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MEMBRANE-BOUND ELECTRON TRANSFER AND ATP SYNTHESIS (taken from
Chapter 18 of Stryer)
FREE ENERGY – MOST USEFUL THERMODYNAMIC
CONCEPT IN BIOCHEMISTRY
Living things require an input of free energy for 3 major purposes
1. Mechanical – Muscle contraction and other cellular movement
2. Active transport of molecules and ions
3. Synthesis of macromolecules and other biomolecules from simple precursors
First law of thermodynamics
“Energy can be neither created nor destroyed”
But, it can be converted from one form into another
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)
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
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
Stages of Catabolism
Citric Acid Cycle
GlycolysisActivated Carriers
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
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
Mitochondrion
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)
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 existing mitochondria are derived from bacterial Rickettsia prowazekii
Oxidative phosphorylation is conceptually simple and mechanistically complex.
Flow of electrons from NADH and FADH2 to
O 2 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.
An example of energy coupling via an electrochemical gradient across a membrane.
REDOX POTENTIAL AND FREE ENERGY CHANGES
The energy stored in ATP is expressed as the phosphoryl transfer potential which is given by G o 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).
Redox couples
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 electrons
1/2 O2/ H2O at +0.82V is a strong oxidising reagent and
poised to accept electrons.
1/2 O2 + NADH +H+ H2O + NAD+
The difference (Eo = 1.14V) is equivalent to -52.6
kcal/mole.
Electrons can be transferrred between groups that are not in contact
THE RESPIRATORY ELECTRON TRANSFER CHAIN CONSISTS OF THREE PROTON PUMPS LINKED BY TWO MOBILE ELECTRON
CARRIERS
Electrons are transferred from NADH to O2 by a
chain of three large transmembrane respiratory chain protein complexes
I
II
III
IV
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
Q- coenzyme Q or ubiquinone
Complex I NADH-Q Oxidoreductase
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 (ubiquinone)
Results in pumping of 4 H+ out of matrix
NADH + Q + 5H+matrix NAD+ +QH2 + 4H+
cytosol
Complex IISuccinate 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
Complex IIIQ-cytochrome c Oxidoreductase
Q-cytochrome c Oxidoreductase Transfers e- from QH2 (2 e- ) cytochrome c
(1 e- ) via heme Mechanism known as Q cycle
QH2 + 2Cyt cox + 2H+matrix Q +2Cyt cred + 4H+
cytosol
Q Cycle
QH2
QH2 + 2Cyt cox + 2H+matrix Q +2Cyt cred + 4H+
cytosol
Complex IVCytochrome c Oxidase
Cytochrome c Oxidase
Proton transport by cytochrome c oxidase
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.
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.
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
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 it’s ability to hydrolyse ATP)
It’s preferred name is the ATP synthase.
Structure of ATPase
ATP synthase nucleotide binding sites are not equivalent
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.
C-ringH+ in
H+ out
Rotates 360ºProducing 3 molecules ofATP
Thus 10 protons required
Requires 3 protons per molecule of ATP
THE COMPLETE OXIDATION OF GLUCOSE YIELDS ABOUT 30 ATP
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
POWER TRANSMISSION BY PROTON GRADIENTS: A CENTRAL MOTIF OF
BIOENERGETICS
Proton gradients power a variety of energy-requiring processes i.e.
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
The most important factor controlling the rate of oxidative phosphorylation is the level of ADP
Regulated by the energy charge.
This regulation of the rate of oxidative phosphorylation by the ADP level is called respiratory control.