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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