220 kJ mol-1
Electron Transport
• Four protein complexes in the inner
mitochondrial membrane
• A lipid soluble coenzyme (UQ, CoQ)
and a water soluble protein (cyt c)
shuttle between protein complexes
• Electrons generally fall in energy
through the chain - from complexes
I and II to complex IV
Standard reduction potentials
of the major respiratory
electron carriers.
Complex I
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•
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•
NADH-CoQ Reductase
Electron transfer from NADH to CoQ
More than 30 protein subunits - mass of 850 kD
1st step is 2 e- transfer from NADH to FMN
FMNH2 converts 2 e- to 1 e- transfer
Four H+ transported out per 2 e-
NADH + H+
NAD+
FMN
FMNH2
Fe2+S
Fe3+S
CoQ
CoQH2
Complex II
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•
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Succinate-CoQ Reductase
aka succinate dehydrogenase (from TCA cycle!)
four subunits
Two largest subunits contain 2 Fe-S proteins
Other subunits involved in binding succinate
dehydrogenase to membrane and passing e- to
Ubiquinone
• FAD accepts 2 e- and then passes 1 e- at a time to
Fe-S protein
• No protons pumped from this step
Succinate
Fumarate
FAD
FADH2
Fe2+S
Fe3+S
CoQ
CoQH2
Q-Cycle
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•
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Transfer from the 2 e- carrier
ubiquinone (QH2) to Complex
III must occur 1 e- at a time.
Works by two single electron
transfer steps taking advantage
of the stable semiquinone
intermediate
Also allows for the pumping of
4 protons out of mitochondria
at Complex III
Myxothiazol (antifungal agent)
inhibits electron transfer from
UQH2 and Complex III.
UQ
UQ.-
UQH2
Complex III
• CoQ-Cytochrome c Reductase
• CoQ passes electrons to cyt c (and pumps H+) in a
unique redox cycle known as the Q cycle
• Cytochromes, like Fe in Fe-S clusters, are oneelectron transfer agents
• cyt c is a water-soluble electron carrier
• 4 protons pumped out of mitochondria (2 from UQH2)
CoQH2
CoQ
cyt b ox
cyt b red
Fe2+S
Fe3+S
cyt c1 ox
cyt c1 red
cyt c red
cyt c ox
Complex IV
• Cytochrome c Oxidase
• Electrons from cyt c are used in a fourelectron reduction of O2 to produce 2H2O
• Oxygen is thus the terminal acceptor of
electrons in the electron transport pathway the end!
• Cytochrome c oxidase utilizes 2 hemes (a and
a3) and 2 copper sites
• Complex IV also transports H+ (2 protons)
cyt c red
cyt c ox
cyt a ox
cyt a red
cyt a3 red
cyt a3 ox
O2
2 H2O
Inhibitors of Oxidative
Phosphorylation
• Rotenone inhibits Complex I - and helps natives
of the Amazon rain forest catch fish!
• Cyanide, azide and CO inhibit Complex IV,
binding tightly to the ferric form (Fe3+) of a3
• Oligomycin and DCCD are ATP synthase
inhibitors
Shuttling Electron Carriers into
the Mitochondrion
• The inner mitochondrial membrane is
impermeable to NADH.
• Electrons carried by NADH that are
created in the cytoplasm (such as in
glycolysis) must be shuttled into the
mitochondrial matrix before they can
enter the ETS
Glycerol phosphate shuttle
malate/aspartate shuttle system
Electron transport is coupled to
oxidative phosphorylation
Uncouplers
• Uncouplers disrupt the tight coupling between
electron transport and oxidative phosphorylation
by dissipating the proton gradient
• Uncouplers are hydrophobic molecules with a
dissociable proton
• They shuttle back and forth across the
membrane, carrying protons to dissipate the
gradient
• w/o oxidative-phosphorylation energy lost as
heat
• Dinitrophenol once used as diet drug, people ran
107oF temperatures
H
NO2
O2N
OH
NO2
O2N
O
Oxidative phosphorylation
Proton Motive Force (p)
• PMF is the energy of the proton concentration gradient
• The chemical (pH= pHin – pHout) potential and the
electrical potential(Y= Yin – Yout) contribute to PMF
•
•
G = nfY and G = –2.303nRT pH
G for transporting 1 H+ from inner membrane space
to matrix = G = nfY –2.303nRTpH
•
p = p = G/nF
•
p = y –(0.059)pH
Proton Motive Force (p)
• What contributes more to PMF, Y or pH?
• In liver Y=-0.17V and pH=0.5
•
p = y –(0.059)pH = -0.17-(0.059)(0.5V)
•
p = -0.20 V
•
Y/p=(-0.17V/-0.20V) X 100% = 85%
• 85% of the free energy is derived form Y
Proton Motive Force (p)
• How much free energy generated from
one proton?
•
G = nFP = (1)(96.48kJ/Vmole)(-0.2V)
= -19 kJ/mole
• To make 1 ATP need 30 kJ/mole.
• Need to translocate more than one
proton to make one ATP
• ETC translocates 10 protons per NADH
ATP Synthase
• Proton diffusion through the protein drives
ATP synthesis!
• Two parts: F1 and F0
Racker & Stoeckenius
confirmed Mitchell’s
hypothesis using vesicles
containing the ATP
synthase and
bacteriorhodopsin
Binding Change Mechanism
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•
•
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ADP + Pi <-> ATP + H2O
In catalytic site Keq = 1
ATP formation is easy step
But once ATP is formed, it binds very
tightly to catalytic site (binding constant
= 10-12M)
• Proton induced conformation change
weakens affinity of active site for ATP
(binding constant = 10-5)
Binding Change Mechanism
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Different conformation at 3 catalytic sites
Conformation changes due to proton influx
ADP + Pi bind to open-site in exchange for ATP
Proton driven conformational change (loose site)
causes substrates to bind more tightly
• ATP is formed in tight-site.
• Requires influx of three protons to get one ATP
ATPase is a Rotating Motor
• Bound a,b,g subunits
to glass slide
• Attached a
fluroescent actin
chain to g subunit.
• Hydrolysis of ATP to
ADP + Pi cause
filament to rotate
120o per ATP.
How does proton flow cause
rotation?
Active Transport of ATP, ADP
and Pi Across Mitochondrial Inner
Membrane
• ATP is synthesized in the matrix
• Need to export for use in other
cell compartments
• ADP and Pi must be imported into
the matrix from the cytosol so
more ATP can be made.
• Require the use of transporters
Transport of ATP, ADP and Pi
• Adenine nucleotide translocator = ADP/ATP antiport.
• Exchange of ATP for ADP causes a change in Y due to
net export of –1 charge
• Some of the energy generated from the proton gradient
(PMF) is used here
• Pi is imported into the matrix with a proton using a
symport.
• Because negative charge on the phosphate is canceled by
positive charge on proton no effect on Y, but effects
pH and therefore PMF.
Transport of ATP, ADP and Pi
• NRG required to export 1 ATP and import 1
ADP and 1 Pi = NRG generated from influx of
one proton.
• Influx of three protons required by ATPase to
form 1 ATP molecule.
• Need the influx of a total of 4 protons for
each ATP made.
P/O Ratio
• The ratio of ATPs formed per oxygens reduced
• e- transport chain yields 10 H+ pumped out per
electron pair from NADH to oxygen
• 4 H+ flow back into matrix per ATP to cytosol
• 10/4 = 2.5 for electrons entering as NADH
• For electrons entering as succinate (FADH2),
about 6 H+ pumped per electron pair to oxygen
• 6/4 = 1.5 for electrons entering as succinate
Regulation of Oxidative
Phosphorylation
• ADP is required for respiration (oxygen
consumption through ETC) to occur.
• At low ADP levels oxidative phosphorylation low.
• ADP levels reflect rate of ATP consumption and
energy state of the cell.
• Intramolecular ATP/ADP ratios also impt.
• At high ATP/ADP, ATP acts as an allosteric
inhibitor for Complex IV (cytochrome oxidase)
• Inhibition is reversed by increasing ADP levels.
Uncouplers
• Uncouplers disrupt the tight coupling between
electron transport and oxidative phosphorylation
by dissipating the proton gradient
• Uncouplers are hydrophobic molecules with a
dissociable proton
• They shuttle back and forth across the
membrane, carrying protons to dissipate the
gradient
• w/o oxidative-phosphorylation energy lost as
heat
• Dinitrophenol once used as diet drug, people ran
107oF temperatures
H
NO2
O2N
OH
NO2
O2N
O
Physiological Uncoupling
• Uncoupling of ETC and Ox-phos occurs in
animals as a means to produce heat =
nonshivering thermogenesis.
• Impt. In hibernating mammals, neborn animals
and mammals adapted to cold
• Occurs in brown adipose tissues (rich in
mitochondria)
• Uncoupling protein (UCP) = channel to allow
influx of protons to matrix (dissipates proton
gradient)
Uncoupling in Plants
• Plants generate heat during fruit ripening
and to emit odors to attach pollinators.
• Plants can by pass normal ATP generating
ETC
• Alternative ETC in plants does not pump
protons, just transfers electron.
• All plant have this pathway, actual
physiological reason not known