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Chapter 14 (part 2)

Chapter 14 (part 2). Oxidative phosphorylation. Proton Motive Force ( D p ). PMF is the energy of the proton concentration gradient The chemical ( D pH= pH in – pH out ) potential and the electrical potential( DY= Y in – Y out ) contribute to PMF D G = nf DY and D G = –2.303nRT D pH

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Chapter 14 (part 2)

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  1. Chapter 14 (part 2) Oxidative phosphorylation

  2. Proton Motive Force (Dp) • PMF is the energy of the proton concentration gradient • The chemical (DpH= pHin – pHout) potential and the electrical potential(DY= Yin– Yout) contribute to PMF • DG = nfDY and DG = –2.303nRT DpH • DG for transporting 1 H+ from inner membrane space to matrix = DG = nfDY –2.303nRTDpH • Dp = Dp = DG/nF • Dp = Dy –(0.059)DpH

  3. Proton Motive Force (Dp) • What contributes more to PMF, DY or DpH? • In liver DY=-0.17V and DpH=0.5 • Dp = Dy –(0.059)DpH = -0.17-(0.059)(0.5V) • Dp = -0.20 V • DY/Dp=(-0.17V/-0.20V) X 100% = 85% • 85% of the free energy is derived form DY

  4. Proton Motive Force (Dp) • How much free energy generated from one proton? • DG = nFDP = (1)(96.48kJ/Vmole)(-0.2V) = -19 kJ/mole • To make 1 ATP need 30 kJ/mole. • Need to translocate more than one proton to make one ATP • ETC translocates 10 protons per NADH

  5. ATP Synthase • Proton diffusion through the protein drivesATP synthesis! • Two parts: F1 and F0

  6. Racker & Stoeckenius confirmed Mitchell’s hypothesis using vesicles containing the ATP synthase and bacteriorhodopsin

  7. Binding Change Mechanism • ADP + Pi <-> ATP + H2O • In catalytic site Keq = 1 • ATP formation is easy step • But once ATP is formed, it binds very tightly to catalytic site (binding constant = 10-12M) • Proton induced conformation change weakens affinity of active site for ATP (binding constant = 10-5)

  8. Binding Change Mechanism • Different conformation at 3 catalytic sites • Conformation changes due to proton influx • ADP + Pi bind to open-site in exchange for ATP • Proton driven conformational change (loose site) causes substrates to bind more tightly • ATP is formed in tight-site. • Requires influx of three protons to get one ATP

  9. ATPase is a Rotating Motor • Bound a,b,g subunits to glass slide • Attached a fluroescent actin chain to g subunit. • Hydrolysis of ATP to ADP + Pi cause filament to rotate 120o per ATP.

  10. How does proton flow cause rotation?

  11. Active Transport of ATP, ADP and Pi Across Mitochondrial Inner Membrane • ATP is synthesized in the matrix • Need to export for use in other cell compartments • ADP and Pi must be imported into the matrix from the cytosol so more ATP can be made. • Require the use of transporters

  12. Transport of ATP, ADP and Pi • Adenine nucleotide translocator = ADP/ATP antiport. • Exchange of ATP for ADP causes a change in DY due to net export of –1 charge • Some of the energy generated from the proton gradient (PMF) is used here • Pi is imported into the matrix with a proton using a symport. • Because negative charge on the phosphate is canceled by positive charge on proton no effect on DY, but effects DpH and therefore PMF.

  13. Transport of ATP, ADP and Pi • NRG required to export 1 ATP and import 1 ADP and 1 Pi = NRG generated from influx of one proton. • Influx of three protons required by ATPase to form 1 ATP molecule. • Need the influx of a total of 4 protons for each ATP made.

  14. P/O Ratio • The ratio of ATPs formed per oxygens reduced • e- transport chain yields 10 H+ pumped out per electron pair from NADH to oxygen • 4 H+ flow back into matrix per ATP to cytosol • 10/4 = 2.5 for electrons entering as NADH • For electrons entering as succinate (FADH2), about 6 H+ pumped per electron pair to oxygen • 6/4 = 1.5 for electrons entering as succinate

  15. Regulation of Oxidative Phosphorylation • ADP is required for respiration (oxygen consumption through ETC) to occur. • At low ADP levels oxidative phosphorylation low. • ADP levels reflect rate of ATP consumption and energy state of the cell. • Intramolecular ATP/ADP ratios also impt. • At high ATP/ADP, ATP acts as an allosteric inhibitor for Complex IV (cytochrome oxidase) • Inhibition is reversed by increasing ADP levels.

  16. Uncouplers • Uncouplers disrupt the tight coupling between electron transport and oxidative phosphorylation by dissipating the proton gradient • Uncouplers are hydrophobic molecules with a dissociable proton • They shuttle back and forth across the membrane, carrying protons to dissipate the gradient • w/o oxidative-phosphorylation energy lost as heat • Dinitrophenol once used as diet drug, people ran 107oF temperatures

  17. Physiological Uncoupling • Uncoupling of ETC and Ox-phos occurs in animals as a means to produce heat = nonshivering thermogenesis. • Impt. In hibernating mammals, neborn animals and mammals adapted to cold • Occurs in brown adipose tissues (rich in mitochondria) • Uncoupling protein (UCP) = channel to allow influx of protons to matrix (dissipates proton gradient)

  18. Uncoupling in Plants • Plants generate heat during fruit ripening and to emit odors to attach pollinators. • Plants can by pass normal ATP generating ETC • Alternative ETC in plants does not pump protons, just transfers electron. • All plant have this pathway, actual physiological reason not known

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