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Oxidative Phosphorylation
1. In Eukaryotes -> Mitochondria
2. Depends on Electron Transfer
3. Respiratory Chain: 4 complexes -> 3 pumps + Link to Citric Acid Cycle
4. Proton Gradient responsible for Synthesis of ATP
5. Shuttles allow movement across membrane
6. Regulation primarily by need for ATP
Oxidation and ATP synthesis are coupled by transmembrane H+ fluxes
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Oxidative Phosphorylation
Oxidation of fuel (glucose, fat) -> formation of proton gradient -> drives synthesis of ATP
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Stages of Catabolism
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The Major Key Players in Oxidative Phosphorylation
1. ATP is the universal currency of free energy in biological systems
2. ATP -> ADP gives ΔGo’ = -7.3 kcal/mol
3. ATP-> AMP gives ΔGo’ = -10.9 kcal/mol
4. ATP hydrolysis drives metabolism by shifting the equilibrium
5. Phosphoryl transfer potential is an important form of cellular energy transfer (Phosphorylated compounds are activated!!!)
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The Major Key Players in Oxidative Phosphorylation
R = H -> NAD+
R = PO32- -> NADP+
Electron carrier for oxidation
!!! NAD+ accepts a H+ and 2 electrons (equivalent to a hydride ion H:-) -> NADH !!!
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The Major Key Players in Oxidative Phosphorylation
FAD+
Electron carrier for oxidation
!!! FAD+ accepts 2 H+ and 2 electrons -> FADH2 !!!
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Oxidative Phosphorylation takes place in the
Inner Membrane of the Mitochondria
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High Energy Electrons: Redox Potentials and Free-Energy Changes
Electron transfer potential of NADH and FADH2 -> Phosphoryl transfer potential of ATP
A 1.14 –Volt potential difference between NADH and O2 drives electron transport and favors formation of a proton gradient
NADH
O2
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The Respiratory Chain
Consists of 4 complexes:
3 proton pumps + link to citric acid cycle
3 proton pumps:
- NADH-Q oxidoreductase
- Q-cytochrome C oxidoreductase
- Cytochrome c oxidase
Link to citric acid cycle:
Succinate-Q reductase
Ubiquinone (Coenzyme Q) also carries electrons from FADH2 (generated by citric acid cycle) generated through succinate-Q reductase
Electron transfer from NADH -> O2
Ubiquinone
Cytochrom c is an electron shuttle
Complex I
Complex II ->
Does not pump protons
Complex III
Complex IV
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Electrons of NADH enter at NADH-Q Oxidoreductase
NADH-Q Oxidoreducatase (Complex I)
- Enormous enzyme (>900 kDa) -> 46 polypeptides
- proton pump
Steps of Electron-Transfer:
1. Binding of NADH and transfer of its electrons to FMN (prosthetic group of complex)
2. Electrons are transfered from FMNH2 to a series of iron-sulfur clusters (prosthetic group of complex) -> 2Fe-2S + 4Fe-4S clusters
3. Electrons are shuttled to coenzyme Q (ubiquinone)
2 Electrons from NADH to Coenzyme Q -> pumping 4 H+ out of matrix of mitochondria
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Oxidation states of flavins
Iron-sulfur clusters
NADH-Q oxidoreductase
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Oxidation state of Quinones (Coenzyme Q)
The reduction of ubiquinone (Q) to ubiquinol (QH2) proceeds through a semiquinone
intermediate (QH.)
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Coupled Electron-Proton Transfer
Reduction of Q -> QH2 results in uptake of 2 protons from matrix
Coenzyme Q has the ability to transfer electrons -> used as an antioxidant (dietary supplement). CoQ10 used for the treatment of -> heart disease (especially heart failure), and also breast cancer
Young people are able to make Q10 from the lower numbered ubiquinones such as Q6 or Q8. -> The sick and elderly may not be able to make enough, thus Q10 becomes a vitamin later in life.
Supplementation of Coenzyme Q10 has been found to have a beneficial effect on the condition of some sufferers of migraine headaches. It is also being investigated as a treatment for cancer, and as relief from cancer treatment side effects.
Some of these studies indicate that Coenzyme Q10 protects the brain from neurodegenerative disease such as Parkinsons and also from the damaging side effects of a transient ischemic attack (stroke) in the brain.
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Ubiquinol is the Entry Point for Electrons from FADH2 of Flavoproteins
FADH2
(citric acid cycle)
Complex II:
- Integral membrane protein (inner mitochondrial membrane)
- Electrons of FADH2 are transfered to Fe-S center and then to Q
- No transport of protons
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Ubiquinol is the Entry Point for Electrons from FADH2 of Flavoproteins
FADH2
(citric acid cycle)
1. Succinate is oxidized to fumarate by the Succinate dehydrogenase A subunit. SDHA contains (FAD) cofactor The oxidized FAD -> reduced to FADH2 in a two electron process. This is part of the citric acid cycle.
2. The electron transfer subunit (SDHB) contains several iron-sulfur centers which relay electrons from SDHA to the membrane domains: a [2Fe-4S] cluster, a [4Fe-4S] cluster and a [3Fe-4S] cluster.
3. SDHC/SDHD dimer, reducing it to ubiquinol (QH2). 4. The resulting ubiquinol molecule is released, free to diffuse through the inner mitochondrial
membrane to interact with subsequent enzymes of the mitochondrial respiratory chain (electron transport chain).
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Electrons Flow from Ubiquinol (QH2) to Cytochrome c Through Q-Cytochrome c Oxidoreductase
Complex III:
- Cytochrome is a electron - transfering protein
- Cytochrome has a prosthetic group -> heme
- Fe in heme group changes between +2 or +3 during e-transport
- Function: catalyse transfer of electrons from QH2 -> oxidized cyt c
- pumps protons out of matrix -> intermembrane space
- Coupling of electron transport from Q -> cyt c and transmembrane proton transport Q cycle
Heme group in cyt c
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The Q Cycle
Electrons that are bound to QH2 are transfered -> trigger uptake of 2 protons from the matrix -> formation of proton gradient
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Cytochrome c Oxidase Catalyzes the Reduction of O2 -> H2O Complex IV:
- Oxidation of cyt c coupled to reduction of O2 -> H2O
- Heme protein
- Heme + other part of active site (CuB) responsible for reduction of O2
- Electron transfer coupled to proton pump
- 8 protons are pumped from the matrix to intermembrane space
Superoxide dismutase deals with toxic derivates (superoxide radicals)
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Toxic Derivates of Oxygen (superoxide radicals) are Scavenged
Superoxide dismutase deals with toxic derivates (superoxide radicals)
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The Electron-Transport Chain
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The Proton Gradient Powers Synthesis of ATP
ATP sythesis mechanism
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ATP Synthase is Composed of a Proton-Conducting Unit and a Catalytic Unit
Proton channel
Bind nucleotides – just β subunit catalysis synthesis (ATPase)
Proton gradient is not used to form ATP but to release ATP
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The World’s Smallest Molecular Motor -> Rotational Catalysis
γ subunit rotates the 3 β-subunits driven by the proton-conducting unit
ATP in tight (T) position -> cannot be released
ATP in open (O) position -> released
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The World’s Smallest Molecular Motor
ATP hydrolysis -> counterclockwise rotation of filament (fluorescence microscope)
Fluorescently labeled actin filaments
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Proton Motion Across the Membran Drives Rotation of the C-Ring
Each proton enters the cytosolic half-channel -> follows a complete rotation of the c-ring -> exits through the other half-channel into the matrix
The difference in proton concentration and potential on the two sides -> leads to different probabilities of protonation through the 2 half-channels -> yields directional rotation motion
c-ring
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Overview of Oxidative Phosphorylation
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Shuttles between Mitochondria - Cytoplasma
1. Regeneration of NAD+ for glycolysis -> in respiratory chain (mitochondria) In Glycolysis -> cytoplasmic NAD+ -> cytoplasmic NADH
Refill of NAD+ in cytosol
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Shuttles between Mitochondria - Cytoplasma
Just in the heart and liver cells !!!
1. Regeneration of NAD+ for glycolysis -> in respiratory chain (mitochondria) In Glycolysis -> cytoplasmic NAD+ -> cytoplasmic NADH
need a shuttle to transfer -> cytoplasmic NADH into mitochondria (cannot just pass membrane)
Transport of generated NADH into the mitochondria !!!
andRefill of NAD+ in cytosol
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Shuttles between Mitochondria - Cytoplasma
2. ATP/ADP transport by ATP/ADP translocase Oxidative phosphorylation generates ATP in the mitochondria -> needed in the cytoplasm
need a shuttle to get -> cytoplasmic ADP into mitochondria (cannot just pass membrane)
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Shuttles between Mitochondria - Cytoplasma
Mitochondrial transporters (carriers)
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Regulation of Respiration -> Primarily by Need for ATP
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Regulation of Respiration -> Primarily by Need for ATP
ATPase inhibited by:
Oligomycin and Dicyclohexylcarbodiimide (DCCD)
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Regulated Uncoupling Leads to the Generation of Heat
Uncoupling of oxidative phosphorylation -> heat generation to maintain body temperature
UCP-1 (uncoupling protein) generates heat by short-circuiting the mitochondrial proton battery
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A Central Motif of Bioenergetics