1. Chapter 14 (part 2) Oxidative phosphorylation

2. Proton Motive Force (  p ) PMF is the energy of the proton concentration gradient The chemical (  pH= pH in – pH out ) potential and the electrical potential(  in  –  out ) contribute to PMF  G = nf  and  G = –2.303nRT  pH  G for transporting 1 H+ from inner membrane space to matrix =  G = nf  –2.303nRT  pH  p =  p =  G/nF  p =  –(0.059)  pH

3. Proton Motive Force (  p ) What contributes more to PMF,  or  pH? In liver  =-0.17V and  pH=0.5  p =  –(0.059)  pH = -0.17-(0.059)(0.5V)  p = -0.20 V  /  p=(-0.17V/-0.20V) X 100% = 85% 85% of the free energy is derived form 

4. Proton Motive Force (  p ) How much free energy generated from one proton?  G = nF  P = (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 drives ATP synthesis! Two parts: F 1 and F 0

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

7. ADP + Pi ATP + H 2 O In catalytic site K eq = 1 ATP formation is easy step But once ATP is formed, it binds very tightly to catalytic site (binding constant = 10 -12 M) Proton induced conformation change weakens affinity of active site for ATP (binding constant = 10 -5 ) Binding Change Mechanism

8. 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  subunits to glass slide Attached a fluroescent actin chain to  subunit. Hydrolysis of ATP to ADP + Pi cause filament to rotate 120 o per ATP.

10. How does proton flow cause rotation?

16. 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

17. Adenine nucleotide translocator = ADP/ATP antiport. Exchange of ATP for ADP causes a change in  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  but effects  pH and therefore PMF. Transport of ATP, ADP and Pi

18. 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. Transport of ATP, ADP and Pi

19. 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 (FADH 2 ), about 6 H + pumped per electron pair to oxygen 6/4 = 1.5 for electrons entering as succinate

20. 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.

21. 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 107 o F temperatures

23. 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)

25. 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