BIOLOGIC OXIDATIONBIOLOGIC OXIDATION

BIOLOGIC OXIDATIONBIOLOGIC OXIDATION

ENERGI (ATP)

Source of ATP :1. Oxidative Phosphorylation.2. Glycolysis.

3. Krebs Cycle.

Ox-red reactions O2 accept single electron

Oxidation-reduction potential

• Oxidation : the removal of electrons• Reduction : the gain of electrons• Redox potential (E0

’) : the free energy change is proportionate to the tendency of reactants to donate or accept electrons

• Redox potential of a system (Eo) is compared with the potential of the hidrogen electrode

• Biologic systems E0’ expressed at PH 7

and electrode potential of H : – 0,42 volts

System E’O volts

H+/H 2NAD+/ NADHLipoate; ox / redAcetoacetate/ 3 – hydroxybutyratePyruvate/ lactateOxaloacetate/ malateFumarate/ succcinateCytochrome b; Fe3+/Fe2+

Ubiquinone; ox/redCytocrome c1; Fe3+/Fe2+

Cytocrome a; Fe3+/Fe2+

Oxygen/ water

-0.42-0.32-0.29-0.27-0.19-0.17+0.03+0.08+0.10+0.22+0.29+0.82

Enzymes in ox-red

• Called oxidoreductases (class I), classified into 4 groups:

- oxidases- dehydrogenases- hydroperoxidases- oxygenases

Oxidases

• Catalyzing the removal hydrogen and using oxygen as a acceptor form water or hydrogen peroxide

• Some oxidases contain copper and others are flavoproteins

• Cytochrome oxidase ( cyt.a.a3 ):heme protein contain Cuterminal component of respiratory chaincontain two molecules of heme as

prosthetic group and Cu

• Flavoprotein enzyms contain FMN or FAD as prosthetic groups

• FMN and FAD are formed in body from riboflavin

• They are tightly bound to their apoenzymes but not covalently

• Exampels: L-amino acid oxidase (in kidney), xanthine oxidase (in intestinal, kidney, liver), aldehyde oxidase (in liver) and glucose oxidase (in fungus)

Oxidases

AH2

A H2O

1/2 O2

OXIDASE

O2

H2O

2

AH2

A

OXIDASE

(Red)

(Ox)

Oxidation of a metabolite catalyzed by an oxidase (A) forming H2O, (B) forming H2O2

A B

Dehydrogenases

• Can not use oxygen as a hydrogen acceptor• Performing two main functions:

1. transfer hydrogen in a coupled oxidation reduction

reactionspecific for their substrates, but utilize common coenzymesuseful in enabling oxidative process to occur in the absence of oxygen2. components in respiratory chain

transfer electron from substrate to oxygen

Dehydrogenases link NAD

• Using NAD+ or NADP+ as a coenzyme• These coenzyme are formed in body from

niacin

- freely and reversibly dissociate from their apoenzymes

- NAD linked D-ase: oxidative pathways of metabolism (glycolysis, kreb’s cycle, respiratory chain)

- NADP linked D-ase: characteristically in reductive synthesis (fatty acid synthesis, steroid synthesis and PMP-shunt)

Dehydrogenases link riboflavin• Using FMN and FAD as a coenzyme

- more tightly bound to their apoenzymes

- most of them are concerned with electron transport in / to resp chain

- NADH D-ase carrier of electrons between NADH and components of higher redox potential

- succinate D-ase, acyl Co-A D-ase, glycerol 3 P D-ase transfer electrons directly from substrate to resp. chain

Cytochromes as dehydrogenase

• Classified as dehydrogenases, except for cytochrome oxidase

- as carriers of electrons from flavoproteins to cytochrome oxidase in the resp chain

- exampels: cyt b, c1, c, a, a3 (resp chain) and cyt P 450, b5 (endoplasmic reticulum)

AH2

A

Carrier(Red)

(Ox)

Oxidation of a metabolite catalyzed by coupled dehydrogenases

(Ox)

Carrier-H2

(Red)

B

BH2

(Red)

(Ox)

DEHYDROGENASESPECIFIC FOR A

DEHYDROGENASESPECIFIC FOR B

Hydroperoxidases

• Using hydrogen peroxide or an organic peroxide as substrate

• Two type: - peroxidase

- catalase• Protecting against harmful peroxides

• Peroxides generate free radicals disrupt membranes and cause cancer and atherosclerosis

• PeroxidasesReducing peroxides using various electron

acceptors (ascorbate, quinones, cyt c):

H2O2 + AH2 2H2O + A

Founding in milk, leukocytes, platelets, erythrocytes and other tissues involved in eicosanoid metabolism

Glutathione peroxidase, containing selenium destruction of H2O2 and lipid

hydroperoxidases protecting membrane lipids and Hb

• Catalase

Using hydrogen peroxide as electron donor and electron acceptor:

2 H2O2 2H2O + O2

In addition to possessing peroxidase activity, it is able to use one of H2O2 as a substrate (electron donor) and another of H2O2 as an oxidant (electron acceptor)

Founding in blood, bone marrow, mucous membranes, kidney and liver

Role of catalase in the destruction of hydrogen peroxie

O2 2H2

OOXIDASE

CATALASE

H2O

2

O2H2O

2

A’H2 A’

AH2 A

Oxygenases

• Catalyzing the direct transfer and incorporation of oxygen into a substrate

• Divided into two subgroups:

1. Dioxygenases / oxygen transferase

Incorporating both atoms of oxygen into substrate: A + O2 AO2

2. Monooxygenases

Mixed function oxidases and hydroxylases incorporate only 1 atom of oxygen into substrate, the other oxygen is reduced to water

MONOOXYGENASES

• Need an additional electron donor / cosubstrate ( Z ):A-H + O2 + ZH2 A-OH + H2O + Z

• Cytochromes P450 are monooxygenases (as cosubstrate ) important for detoxification of many drugs and for hydroxylation of steroids

• NADH and NADPH donate reducing equivalents for the reduction of cyt P450

Cytochrome P 450• Mitochondrial cyt P450 systems in

steroidogenic tissues biosynthesis of steroid hormones from cholesterol

• Mitochondrial cyt P450 systems in kidney metabolism of vitamin D

• Mitochondrial cyt P450 systems in liver biosynthesis of bile acid

Superoxide free radicals (O2-)

• Generated from transfer of a single electron to O2

• It is formed reduced flavin, are reoxidized univalently by molecular oxygen

• Superoxide dismutase in aerobic organisms removal O2

- , the reaction:

O2- + O2

- + 2H+ H2O2 + O2

• Superoxide can reduce oxidized cyt c:

O2- + cyt c (Fe3+) O2 + cyt c (Fe2+)

• Exposure to an atmosphere of 100% oxygen causes an adaptive increase in superoxide dismutase

OXIDATIVE OXIDATIVE PHOSPHORYLATIONPHOSPHORYLATION

OXIDATIVE OXIDATIVE PHOSPHORYLATIONPHOSPHORYLATION

Oxidative phosphorylation

• Oxidative reaction Coupled by phosphorylation to the generation of high energy intermediate (ATP or other high phosphagen)

• Oxidative phosphorylation at resp chain level via NAD D-ases form 3 mol ATP and via flavoprotein D-ases form 2 mol ATP

• Phosphorylations at the substrate level captured smaller energy eg:a) High energy phosphates are captured in kreb’s cycle during the conversion of succinyl Co-A to succinate. And b) in glycolytic reactions on cytoplasmic.

Respiratory chain• Enzyme complexes in mitochondria

collects and transports reducing equivalents directing them to final reaction with oxygen form water and ATP

• Reducing equivalents flow through from redox potential negative to positive

• There are 4 enzyme complexes:- NADH-Q dehydrogenase / I- Succinate-Q dehydrogenase / II- Cytochromes dehydrogenase / III- Cytochrome oxidase / IV

DEHYDROGENASE

AH2

(Red)

A(Ox)

Carrier1

(Ox)

Carrier-H2

1(Red)

Carrier2

(Red)

Carrier2

(Ox)

Carrier3

(Ox)

Carrier-H2

3(Red)

H2O

1/2 O2

OXIDASEDEHYDROGENASE

DEHYDROGENASE

Oxidation of a metabolite by dehydrogenases and finally by an oxidase in a respiratory chain

Transport of reducing equivalents through the respiratory chain

NAD+

FpH2

NADH Fp

AH2

A

2Fe3

+

2Fe2

+

H2O

1/2O

2

Substrate Cytochromes

Flavoprotein

H+ H+ 2H+ 2H+

Mitochondrial • Powerhouses of the cell most of energy

captured takes place inside it• Outer membrane permeable to most

metabolites, contain various enzym (acyl Co-A synthetase, glycerolphosphate acyltransferase )

• Inner membrane selectively permeable• Matrix contain phospholipid cardiolipin

together with enzymes of resp chain• Intermembrane space has similar

composition with cytoplasmic and contain adenylyl kinase and creatine

kinase

Phosphorylatingcomplexes

OUTER MEMBRANE

INNERMEMBRAN

E

MATRIX

Cristae

B

A

OUTERMEMBRANE

INNERMEMBRANE

MATRIXF1 subunitsF0 subunits

Submitochondrial particel

Formed from fragments of the inner membrance

Sonication

B

Respiratory chain

• Not all substrates are linked to resp chain through NAD-D-ase

• Co-Q (ubiquinone) mobile component, collects reducing equivalents from flavoprotein complexes and passes them on to cytochrome b (the lowest redox pot)

• Cytochrome oxidase has a very high affinity for oxygen resp chain to function at maximum rate until tissue depleted of O2 irreversible reaction

Glycerol 3-phosphate

Pyruvate

- Ketoglutarate

Cyt aa3Cu

O2

FeS : Iron-sulfur protein

ETF : Electron- transferring

flavoproteinFp : FlavoproteinQ : UbiquinoneCyt : Cytochrome

Proline3-Hydroxyacyl-

CoA3-Hydroxybutyrate

GlutamateMalate

Isocitrate

Acyl-CoASarcosine

Dimethylglycin

Fp(FAD)FeS

Lipoate Fp(FAD) NAD

Fp(FMN)FeS

Succinate

Choline

Fp(FAD)FeS

FeSETF

(FAD)

Fp(FAD)

Q Cyt bFeS

Cyt c1 Cyt c

Resp chain & oxd phos inhibitors• Inhibitors of resp chain1. Blocking electrons transfer from Fe-S to

co-Q , ie: barbiturates , pierisidin-A , rotenon , carboxine ,succinate D’ase competitive inhibitor: malonate

2. Blocking electrons transfer from cty b to cyt c, ie: dimercaprol , antimycin A

3. Inhibitors of cytochrome oxidase: H2S , CO and CN

Resp chain & oxd phos inhibitors

• Inhibitors of oxidative phosphorylation, ie:oligomycine, atractyloside

• Un-couplers (dissociate oxidation in resp chain from phosphorylation) respiration to become uncontrolled, ie: dinitrophenol, dinitrochressol, pentachlorophenol, chloro carbonyl cyanide phenilhydrazon (cccp)

Oligomycin

O2

SuccinateFADFeS

FMN, FeSNADH

BALAntimycin

A

Complex III

Cyt b, FeS, Cyt C1

Cyt c Cyt a Cyt a3

Cu CuQ

Uncouplers

ADP + P1

ADP + P1

ATPATP ADP + P1

Uncouplers

ATP

Piericidin A

Amobarbital

Rotenone

Complex I

Complex IV

H2SCOCN -

Oligomycin

Mechanism of oxidative phosphorylation

• Mitchell’s chemiosmotic theory:- energy from oxidation in resp chain translocation of H+ (protons) electrochemical potential difference in matrix and intermembrane space drive the mechanism of responsible for the formation of ATP (ATP synthase)

Mechanism of oxidative phosphorylation

• Complexes I, III and IV of resp chain is a proton pump

• Pi + ADP ATP, by ATP synthase

• ATP synthase is a complex enzyme consist of several protein subunits (F1), which attached to membrane protein complex (F0)

• F1 project into matrix and contain the phosphorylation mechanism

F0 spans the membrane and forms the proton channel

Exchange metabolites at inner mitochondrial membrane

- Exchange of anions against OH- ions and cations against H+ ions for transport of ionized metabolites

- Freely permeable to uncharged small molecules O2 , H2O , CO2 , NH3 monocarboxylic acids (3 hydroxy butyric, acetoacetic, acetic)

- Long chain fatty acids need carnitine system

- Symport pyruvate - H+

Exchange metabolites at inner mitochondrial

membrane

• Dicarboxylate and tricarboxylate anions require specific carrier linked to inorganic phosphate (H2PO4

- )

• Exchange ATP / ADP by adenine nucleotide transporter

• Transport of oxaloacetate need transamination process

Oxidation of extramitochondrial NADH

- NADH cannot penetrate mitochondrial membrane produced continuously in cytosol by 3 phosphoglyceraldehyde D-ase

- Aerobic conditions: not accumulated be oxidized by resp chain

- Transfer of reducing equivalents from cytosol to mitochondrial require substrate pairs, linked by suitable D-ase

Oxidation of extramitochondrial NADH- The mechanism:

1. Glycerophosphate shuttle only 2 mol ATP are formed per atom oxygen consumed present in brain, muscle, adipose, liver but deficient in heart muscle2. Malate shuttle more universal utility more complex, due to the impermeability of mitochondrial membrane to oxaloacetate

INNERMEMBRANE

FAD

FDH2

Respiratory Chain

GLYCEROL-3-PHOSPHATE

DEHYDROGENASE(MITHOCONDRIAL)

GLYCEROL-3-PHOSPHATE

DEHYDROGENASE(CYTOSOLIC)

NAD+

NADH + H+

Dehydroxyacetone

phosphate

Dehydroxyacetone

phosphate

Glycerol 3-phosphate

Glycerol 3-phosphate

CYTOSOLMITOCHONDRION

OUTER MEMBRAN

E

Glycerophosphate shuttle for transfer of reducing equivalents from the cytosol into the mitochondrion

MALATE DEHYDROGENASE

MALATE DEHYDROGENASE

TRANSAMINASE

TRANSAMINASE

INNER MAMBRA

NE MITHOCONDRION

Malate

Oxaloacetate

Glutamate

-KG

Asp

NAD+

NADH+H+

H+H+

Glutamate

-KG

Asp

Malate

Oxaloacetate

NAD+

NADH

+H+

CYTOSOL

1

2

Malate shuttle for transfer of reducing equivalents from the cytosol into the mitocondrion. 1. Ketoglutarate transporter, 2. glutamate-aspartate transporter (note the proton symport with glutamate)

Creatine phosphate shuttle• Facilitating transport of high energy

phosphat from mitochondria in active tissues

• Isoenzyme of creatine kinase (CKM), in intermembrane space catalyzing transfer ~ P (ATP) to creatine:~ P(ATP) + creatine creatine-P , transported into cytosol via protein pores available for generation of

extramitochondrial ATP

CREATINEKINASE

NH

H2N

C

NH3C

COO-

NH

H

N

C

N

COO-

P

Creatinephosphate Creatine

ΔGO’ = 12.6 kJ/mol

Clinical aspects

• Fatal infantile mitochondrial myopathy and renal dysfunction due to severe diminution / absence of most oxidoreductase

• MELAS (mitochondrial encephalopathy, lactic acidosis and stroke) due to complex I or complex IV deficiency mutation in mt DNA