Dr. Faisal Al-Khteeb Sheet Biochemistry 9 Tala Al-Hyasat

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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.

BACKGROUND

Dr. Faisal Al-Khteeb Sheet Biochemistry 9 Tala Al-Hyasat

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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 lipid-

soluble electron carriers and water-soluble electron carriers.

The overall electron transport chain:

NADH → Complex I → Q → Complex III → cytochrome c → Complex IV → O2

↑

Complex II

↑

FADH

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.

Mitochondrial redox carriers

Dr. Faisal Al-Khteeb Sheet Biochemistry 9 Tala Al-Hyasat

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Last lecture we talked about Flavin Mononucliotide , Iron Sulfur Centers and

Coenzyme Q.

Now ,

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 )

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 .

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 .

cytochromes

(Heme- containing protein)

cytochrome

pyrrole

Dr. Faisal Al-Khteeb Sheet Biochemistry 9 Tala Al-Hyasat

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- 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 )

- 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 )

- 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.

Dr. Faisal Al-Khteeb Sheet Biochemistry 9 Tala Al-Hyasat

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-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).

- 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

Complex l

Complex lll

FMN

Co Q

Dr. Faisal Al-Khteeb Sheet Biochemistry 9 Tala Al-Hyasat

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• 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.

Dr. Faisal Al-Khteeb Sheet Biochemistry 9 Tala Al-Hyasat

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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 .

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.

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

Dr. Faisal Al-Khteeb Sheet Biochemistry 9 Tala Al-Hyasat

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. 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.

Dr. Faisal Al-Khteeb Sheet Biochemistry 9 Tala Al-Hyasat

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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 .

Dr. Faisal Al-Khteeb Sheet Biochemistry 9 Tala Al-Hyasat

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- 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 → O2

↑

Complex II

↑

FADH

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.

Dr. Faisal Al-Khteeb Sheet Biochemistry 9 Tala Al-Hyasat

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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 .

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.

From the reduction potential we

can put the different reactions in

the right order.

Dr. Faisal Al-Khteeb Sheet Biochemistry 9 Tala Al-Hyasat

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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.

Dr. Faisal Al-Khteeb Sheet Biochemistry 9 Tala Al-Hyasat

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*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

Dr. Faisal Al-Khteeb Sheet Biochemistry 9 Tala Al-Hyasat

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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

Dr. Faisal Al-Khteeb Sheet Biochemistry 9 Tala Al-Hyasat

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DONE BY : TALA AL- HYASAT

NO ROAD IS LONG …

WHEN DREAMS ARE BIG …