Reactions of the citric acid cycle'

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Reactions of the citric acid cycle.
Acetyl-CoA GDP Pi 3 NAD Q ? 2 CO2 CoA
GTP 3 NADH QH2

• - Acetyl-CoA enter the cycle
• Highly exergonic (as GTP and reduced cofactros)

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The citric acid cycle is regulated at three
steps. three metabolically irrreversible steps
Regulation of the citric acid cycle.
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The citric acid cycle is both catabolic and
anabolic Citric acid cycle intermediates are
precursors of other molecules Intermediates of
CAC can be siphoned off to form other compounds

Acetly-CoA
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Ch 12. Oxidative Phosphorylation

• Oxidation of metabolic fuels produces the
  reduced NADH and ubiquinol (QH2)
• Oxidative Phosphorylation
• ? synthesis of ATP by generated energy during
  oxidation of the reduced cofactors by oxygen
• ? indirect process in which free energy is
  temporarily stored as a form of proton gradient

Figure 12.01 Oxidative phosphorylation in context.
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1. The thermodynamics of oxidation-reduction
reactions
Redox reactions electrons are transferred.
Aoxidized Breduced ? Areduced Boxidized
(A oxidizing agent, B reduing agent) Reduction
potential indicates a substances tendency to
accept electrons Standard reduction potential
(e??) the affinity of a substance for electrons
(volts) The bigger e??, the greater the
tendency of the oxidized form of the substance to
accept electrons Nernst equation Half
reaction Table 12-1
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Changes in reduction potential and free energy
changes ? reduction potential to predict the
movement of electrons between two substances ?
electrons move from the substance with lower
reduction potential to the substance with higher
reduction potential The larger the
difference in e values, the greater the tendency
of electrons to flow one substance to the
other ? ?G - nF ? e Sample calculation 12-1
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2. Mitochondrial electron transport
Respiration a process to reoxidize the NADH and
ubiquinole produced by flycolysis and CAC by
molecular oxygen, electrons are shuttled in
multiple steps Oxygen the most effective
oxidizing agent Mitochondrial anatomy ?Outer
membrane similar to bacteria outer membrane,
porous ?intermembrane space similar to cytosol
ionic composition ? inner membrane enclose
mitochondrial matrix, impermeable to ions and
small molecules ?Cristae a system of baffles
formed by innemrmembranes ? highly variable
structure thousand rod-like structure or one
tubular organell
Figure 12.02 Model of mitochondrial structure.
Figure 12.03a Images of mitochondria.
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Figure 12.03b Images of mitochondria.
Electron tomography(?? ?????) a tomography
technique for obtaining detailed 3D structures of
macromolecular objects. As part of an electron
microscope, information is collected and used to
assemble a three dimensional image of the target.
Current resolutions of ET systems are in the 5-20
nm range, suitable for examining supra-molecular
multi-protein structures, although not the
secondary and tertiary structure of an individual
protein or polypeptide
Micrograph of a mitochondrial network. (tubular
form vs bacteria-shape form))
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Mitochondrial anatomy ? contains its own genomic
DNA, tRNAs and rRNAs.
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• Mitochondrial anatomy
• ? contain its own genome (encoding 13
  mitochondrial proteins) and protein synthesizing
  machinery other mitochondrial proteins?
• ? locations
• reduced cofactors generation mitochondria matrix
• electron transport chain inner mitochondria
  membrane
• ? transport system
• i) the malate-aspartate shuttle system
• ii) adenine nucleotide translocase exports
  ATP and import ADP
• iii) Pi, H symport protein import Pi and H

Figure 12.05 The malate-aspartate shuttle system.
Figure 12.06 Mitochondrial transport systems.
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Mitochondrial Transport Systems

• Outer membrane porins,molecules lt10 kD freely
  pass
• Inner membrane
• 75 protein, impermeable to most ions
• needs specific shuttle to transport molecules

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(1) Malate-Aspartate shuttle

• This shuttle for transporting reducing
  equivalents from cytosolic NADH into the
  mitochondrial matrix is used in liver, kidney and
  heart.

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(2) Glycerol phosphate shuttle

• This alternative means of moving reducing
  equivalents from the cytosol to the mitochondrial
  matrix operates in skeletal muscle and the brain.

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(3) ADP-ATP Translocator (Adenine nucleotide
translocase)

• The proton-motive
• force drives ATP-
• ADP exchange.
• ADP (net charge 3),
• ATP (net charge 4)

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Separation of functional complexes of oxidative
phosphorylation (respiratory chain)
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Complex I transfers electrons from NADH to
ubiquinone
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4
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• Complex I transfers electrons from NADH to
  ubiquinone
• Complex I NADHubiquinone oxidoreductase, NADH
  dehydrogenase
• NADH H Q ? NAD QH2
• the largest electron transport proteins in the
  chain (43 proteins, 900kD)
• 2) structure (cryoelectron crystallography)
  L-shaped, a soluble globular arm connected by a
  narrow stalk to a membrane-embeded foot

Structure of Complex I determined by
cryoelectron microscopy.
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Complex I transfers electrons from NADH to
ubiquinone 3) contains prosthetic groups ? redox
center, their redox potential between NAD and
ubiquinione ? electron move between redox centers
by tunneling through the covalent bonds of the
protein ? flavin mononucleotide the first redox
center ? Fe-S cluster the second redox center,
the most ancient electron carrier?, 6-8 Fe-S
prosthetic groups, one electron carrier
Figure 12.08 Flavin mononucleotides (FMN).
Figure 12.09 Iron-sulfur clusters.
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Complex I transfers electrons from NADH to
ubiquinone 4) Trasnfers four protons from the
matrix to the intermembrane space (proton wire
like proton jumping) 5) Electron flow NADH (2e)
? FMN(2e) ? Fe-S(1e) ? Q (2e)
Figure 12.10 Complex I functions.
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Other oxidation reactions contribute to the
ubiquinone pool

• Reduced quionone by the Complex I joins a
  quinone pool soluble in inner mitochondrial
  membrane
• Succinate dehydrogenase Succinate Q ?
  fumarate QH2
• ? the only CAC enzyme located in inner
  mitochondrial membrane (called as complex II)
• ? augments pool of reduced quinones
• ? contains several redox centers (FAD group)
• ? DO NOT directly contribute the free energy for
  ATP synthesis
• 3) Cytosolic and mitochondrial glycerol-3-phosphat
  e dehydrogenase
• ? electrones from cytosolic NADH enter the
  mitochondrial ubiquinol pool
• ? bypass the Complex I (different numbers of
  ATPs for 1 NADH)

Reactions that contribute to the ubiquinol pool.
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• Complex III transfers electrons from ubiquinol to
  cyctochrome c
• the Complex III (ubiquinolcytochrome c
  oxidoreductase, cytochrome bc1) integral
  membrane proteins (11 x 2 monomeric subunits)
• Transfer electrons to the peripheral protein,
  cytochrome c
• cytochrome
• ? proteins with heme prosthetic group
• ? cell color (mitochondria color, purple)
• ? named by extra structure of porphyrin ring of
  heme ( a, b, c)
• ? structure of heme and surrounding proteins
  determines proteins absorption spectrum,
  reduction potential (-0.080 V to 0.384 V)
• ? reversible one electron reduction (Fe2 ?
  Fe3)
• QH2 2 cytochrome c3 ? Q 2
  cytochrome c2 2 H
• 4) 2 cytochrome (b, c1) and an iron-sulfur
  protein (the Rieske protein, the one present in
  bacteria)
• 5) Each monomer anchored in the membrane by 13
  transmembrane domains

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The heme group of a cytochrome.
X-Ray structure of mammalian Complex III.
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Figure 12.14 The Q cycle.
Figure 12.15 Complex III function.
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• Complex IV oxidizes cyctochrome c and reduces O2
• cytochrome c soluble in intermembrane space,
  linked by several Lys residues to the Complex III
  and IV
• the Complex IV (cytochrome c oxidase)
• ? the last enzyme in electron transport
• ?four electrons from cytochrome c to molecular
  oxygen
• 4 cytochrome c2 O2 4 H ? 4
  cytochrome c3 2H2O
• ? 13 subunits/monomer, dimer complex
• ? contains heme group and copper ion (CuA CuB
  redox center)
• ? electron transfer cytochrome c ? CuA ? heme
  a ? Fe-Cu binuclear center (heme a3 CuB)
• ? 4 protons translocated from the matrix to
  intermembrane space
• 3) protein complex contains two proton wires,
  spanning the 50A? distance, Arg, Glu are active
  sites
• 4) Proton and water relays form a proton
  gradient

Figure 12.18 A proposed model for the cytochrome
c oxidase reaction.
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Figure 12.16 Cytochrome c from tuna.
Lysune
Figure 12.19 Complex IV function.
Figure 12.17 X-Ray structure of cytochrome c
oxidase.
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3. Chemiosmosis Free energy Table 12-2, each
step generates energy enough to synthesize
ATP Chemiosmosis links electron transport and
oxidative phosphorylation Peter Mitchells
Chemiosmotic theory, protonmotive force each
pair of electrons from Complexes I, III and IV
10 proton are translocated from matrix to
intermembrane space
The proton gradient is an electrochemical
gradient ?Imbalance of protons associated free
energy (the force to restore the system to
equilibrium) ? Two components chemical
concentration, electrical charge ?
electrochemical ? free energy to translocate
one proton 20 kJ/mol 10 protons -200 kJ/mol,
enough for several ATP synthesis Sample
calculation 12-2
Figure 12.20 Generation of a proton gradient.
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4. ATP synthase F1F0ATP synthase (Complex V)
F0functions as a transmembrane channel for
proton, F1 catalyze the reaction of ADP
phosphorylation The structure of ATP synthase ?
highly conserved ? Membrane embeded F0
component a subunit, two b subunits, gt9 c
subunits, Proton transport involves c ring past
a subunit ? Soluble F1 component three a and ß
subunits, ? (linker to F0 component), d subunit ?
interaction of ? subunit with 3 aß subunits ?
rotation of ? subunit
Figure 12.21 ATP synthase function.
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4. ATP synthase The structure of ATP synthase ?
highly conserved ? Membrane embeded F0
component a subunit, two b subunits, gt9 c
subunits, Proton transport involves c ring past
a subunit ? Soluble F1 component three a and ß
subunits, ? (linker to F0 component), d subunit ?
interaction of ? subunit with 3 aß subunits ?
rotation of ? subunit
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Figure 12.23 Model for proton transport by Fo.
Figure 12.24 Structure of the F1 component of ATP
synthase.
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• 4. ATP synthase
• The binding change mechanism
• ? Rotation driven conformation changes (open,
  loose, tight) alter affinity of each aß
  subunits for adenine nucleotides
• Evidence isolation of F1 components
  functions as ATPase
• ? ATP synthase can function reversible
• Uncoupler (Box 12A)
• Stoichiometric considerations of oxidative
  phosphorylation
• 1 ATP synthesis for 3-4 proton translocated
  through F0
• Chemical energy ? protonmotive force ?
  mechanical movement of a rotatory engine ?
  chemical energy
• PO ratio the number of phosphorylations of ADP
  relative to the number oxygen atoms reduced
• Regulation of oxidative phosphorylation
• Cyt. c oxidase highly exergonic? regulatory
  step?, but no known effectors
• Availability of ADP and Pi?

The binding change mechanism.
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• 4. ATP synthase
• The binding change mechanism
• ? Rotation driven conformation changes (open,
  loose, tight) alter affinity of each aß
  subunits for adenine nucleotides
• Evidence isolation of F1 components
  functions as ATPase
• ? ATP synthase can function reversible
• Uncoupler (Box 12A)
• Stoichiometric considerations of oxidative
  phosphorylation
• 1 ATP synthesis for 3-4 proton translocated
  through F0
• Chemical energy ? protonmotive force ?
  mechanical movement of a rotatory engine ?
  chemical energy
• PO ratio the number of phosphorylations of ADP
  relative to the number oxygen atoms reduced
• Regulation of oxidative phosphorylation
• Cyt. c oxidase highly exergonic? regulatory
  step?, but no known effectors
• Availability of ADP and Pi?

The binding change mechanism.
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Stoichiometry of O2 consumption and ATP synthesis

• ADP Pi x(1/2)O2 xH xNADH
• ? ATP xH2O xNAD
• The value of x P/O ratio or P/2e ratio
• The consensus values for protons pumped out per
  pair of electrons are 10 for NADH and 6 for
  succinate.
• The most widely accepted experimental value for
  number of protons required to drive the synthesis
  of an ATP molecule is 4 (or 3?).
• P/O ratio is 2.5 (or 3) for NADH and 1.5 (or 2)
  for succinate

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4 or 6
6
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4
36 or 38
PO ratio(NADH/FAD) 3 /2
2/1.5 H/ATP
3 4
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(2) Glycerol phosphate shuttle

• This alternative means of moving reducing
  equivalents from the cytosol to the mitochondrial
  matrix operates in skeletal muscle and the brain
  .

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Uncoupling oxidative phosphorylation

• Uncoupler ADP ??? ??? ?????? ?? ??? ??? ?????
  ATP ??? ??? ?? ??
• Proton ionophore (?????)? ????
  ?????? ??? ????? proton gradient? ???? ATP
  synthase? ?? ATP ?? ??
• 1. 2,4-dinitrophenol (DNP), diet pills in the
  1920s with fatal side effects
• 2. ? ? dicoumarol, carbonyl cyanide ?
• - ???, aromatic ring? acidic group? ??

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2,4-dinitrophenol
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Uncoupling in Brown Adipose Tissue

• white fat/ brown fat
• Most newborn mammals, including humans, have a
  type of adipose tissue called brown fat in which
  fuel oxidation serves not to produce ATP, but to
  generate heat to keep the newborn warm.
• brown because of the presence of large numbers of
  mitochondria and thus large amounts of
  cytochrome.
• nonshivering thermogenesis
• uncoupling protein (UCP thermogenin)

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Key points

• 1. Oxidative phosphorylation needs intact inner
  mitochondrial membrane.
• 2. Impermeability of Inner mitochondrial membrane
  against H, OH-, K, Cl-
• 3. H electrochemical gradient (protonmotive
  force) in mitochondrial membrane resulted from
  oxidative phosphorylation
• 4. Uncoupler makes inner mitochondrial membrane
  permeable to H
• ? electron transport still occurs, but no ATP
  synthesis

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Experiment

• ION EXCHANGE CHROMATOGRAPHY

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The major problem in protein purification
Maximize yield get as many red marbles as
possible High purity take only the red
marbles. These two are opposing forces.
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Exploiting chemical properties of
proteins Purification

• Purification procedures attempt to maintain the
  protein in native form. Although some proteins
  can be re-natured, most cannot!
• To purify a protein from a mixture, biochemists
  exploit the ways that individual proteins differ
  from one another. They differ in
• Size, charge, tag
• Thermal stability

Precipitation with ammonium sulfate (salting out)
solubility
For most protein purifications, all steps are
carried out at 5C to slow down degradative
processes.
Ammonium sulfate precipitation is cheap, easy,
and accommodates large sample sizes. It is
commonly one of the first steps in a purification
scheme.
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Size exclusion chromatography
Porous beads made of different but controlled
sizes. Smaller proteins go in and out of beads
and will be retained in the resin. Large
proteins will only go into large beads and will
be retained less. Very large proteins will not
go into any of the beads (exclusion limit). Can
be used as a preparative method or to determine
the molecular weight of a protein in solution.
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Affinity chromatography
A ligand with high affinity to the proteinis
attached to a matrix. Protein of interest bin ds
to ligand and is retained by resin. Everything
else flows through. Can use excess of the
soluble ligand to elute the protein.
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Ion exchange chromatography
Anion exchange Column is postively charged
(can bind negativey charged proteins). Cation
exchange Column is negativey charged (can bind
negatively charged proteins).
Exploit the isoelectric point of a protein to
separate it from other macromolecules.
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• Ion-Exchange Processes
• Based on exchange equilibria between ions in
  solution and
• ions of like charge on surface of essentially
  insoluble, high-
• molecular weight solid.
• Most common cation exchangers
• The strong acid sulfonic acids, SO3-H
• The weak acid carboxylic acids, COOH
• Most common anion exchangers
• The strong base ternary amines, -N(CH3)3OH-
• The weak base primary amines, -NH3OH

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Ion-exchange chromatography
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Dissociation of RNAP and purification of s by
ion-exchange chromatography
Carboxymethyl- (-CO2-2) or phospho- (-PO3-2)
cellulose
Fraction number
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• IEC in practice
• Choose the matrix according to your target
  protein
• 2. Equilibrate (low salt, lt 20 mM )
• 3. Inject protein sample (in low salt), balance
  (wash)
• 4. Apply gradient (increasing salt) to elute
  proteins
• Obey buffer instructions
• a. gradient increasing salt gradient (0-1M NaCl
  in 20 mM buffer)
• or pH gradient (ampholytes in
  chromatofocussing)
• b. type of gradient linear gradient /step wise

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????-???????? ?? ????? ??
??? ????-???????? ????? ???? ?? ??? ??, ? ?????
(ion exchanger)? ?? ?? ?? ?? ?? ???? ????? ????
??
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????-???????? ?? ????? ??
?Ninhydrin ?? Ninhydrin (triketohydrindene
hydrate)? ????? ???? ???? ?? ??? ???. ?? ???
????? ??? ???? ???. ? ???? NH3? CO2?
???. Ninhydrin (triketohydrindene hydrate) is a
chemical used to detect ammonia or primary and
secondary amines. When reacting with these free
amines, a deep blue or purple color known as
Ruhemann's purple is evolved. Ninhydrin is most
commonly used to detect fingerprints, as amines
left over from peptides and proteins ( or lysine
residues) sloughed off in fingerprints react with
ninhydrin
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????-???????? ?? ????? ??

• ??? ?? ? ??
• 0.1 N HCl, 0.2 M pH 8.2 Tris-HCl (????), ????
  ??(0.2 acetone ??), ???????? ?, ?? ?? ??, ???,
  ??, ?? ???, ?????, ??, ???? ?? ( Aspartate,
  Methionine, Arginine )
• ?????
• 1. 0.1 N HCl 10ml? ??? ????? ??? ?? 12cm ????
  ???.
• ( ??? ?? ??? ?? ??? ?? ??? ?? ??? ?? ??? ??.)
• 2. 0.2 ml? ???? ????? ?? ? ??? ??? ??? ???? ??.
• 3. 0.2 ml? 0.1 N HCl? ??? ???? ?? ??? ??, ? ??? ?
  ? ????.
• 4. 2 ml? 0.1 N HCl? ?? ? ?? ??? 0.1 N HCl 500 ml?
  ?? ???? ????, ???? ?? ???? ??? ???.
• 5. ????? ???? ??? ??? 5?? ???? ??? ????? ??? ??
  ????? ??? ??? ?? ???? ??? ????? ????.
• 6. 0.2 M Tris-HCl ???? 2ml? ?? ???? ? ?? ????? ?
  ?? ????? ??? ??? ??? ????? ?? ????.
• 7. ???? ??? ?? ????? ????.