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. 1 BIOLOGICAL OXIDATION DR BUSHRA FIZA PROFESSOR, DEPT. OF BIOCHEMISTRY, MGMCH BIOLOGICAL OXIDATION The transfer of electrons from the reduced co-enzymes (NADH and FADH 2 ) through the respiratory chain to O 2 is known as biological oxidation. Energy released during this process is trapped as ATP. This coupling of oxidation with phosphorylation is called oxidative phosphorylation. In the body, this oxidation is carried out by successive steps through successive dehydrogenases, together known as electron transport chain (ETC). The electrons are transferred from higher potential to lower potential. Mitochondrion and the electron transport chain Metabolic oxidations generate reduced electron carriers, such as NADH and FADH 2 e.g. from TCA Cycle. Oxidation of these electron carriers in the mitochondrion generates most of the energy needed for ATP synthesis. Most vertebrate cells contain several hundred mitochondria, but the number varies. Mitochondrion (power house of cell) has an outer membrane, an inner membrane and an intermembrane space a matrix, located within the inner membrane. Structure of mitochondria Components of mitochondria The outer membrane is simple, porous, permeable to ions and small molecules (MW <5,000) which move freely through transmembrane channels, formed by transmembrane proteins called porins. The inner membrane is much tighter, impermeable to most small ions, including protons (H + ); only species which cross the inner membrane are those for which there are specific transporters. highly folded into cristae, which project into the interior of the mitochondrion (F-particle) Matrix is the space surrounded by the inner membrane

Mitochondrion and the electron transport chain

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BIOLOGICAL OXIDATION

DR BUSHRA FIZA

PROFESSOR, DEPT. OF BIOCHEMISTRY, MGMCH

BIOLOGICAL OXIDATION

The transfer of electrons from the reduced co-enzymes (NADH and FADH2) through the respiratory chain to O2

is known as biological oxidation.

Energy released during this process is trapped as ATP. This coupling of oxidation with phosphorylation is called oxidative phosphorylation.

In the body, this oxidation is carried out by successive steps through successive dehydrogenases, together known as electron transport chain (ETC).

The electrons are transferred from higher potential to lower potential.

Mitochondrion and the electron transport chain

Metabolic oxidations generate reduced electron carriers, such as NADH and FADH2 e.g. from TCA Cycle.

Oxidation of these electron carriers in the mitochondrion generates most of the energy needed for ATP synthesis.

Most vertebrate cells contain several hundred mitochondria, but the number varies.

Mitochondrion (power house of cell) has

an outer membrane,

an inner membrane and an intermembrane space

a matrix, located within the inner membrane.

Structure of mitochondria Components of mitochondria

The outer membrane is simple, porous, permeable to ions and small molecules (MW <5,000) which move freely through transmembrane channels, formed by transmembrane proteins called porins.

The inner membrane is much tighter, impermeable to most small ions, including protons (H+); only species which cross the inner membrane are those for which there are specific transporters.

highly folded into cristae, which project into the interior of the mitochondrion (F-particle)

Matrix is the space surrounded by the inner membrane

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Inner membrane contains……..

ATP synthase complexes (Complex V)

The components of the electron transport chain (ETC) and oxidative

phosphorylation

Cytochromes - protein electron carriers - are embedded within the

inner membrane. They are assembled in the form of four

multiprotein complexes, named I, II, III, IV

Smaller carriers, e.g. coenzyme Q and cytochrome c, also participate

in carrying electrons (mobile electron carriers)

Matrix is the space surrounded by the inner membrane

Processes occurring inside the mitochondrial matrix include all the

pathways for fuel oxidation except glycolysis (occurs in cytosol) :

Pyruvate oxidation

Fatty acid oxidation (-oxidation)

Citric acid cycle

Pathways of amino acid oxidation

Synthesis of urea and haem

In addition, it contains NAD, FAD, and ADP and Pi, which

are used to produce ATP

Location of enzymes

in mitochondria

Outer membrane :

Monoamine oxidase

Acyl CoA synthase

Phospholipase A2

Between outer and inner membrane

Adenylate kinase

Creatine kinase

Inner membrane - Outer surface

Glycerol-3-phosphate dehydrogenase

Inner membrane – Inner surface

Succinate dehydrogenase

Enzymes of ETC

Soluble matrix

Enzymes of TCA cycle

Enzymes of β-oxidation of fatty acids

Components of the electron transport chain

With the exception of coenzyme Q, all members of this chain are

proteins.

Coenzyme Q or ubiquinone is a lipid-soluble benzoquinone with a

long isoprenoid side chain [e.g.CoQ10 has 10 isoprenoid side chains]

The cytochromes are proteins with iron-containing haem prosthetic

groups. Cytochromes of type a and b are integral proteins of the

mitochondrial membrane while mitochondrial cytochrome c is a

soluble protein and hence “mobile”.

The iron-sulphur proteins in which the iron present is not in

association with haem but in association with inorganic

sulphur atom or with sulphur of cysteine residues in the

protein.

Components of the respiratory chain are arranged in order of

increasing redox potential (more negative to more positive)

Reducing equivalents are passed down the chain and

ultimately oxidised by oxygen to form water.

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Organization of the Electron Transport chain (ETC)

Five separate enzyme complexes, called complex I, II, Ill, IV and V.

Complex I : NADH Dehydrogenase – Coenzyme Q (CoQ)

Reductase

Complex II : Succinate dehydrogenase- CoQ Reductase

Complex III : CoQ-Cytochrome c reductase

Complex IV : Cytochrome oxidase

Complex V : ATP Synthase

Reactions of the Components of ETC

Complex I (NADH Dehydrogenase)

Complex I accepts electrons from NADH which is generated by numerous dehydrogenases in the cell.

-NADH is reoxidized to NAD+ by complex I. -NADH dehydrogenase contains flavin mononucleotide (FMN) as a tightly bound prosthetic group and catalyzes the following reaction

NADH + H+ + FMN NAD+ + FMNH2

Complex I contains iron-sulfur centers, which transfer electrons from FMNH2 tothe next carrier, coenzyme Q.

Thus, Complex I is also called NADH DH-coenzyme Q reductase because the electrons are used to reduce coenzyme Q.

Complex II (Succinate dehydrogenase)

Complex II is not in the path travelled by electrons from Complex I.

Instead, it is a point of entry of electrons from FADH2 produced by

succinate dehydrogenase in TCA cycle

acyl CoA dehydrogenase in β-oxidation of fatty acids.

Mitochondrial glycerol phosphate dehydrogenase

Both complexes I and II donate their electrons to the same acceptor, coenzyme Q.

Complex II is also called succinate-coenzyme Q reductase.

Complex II contains iron-sulfur proteins, which participate in electron transfer.

Succinate Q Reductase (Succinate dehydrogenase)

Is the only membrane bound enzyme in the TCA cycle and

Contains FAD, Fe-S

Complex II → electrons → UQ

2H+ + 2 e-

a lipid in inner membrane

carries electrons

polyisoprene tail

moves freely within membrane

Coenzyme Q = Ubiquinone

Complex III (Cytochrome bc1 complex)

Complex III contains cytochrome b, cytochrome c1 and an Fe-S center. (Cytochrome b and c1 contain a haem group which is the electron acceptor)

Couples the transfer of electrons from CoQ to cytochrome c.

Also known as ubiquinone:cytochrome c oxidoreductase

The cytochrome iron atom in haem is reversibly converted from its ferric (Fe3+) to its ferrous (Fe2+) form as a normal part of its function as a reversible carrier of electrons

Cytochrome c is a soluble protein of the intermembrane space. Accepts an electron from Complex III, move to complex IV to donate its electron

Complex IV (Cytochrome oxidase)

Electrons are passed down the chain from Complex III to cytochrome c and then to cytochrome oxidase

Complex IV, made up of cytochrome a and a3 (cytochrome oxidase), contains bound copper ions which are essential for electron transfer. During electron transfer, the copper shuttles between Cu+ and Cu++ states

Cytochrome a + a3 is the only electron carrier in which the haem iron has a free ligand that can react directly with molecular O2.

At this site the transported electrons, molecular O2, and free protons are brought together to produce water

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Organization of the Electron Transport Chain

NADH

NAD

FMN

FMNH2

Fe2+S

Fe3+S

CoQ

CoQH2

Cyt b

Fe2+

Cyt b

Fe3+

Fe3+S

Fe2+S

Cyt c1

Fe2+

Cyt c1

Fe3+

Cyt c

Fe3+

Cyt c

Fe2+

Cyt a

Fe2+

Cu+

Cyt a

Fe3+

Cu2+

Cyt a3

Fe3+

Cu2+

Cyt a3

Fe2+

Cu+

H2O

½O2

FADH2 FAD

Fe3+ S Fe2+S

Complex II

Succinate dehydrogenase

Complex I

NADH DehydrogenaseComplex III

Cytochrome bc1 complex

Complex IV

Cytochrome oxidase

H+H+

H+

Complex V not shownInter-membrane space

Mitochondrial matrix Energy Release from an Electron Transport System

In an ETC system, each complex accepts or donates electrons to relatively mobile electron carriers e.g. coenzyme Q and cytochrome c.

Each carrier of the ETC can receive electrons from an e-donor and can donate electrons to the next carrier in the chain, ultimately to combine with O2 and protons to form water.

H+ and electrons flow through the chain in steps from the components of more negative redox potential to components of more positive redox potential.

During each transfer, energy is released .

Oxidative Reactions that Drive ATP Synthesis

Energy required to drive the synthesis of ATP from ADP + Pi is 31

kJ/mol (7.3 kcal/mol) [1 kcal = 4.2 kJ]

Only three reactions in the ETC which yield ∆Eo that will generate

∆Go greater than that value

Oxidation of FMNH2 by coenzyme Q (Complex I)

Oxidation of cytochrome b by cytochrome c1 (Complex III)

Cytochrome oxidase reaction (Complex IV)

Each of these sites is the coupling site for ATP synthesis – site

whereby ATP synthesis is driven directly by energy released from the

reaction

Chemiosmotic TheoryProposed by Peter Mitchell in1961 and further elaborated in1966

Mitchell proposed that electron transfer directly produced an electrochemical

gradient of protons across the coupling membrane that was subsequently used to

drive ATP synthesis.

The chemiosmotic hypothesis is named because it is postulated to involve both

a) chemical reactions, the transfer of chemical groups (electrons,protons and O22-

) within the membrane

b) osmotic reactions, the transport of a solute (protons) across the membrane

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3 molecules of ATP are synthesized when one molecule of

NADH is oxidised.

NADH = 3 ATPs (P:O ratio = 3)

FADH2 = 2 ATPs (P:O ratio = 2)

The P:O ratio is defined as the number of inorganic

phosphate molecules incorporated into ATP for every atom

of oxygen consumed.

Significance of P:O ratioSite specific Inhibitors of ETC/ Respiratory chain

Site 1 (Complex 1)

Alkylguanides (guanethide), hypotensive drug

Rotenone, insecticide and fish poison

Barbiturates (amobarbital), sedative

Site 2 (Complex 2)

BAL (British anti lewisite), antidote of war gas

Napthoquinone

Site 3 (Complex 3)

Carbon monoxide, CO, inhibits cellular respiration

Cyanide

Azide

Site between Succinate dehydrogenase and Co-Q

Carboxin, inhibits transfer of ions from FADH2

Malonate, competitive inhibitor of succinate dehydrogenase

Uncouplers Uncouplers uncouple electron transport from

oxidative phosphorylation.

They collapse the chemiosmotic gradient by dissipating protons across the inner mitochondrial membrane.

Uncouplers 2,4-Dinitrophenol, dicumarol and

carbonyl cyanide-p-trifluorocarbonyl-cyanide methoxyphenyl hydrazone (FCCP) all have hydrophobic character making them soluble in the bilipid membrane.

All of these decouplers also have dissociable protons

allowing them to carry protons from the intermembrane space to the matrix which collapses

the pH gradient.

The potential energy of the proton gradient is lost as heat.

UncouplersThermogenin

A protein found in mitochondria of Brown adipose tissue

It decreases the proton gradient generated in oxidative phosphorylation by increasing the permeability of the inner mitochondrial membrane, allowing protons that have been pumped into the intermembrane space to return to the mitochondrial matrix.

Energy is released as heat.

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NADH is generated during glycolysis and by other cytosolic dehydrogenases

NADH cannot traverse the mitochondrial membrane to be oxidised by respiratory

chain

Reducing equivalents are shuttled into the inner mitochondrial membrane to be

oxidised by the respiratory assemblies

Two shuttle mechanisms exist. Relative proportion of the activities of the two

shuttles varies from tissue to tissue

Malate-aspartate shuttle – particularly active in liver, kidney and heart

Glycerol phosphate shuttle – particularly active in brain, skeletal muscle

and flight muscles of insects

NADH Generation

Malate Aspartate Shuttle

Malate dehydrogenase and transaminase

3 ATPs

Glycerol-3-phosphate Shuttle

2 ATPs