Subject: biochemistry
Lec# 10
Dr. Karadsheih
Date: 3-3-2011
Continuing with oxidative phosphorylation from last lecture:
Last time we talked about electron flow which is utilized to pump
H+ from the matrix to the intermembrane space. As a result,
proton motor force was created resulting from the electrical and
chemical tension that was created at both sides of the inner
membrane.
Protons have no other way to cross the inner membrane from the
intermembrane space back to the matrix than ATP-Synthase
complex, producing ATP in the process.
ATP-Synthase Complex is formed from multi-subunits (node,
rotator, ….)
In order for the rotator to rotate once complete cycle, it will need
12 protons to pass through it, inducing conformational changes
and releasing 3 ATP molecules in the process.
So we conclude, the proton motive force is behind ATP synthesis.
We know that respiration is an oxidation reaction of the food.
Oxidation is always accompanied by reduction.
Reduction is removal of electrons that are accepted by co-factors. Co-factors like: NADH
(mainly) or FADH2.
Oxidation happens mainly in the mitochondria (Krebs cycle) and some of it in the
cytoplasm (glycolysis)
NADH is produced in the cytoplasm.
P.S. the inner membrane of the mitochondria is impermeable to NADH, so there should
be special mechanism to transport NADH from cytoplasm to inside the mitochondria.
Glycolysis in the cytoplasm produces NADH carrying electrons which should be
transported to the electron transport chain inside the mitochondria.
Each glycolysis produces  2 NADH
Each NADH produces  3 ATP
Q. How does the NADH that was produced in the cytoplasm reach inside the
mitochondria?!
A. We have two shuttle mechanisms:
1- The Glycerol-3-phospahte shuttle:
(Operated mainly in the skeletal muscles and brain)
Electrons from NADH are transported to  dihydroxyacetone phosphate (a
glycolitic intermediate) which is reduced to  glycerol-3-phosphate with the help
of an enzyme called glycerol-3-phosphate dehydrogenase (cytoplasmic enzyme)
Now glycerol-3-phosphate enters the outer-membrane of the mitochondria
towards the inner-membrane.
On the inner membrane there is a membrane bound enzyme called glycerol-3phosphate dehydorenase (very similar to the previous one, but this one is a
membrane bound enzyme). It re-oxidizes glycerol-3-phosphate back to
dihydroxyacetone phosphate. Then electrons are transported to FAD reducing it
to FADH2 and then electrons are transported to Coenzyme Q (part of the
electron transport chain)
Note: Because the electrons using the glycerophosphate shuttle enter the
electron transport pathway at the level of Coenzyme Q , the electrons can
only be used to synthesize a maximum of two ATP, instead of the maximum of
three ATP derived from NADH formed inside the mitochondria.
2- Malate Aspartate Shuttle:
(operated mainly in the liver and heart)
Involves malate and aspartate to transport electrons across the mitochondrial
inner membrane. Oxaloacetate in the cytoplasm is converted to malate by
malate dehydrogenase, oxidizing NADH to NAD+.. The malate enters the
mitochondria using an exchanger protein that must also transport α-ketoglutarate
in the opposite direction (this protein is called antiporter). The malate is then
oxidized to oxaloacetate by the mitochondrial malate dehydrogenase, resulting in
formation of NADH, which can then enter the electron transport pathway.
This shuttle conserves all of the energy in the NADH electrons, and allows the
synthesis of three ATP (under optimum conditions) from NADH generated in the
cytoplasm.
Note: antiporters are special proteins that allow the entry of one molecule in
exchange of another.
Return of the oxaloacetate to the cytoplasm requires a separate transporter,
which exchanges aspartate for glutamate. Oxaloacetate is transformed to become
aspartate by an enzyme called aspartate amino-tansferase.
P.S. we will talk about these reactions later in more details when we study
metabolism 
The main difference between both shuttles is how many ATP each produces:
The Glycerol-3-phospahte shuttle  2( FADH2)  2 ATP
Malate Aspartate Shuttle 2( NADH) 3 ATP
Poisons and Inhibitors of the Electron Transport Chain:
1- Rotenone: fish poison and insecticide ->inhibits electron transport between
NADH and coQ ( complex I)
2- Antimycin A: antibiotic (inhibits complex III)
3- Carbon Monoxide (MO): inhibits cytochrome C oxidase (complex IV)
4- Cyanide: very lethal (complex IV)
One way Cyanide poising could be treated is to oxidize some hemoglobin to
give cyano-hemoglobin which prevents cyanide from reaching cytocrome C
oxidase.
5- Oligomycin: blocks ATP-synthase, as a result protons can’t pass and ATP
synthesis is inhibited.
Protons concentration will increase dramatically in the intermembrane space
and a steep gradient is established. Because protons continue to accumulate in
the intermembrane space and their passage back to the matrix is blocked,
oxidation phosphorylation is blocked as well, and the electron transport chain
doesn’t function anymore.
6- Atractyloside: inhibits antiporter on the inner-membrane that permits ATP to
exit the mitochondria.
Uncoupling agents:
Uncoupling is defined as a condition in which the rate of electron transport can no
longer be regulated by an intact chemiosmotic gradient. The electron transport system
is uninhibited due to complete and irreversible dissipation of the chemiosmotic
gradient. it involves some kind of leakage of protons from intermembrane space back
to the matrix without having to pass through ATP-synthase. It causes hyperthermia
(increasing body temperature).
1- 2,3-dinitrophenol: when it enters the intermembrane space of the mitochondria,
it gets protonated thus making it impossible to maintain a proton gradient. The
uncoupling of electron transport from ATP synthesis allows rapid oxidation of
Krebs substrates, promoting the mobilization of carbohydrates and fats, since
regulatory pathways are programmed to maintain concentrations of those
substrates at set levels. Since the energy is lost as heat, biosynthesis is not
promoted, and weight loss is dramatic. This made it possible for this substance to
be used as a drug for losing weight, but studies have shown that it might cause
blindness to some patients, so it was withdrawn from the market.
2- Uncoupling Proteins: found in very small quantities on the inner-membrane of
the mitochondria. Those proteins are present in high concentration in newborn
babies in brown fat tissues (around the neck) for the purpose of producing heat
Brown fat (thermogenic) is also found in hibernating animals to help them stay
warm during hibernating season.
Most of the proteins of the mitochondria are created from nuclear DNA.
In Oxidative Phosphorylation there are almost 120 polypeptides, most created from
nuclear DNA and 13 polypeptides from Mitochondrial DNA.
Those 13 polypeptides are divided as follows: 7 in complex I, 1 in complex III, 3 in
complex IV, 2 in ATP-synthase complex).
Note: we all inherit our mitochondria from our mothers because mitochondria of sperm
is excluded out.
Diseases & Disorders Associated with Mitochondria:
Most diseases that are related to the mitochondria are also related to oxidative
phosphorylation and are called mitochondrial myopathy. Mitochondrial myopathy is
caused by defects in the 13 polypeptides created from mitochondrial DNA.
-Apoptosis:
Cell’s programmed Auto-destruction.
One of the causes of apoptosis is leakage of the outer-membrane of the mitochondria.
Cytochrome C from the inner-membrane migrates to the cytoplasm and activates
proteolytic enzymes that degrade most proteins of the cell which initiate programmed
cell death.
Best of luck,
Osama Afaneh 