Biological Oxidation

Dr.S.Chakravarty, MD

Home assignment Due date :-21st Sep 2015

Instructions : Handwritten submissions only(i.e pen and paper)

Draw diagrams wherever required You can contact me if you face any difficulties.

1.Describe the factors that influence enzyme activity .Write a short note on Vmax and Km

(8+2 = 10 marks )

2. Describe competitive and non- competitive inhibition with examples. (10 marks )

Oxidation is defined as the removal of electrons and reduction as the gain of electrons.

Oxidation is always accompanied by reduction of an electron acceptor.

Oxidation : Reduction :

Oxidation and reduction:

• Loss of electrons

• Loss of hydrogen

• Gain of oxygen

• Gain of electrons

• Gain of hydrogen

• Loss of oxygen

REDOX POTENTIAL

• When a substance exists both in the reduced state and the oxidized state , the pair is called a REDOX COUPLE.

• The redox potential of this couple is estimated by measuring the EMF of a sample half cell connected to a standard half-cell.

1M H + ~ H gas @ 1 Atmospheric pressure

E0’ =0 meV

Salt bridge

• WHEN A SUBSTANCE HAS LOWER AFFINITY FOR ELECTRONS THAN HYDROGEN IT HAS A NEGATIVE REDOX POTENTIAL

• ELECTRONS MOVE ALWAYS FROM MORE ELECTRONEGATIVE TO ELECTROPOSITIVE

LOSS OF FREE ENERGY

(THIS ALWAYS ENSURES THAT FREE ENERGY DECREASES)

Redox Potentials

Oxidant Reductant E0’ ( in V)

NAD + NADH + H + -0.32

Cytochrome b+++ Cytochrome b ++ +0.07

Co-eneyme Q Co-eneyme QH2 +.010

Cytochrome c +++ Cytochrome c ++ +0.22

Cytochrome a +++ Cytochrome a +++ +0.29

½ O2 +2H H2O +0.82

• ½ O2 + 2H+ H2O (E0’ = +0.82 )

• NAD+ + H + NADH (E0’ = -0.32)• E0’ = +0.82 – (-0.32 ) = 1.14

• COMBINE BOTH OF THESE :-

½ O2 + NADH + H+ H2O + NAD+

Δ G0 = -nFΔE0

= - 2 x 23.06 x 1.14 = -52.6 kcal/mol

02 H2O

FOOD(REDUCED )

SMALLER MOLECULES

REDUCED COENZYMES

e.g NAD

ENERG

Y

BIOLOGIOCAL OXIDATION

e-

ELECTRON TRANSPORT CHAIN

MITOCHONDRION

Harper’s Illustrated Biochemistry

With the help of successive reductions in the electron transport chain assembly , this energy change is released in small increments so that the body can utilize it .

HIGH ENERGY COMPOUNDS

• THESE COMPOUNDS WHEN HYDROLYSED RELEASE A LARGE AMOUNT OF ENERGY

• INDICATED BY SQUIGGLE (~)

• FREE ENERGY VARIES FROM -7 TO -15 kcal/mol

Substrate level Phosphorylation• Defn :- When the energy of high energy compound is directly transferred

to nucleoside diphosphate to form a triphosphate without the help from electron transport chain.

• Examples :-

1.Bisphosphoglycerate kinase ( Glycolysis)(1,3 bisphosphoglycerate 3-phosphoglycerate)

2. Pyruvate kinase (Glycolysis)(Phosphoenol pyruvate Pyruvate)

3. Succinate thiokinase (TCA cycle) (Succinyl CoA Succinate ) usmle

Impermeable to ions and most other compounds

MITOCHONDRIA The mitochondrion contained the enzymes responsible for electron transport and oxidative phosphorylation

USMLE concept!

The MITOCHONDRION – powerhouse of the cell

Harper’s Illustrated Biochemistry

IMPORTANT MITOCHONDRIAL TRANSPORTERS

Pyruvate PyruvateHydrogen Hydrogen

Malate MalateCitrate Citrate

ADP ADPATP ATP

INNER MITOCHONDRIAL MEMBRANE

The mitochondrial membrane is impermeable to NADH….. So, how does cytosolic NADH reach there ??

Malate –Aspartate shuttle

Operates in Liver , Kidney and Heart

Glycerol -3 –phosphate shuttle

Operates mainly in muscle and Brain.

• The flow of electrons occurs through successive dehydrogenase enzymes in mitochondria , together known as the electron transport chain (ETC).

(the electrons are transferred from lower to higher redox potential)

Electron transport chain

Electron Carriers- The transfer of electrons is not directly to oxygen but through coenzymes

NAD+

FMN

FeS

ubiquinoneFAD FeS

Cyt b

FeS Cyt c1 Cyt c Cyt a Cyt a3

1/2 O2

ubiquinone

Protein complexes:

• NADH-CoQ Dehydrogenase (Complex I)

• Succinate-CoQ Dehydrogenase (Complex II)

• CoQ-cytochrome c Reductase (Complex III)

• Cytochrome c Oxidase (Complex IV)

• Mobile complexes:1. Co-enzyme Q or ubiquinone2. Cyt C

COMPLEX IFMN-FeS

Complex IIIFeS-Cytb-Cyt c1Co Q

Complex IV

Cyt a-a3(Fe+3,Cu+2)

Cyt C

COMPLEX IIFeS

8.Succinate9. Fatty Acyl CoA 10. Glycerol 3-PFAD

Mitochondrial Matrix

NAD

1.Glyceraldehyde -3P2.Isocitrate3.Malate4.Glutamate5.β-OH-acyl CoA

2H +

½ O2

H2O

+

SITE 1 4 PROTONS PUMPED OUT

SITE 24 PROTONS PUMPED OUT

SITE 32 PROTONS PUMPED OUT

Fp(FAD) lipoate 6.Pyruvate 7.α-ketoglurarate

Inner mitochondrial membrane

USMLE !

1

2

3 4

Harper’s Illustrated Biochemistry

COMPLEX I-NADH CoQ REDUCTASE

• Also known: NADH dehydrogenase complex.

• Tightly bound to the inner membrane.

• Contains a flavoprotein (Fp)- FMN (Favin mono nucleoide): prosthetic group and an iron-sulphur protein (Fe-S).

Complex 1 NADH FMN Reduced FeS Q

NAD FMNH2 Oxidized FeS QH2

• Large negative free energy change; energy released is 12 kcal/mol. This is utilized to drive 4 protons out of the mitochondria.

• 1 ATP is produced.

COMPLEX II:SUCCINATE-CoQ-REDUCTASE

• The electrons from FADH2 enter ETC at the level of CoQ.

• This step does not liberate enough energy to produce ATP.

• The 3 major enzyme systems that transfer their electrons directly to ubiquinone are:

USMLE !! a. Succinate dehydrogenase( TCA cycle )b. Fatty acyl CoA dehydrogenase( Fatty acid oxidation)c. Mitochondrial glycerol phosphate dehydrogenase.

Succinate FAD Reduced FeS Q

Fumarate FADH2 Oxidized FeS QH2

USMLE !

Iron-sulfur centers (Fe-S) are prosthetic groups containing 1-4 iron atoms

Iron-sulfur centers transfer only one electron, even if they contain two or more iron atoms.

E.g., An Fe center might cycle between redox states:Fe+++ (oxidized) + 1 e- Fe++

(reduced)

Iron-sulfur Centers (clusters)

COENZYME Q• The ubiquinone is reduced

successively to semiquinone (QH) and finally to quinol (QH2)

It accepts a pair of electrons from NADH or FADH2 through complex I or complex II respectively.

Co-enzyme Q is a quinone derivative having long isoprenoid tail.

2 molecules of cytochrome c are reduced.

The Q cycle thus facilitates the switching from the 2 electron carrier ubiquinol to the single electron carrier cytochrome c.

This is a mobile carrier.

COMPLEX III: CYTOCHROME REDUCTASE• Cluster of iron-sulphur proteins, cytochrome b and cytochrome c1• Prosthetic group in both- heme• Iron in heme shuttles between Fe3+ and Fe2+ forms.• Free energy change: -10kcal/mol• 4 protons pumped out.

QH2 FeS Cyt b Cyt c1 Cyt C(Red) (Fe+3) (Fe+2) (Fe+3) (Fe+2)

Q FeS Cyt b Cyt c1 Cyt C(oxi) (Fe+2) (Fe+3) (Fe+2) (Fe+3)

Cytochrome C

• Contains one heme prosthetic group .

• It collects electrons from Complex III and delivers to Complex IV.

• It is also the mediator of apoptosis (programmed cell death)

COMPLEX IV: CYTOCHROME OXIDASE• Contains cytochrome a and

cytochrome a3

• Tightly bound to inner mitochondrial membrane.

• 2 protons are pumped out.

• Cytochrome oxidase- 2 heme groups and 2 copper ions.

• Functional unit of enzyme: cytochrome a-a3.

Cyt C Cyt a-a3 2H2O

(Fe +2) (Fe+3, Cu +2)

Cyt C Cyt a-a3 O2

(Fe +3) (Fe+2, Cu +3)FINAL ELECTRON

ACCEPTOR

CHEMIOSMOTIC THEORY(Peter Mitchell. N.P 1978)

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

• Protons accumulate outside the membrane creating an electrochemical potential.

• This drives the synthesis of ATP by ATP synthase .

Harper’s Illustrated Biochemistry

Harper’s Illustrated Biochemistry

• The pH outside is 1.4 units lower than inside . • The outside is positive 0.14V relative to inside.• The proton motive force (PMF ) IS 0.224 v

corresponding to a free energy change of 5.2 kcal/mol of protons.

• ENERGETICS OF ATP SYNTHESIS

– ENERGY RELEASED = 52kcal/mol– Synthesis of 1ATP and Pi requires 7.3 ATP

molecules– Chemical energy trapped = 7.3 x 3 = 21.9kcal =

40%– Rest 60% energy is dissipated as HEAT !!

F0 – F1 complex :Fo complex: –INHIBITED BY OLIGOMYCIN Made of 12 subunits . H+ passes through each subunit

from membrane space to inner space rotating the Fo complex (turbines).

F1 complex: Has 9 polypeptide chains ,(3 alpha , 3 beta , 1 gamma , 1 sigma , 1 epsilon)

the α chains have binding sites for ATP and ADP and beta chains have catalytic activity.

ATP SYNTHESIS NEEDS Mg +2 IONS• ADP and Pi bind the alpha subunit• Binding change mechanism - conformation change of beta

subunits causes release of ATPs from the complex.

• ATPs formed in the mitochondrial matrix are translocated to cytosol by ATP/ADP translocase

1 ) ADENINE NUCLEOTIDE TRANSPORTER2)H+/Pi SYMPORT

Harper’s Illustrated Biochemistry

EARLIER ESTIMATES WERE WRONG!!

• According to recent estimates , NADH may generate only 2.5 ATPs while FADH2 may generate only 1.5 ATP.

• So , instead of 38 ATP , only 32 ATPs are generated from glucose .

ATP IS THE ENERGY CURRENCY

INHIBITORS OF ATP SYNTHESIS

• 1. SITE SPECIFIC INHIBITORS:-

– Site : COMPLEX 1 to Co Q• Alkylguanide(guanethide), hypotensive drug • Rotenone ( insecticide and rat poison)• Chlorpromazine (tranquilizer)• Barbiturates (sedative) • Pericidin (antibiotic)

USMLE !

• Site :COMPLEX III• BAL –BRITISH ANTI LEWISITE – WAR GAS• Napthoquinone

• Site : Complex IV inhibitors– Carbon monoxide – Cyanide– Azide – Hydrogen Sulphide

• Site : between succinate dehydrogenase and CoQ– Carboxin– Malonate : competetive inhibitor of Succinate DH

USMLE !

USMLE ! Lewisite is an organoarsenic compound. It was once manufactured in the U.S., Japan, and Germany for use as a chemical weapon, acting as a vesicant (blister agent) and lung irritant.Later causes liver necrosis and death.

(Dimercaprol)

(Thenoyltrifluoroacetone)

(Insecticide)

• Site specific inhibitors of Oxidative Phosphorylation– Actractlyoside inhibits translocase whereas

oligomycin acts through one of the proteins present in Fo-Fi stalk.

– Ionophores are lipid soluble compunds that increase the permeability of lipid bilayers to certain ions.• TYPES

– MOBILE ION CARRIERS eg.Valinomycin( allows K+ to permeate mitochondria and dissipate proton gradient)

– CHANNEL FORMERS eg.Gramicidin

UNCOUPLERS

• ALLOW OXIDATION TO PROCEED BUT THE ENERGY IS DISSIPATED AS HEAT .– EG. • 2,4 dinitro phenol, • 2,4 dinitro cresol • pentachlorophenol• CCCP (Chloro carbonyl cyanide phenyl hydrazone)• THERMOGENIN in brown adipose tissue• Thyroxine

TOXICITY : PRESENTS WITH INCREASED BODY TEMPERATURE

Mitochondrial inherited diseases :

• Heteroplasmy – proportion of somatic cells which are affected to normal cells.

• Threshold – the amount of affected DNA should reach threshold before it can manifest.

• Affects mainly Neurons and muscle – which require high ATPs for metabolism.

MELAS ( complex 1 or complex 1 and 4 defect)

• Mitochondrial encephalopathy• Lactic acidosis• Stroke like episodes.

• Cause : mutation in the mitochondrial DNA• Abnormality in the t-RNA ( A G mutation)• Deficiency of complex proteins impaired oxidative

phosphorylation.• OXPHOS – transient oxidative phosphorylation in brain

stroke like symptoms.

Leber’s Hereditary Optic Neuropathy – LHON complex -3 defect

• Degeneration of Retinal ganglion layer.

• Specially maculo-papular bundle.

• Central loss of vision in mid 20’s.

• Increased reactive oxygen species apoptosis of cells.

Kearns-Sayre syndrome

• Oculocraniosomatic neuromuscular disease with ragged red fibers

• Triad of :1. Chronic progressive external ophthalmoplegia

2. Bilateral pigmentary retinopathy – salt and pepper appearance of retina

3. Cardiac conduction abnormalities

• Other defects include short stature and ataxia.

Leigh’s disease

• Subacute Necrotizing Encephalomyelopathy (SNEM)

• lactic acidemia, Developmental delay, seizure, extraocular palsies, and hypotonia

• Fatal by age of 2 years.

• Complex -4 defect

• A comatose laboratory technician is rushed into the emergency room. She dies while you are examining her. Her most dramatic symptom is that her body is literally hot to your touch, indicating an extremely high fever. You learn that her lab has been working on metabolic inhibitors and that there is a high likelihood that she accidentally ingested one. Which one of the following is the most likely culprit?

• Barbiturates• Piericidin A• Dimercaprol • Dinitrophenol • Cyanide

• A 53-year-old, previously successful man recently lost his job and is under investigation for racketeering. His wife returns home to find him slumped over the steering wheel of his idling car in the closed garage. He is unresponsive and has a cherry color to his lips and cheeks. Which of the following is inhibited ?

A. Complex I of the ETCB. Cytochrome oxidaseC. The ATP-ADP antiporterD. The F0 component of the F0-F1 ATPase

E. The F1 component of the F0-F1 ATPase

Thank you