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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