Oxidative Phosphorylation

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Oxidative Phosphorylation

1. In Eukaryotes -gt Mitochondria
2. Depends on Electron Transfer
3. Respiratory Chain 4 complexes -gt 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) -gt formation of
proton gradient -gt 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 -gt ADP gives ?Go -7.3 kcal/mol
3. ATP-gt AMP gives ?Go -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
Electron carrier for oxidation
R H -gt NAD R PO32- -gt NADP
!!! NAD accepts a H and 2 electrons
(equivalent to a hydride ion H-) -gt NADH !!!
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The Major Key Players in Oxidative Phosphorylation
Electron carrier for oxidation
FAD
!!! FAD accepts 2 H and 2 electrons -gt 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 -gt
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
1.14 Volt
O2
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The Respiratory Chain
Electron transfer from NADH -gt O2

• 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

Complex I
Complex II -gt Does not pump protons
Ubiquinone
Complex III
Cytochrom c is an electron shuttle
Complex IV
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Electrons of NADH enter at NADH-Q Oxidoreductase

• Enormous enzyme (gt900 kDa) -gt 46 polypeptides
• proton pump

• Steps of Electron-Transfer
• Binding of NADH and transfer of its electrons to
  FMN (prosthetic group of complex)
• Electrons are transfered from FMNH2 to a series
  of iron-sulfur clusters (prosthetic group of
  complex) -gt 2Fe-2S 4Fe-4S clusters
• Electrons are shuttled to coenzyme Q (ubiquinone)
• ? 2 Electrons from NADH to Coenzyme Q -gt
  pumping 4 H out of matrix of mitochondria

NADH-Q Oxidoreducatase (Complex I)
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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 -gt QH2 results in uptake of 2
protons from matrix
Coenzyme Q has the ability to transfer electrons
-gt used as an antioxidant (dietary supplement).
CoQ10 used for the treatment of -gt 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.
-gt 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)

• Succinate is oxidized to fumarate by the
  Succinate dehydrogenase A subunit. SDHA contains
  (FAD) cofactor The oxidized FAD -gt reduced to
  FADH2 in a two electron process. This is part of
  the citric acid cycle.
• 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.
• SDHC/SDHD dimer, reducing it to ubiquinol (QH2).
• 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 -gt heme
• Fe in heme group changes between 2 or 3 during
  e-transport
• Function catalyse transfer of electrons from
  QH2 -gt oxidized cyt c
• pumps protons out of matrix -gt intermembrane
  space
• Coupling of electron transport from Q -gt 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 -gt
trigger uptake of 2 protons from the matrix -gt
formation of proton gradient
1st half of Q cycle
2nd half of Q cycle
Q pool
Q pool
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Cytochrome c Oxidase Catalyzes the Reduction of
O2 -gt H2O

• Complex IV
• Oxidation of cyt c coupled to reduction of O2 -gt
  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

Transfer of 4 e- leads to safe product -gt H2O
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Reduction of O2 -gt H2O can be toxic -gt when
single e- transfered
In Peroxisomes -gt catalase
Superoxide dismutase deals with toxic derivates
(superoxide radicals)
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The Electron-Transport Chain
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Mitochondrial electron transport chain -gt drives
ATP production
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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 gradient is not used to form ATP but to
release ATP
Proton channel
Bind nucleotides just ß subunit catalysis
synthesis (ATPase)
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The Worlds Smallest Molecular Motor -gt
Rotational Catalysis
? subunit rotates the 3 ß-subunits driven by the
proton-conducting unit ATP in tight (T) position
-gt cannot be released ATP in open (O) position -gt
released
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The Worlds Smallest Molecular Motor
Fluorescently labeled actin filaments
ATP hydrolysis -gt counterclockwise rotation of
filament (fluorescence microscope)
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Proton Motion Across the Membran Drives Rotation
of the C-Ring
Each proton enters the cytosolic half-channel -gt
follows a complete rotation of the c-ring -gt
exits through the other half-channel into the
matrix The difference in proton concentration
and potential on the two sides -gt leads to
different probabilities of protonation through
the 2 half-channels -gt yields directional
rotation motion
c-ring
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Fagellum -gt another example for a H driven
rotary motor
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Overview of Oxidative Phosphorylation
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Shuttles between Mitochondria - Cytoplasma
1. Regeneration of NAD for glycolysis -gt in
respiratory chain (mitochondria) In
Glycolysis -gt cytoplasmic NAD -gt cytoplasmic
NADH
Refill of NAD in cytosol
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Shuttles between Mitochondria - Cytoplasma
1. Regeneration of NAD for glycolysis -gt in
respiratory chain (mitochondria) In
Glycolysis -gt cytoplasmic NAD -gt cytoplasmic
NADH need a shuttle to transfer -gt
cytoplasmic NADH into mitochondria (cannot just
pass membrane)
Transport of generated NADH into the mitochondria
!!! and Refill of NAD in cytosol
Just in the heart and liver cells !!!
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Shuttles between Mitochondria - Cytoplasma
2. ATP/ADP transport by ATP/ADP translocase
Oxidative phosphorylation generates ATP in
the mitochondria -gt needed in the cytoplasm
need a shuttle to get -gt 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 -gt Primarily by Need
for ATP
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Regulation of Respiration -gt 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 -gt heat
generation to maintain body temperature Thermogen
in UCP-1 (uncoupling protein) generates heat by
short-circuiting the mitochondrial proton
battery -gt Special adipose tissue called Brown
fat (cells with high content of mitochondria ,
cytochrome -gt brownish color) -gt in these cells
mainly heat generation (babies have a high amount
of them)
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A Central Motif of Bioenergetics