biology oxidation.ppt [read-only]
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Source of ATP :1. Oxidative Phosphorylation.2. Glycolysis.3. Krebs Cycle.
Ox-red reactions O2 accept single electron
Ox-red reactions O2 accept single electron
ROS (radical or non radical)
Cell damage linked to at least 100 deseases
Ca, CV disorders, neurologic disorders
Anti oxidantNatural & vitamins
BIOLOGIC OXIDATION
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+/H2NAD+/ 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
H2O2
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 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, 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 redoxpotential
- 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
• CatalaseUsing 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 2H2OOXIDASECATALASEH2O2
O2H2O2
A’H2 A’
AH2 A
Oxygenases• Catalyzing the direct transfer and
incorporation of oxygen into a substrate• Divided into two subgroups:
1. Dioxygenases / oxygen transferaseIncorporating both atoms of oxygen into substrate: A + O2 AO2
2. MonooxygenasesMixed 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-H21
(Red)
Carrier2
(Red)
Carrier2
(Ox)
Carrier3
(Ox)
Carrier-H23
(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
1/2O2
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
INNERMEMBRANE
MATRIX
Cristae
B
A
OUTERMEMBRANE
INNERMEMBRANE
MATRIXF1 subunitsF0 subunits
Submitochondrial particelFormed 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 flavoprotein
Fp : FlavoproteinQ : UbiquinoneCyt : Cytochrome
Proline3-Hydroxyacyl-
CoA3-
HydroxybutyrateGlutamate
MalateIsocitrate
Acyl-CoASarcosine
Dimethylglycin
Fp(FAD)FeS
Lipoate Fp(FAD) NAD
Fp(FMN)FeS
Succinate
CholineFp
(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
Succinate FADFeS
FMN, FeSNADH
BALAntimycin A
Complex III
Cyt b, FeS, Cyt C1
Cyt c Cyt a Cyt a3Cu CuQ
Uncouplers
ADP + P1 ADP + P1 ATPATP ADP + P1
Uncouplers
ATP
Piericidin AAmobarbitalRotenone
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 , NH3monocarboxylic 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+
Dehydroxyacetonephosphate
Dehydroxyacetonephosphate
Glycerol 3-phosphate
Glycerol 3-phosphate
CYTOSOL MITOCHONDRION
OUTER MEMBRANE
Glycerophosphate shuttle for transfer of reducing equivalents from the cytosol into the mitochondrion
MALATE DEHYDROGENASE
MALATE DEHYDROGENASE
TRANSAMINASE
TRANSAMINASE
INNER MAMBRAN
E MITHOCONDRION
Malate
Oxaloacetate
Glutamate
-KG
Asp
NAD+
NADH+H+
H+H+
Glutamate
-KG
Asp
Malate
Oxaloacetate
NAD+
NADH+H+
CYTOSOL1
2
Malate shuttle for transfer of reducing equivalents from the cytosolinto 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