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ط       Electron Transport chain, structure and Organization

Electron transfer chain. ط       Electron Transport chain, structure and Organization ط      Redox potentials and free energy changes ط       Coupling of oxidative phosphorylation to electron transferer                                         D4 246-261. Introduction.

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ط       Electron Transport chain, structure and Organization

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  1. Electron transfer chain ط      Electron Transport chain, structure and Organization ط     Redox potentials and free energy changes ط      Coupling of oxidative phosphorylation to electron transferer                                         D4 246-261

  2. Introduction 1. Mitochondrial Structure: fig6.25, fig6.26, Outermembrane, Intramembrane space, innermembrane (F1F0-ATPase), Matrix (actin filaments) 2. In Glycolysis: NADH produced in cytoplasm by GAPDH, a. Reoxidized in cytoplasm to NAD+ by LDH (in RBCs, anaerobic) b. fig 6.28, 7.9, Transported into mitoch by substrate shuttle (aerobic), * Gly-phopsh: muscle mitoch inner memb, 2 ATP (FADH2) * Mal-Asp: liver mitosol, 3 ATP (NADH) 3. In CAC, FA & KB oxid: NADH & FADH2 produced in cytoplasm by GAPDH, Yields ATP in presence of O2 during electron transport sequence (Respiratory Chain)

  3. Oxidation-Reduction Reaction § Electron transfer: Electron donor (red)  electron donor (oxid): AH2 + B A + BH2 § In reducing equivalents, both electrons & protons are transferred: NADH + H+ + FAD+NAD+ + FADH2 § In cytochromes, only electrons are transferred: cyt c (Fe2+) + cyt a (Fe3+) cyt c (Fe3+) + cyt a (Fe2+) § Oxid form & red form are referred as REDOX couple/pair § Quantitative elect transf facilitated as oxid-red POTENTIAL, measured as electromotive force (emf) § table6.6, the lower the negative emf (–) the more energy produced (ATP) & the higher positive emf (+) the more energy utilized

  4. Free-Energy Change § Oxid-red POTENTIAL (with 2 REDOX pair) = Free-Energy Change (in chem. react) (NAD+/NADH : ½O2/H2O) depends on concentration of reactant & product § In electectron transfer chain, the Free-Energy from oxid-red POTENTIAL (NADH : O2) generates energy (synthesis of ATP from reducing equivalent) if necessary enzs are present

  5. Multi-component System Mitoch. electectron trasnport (resp chain) major components (oxid-red react): 1. NAD+-linked dehydrogenases (NAD+-DHs) 2. Flavin-lined dehydrogenases (Flv-DHs) 3. Iron-Sulfur Protiens (Fe-S centre) 4. Coenzyme Q (CoQ) 5. Cytochromes (Cyt)

  6. 1. NAD+-linked DHs In CAC, FA oxid: NAD+ (oxidized form) is reduced to NADH 1. Cytoplasm: -  GAPDH -  G6PDH, NAD(P)+: NADP+ is not a substrate in mitoch resp chain but     Used in reductive biosynthesis (FA, Sterols) 2. Mitochondria: -  IsoCDH: Isocitrate + NAD(P)+ α-KG + CO2 + NAD(P)H + H+ -  MDH: L-Malate + NAD+ OA + NADH + H+ § NAD+ can be measured by spectrophotometer (light absorbance & fluorescence emission) § NAD+ enzyme assay for determination of changes in substrate or O2 concentration or during drug or hormone addition (function of metabolic condition)

  7. 2. Flavin-linked DHs fig6.34, Flavin (i.e. FAD+, FMN) derived from riboflavin as elect acceptor (oxid form) table6.8, Flavin-linked Enzymes: e.g. SDH, DHLDH, NADHDH § Flavin (FAD+, FMN) binds tightly noncovalently to their enzyme or protien § Flavoproteins: -  Flv-DHs: reduce flavin is reoxidized by electron carriers (other flavins, CoQ) -  Oxidase: reduced flavin is reoxidized by O2 as electron acceptor and yields H2O2 (2H2O2catalase 2H2O + O2)

  8. 3. Fe-S Center § fig6.35, In flavin-linked enzymes, containing non-heme iron (Fe-S Center), iron is converted from oxid form (Fe3+) to the reduced form (Fe2+) during transform of electron from reducing equivalents § SDH & NADHDH contain Fe-S centers found in all species (microorganism & mammals)

  9. 5. Cyts § The aerobic (O2) energy-generating function possess Cyts involved in elect trasnf system § fig6.37, Cyts are prts possess bound iron-containing Heme group § unlike hemoglobins or myoglobins Heme Iron (Fe), the Heme Fe of Cyst oxid (Fe3+) or red (Fe2+) acts as a function of electron transport chain § fig6.36, Type of Cyts are: a, b & c (c smallest subunit) depends on α band of absorption spectrum and type of Heme § fig6.37, since Fe of Heme fill coordination position of Cyt (except Cyt a3), direct O2 binding to Fe can be prevented by inhibitors (cyanide, azide, CO)

  10. Respiratory Chain Resp chain is located in inner membrane of mitoch fig6.40,fig6.44, Elect trasp is in a specific sequence: § -  NAD+-linked Enzs (CAC)  Reducing Equivalents (electrons) electronComplex I (NADHDH + FMN-linked, Fe-S center), & ejects H+ or -  FAD-lined Enzs (FA, KB oxid) Reducing Equivalents (electrons) electronComplex II (SDH) § Complex I / II electCoQ (mobile carrier) elect Complex III (Cyt b, c1, Fe-S center), & ejects H+ § Complex III electCyt celectComplex IV (Cyt oxidase), & ejects H+ § ½ O2 is electron acceptor H2O (O2 consumption) proton (H+) ejected from matrix to space are translocated back to matrix by F1F0-ATPase (ATP synthetase process, oxid-phosph): ADP + Pi ATP

  11. Respiratory Chain

  12. Respiratory Chain

  13. Respiratory Chain

  14. Respiratory Chain

  15. The  respiratory chain and oxidative-phosphorylation ط      Chemiosmotic theory, ATP synthetase ط     Inhibitors of respiratory chain ط     Uncouplers: Dinitrophenol, Valinomycin, Thermogenin                                      D4 261-263  ,     L2  555-563

  16. Inhibitors § fig6.40, The fish poison (retenone) & Barbiturate (amytal) inhibit at NADHDH/flavorprotein § The antibiotic (antimycin A) inhibit at Cyt b § Cyanide (CN), azide combine with Fe3+ (oxid Heme iron form) at Cyt a and a3, which prevents reduction of Heme iron from Cytc § Carbonmonoxide (CO) binds to Fe2+ (red Heme iron form) at terminal step (Cyt c) of resp chain, which inhibit catalyzation by Cyt oxidase (complex IV) If electrons are not oxidized causing results in impairment of energy-generation function (ATP synthatase), which leads tissue asphyxia and death

  17. Reversibility of Electron Transfer Reducing equivalents from S-ate can be transferred to NADH with hydrolysis of ATP fig6.46, the electron transport can be reversible: S-ate  F-ate (elect donor)  +  FAD  FADH2 ……

  18. Coupling of Oxidative Phosphorylation to Electron Transport § fig6.47, Succinate (elect donor) + ADP (phosphate acceptor) ==> active state (rapid) § In the active state, -  electrons are transferred (succinate  fumarate), elect transp -  O2 is consumped (O2 H2O), oxid -  ATP is synthesized (ADP  ATP), phosph § This continues till ADP concentration is zero (0) ==> resting state § In the resting state, -  all stops: elect transp, O2 is consumption & ATP is synthesis § Once ADP is added ==> all continue including ATP synthesis continues till O2 concentration is zero (0) *  Integrity of mitoch. is required for tight coupling

  19. Uncouplers § fig6.48, Succinate + ADP ==> active state (rapid O2 consumption) § This continues till Oligomycin (inhibitor of phoph, F1F0-ATPase) is added ==> resting state (all stop because of coupling) § When DNP (an uncoupler) is added ==> rapid O2 consumption § This continues till O2 concentration is zero (0) * Other uncouplers: FCCP, Valinomycin, Thermogenin * Uncouplers of resp and phosph act by pumping protons from space back to matrix.  This alters the normal flow of protons through F1F0-ATPase, which lead to no ATP synthesis

  20. Mechanism of Coupling • Chemical-coupling hypothesis (substrate-level phoph) • 2. Conformation-coupling hypothesis • 3. Chemiosmotic-coupling Mechanism (storage of energy)

  21. Mechanism of Coupling • Chemical-coupling hypothesis (substrate-level phoph): • § GAPDH (glycolysis): • -  GAP oxid to a high-energy intermediate product (1,3BPG) • -  ATP is synth in the next step by PGK • § SCoAS-tase (CAC):     the high-energy product SCoA generates GTP

  22. Mechanism of Coupling 2. Conformation-coupling hypothesis § In muscle contraction a) ATP hydrolysis drives conformational change in myosin head group b) This change disrupts cross-bridge to actin thin filaments § In elect ransf a) Elect transport through inner membrane causes membrane protein conformational change b) ATP synthesis during change in membrane protein from high-energy form to low * given evidence that conformational integrity is involved in the mechanism of ATP synthesis

  23. Mechanism of Coupling 3. Chemiosmotic-coupling Mechanism (storage of energy) § fig6.49, Electrochemical gradient (protons) between inner membrane during electron transport § This gradient is formed by pumping protons from matrix to space § Protons (H+) allowed back to the matrix during ATP synthetase process (F1F0-ATPase) § One ATP is synthesized per one proton pumped back from space to matrix

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