MEMBRANE-BOUND ELECTRON TRANSFER AND ATP SYNTHESIS (taken from Chapter 18 of Stryer)

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MEMBRANE-BOUND ELECTRON TRANSFER AND ATP
SYNTHESIS (taken from Chapter 18 of Stryer)
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FREE ENERGY MOST USEFUL THERMODYNAMIC CONCEPT
IN BIOCHEMISTRY

• Living things require an input of free energy for
  3 major purposes
•  
• Mechanical Muscle contraction and other
  cellular movement
• Active transport of molecules and ions
• Synthesis of macromolecules and other
  biomolecules from simple precursors
•  

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First law of thermodynamics

• Energy can be neither created nor destroyed
• But, it can be converted from one form into
  another

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Free energy for these processes comes from the
environment Phototrophs - obtained by trapping
light energy Chemotrophs energy by oxidation
of foodstuffs Free energy donor for most
energy requiring processes is Adenosine
triphosphate (ATP)
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Large amounts of free energy is liberated when
ATP is hydrolysed to ADP Pi or AMP PPi ATP
is continuously formed and consumed Phototrophs
harness the free energy in light to generate ATP
- Photosynthesis  Chemotrophs form ATP by
oxidation of fuel molecules Oxidative
phosphorylation
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OXIDATIVE PHOSPHORYLATION
Glucose is converted to pyruvate   And under
aerobic conditions undergoes oxidative
decarboxylation to form AcCoA which is then
oxidised to CO2 by the citric acid cycle
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Stages of Catabolism
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Activated Carriers
Glycolysis
Citric Acid Cycle
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These pathways along with fatty acid oxidation
produce energy rich molecules NADH and FADH2 as
well as small amounts of ATP  Chemotrophs derive
energy from oxidation of fuel molecules and in
aerobic organisms the ultimate electron acceptor
is O2 Electron is not transferred directly
Electron is transferred through special
carriers, Pyridine nucleotides   Electron
acceptor Electron donor   NAD NADH   FAD
FADH2  
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Respiratory electron transfer is the transfer of
electrons from the NADH and FADH2 (formed in
glycolysis, fatty acid oxidation and the citric
acid cycle) to molecular oxygen, releasing
energy. Oxidative phosphorylation is the
synthesis of ATP from ADP and Pi using this
energy.   Both processes are located on the IMM
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Mitochondrion
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• Outer membrane
• Permeable (12000da)
• Porin 30-35kd pore forming protein
• Inner membrane
• Impermeable all ions and polar molecules
• Possess family of transporter molecules (for
  ATP/ADP , Pi , pyruvate, citrate , etc.).
• Matrix side (N-negative), cytosolic side
  (P-postive)

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Mitochondria are the result of an Endosymbiotic
event
Organelles contain their own DNA which encode 13
respiratory chain proteins Many proteins encoded
by cell nuclear DNA Cells depend on organelle for
oxidative phosphorylation , mitochondrion depend
on cell for their very existence Suggested that
all extant mitochondria are derived from
bacterial Rickettsia prowazekii
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Oxidative phosphorylation is conceptually simple
and mechanistically complex. Flow of electrons
from NADH and FADH2 to O2 occurs via protein
complexes located in the IMM Leads to the pumping
of protons from the matrix to the cytosol across
the IMM. ATP is synthesised when protons flow
back into the matrix via a protein complex in the
IMM.
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An example of energy coupling via an
electrochemical gradient across a membrane.
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REDOX POTENTIAL AND FREE ENERGY CHANGESThe
energy stored in ATP is expressed as the
phosphoryl transfer potential which is given by
?Go for hydrolysis of ATP (-7.3kcal/mol)The
electron transfer potential of NADH is
represented as Eo the redox potential ( or
reduction potential or oxidation-reduction
potential) which is an electrochemical
concept.Redox potential is measured relative to
the H H2 couple which has a defined redox
potential of 0V (Volts).
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A negative redox potential means that a substance
has a lower affinity for electrons than H2 . A
positive redox potential means a substance has a
higher affinity for electrons than H2. NAD/
NADH at -0.32V is a strong reducing agent and
poised to donate electrons1/2 O2/ H2O at 0.82V
is a strong oxidising reagent and poised to
accept electrons.The difference (?Eo 1.14V)
is equivalent to -52.6 kcal/mole.
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Electrons can be transferrred between groups that
are not in contact
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THE RESPIRATORY ELECTRON TRANSFER CHAIN CONSISTS
OF THREE PROTON PUMPS LINKED BY TWO MOBILE
ELECTRON CARRIERS
I
Electrons are transferred from NADH to O2 by a
chain of three large transmembrane respiratory
chain protein complexes
II
III
IV
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These are a) Complex I also known as
NADH-Ubiquinone (UQ) oxidoreductase NADH-Q
reductase b) Complex III also known as Ubiquinol
(UQH2)-Cytochrome c oxidoreductase Cytochrome
reductase  c) Complex IV also known as
Cytochrome c- Oxygen oxidoreductase Cytochrome
oxidase
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NADH-Q Oxidoreductase
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NADH-Q reductase

• NADH transfer of e- to flavin mononucleotide to
  produce FMNH2
• e- from FMNH2 transferred to iron sulfur clusters
• e- from iron sulfur (Fe-S) clusters shuttle to
  coenzyme Q
• Results in pumping of 4 H out of matrix

NADH Q 5Hmatrix NAD QH2
4Hcytosol
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Succinate Q reductase

• FADH2 already part of complex, transfers
  electrons to Fe-S centres and then to Q
• This transfer does not result in transport of
  protons

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Q-cytochrome c Oxidoreductase

• Transfers e- from QH2 cytochrome c via heme
• Mechanism known as Q cycle

QH2 2Cyt cox 2Hmatrix Q 2Cyt cred
4Hcytosol
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Cytochrome c Oxidase
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Cytochrome c Oxidase
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Proton transport by cytochrome c oxidase
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Electrons are carried from Complex I to Complex
III by UQH2, the hydrophobic quinol (reduced
quinone) diffuses rapidly within the IMM.
Electrons are carried from Complex III to
Complex IV by cytochrome c, a small hydrophilic
peripheral membrane protein located on the
cytosolic or P side of the IMM. Complex II
(Succinate-UQ oxidoreductase) is membrane bound
and contains the FADH2 as a prosthetic group . So
electrons from FADH2 feed in to UQH2. These
respiratory chain complexes contain redox
groups to carry the electrons being transferred
through them. These are flavins, iron-sulfur
clusters, haems and copper ions.
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PROTON PUMPS AND THE ATP SYNTHASE
The free energy change of the reactions catalysed
by Complexes I, III and IV is large enough for
them to pump protons from the matrix or N side of
the IMM to the cytosolic or P side of the IMM.
  There is not enough energy released in Complex
II, so no proton pumping occurs in this complex.
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OXIDATION AND PHOSPHORYLATION ARE COUPLED BY A
PROTON-MOTIVE FORCE
This is the chemiosmotic hypothesis put forward
by Peter Mitchell in 1961. Transfer of electrons
from NADH (or FADH2) to oxygen leads to the
pumping of protons to the cytosolic side of the
IMM. The H concentration (pH) becomes higher
(lower pH) on the cytosolic side, and an
electrical potential (membrane potential) with
the cytosolic side of the IMM positive is
generated
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So a proton-motive force (?p) is generated which
consists of both a ?pH and a ??. Mitchell
proposed that this proton-motive force drives the
synthesis of ATP by another transmembrane protein
complex, as the protons return back across the
IMM through this protein complex.   This protein
complex is called the ATPase (because like any
enzyme it is reversible and was first discovered
by its ability to hydrolyse ATP) Its preferred
name is the ATP synthase.
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It is now thought that the proton-motive force
induces a conformational change in the ATP
synthase, which allows the release of tightly
bound ATP (the product) from the enzyme, and thus
catalyses ATP synthesis.
So this is an example of energy coupling via an
activated protein conformation.
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THE COMPLETE OXIDATION OF GLUCOSE YIELDS ABOUT 30
ATP
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Net Yield per glucose   Glycolysis
2 ATP   Citric Acid cycle 2
ATP (GTP)   Oxidative phosphorylation 26
ATP   Most of the ATP is generated by oxidative
phosphorylation
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POWER TRANSMISSION BY PROTON GRADIENTS A CENTRAL
MOTIF OF BIOENERGETICS
Proton gradients power a variety of
energy-requiring processes i.e.
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IT IS EVIDENT THAT PROTON GRADIENTS ARE A CENTRAL
INTERCONVERTIBLE CURRENCY OF FREE ENERGY IN
BIOLOGICAL SYSTEMS. THE RATE OF OXIDATIVE
PHOSPHORYLATION IS DETERMINED BY THE NEED FOR
ATP Under most physiologic conditions, electron
transfer is tightly coupled to phosphorylation.
Electrons do not usually flow through the
electron transfer chain unless ADP is
simultaneously phosphorylated to ATP. Oxidative
phosphorylation and thus electron transfer
require a supply of NADH O2 ADP and Pi  
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The most important factor controlling the rate of
oxidative phosphorylation is the level of
ADPRegulated by the energy charge. This
regulation of the rate of oxidative
phosphorylation by the ADP level is called
respiratory control.