Download Electron transport chains in mitochondria

Dr. Faisal Al-Khteeb
Sheet Biochemistry 9
Tala Al-Hyasat
BACKGROUND
Electron transport chains in mitochondria
Most eukaryotic cells have mitochondria, which produce ATP from products of the
citric acid cycle, fatty acid oxidation, and amino acid oxidation. At the mitochondrial
inner membrane, electrons from NADH and succinate pass through the electron
transport chain to oxygen, which is reduced to water.
* Electrons from NADH pass to complex 1 , Electrons from succinate pass to
complex 2
The electron transport chain comprises an enzymatic series of electron donors and
acceptors. Each electron donor passes electrons to a more electronegative acceptor,
which in turn donates these electrons to another acceptor, a process that continues
down the series until electrons are passed to oxygen, the most electronegative and
terminal electron acceptor in the chain.
*electrons flow from low to large reduction potential
Passage of electrons between donor and acceptor releases energy, which is used to
generate a proton gradient across the mitochondrial membrane by actively “pumping”
protons into the intermembrane space, producing a thermodynamic state that has the
potential to do work. The entire process is called oxidative phosphorylation, since
ADP is phosphorylated to ATP using the energy of hydrogen oxidation in many steps.
A small percentage of electrons do not complete the whole series and instead directly
leak to oxygen, resulting in the formation of the free-radical superoxide, a highly
reactive molecule that contributes to oxidative stress and has been implicated in a
number of diseases and aging.
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Dr. Faisal Al-Khteeb
Sheet Biochemistry 9
Tala Al-Hyasat
Mitochondrial redox carriers
Energy obtained through the transfer of electrons down the ETC is used to pump
protons from the mitochondrial matrix into the intermembrane space, creating an
electrochemical proton gradient across the mitochondrial inner membrane (IMM) .
This electrochemical proton gradient allows ATP synthase (ATP-ase) to use the flow
of H+ through the enzyme back into the matrix to generate ATP from adenosine
diphosphate (ADP) and inorganic phosphate.
Complex I (NADH coenzyme Q reductase; labeled I) accepts electrons from the Krebs
cycle electron carrier nicotinamide adenine dinucleotide (NADH), and passes them to
coenzyme Q (ubiquinone; labeled UQ), which also receives electrons from complex II
(succinate dehydrogenase; labeled II).
UQ passes electrons to complex III (cytochrome bc1 complex; labeled III), which
passes them to cytochrome c (cyt c).
Cyt c passes electrons to Complex IV (cytochrome c oxidase; labeled IV), which uses
the electrons and hydrogen ions to reduce molecular oxygen to water.
Four membrane-bound complexes have been identified in mitochondria. Each is an
extremely complex transmembrane structure that is embedded in the inner membrane.
Three of them are proton pumps. The structures are electrically connected by lipidsoluble electron carriers and water-soluble electron carriers.
The overall electron transport chain:
NADH → Complex I → Q → Complex III → cytochrome c → Complex IV
↑
Complex II
↑
FADH
→ O2
In conclusion , electron transport chain consist of 3 parts :
1- Electron doners : for example ( electrons from NADH pass to complex 1 ,
Electrons from succinate pass to complex 2 )
2- Electron carriers:
• Flavin Mononucliotide
• Iron Sulfur Centers
• Coenzyme Q
• Cytochromes
• Cupper
3- Membrane proteins : the protein complexes.
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Dr. Faisal Al-Khteeb
Sheet Biochemistry 9
Tala Al-Hyasat
Last lecture we talked about Flavin Mononucliotide , Iron Sulfur Centers and
Coenzyme Q.
Now ,
cytochromes
Protein contains a heme group (made of a porphyrin ring
plus iron). Unlike the heme groups of hemoglobin, the
cytochrome iron atom is reversibly converted from its ferric
(Fe+3) to its ferrous (Fe+2) form as a normal part of its
function as a reversible carrier of electrons. Electrons are
passed along the chain from coenzyme Q to cytochromes
bc1 (complex lll) ,c , and a + a3 ( complex lV )
(Heme- containing protein)
cytochrome
Notes :
- Cytochrome c is associated with the outer face of the inner membrane.
- Cyto means cell ,, chrome means pigment ( they are colored
because of the presence of the heme group which: has iron , show
alternation of single – double bonds ).
- The basic unit of heme group is tetrapyrrole each one is formed by
5 members : 4C and 1N , each N atom is bound to the iron which
locates at the centre .
pyrrole
ABSORPTION SPECTRA
-
Absorption of light can be measured by spectrophotometer (in absorber unit).
More concentrated molecule , more absorption of light .
Every compound has characteristic absorption spectra .
Compounds differ in : 1) their ability to absorb light 2) in the wave length at
which the light absorption occur (red, green , …)
- If you measure the light absorption at various wavelength at the visible region (
350 – 700 nm ) , you will find that it's differ according to the change of the
wave length.
- You can change the light absorption by changing the wave length.
- Cytochroms can absorb light because of the alternation of single – double
bonds .
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Dr. Faisal Al-Khteeb
Sheet Biochemistry 9
Tala Al-Hyasat
- Reduced and oxidized form of cyt c have different absorption spectra (curve
for each one ) .
Cytochrome c was isolated and purified.
Absorption spectra of both the oxidized and the
reduced cytochrome c are shown in the figure . (
blue- reduced , red- oxidized )
- At this wave length : absorption is
maximum for reduced form , while there
is no absorption for the oxidized form
(approximately )
- We can mesure the percentage of the
reduced form in the mixture after
measuring the absorption at this wave
length.
- Three classes of cytochromes a,b and c , they differ in the heme group .
- different types of the heme group have different side chains attached to the
tetrapyrrole ring ( minor differences )
-can be distinguished by differences in their light-absorption( different absorption
spectra )
- Each one has different reduction potential
- we can distinguish between the oxidized and reduced form .
- Absorption spectra is affected by the protein environment ( isolated, pure , free
form )
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Dr. Faisal Al-Khteeb
Sheet Biochemistry 9
Complex l
Tala Al-Hyasat
FMN
-NADH Dehydrogenase ( according to its function : remove
the hydrogen from the substrate ( NADH ))
-Membrane- Spanning.
-binding sites to NADH .
-More than 25 polypeptide chain
- Tightly bound FMN group ( can't destroy the structure of complex l )
-Seven Fe-S centers of at least two different types ( involved in the electron transfer )
- Drop in energy≈ -13 to 14 kcal when electrons pass from NADH to Co Q , this
energy is more than enough to synthesis ATP .
NOTE : Co Q is lipid soluble
The free proton plus the hydride ion carried by NADH are transferred to NADH
dehydrogenase, a protein complex ( complex l ) embedded in the inner
mitochondrial membrane . complexl has a tightly bound molecule of flavin
mononucleotide (FMN, a coenzyme structurally related to FAD) that accepts the
two hydrogen atoms ( 2 H + and 2 e- ) becoming FMNH2. NADH dehydrogenase
also contains several iron atoms paired with sulfur atoms to make iron-sulfur
centers . These are necessary for the transfer of the hydrogen atoms to the next
member of the chain, ubiquinone (known as coenzyme Q).
Complex lll
- The coenzyme Q - cytochrome c — oxidoreductase( the donner or the
substrate is Co Q , the acceptor is cyt c )
- sometimes called the cytochrome bc1 complex ( the common name )
- or Cytochrome reductase and at other times complex III, is the third
complex in the electron transport chain
• Catalyzes the transfer of electrons from QH2 to cytochrome c
• 11 subunits including two cytochrome subunits
• Contain iron sulfur center
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Co Q
Dr. Faisal Al-Khteeb
Sheet Biochemistry 9
Tala Al-Hyasat
• Contain three heme groups in two cytochrome subunits
– bL and bH in cytochrome b ( L means low ability , H means high ability )
– c type in cytochrome c1
• Contain two CoQ binding sites ( 2 molecules bind at the same time )
THE
Q
CYCLE
The Q cycle describes a series of reactions that describe how the sequential oxidation
and reduction of the lipophilic electron carrier, ubiquinol-ubiquinone ( Coenzyme Q),
can result in the net pumping of protons across a lipid bilayer ( the inner
mitochondrial membrane).
Round 1:
1. Cytochrome b binds a ubiquinol and a ubiquinone.
2. The 2Fe/2S center and BL heme ( cyt bL in the slide ) each pull an electron off
the bound ubiquinol, releasing two hydrogens into the intermembrane space.
3. One electron is transferred to cytochrome c1 from the 2Fe/2S centre, while
another is transferred from the BL heme to the BH Heme( cyt bH in the slide ) .
4. Cytochrome c1 transfers its electron to cytochrome c (not to be confused with
cytochrome c1), and the BH Heme transfers its electron to a nearby
ubiquinone, resulting in the formation of a ubisemiquinone.
5. Cytochrome c diffuses. The first ubiquinol (now oxidised to ubiquinone) is
released, while the semiquinone remains bound.
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Dr. Faisal Al-Khteeb
Sheet Biochemistry 9
Tala Al-Hyasat
Round 2:
1. A second ubiquinol is bound by cytochrome b.
2. The 2Fe/2S center and BL heme each pull an electron off the bound ubiquinol,
releasing two hydrogens into the intermembrane space.
3. One electron is transferred to cytochrome c1 from the 2Fe/2S centre, while
another is transferred from the BL heme to the BH Heme.
4. Cytocrome c1 then transfers its electron to cytochrome c, while the nearby
semiquinone picks up a second electron from the BH Heme, along with two
protons from the matrix.
5. The second ubiquinol (now oxidised to ubiquinone), along with the newly
formed ubiquinol ( fully reduced ) are released.
_ I copied everything related to ( the Q cycle ) from wiqipedia .
Remember that :
-the oxidized form of coenzyme Q ( Q ubiquinone )
- the reduced form of coenzyme Q ( QH2 ubiquinol )
Simple way to memorize it (OL = all electrons so it's the reduced form)
Complex lV
The enzyme cytochrome c oxidase or Complex IV, is a large transmembrane protein
complex.
It receives an electron from each of four cytochrome c molecules, and transfers them
to one oxygen molecule, converting molecular oxygen to two molecules of water. In
the process, it binds four protons from the inner aqueous phase to make water, and
in addition translocates four protons across the membrane, helping to establish a
transmembrane difference of proton electrochemical potential that the ATP synthase
then uses to synthesize ATP.
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Dr. Faisal Al-Khteeb
Sheet Biochemistry 9
Tala Al-Hyasat
. Passes electrons from Cytocrome c to oxygen ( cyt c is a small protein found in the
periphery of the membrane )
• Contains cytochrome a and a3
• Contains two copper ( e- carrier group )
• Contains oxygen binding sites
• O2 must accept 4 electrons to be reduced to two H2O ( 4cyt c to reduce 1 O2 )
• Cytochrome c is one electron carrier
4Cyt cred + 4H+ + O2 → 4Cyt cox + 2H2O
• Partial reduction of O2 is hazardous ( if the o2 take 1 e- , radicals will be formed,
they can oxidize cellular components like lipids … )
 Parialy reduced O2 shouldn't be realesed
The dr said that we didn't have to memorize these steps.
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Dr. Faisal Al-Khteeb
Sheet Biochemistry 9
Tala Al-Hyasat
NOTS: ( mentioned by the dr )
-
Partial reduction is avoided
Partially reduced oxygen doesn't release ( doesn't occur )
Notice the site of oxygen , between Cu B and iron in the cytochrome .
Iron has one more ( new ) oxidative state (Fe+4 )
Complete reduction of oxygen by forming Cyt cs .
in this figure:
- 4 complexs
- Complex V is the ATP synthease ( not shown here )
- 2 electron carriers : UQ which is lipid soluble , it transfer electrons from
complex l to lll
Cyt C mobile protein , it's not an integral part of the membrane , peripheral
protein , can be separated by saline or by water and salt .
- Complex l oxidize NADH then the electrons will move towards UQ , complex lll ,
cyt c then complex lV
- Complex ll ( succinate dehydrogenase ) oxidize succinate into fumarate .
- FADH2 can produced by fatty acid oxidation .
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Dr. Faisal Al-Khteeb
Sheet Biochemistry 9
Tala Al-Hyasat
- Complex ll doesn't pump protons because it doesn't span the membrane (
doesn't found in the both sides ) , also the energy result from the oxidation of
succinate into fumarate is not sufficient to pump protons .
- Complexes l , lll and lV can pump protons.
- Again :
NADH → Complex I → Q → Complex III → cytochrome c → Complex IV
↑
Complex II
↑
FADH
→ O2
How can we know the
function of the complexes or
the electron carriers ?
Protein purification
1) Treatment with digitonin : Remove the
outer membrane of the mitochondria by
digitonin in a test tube .
2) Osmotic rupture : by putting the
mitochondria in low salt concentration ,
so the water will enter , it will lyses.
3) Inner membrane fragments have
different complexes .
4) Solubilization with detergent followed
by ion exchange chromatography .
5) Separate the complexes in test tubes
By fractionation , we can identify the substrate of each complex and the direction of
the flow of electrons.
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Dr. Faisal Al-Khteeb
Sheet Biochemistry 9
Tala Al-Hyasat
Notes:
In the test tube V we can see ATP hydrolysis not ATP synthesis !!! WHY?
-Because there is no ETC in the test tube .
-There are enzymes , reactants and products.
- the reaction occur as a hydrolysis reaction ,
because it is favorable .
From the reduction potential we
can put the different reactions in
the right order.
Each component of the chain taks electrons from the substrate and move
them to the product, until it reach the oxygen
the inhibitors of the Electron Transport Chain
the inhibitors of the Electron Transport Chain are substances that bind to some of the
components of the ETC blocking its ability to change in a reversible form from an
oxidized state to a reduced state.
This inhibition results in the accumulation of reduced forms before the inhibitor
point, and oxidized forms of the components of the ETC downstream (ahead) the
inhibition point.
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Dr. Faisal Al-Khteeb
Sheet Biochemistry 9
Tala Al-Hyasat
Some substances if they are added to ETC or to the mitochondria ,electron flow will
stop like:
1- Amytal rotenone.
2- Antimycin A.
3- CN- CO sodium azid.( block the electron flow at the last complex )
 In the absence of oxygen , electron flow stops ( unless artificial electron
accepter was used )
 Blocking ,by any one of the inhibitors ,stops the flow of electrons from
the substrate to the oxygen.
 Before and after blocking , reduced or oxidized state ?
Before blocking , reduced
After blocking , oxidized
In the absence of O2 , all components of the ETC are in the reduced form ( we can
recognize them by different absorption spectra) , if we add O2 suddenly , the last
molecule ( proximal to the oxygen) will oxidized first, then the one before ( toward
the left ) , so we can know the order of the ETC.
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Dr. Faisal Al-Khteeb
Sheet Biochemistry 9
Tala Al-Hyasat
*Coupling of oxidation and phosphorylation is due to proton pump.
*ATP synthesis driven by proton-motive force:
1) ph gradiant
2) electrical potential
*protons can return to the matrix only by ATP synthase.
*if we block the transfer of electrons by an inhibitor , ATP synthesis will stop because
no protons are pumped.
How to prove that ATP synthesis depends on proton pump ?
1) Chemiosmotic hypothesis:
a) Airtight vessel initially contains
mitochondria , in a medium with
ADP, Pi and succinate but no
oxygen.
b) Electrode to measure PH
c) At time zero we injected O2
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Dr. Faisal Al-Khteeb
Sheet Biochemistry 9
Tala Al-Hyasat
d) Initiate H+ pumping , ph will decrease
 When the electron transport start , ph will decrease.
2) Bacteriorhodopsin (protein from the
bacteria)
Purple membrane protein from halobacter.
Pumps proton when illuminated .
*They took the Bacteriorhodopsin and put it in
synthetic vesicle
*they put mitochondrial ATPas in the same vesicle
Now, we have vesicle (artificial system of oxidative phosphorylation )
which has:
Proton pump ( depends on elimination )
ATP synthase ( depends on H+ concentration )
When it's illuminated
ATP synthesis
Demonstration that ATP synthesis depends on proton gradiant
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Dr. Faisal Al-Khteeb
Sheet Biochemistry 9
DONE BY : TALA AL- HYASAT
NO ROAD IS LONG …
WHEN DREAMS ARE BIG …
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Tala Al-Hyasat