BIOLOGIC 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

EO volts

H+/H2 NAD+/ NADH Lipoate; ox / red Acetoacetate/ 3 hydroxybutyrate Pyruvate/ lactate Oxaloacetate/ malate Fumarate/ succcinate Cytochrome b; Fe3+/Fe2+ Ubiquinone; ox/red Cytocrome 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 Cu terminal component of respiratory chain contain two molecules of heme as prosthetic group and Cu

Oxidases
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)

AH2

1/

O2

AH2

O2

(Red)

OXIDASE

OXIDASE

H2O

(Ox)

H2O2

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

Dehydrogenases
Can not use oxygen as a hydrogen acceptor Performing two main functions: 1. transfer hydrogen in a coupled oxidation reduction reaction specific for their substrates, but utilize common coenzymes useful in enabling oxidative process to occur in the absence of oxygen 2. 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, krebs 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

Carrier
(Ox)

(Red)

(Red)

BH2

(Ox)

Carrier-H2
(Red)

B
(Ox)

DEHYDROGENASE SPECIFIC FOR A

DEHYDROGENASE SPECIFIC FOR B

Oxidation of a metabolite catalyzed by coupled dehydrogenases

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

 Peroxidases
Reducing 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

AH2 AH2 A

O2

OXIDASE

H2O2

CATALASE

2H2O

H2O2

O2

Role of catalase in the destruction of hydrogen peroxie

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 PHOSPHORYLATION

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 krebs cycle during the conversion of succinyl CoA to succinate. And b) in glycolytic reactions on cytoplasmic.

 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

Respiratory chain

AH2

NAD+

FpH2 Flavoprotein

2Fe3
+

H2O

Substrate
A

Cytochrome s 2Fe2
+

NADH H+ H+

Fp
2H+

1/

2H+

O2

Transport of reducing equivalents through the respiratory chain

 Powerhouses of the cell most of energy captured takes place inside it Outer membrane permeable to most metabolites, contain various enzym (acyl CoA 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

Mitochondrial

A B Phosphorylating complexes

B
OUTER MEMBRANE INNER MEMBRANE

MATRIX

F1 subunits F0 subunits

MATRIX

Cristae

Sonication

INNER MEMBRANE

OUTER MEMBRANE

Submitochondrial particel Formed from fragments of the inner membrance

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

Pyruvat e

Proline 3-HydroxyacylCoA 3Hydroxybutyrate Glutamate Malate Isocitrate Fp (FAD) NAD

Succin ate Cholin e Fp (FAD) FeS Q FeS ETF (FAD) Fp (FAD)

Lipoate

Fp (FMN) FeS

Cyt b FeS

Cyt c1

Cyt c

Cyt aa3 Cu

O2

Ketoglutarate

Fp (FAD) FeS

FeS ETF

Glycerol 3-phosphate

Acyl-CoA Sarcosine Dimethylglycin

Fp Q Cyt

: Iron-sulfur protein : Electrontransferring flavoprotein : Flavoprotein : Ubiquinone : Cytochrome

Resp chain & oxd phos inhibitors

1. Inhibitors of resp chain Blocking electrons transfer from Fe-S to co-Q , ie: barbiturates , pierisidin-A , rotenon , carboxine , succinate Dase 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)

Succinate

FAD FeS
BAL Antimycin A

H2S CO CN -

Complex I
NADH FMN, FeS Q

Complex III
Cyt b, FeS, Cyt C1
Cyt c

Complex IV
Cyt a Cu Cyt a3 Cu O2

Piericidin A Amobarbital Rotenone

Uncouplers

Uncouplers
Oligomycin ATP

Oligomycin ADP + P1

ADP + P1

ATP

ADP + P1

ATP

Mechanism of oxidative phosphorylation

Mitchells 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 muscle 2. Malate shuttle more universal utility more complex, due to the impermeability of mitochondrial membrane to oxaloacetate

OUTER MEMBRANE

INNER MEMBRANE

CYTOSOL NAD+
Glycerol 3phosphate GLYCEROL-3PHOSPHATE DEHYDROGENASE (CYTOSOLIC)

MITOCHONDRION

Glycerol 3phosphate GLYCEROL-3PHOSPHATE DEHYDROGENASE (MITHOCONDRIAL)

FAD

NADH + H+

Dehydroxyacetone phosphate

Dehydroxyacetone phosphate

FDH2
Respiratory Chain

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

CYTOSOL NAD
+

Malat e MALATE DEHYDROGENASE Oxaloacetat e TRANSAMI NASE Glutamate

INNER MAMBRAN MITHOCOND E RION 1

Malate

NAD
+

NADH +H+

MALATE DEHYDROGENASE

-KG

-KG

Oxaloacetat e TRANSAMI NASE

NADH +H+

Asp 2 H+

Asp

Glutamate

H+

Malate shuttle for transfer of reducing equivalents from the cytosol into the mitocondrion. 1. Ketoglutarate transporter, 2. 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

N
C NH

CREATINE KINASE

H2N

C H3C N

NH

COO-

GO = 12.6 kJ/mol

COO-

Creatine phosphate

Creatine

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