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2010-11-11 13:44:24
Biomolecular Science

Metabolism- Oxidative phosphorylation
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  1. Tissue location of Ox Phos
    • only in tissues w/ mitochondria (liver, muscle, heart, kidney, CNS etc.
    • NOT in RBC
    • Tissue w/ more mitochondria use more aerobic energy
    • Brown adipose tissue: many mitochondria, in neonatal and hibernating animals- energy used for heat more than ATP
  2. Cellular location of Ox Phos
    • oxidative phosphorylation takes place in the mitochondria
    • ox phos is a membrane process (inner)
    • Enzymes/proteins on inner mitochondrial membrane
  3. Ox Phos and Inner membrane
    oxidative phosphorylation has 2 parts
    • oxidative phosphorylation has 2 parts:
    • 1) electron transport chain:
    • series of proteins in membrane
    • oxidation of cofactors
    • transfer of protons from matrix to inter-membrane space
    • generation of proton gradient
    • 2)phosphorylation of ADP by ATPsynthase:
    • proton gradient causes flow of protons back into matrix
    • energy of proton motive force drives (ADP + phosphate --> ATP)
  4. Functions of Ox Phos
    • production of useful energy (ATP from ADP and Phos)
    • Oxidation of reduced cofactors (NADH + H+ and FADH2)
    • restoration of oxidised cofactors (NAD+ and FAD)
    • Production of H20 (from 2 x H+ and 1/2 O2)
  5. Substrates of Ox Phos
    • Electron transport chain:
    • reduced cofactors frm TCA cycle and B-oxidation and from cytoplasm (glycolysis)
    • oxygen from circulating haemoglobin in RBC- local store on myoglobin
    • protons (H+) from environment
    • ATPase synthetase:
    • Protons in proton gradient across inner membrane
    • ADP
    • phosphate (have to enter mitochondria)
    • NAD/NADH (oxidised form)
    • FAD (oxidized)/FADH2 (reduced)
    • *oxidation yields energy, reduction requires energy
    • reduced cofactors are energy transporters
  6. origin of reduced cofactors (NADH and FADH2)
    • TCA (krebs) cycle: using acetyl CoA from glycolysis, B-oxidation and amino acid degradation
    • B-oxidation of fatty acid: cofactors during degradation of fatty acid to acetyl CoA
    • Glycolysis: NADH produced during glycolysis has to be removed or it inhibits glycolytic enzymes
  7. Products of Ox Phos
    • Final product: adenine triphosphate (ATP)
    • provides useful and controllable energy
    • muscle and movement, biosynthesis, nerve impoulse membrane transport etc
    • Products of electron transport chain
    • water, H2O: enters tissue pool of water
    • oxidised cofactors (NAD, FAD): recycled to TCA, B-oxidation and glycolysis
    • ATP has 2 high energy bonds
    • energy required to synthesise bonds
    • repulsion of - charge, products of hydrolysis more stable
    • energy released on hydrolysis is coupled to other reactions
  8. physiological effects on Ox Phos
    • increases in response to energy demand (muscle movement, CNA to maintain Na/K pump, growth and repair)
    • reduced when no or little demand for energy (sleeping, hibernating)
  9. Control of Ox Phos
    • Respiratory control: limited bt oxygen supply
    • dependent on level of ADP: in provision of energy (ATP-> ADP and P)
    • high levels of ADP when cells ATP has been used ie when cell is using a lot of energy
    • ADP along with phos activates Ox phos
    • absence of ADP and P inhibits Ox phos
  10. Pathophysiology of Ox Phos
    • essential for most cellular func.
    • DZ process is affecting ox phos are fatal
    • inhibitors of the process may be acute toxins (cyanide, 2,4-dinitrophenol, rotenone, antimycin A, oligomycin)
  11. Mechanism of Ox Phos Chemiosmotic process
    • Electron Transport Chain: electrons from reduced cofactors transferred by series of rxns to oxygen, oxidation of reduced cofactors releases free energy, energy is used to pump protons from matrix to intermembrane space of mitochondria
    • formation of ATP from ADP + P: as protons flow back into matrix, via ATPsynthase, energy is released and used to form ATP
  12. Components of Electron Transport chain- Complex I
    • integral membrane protein
    • flavoprotein with FMN as cofactor
    • has iron sulphur centres
    • non haem
    • NADH-Q reductase
    • oxidation rxn is NADH + H+ --> NAD+ + 2H+ + 2e-
    • protons pumped from matrix (produces 1 ATP)
    • electron then passes through further complexes
    • passes first to Ubiqinone (coenzyme Q)
  13. Components of Electron Transport chain- Complex II
    • peripheral membrane protein
    • flavoprotein with FMN as cofactor
    • has iron-sulphur centres
    • non haem
    • succinate DH aslo succinate Q reductase
    • oxidation rxn is FADH2 --> FAD 2H+ + 2e-
    • NOT a proton pump
    • electron then passes through further complexes
    • passes first to Ubiqinone (coenzyme Q)
  14. Ubiquinone
    • Transport molecule
    • long chain isoprene lipid
    • soluble in phospholipid membrane (hydrophobic)
    • reduced by complex I & II
    • present in virtually all living organisms
    • also reduced by: fatty acyl CoA DH, Glycerol 3-phos DH
    • Transports from TCA
  15. Components of Electron Transport chain- Complex III
    • Integral membrane protein
    • Cytochrome b and c1
    • proteins contain haem
    • also has iron-sulphur centres
    • cytochrome C reductase
    • oxidises ubiquinone/reduces cytochrome c
    • protons pumped from matrix (produces 1 ATP)
    • electron passed to cytochrome C
  16. Cytochrome C
    • small peripheral protein
    • transports e- to complex IV
  17. Components of Electron Transport chain- Complex IV
    • cytochrome C oxidase
    • integral membrane protein
    • has cytochrome a & a3
    • has copper centres
    • oxidises cyt. C/reduces oxygen
    • protons pumped from matrix (producess 1 ATP)
    • electron passes to oxygen and water formed
  18. Electron Transport
    • protons are translocated across the membrane from the matrix to the intermembrane space
    • electrons are transported alon the membrane, through a series of protein carriers
    • oxygen is the terminal electron acceptor, combining w/ electrons and H+ ions to produce water
    • As NADH delivers more H+ and electrons into the ETS, the proton mitochondrial membrane, and OH- inside the membrane
    • (lower pH intermembrane space)
  19. Fo F1-ATPsynthase
    • Fo unit: transmembrane proton channel, inhibited by oligomycin
    • F1-ATPsynthase: central y subunit, 3a 3B subunits, protons pass through channel, a & B stalk rotate round y subunit, ADP + Pi bind to B subunit and energy of spinning used to form ATP
    • Repiratory control:
    • ADP/ATP ration
    • Hi ADP and Pi/lo ATP (Ox phos increase, ATP produced)
    • Lo ADP and Pi/Hi ATP (no need for ATP, Ox Phos stops)
    • No ADP/Pi on complex, proton channel closed
  20. Control of Ox Phos
    • respiratory control (limited by oxygen supply)
    • Dependent on the level of ADP:
    • in provision of energy (ATP --> ADP and P)
    • high levels of ADP when cell's ATP has been used
    • ADP along with phos activates Ox phos (ATP syns)
    • Absence of ADP and P inhibits Ox Phos
  21. Membrane Transport
    • ATP: ADP translocase, ATP out and ADP in
    • Phosphate in and OH- out by transporter
  22. Summary of Ox Phos
    • reduced cofactors NADH/FADH2 are high energy intermediates
    • electron transfer chain is series of oxidation/reduction reactions
    • at 3 sites as energy content (Gibbs free energy) is recuced, protons pumped across membrane
    • proton gradient produced
    • protons pass through FoF1ATPsynthase
    • ATP produced using energy of proton movement
  23. transport systems (of cytoplasmic NADH from glycolysis to Ox phos)
    • Malate-aspartate shuttle
    • in liver and heart
    • for transfer of NADH + H+ from glycolysis into mitochondria for aerobic respiration
    • cytoplasmic rxn:
    • oxaloacetate to malate
    • malate can enter mitochondria
    • malate back to oxaloacetate + NADH to complex I --> 3 ATP
  24. Glycerol Phosphate Shuttle
    • other tissues
    • dihydroxyacetone phosphate to glycerol 3 phosphate
    • NADH to NAD in cytoplasm
    • Glycerol 3 phos DH reverses reaction in mitochondrial membrane (oxidises Ubiquinone --> 2 ATP)
    • *less efficient*
  25. Disruption of Ox Phos
    • Uncouplers: allow protons to cross membrane (dinitrophenol)
    • Brown adipose tissue: natural uncoupling, energy released as heat, thermogenin (inner membrane protein)
    • used for hibernation/neonatal
    • inhibition of specific rxns
    • rotenone (insecticide, inhibits complex I)
    • Antimycin A (AB, inhibits complex III)
    • Cyanide and CO (poisons, inhibit complex IV)
    • Oligomycin (AB, inhibits proton channel of Fo subunit)
    • Bongkrekig acid & atractyloside (plant toxins, inhibit ATP= ADP translocase)