Biological oxidation Biological oxidation and Electron and Electron transport chaintransport chain

Dr. SaidunnisaProfessor and chairpersonBiochemistry (3 lectures)

Matrix H+

+ NADH NAD+ + 2H+ 2H+

+ ½ O2 H2O 2 e

I Q III IV

Intermembrane Space cyt c

Learning objectivesLearning objectives At the end of the session the student

shall be able to define, describe, enumerate, analyze and list:

1. Location of ETC2. Components of ETC3. Sites of ATP production4. Oxidative phosphorylation5. Chemiosmotic theory 6. Energetics of ETC(Chapter 19 pages: 223-235 in (Text

Book of Biochemistry DM Vasudevan)

I. Case:I. Case:A 35-year old male is rescued from

enclosed fire. Upon arrival in the emergency

department, he is disoriented and in moderate distress. He is coughing up soot, and has difficulty breathing.

Initial vital signs: BP 90/60, HR 120, RR 30, O2 sat 95%On examination: burned nasal hair, soot

around mouth, burns to face, arms and back.

What are the immediate concerns?

1Case conti…1Case conti…We have a tendency to focus on CO

as the diagnosis in victims of smoke inhalation.

Pitfall – CN exposure is frequently

overlooked. Consider that CN can be produced from the combustion of paper, silk, wool, plastic, and cotton.

The probability of CN exposure in fires is therefore high.

1. Case conti……1. Case conti……Lets us unfold the mystery of CO

and CN poisoning on human health while studying respiratory chain.

Stages of oxidationStages of oxidation of food stuffs of food stuffs

First stage: Digestion in the GIT converts the macro molecules into small units. (carbohydrates into glucose, lipids into fatty acids, proteins into amino acids). This is called primary metabolism.

Second stage: These products are absorbed in mitochondrial citric acid cycle to generate reducing equivalents NADH or FADH2.This is called secondary or intermediary metabolism.

Third stage: These reducing equivalents enter into the electron transport chain or respiratory chain where energy is released (ATP). This is tertiary metabolism or internal or cellular respiration.The energy (ATP) is then used for body synthetic purpose.

ATP-Universal currency of ATP-Universal currency of energy in living cellsenergy in living cells

The energy released from the hydrolysis of ATP is utilized for

Mechanical -muscle contractionTransport work -Sodium potassium

ATPase pumpBiochemical work -Initial steps of

GlycolysisAnabolic pathways -TAG, DNA, Protein

synthesisDetoxification(urea cycle),formation of

active intermediates like UDP glucose

HEAT

Chemistry of ATP hydrolysisChemistry of ATP hydrolysis

Two high energy bonds in ATP which are represented by a squiggle bond ( ˜ ) on hydrolysis each releasing -7.3kcal/ mole.

More than 90% of ATP is formed through ETC (Oxidative phosphorylation)

Remaining in creatine phosphate shuttle, and Substrate level phosphorylation

High energy compoundsHigh energy compounds

High energy compounds:

ATP, GTP, UTP,PEPCarbamoyl phosphatecAMP 1-3

BisphosphoglycerateCreatine PhosphateAcetyl Co ASAM

Low energy compounds

AMPGlucose-1

phosphateFructose-1-

phosphate

Any bond that can be hydrolyzed with the release of same energy as ATP hydrolysis is called as “high energy bonds”.

Redox potentialsRedox potentialsOxidation: loss of

electronsReduction: gain of

electronsOxidation is always

accompanied by reduction.

Redox couple: when a substance exists both in the reduced and oxidized state.

Redox potentials: electron transfer potential E0’.

Expressing Redox reactions as Expressing Redox reactions as half reactionshalf reactions

E.g. Fe 2+ + Cu 2+ = Fe 3+ + Cu +

which can be expressed in the form of 2 half reactions

Fe 2+ = Fe 3+ + e- / Fe 2+

Cu / Cu 2+ + e-

They together make a conjugate redox pair.

Substrate level Substrate level phosphorylationphosphorylationEnergy from a high energy

compound is directly transferred to ADP or GDP to form ATP or GTP without the help of electron transport chain.

Examples:1.Bisphosphoglycerate kinase

(glycolysis)2.Pyruvate kinase (glycolysis)3.Succinate thiokinase (TCA cycle)

Biological oxidationBiological oxidation

Definition: Transfer of electrons from reduced coenzymes through ETC to oxygen.

Energy released during this process is trapped as ATP.

This coupling of oxidation with phosphorylation is called oxidative phosphorylation.

In the body this is carried out by dehydrogenations.

Oxidoreductases Oxidoreductases

1. Oxidases2. Aerobic dehydrogenases3. Anaerobic dehydrogenases4. Hydro peroxidases5. Oxygenases

OxidasesOxidases

Removal of hydrogen from substrates but only oxygen can act as acceptor of hydrogen so that water is formed.

Example: cytochrome oxidase (terminal component of ETC), MAO, tyrosinase, etc.

Aerobic dehydrogenasesAerobic dehydrogenases

Removal of hydrogen from substrates but only oxygen can act as acceptor of hydrogen product H2O2 (hydrogen peroxide) is formed.

Example: Flavoproteins (FMN FAD), Xanthine oxidase

Anaerobic dehydrogenasesAnaerobic dehydrogenases

NAD+ dehydrogenasesNADP+ dehydrogenasesFAD+ dehydrogenasesCytochromes

NADNAD+ + Linked Linked dehydrogenases dehydrogenases

NAD+ is derived from nicotinic acid a Vitamin B-complex.

The electron is also accepted by the NAD+ so as to neutralize the charge on the co-enzyme.

H2 H + H+ + e-

AH2 + NAD+ A + NADH + H+

When NAD+ accepts the two hydrogen atoms, one of the hydrogen atom is removed from the substrate as such the other is split into one hydrogen ion and one electron.

NADNAD+ + Linked Linked dehydrogenases dehydrogenases Examples: 1.Glyceraldehyde -3-phosphate

dehydrogenase.2.Isocitrate dehydrogenase3.Glutamate dehydrogenase4.Pyruvate dehydrogenase5.Alpha ketoglutarate

dehydrogenaseNADP+ Linked dehydrogenases takes part in reductive biosynthesis.

FADFAD Linked Linked dehydrogenases dehydrogenases

FAD is derived from riboflavin a Vitamin B-complex.

Examples:1.Succinate

dehydrogenase2.Fatty acyl CoA

dehydrogenase.

AH2 + FAD

FADH2

Both the hydrogen atoms are attached to the flavin ring.

HydroperoxidasesHydroperoxidasesIncludes 2 sets of

enzymes : catalase and peroxidases

Peroxisomes are rich in oxidases and catalases

H2O2 + AH2 2H2O + A

2 H2O2

2H2O Catalase uses H2O2 as electron acceptor & electron donor

Peroxidases reduce H2O2 which is a free radical

Oxygenases Oxygenases Consists of two

sets of enzymes Dioxygenases :

Incorporate both atoms of oxygen into the substrate:

A + O2 AO2

e.g. Homogentisic acid oxidase

Monooxygenases : Incorporates one atom of oxygen into the substrate & the other is reduced to water

A – H + O2 + ZH2

A – OH + H2O + Z e.g. phenylalanine

hydroxylase

Energetics of oxidative Energetics of oxidative phosphorylationphosphorylationFree energy change between

NAD+ and water is equal to 53kcal/mol.

This is so great that if this much energy is released at one stretch body cannot utilize it hence the ETC assembly the total energy released in small increments so that energy can be trapped as chemical bond energy ATP.

Mitochondrial Mitochondrial OrganizationOrganization

Electron Transport and Electron Transport and Oxidative PhosphorylationOxidative Phosphorylation

Note: Electrons ultimately combine with oxygen and protons to form water.

Components of ETCComponents of ETC5 Complexes1. Enzyme complex I, (NADH

dehydrogenase)2. Enzyme complex II (Succinate

dehydrogenase)3. Enzyme complex (III)

Cytochrome reductase 4. Enzyme complex (IV)

Cytochrome oxidase. 5. ATP Synthase (V)

Components of ETCComponents of ETC

Two mobile carriers These are connected by two mobile

carriers Coenzyme Q, Cytochrome C.

Coenzyme Q, connects complex-1 and 11.

Cytochrome C connects complex 111 and 1V

Electrons flow from more electronegative to electropositive components.

Complex-I Complex-I NADH NADH Dehydrogenase Dehydrogenase

NAD+ is reduced to NADH+H+ by dehydrogenases with the removal of two hydrogen atoms from the substrate (AH2).

AH2 + NAD+ A + NADH+H+

It has binding sites for NADH, FMN and Fe-S proteins and for Co Q.

FMN accepts two electrons and protons from NADH and pass electrons to Fe-S and pass to CoQ.

Complex-II-Complex-II-Succinate Succinate dehydrogenase dehydrogenase

The coenzymes FAD is derived from vitamin riboflavin.

FAD accepts (2H+ and 2electrons) from Succinate fumarate, and beta oxidation to form FADH2.

They pass electrons to CoQ.

Iron sulfur proteinsIron sulfur proteinsSeveral iron atoms

paired with sulfur atoms to make iron sulfur centers. They exist in the oxidized (Fe+3) or reduced (Fe+2) on accepting an electron.

Following FeS types are normally present:

FeS: Single Fe coordinated to the side chain SH groups of four cysteine residues

Fe2S2, Fe4S4.One FeS participates in the transfer of electrons from FMN to coenzyme Q.Other FeS proteins transfer electrons from Cyt.c, b1 to cyt,c.

Coenzyme QCoenzyme Q

Is a Quinone derivative with a long isoprenoid tail. It is also called ubiquinone because it is ubiquitous in biologic systems. It is lipophilic electron carrier. It can accept hydrogen atoms both from FMNH2 and FADH2

CytochromesCytochromes

Cytochrome C is a mobile component of ETC. Are conjugated proteins containing heme group having porphyrin ring and iron atom.Iron in cytochromes is alternatively oxidized (Fe+3) and reduced (Fe+2) in contrast to iron of hemoglobin and myoglobin which remains in (Fe+2) state.The electrons are transported from coenzyme Q to cytochromes in the order b,c1,c, a and a3 during electron transport.

Complex-III (Cytochrome Complex-III (Cytochrome b-c1)b-c1)

Complex- IVComplex- IVCytochrome oxidase Cytochrome oxidase

Cytochrome a and a3 electron carrier that can react with molecular oxygen, protons and electrons to form water.

This also contain copper that undergoes oxidation-reduction (Cu+2 Cu+)

Components of ETCComponents of ETC

Complex-V ATP synthaseComplex-V ATP synthaseEnzyme that generates

ATP sometimes referred as complex-V is a multi subunit having 9 poly peptide chains ( 3alpha, 3 beta,1 gamma,1sigma ,1 epsilon). The alpha chains have binding sites for ATP and ADP.

It has two functional subunits Fo (oha) portion embedded in the IMM where as F1 portion

protrudes into the mitochondrial matrix.

ATP synthase conti…..ATP synthase conti…..The Fo contain a central pore (proton

channel) this is because the IMM is impermeable to protons and so the extruded protons in the intermembrane space can reenter the mitochondrial matrix through this proton channel.

A proton pair attacks one Oxygen of Pi to form H2Oand an active form of pi which immediately combines with ADP to form ATP.

Chemiosmotic theoryChemiosmotic theoryPeter Mitchell proposed this theory

to explain the oxidative phosphorylation.

The transport of electrons from inside to outside of IMM is accompanied by the generation of a proton gradient across the membrane.

Protons (H+) accumulate outside the membrane, creating an electrochemical potential difference.

Chemi-osmotic theory Chemi-osmotic theory conti…conti…The proton pumps (complexes -I,

III, IV) expels H+ from inside to outside of the membrane.

So there is high H+ concentration outside.

This causes H+ to enter into mitochondria through the channels (Fo –F1complex ) , this proton influx binds to oxygen of pi+ADP to form ATP

Chemi-osmotic theory Chemi-osmotic theory conti….conti….

Current concept of ATP Current concept of ATP synthesissynthesis

Energy of electron transfer is used to drive protons out of the matrix by the complexes 1, 111 and IV which are proton pumps.

Proton gradient is created across the IMM till the electrons are transferred to oxygen to from water.

This electrochemical potential of this gradient is used to synthesize ATP.

Around 3 protons are required per ATP synthesized.

When 1 NADH transfers its electrons to oxygen, 10 protons are pumped out this accounts for approximately 3 ATP synthesis.

Oxidation of 1 FADH2 is accompanied by the pumping of 6 protons accounting for 2 molecules of ATP.

Peter Hinkle proved that actual energy production is less because there is always leakage of protons.

According to recent findings:1 NADH generates- 2.5ATP1 FADH2 generates- 1.5 ATP

Recent concept of: Recent concept of: Sites of ATP synthesisSites of ATP synthesis

Now ATP synthesis occurs when proton gradient is dissipated and not when protons are pumped out.

Traditionally Between complex-I and coenzyme-Q –First site.

Between complex -III and cytochrome c

Second site. At complex-IV – third site.

Oxidative PhosphorylationOxidative Phosphorylation

The process of synthesizing ATP from ADP and Pi coupled with the electron transport chain is known as oxidative phosphorylation.

The complex V of the IMM is the site of O.P

Inhibitors of oxidative Inhibitors of oxidative phosphorylationphosphorylation1. Oligomycin: an antibiotic, used

as anti-fungal drug, prevents the cell from using the established H+-gradient, to make ATP.

2. Atractyloside: Plant toxin

Inhibitors of ETCInhibitors of ETCThe inhibitors bind to one of the

components of ETC and block the transport of electrons.

This causes the accumulation of reduced components before the blockade step and oxidized components after that step.

The synthesis of ATP is dependent on ETC.

Hence, all the site specific inhibitors of ETC also inhibit ATP formation.

Inhibitors of ETCInhibitors of ETC• ETC inhibitors prevent: 1. The reduction of oxygen to

water, 2. The build-up of the H+-gradient

and 3. Finally the synthesis of ATP

Site- of inhibitorsSite- of inhibitorsSite –I Between complex-I and Co-Site –I Between complex-I and Co-Q: Q:

1. Rotenone : a potent, plant-derived and widely used pesticide.

2. Amytal : a barbiturate sedative drug

3. Piercidin A : antibiotic

Site- of inhibitors Site- of inhibitors

Site-IISite-II cytochrome b and c1:cytochrome b and c1:

1. Antimycin A : antibiotic

2. BAL (British antilewisite): an antidote used against war-gas

Site- of inhibitors Site- of inhibitors Site-III At complex-IV ( cytochrome Site-III At complex-IV ( cytochrome oxidase):oxidase):

1. Carbon monoxide: - odor-less, toxic gas frequently released during in-complete combustion reactions, e.g. in car engines or during coal gasification

2. Cyanide: most potent inhibitor of ETC, an extremely toxic compound; low doses are lethal to humans

3. Used by the Nazis during WWII in form of “Zyklon B” in the 1940s to commit mass murder of the Jewish population imprisoned in the concentration camps.

4. In 1984, in one of the worst industrial accidents in human history, the toxic cyanide derivative methyl isocyanate killed 3,000 people and injured more than 100,000 humans after a catastrophic gas leak in a chemical factory in Bhopal, India.

3. Hydrogen Sulfide:

UncouplersUncouplers These increase the permeability of IMM to protons (H+).

Thus an Uncoupler allows ETC but blocks the establishment of proton gradient across the IMM.

The result is that ATP synthesis does not occur.

The energy linked with the transport of electrons is dissipated as heat.

Compounds that can uncouple or delink the electron transport chain from oxidative phosphorylation, such compounds are known as Uncouplers.

Chemical UncouplersChemical Uncouplers

Chemical UncouplersChemical Uncouplers1.2,4-dinitrophenol ( has been extensively studied).

2.Dinitrocresol.

3.Pentachlorophenol

4.Tri fluoro carbonyl cyanide phenyl hydra zone (FCCP).

5.Aspirin (high doses)

Physiological Physiological UncouplersUncouplers

1. Thyroid hormones.2. Long chain fatty

acids.3. Unconjugated

Bilirubin. These act as

Uncouplers only at high concentration.

Significance of UncouplingSignificance of Uncoupling Brown adipose tissue

present in the upper back and neck portions and around kidney is rich in mitochondria and carry oxidation uncoupled from phosphorylation.

This causes liberation of heat when fat is oxidized in this tissue.

Significance of UncouplingSignificance of Uncoupling Examples :

1. New born infant, (Non-shivering Thermogenesis)

2. Hibernating animals

Brown adipose tissue is located in the neck area and is more physiologically active in the woman than in the man.

1. In certain individuals due to presence of this brown adipose tissue it is believed to protect them from becoming obese.

2. The excess calories consumed by this people are burnt and liberated as heat , instead of being stored as fat.

However, research, that was published in the New England Journal of Medicine, is showing that brown adipose tissue helps adults burn more calories than white adipose tissue.

Disorders of Oxid.Phos.Disorders of Oxid.Phos.DNA is present in mitochondria

(mtDNA) and nucleus.mtDNA is maternally inherited

since mitochondria from the sperm do not enter the ovum.

mtDNA is about 10times more susceptible to mutations than nuclear DNA.

It is present in tissues like CNS, Skeletal , heart muscle and liver.

Diseases associated with Diseases associated with oxida . phosoxida . phosLHON : Lebers hereditary optic

neuropathyMELAS: Mitochondrial

encephalopathy Lactic Acidosis Stroke

Case-1Case-1Mr. X 26yrs old male noted heat

intolerance, with profuse sweating . He is loosing weight in spite of good appetite . On physical Examination thyroid swelling was present and T3 and T4 are increased. Give biochemical explanation for above symptoms.

An excess thyroid hormones affect the efficiency of ATP production resulting in fewer ATP production.

What about NADH made in cytosolCan’t get into matrix of mitochondrion?

• By 2 Shuttle pathways:

– In muscle and brain• Glycerol phosphate shuttle

– In liver and heart• Malate / aspartate shuttle

Glycerol Phosphate Glycerol Phosphate shuttleshuttle

◦In muscle and brain

Malate – Aspartate ShuttleMalate – Aspartate Shuttle

In liver and heart

ApoptosisApoptosis

Cytochrome C is a mediator of apoptosis in response to oxidant stress due to ROS or free radicals.