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Chapter 19. Oxidative Phosphorylation Part 2. Oxidative Phosphorylation Part 2. Key Topics: To Know How cells deal with reactive oxygen species (ROS). Calculating Δ G o ’ of the Proton Motive Force. Membrane ATPase and how it works. Cytoplasmic NADH getting into the mitochondria. - PowerPoint PPT Presentation

Transcript of Chapter 19

  • Oxidative Phosphorylation

    Part 2Chapter 19

  • Oxidative Phosphorylation Part 2Key Topics: To Know

    How cells deal with reactive oxygen species (ROS).Calculating Go of the Proton Motive Force.Membrane ATPase and how it works.Cytoplasmic NADH getting into the mitochondria. Adenylate Control. Mitochondia and apoptosis.PMF can be used to ?

  • ROS : Reactive Oxygen Species

  • G of the Proton Motive Force

  • Nobel Prize for Chemiosmotic Model = PMF

  • Converting PMF to ATP

  • Mitochondrial Respiration+PhosphorylationEOC Problem 9: Compartmentalization of Citric Acid Cycle.

  • Adjusting the pH Generates ATP

  • Molecules that Collapse the PMF

  • DNP is an UncouplerEOC Problem 6: Figuring out what happens with uncouplers.

  • Odorous Flowers that Heat Up Using Uncoupled Respiration

  • Worked out F1 ATPase kinetic mechanism + First purified the FoF1 ATPase

  • ATP Synthase

  • ATP Synthase Catalyzes by Steps

  • F1 Fo ATP synthase

  • F1 Complex

  • Used to Inhibit ATP Synthase Reaction

  • F1 ATPase Spinning

  • ATP Synthase Seen Rotating !

  • ATP Synthase Substrate TransportADP and Pi are products of AnabolismEOC Problem 11 is the effect of ADP and Pi on ATP Synthesis.

    Get Ready, NADH formed in Cytoplasm in Next

  • Glycolysis NADH Enters the Mitochondria by the Malate Aspartate Shuttle

  • NADH from Glycolysis Can Also Get in by Glycerol Phosphate DHAlso has a Anabolic Role

  • EOC Problems 13 and 14: Get in to the Pasteur Effect and Petites in Yeast Colonies. Fun stuff.

  • Adenylate Control of Glycolysis/CAC/e-transportAcceptor Control: availability of ADP + Pi

    Mass action ratio: [ATP]/([ADP] [Pi])

    EOC Problem 17 Gets into the rate of ATP turnover in heart musclethe muscle that always has to be on.EOC Problem 18 Gets into the same in insect flight muscle.

  • Inhibitory Protein IF1 Stops Loss of ATP During Ischemia that is HypoxiaIF (red and white) forms dimers at pH 6.5 to Stop Rotation of ATP Synthase

  • Thermogenin Protein Uncoupler in Brown Fat

  • Mitochondria in Adrenal Gland: P-450 Oxygenases Specialized for Steroid Synthesis

  • Electron Flow to P-450

  • Apoptosis is Regulated by the MitochondriaInitiates a series of proteasessStress, DamageEOC Problem 19: relates mitochondrial function to cancer. Hint think about the first couple of slides.

  • Escherichia coli Electron Transport

  • Bacterial Quinone

  • Bacteria Use the PMF to Rotate Their Flagella

  • Proton Motive Force Functions ToSynthesize ATP (from ADP + Pi).Active Transport (Symports, Antiports, Uniports; review Chapter 11).Rotate Bacterial Flagella.Reversed Electron Transport (some Photosynthetic and Chemoautotrophic Bacteria)

  • Things to Know and Do Before ClassHow cells deal with reactive oxygen species (ROS).

    Calculating Go of the Proton Motive Force.

    Membrane ATPase and how it works.

    Cytoplasmic NADH getting into the mitochondria.

    5. Adenylate Control.

    Mitochondria and apoptosis.

    7. PMF can be used to ?

    8. EOC Problems: 6, 9, 11,13,14, 17, 18, 19.

    ***ROS formation in mitochondria and mitochondrial defenses. When the rate of electron entry into the respiratory chain and the rate of electron transfer through the chain are mismatched, superoxide radical (O2) production increases at Complexes I and III as the partially reduced ubiquinone radical (O) donates an electron to O2. Superoxide acts on aconitase, a 4Fe-4S protein, to release Fe2+. In the presence of Fe2+, the Fenton reaction leads to formation of the highly reactive hydroxyl free radical (OH). The reactions shown in blue defend the cell against the damaging effects of superoxide. Reduced glutathione (GSH; see Figure 22-27) donates electrons for the reduction of H2O2 and of the oxidized Cys residues (SS) of enzymes and other proteins, and GSH is regenerated from the oxidized form (GSSG) by reduction with NADPH.

    Look at glutathione peroxidaseis there something chemically out of balance in this cartoon.*Mitchell and Moyle proposed this model in 1960. At that time most believed (not a good word to use in science) that electron transport itself somehow made ATP (ADP + Pi ATP) by some component of the electron transport system itself (quinones? Cytochromes? Iron-sulfur complexes). It took almost 10 years to realize Mitchell and Moyle had it all figured out.*ATP synthesis driven by PMF mediated by the membrane bound (the Fo portion is the transmembrane portion) ATPase. Lets look at the evidence.*In these experiments we can measure oxygen uptake (oxygen electrode) and amount of ATP produced (by various methods) to purified mitochondria. Here adding ADP and Pi doesnt do much, but when succinate is added (starting electron transport from succinate DH, Complex II) oxygen is consumed and ATP is being made. Then CN- is added which blocks electron transport at Cyt-a, and oxygen uptake is halted along with ATP synthesis. Thus: oxygen uptake and ATP synthesis are COUPLED. Lets see the Mitchell and Moyle experiment next.*What they did with isolated mitrochondria was to gently raise the pH to 9 in an almost non-buffered state but with 0.1 M KCl, then add valinomycin (collapses any K+ gradient) and then added a good concentration of a pH 7 buffer which made the outside proton concentration jump 100Xand, this generated ATP without electron transport or a K+ gradient. **Two chemical uncouplers of oxidative phosphorylation. Both DNP and FCCP have a dissociable proton and are very hydrophobic. They carry protons across the inner mitochondrial membrane, dissipating the proton gradient. Both also uncouple photophosphorylation, next slide.This is a little change: add succinate without ADP and Pi, not much happens until you add the ADP and Pi and coupled respiratioin-ATP synthesis takes off. Then add DNP which allows electron transport to go on (oxygen being consumed) but collapses the PMF gradient so no ATP can be made.*Some organisms use uncoupled respiration to generate heat.**These experiments used labeled oxygen. At the right is the active site of ATPase, the incoming phosphate is almost 90o to the viewer. Lets look at the energetics.*What is amazing is that this is done largely through protein conformational change as the F1 rotates ! Rotation is driven by the PMF.*Next two slides show the protein structure changes of the F1 subunits. Here it is hinted at by color shading.*On the left is a partical look at with an ADP bound as it is rotated by being driven by the PMF it changes conformation catalyzing the reaction and making ATP, next a top down view of the whole complex*ATPase can spin in one direction and make ATP, or in the other direction and use ATP to pump proton out of the matrix.***How do we know it is spinning ? Can this be seen ?*We can not see the ATPase with light microscopy, but if we fix it to a nickel complex on a glass slide and have a long actin filament attached to it, then add ATPthe time lapse photographs next slide.*And rotating counter-clockwise. Now lets consider what we have done: the mitochondrion has done electron transport and made ATP in the matrix. How can this be when the mitochonria are the Powerhouse of the cell. Somehow we have to get the ATP to the rest of the cell.***The products of cytoplasmic metabolism (mainly anabolism using ATP) are ADP and Pi need to get in the mitochondria and the ATP needs to get out. The ATP/ADP antiporter works stoichimetically and the phosphate gets in as a PMF driven symport. Anabolism uses NTPs which can be made from ATP and also needs NADPH (which is made in the cytoplasm, remember your PPP). But, the cytoplasm does glycose catabolism producing NADHhow does this get IN to the mitochondria.*This looks a little complex, but it is all done with molecules we know and LOVE. Cytoplasmic NADH converting OAA to malate, malate in and KG out (antiporter). Malate to OAA (mitochondria CAC) produces NADH, OAA by a Aspartate amino transferase (whats the coenzyme?) to D picking up the E amino converting E to KG . D out and E in through another antiporter. In the cytoplasm then D to OAA giving up Ds amino group of KG making E for the glutamate-aspartate antiporter. Note that this works well: remember the thermodynamics of malate DH. In the cytoplasm malate DH is in its exothermic mode and in the mitochondria it has to be driven by the exothermic side (citrate synthase.to KG DH) of CAC. All the other enzymes of this system are just about at equilibrium.

    Wait a minute, does NADH have another way to get in? Next slide.*NADH can donate its electrons to electron transport at the FAD level to Q. What this means is that when NADH uses this way it generates less PMF than having NADH putting in its electrons at Complex I. Thus, this is why this NADH is worth only 1.5 ATP while on the matrix side NADH is worth 2.5 ATP. This affects the calculation of energy yield from glucose, next slide.**This looks mainly at adenylate control (see pfk-1, they left out a major regulator) operates as the main regulator of oxidative phosphorylation (see slide 9 for the evidence!)*If IF didnt do this, ATP synthase could go the wrong way and deplete heart cells of ATP.*Babies have brown fat. Most fat is whitish in color. Brown fat has more mitochondria (thereby making a brownish color) which contain an aptly named uncoupling protein, thermogenin which collapses the PMF releasing the energy as heat. This keeps babies warm (besides being cute). ***What is important to know is that these enzymes in the liver oxidize hydrophobic xenobiotics to make them more water so