For each of the following experiments, tell me whether you would expect the rate of O₂ consumption and ATP synthesis to increase, decrease or remain the same (respectively). Assume there is plenty of O₂ around. (a) Succinate is suddenly added to mitochondria that have no other oxidizable substrates (Assume that there are ADP and Pi in mitochondria). (b) ADP is not allowed to pass into the mitochondrial matrix. (c) 2,4 Dinitrophenol (an uncoupler) is added.
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- When the antibiotic X is added to actively respiring mitochondria, several things happen: the yield of ATP decreases, the rate of O2 consumption increases, heat is released, and the pH gradient across the inner mitochondrial membrane increases. Does X act as an uncoupler or an inhibitor of oxidative phosphorylation? Explain the experimental observations in terms of the antibiotic’s ability to transfer K+ ions across the inner mitochondrial membrane.Calculate the number of usable ATP molecules produced per pair of electrons transferred from NADH to oxygen, if (i) five protons are pumped across the inner mitochondrial membrane for each electron passed through the three respiratory enzyme complexes, (ii) three protons must pass through the ATP synthase for each ATP molecule that it produces from ADP and inorganic phosphate inside the mitochondrion, and (iii) one proton is used to produce the voltage gradient needed to transport each ATP molecule out of the mitochondrion to the cytosol where it is used.In the early days of “mitochondriology,” P/O ratios were determined from measurements of volume of O2 taken up by respiring mitochondria and chemical assays for disappearance of inorganic phosphate. Now, however, it is possible to measure P/O ratios simply with a recording oxygen electrode. How might this be done?
- 1 a) What is meant by the ATP currency exchange ratio? Why does the oxidation of mitochondrial FADH2 generate one less ATP than oxidation of mitochondrial NADH? b) If 12 H+ are moved across the inner mitochondrial membrane by NADH oxidation, and each ATP synthesized requires 3 H+ to move through ATP synthase, why are only 3 ATP molecules produced by oxidation of each NADH?In the early days of “mitochondriology,” P/O ratios were determined frommeasurements of volume of O2 taken up by respiring mitochondria andchemical assays for disappearance of inorganic phosphate. Now, however, itis possible to measure P/O ratios simply with a recording oxygen electrode.How might this be done?Mitochondrial oxidation of fatty acids is a major source of ATP, yet fatty acids can be oxidized elsewhere. What organelle, besides the mitochondrion, can oxidize fatty acids? What is the fundamental difference between oxidation oc- curring in this organelle and mitochondrial oxidation?
- If actively respiring mitochondria are exposed to an inhibitor of ATPADP translocase, the electrontransport chain ceases to operate. Why?Which step(s) of cellular respiration would be directly inhibited by high concentrations of NADH? How would this affect other steps of cellular respiration?What is the minimum voltage drop for individual electrontransfer events in the mitochondrial electron transport systems that is necessary for ATP synthesis?
- If the mitochondrial inner membrane potential was depolarized (switched from + to -), what effect would this have on protein translocation into the mitochondria? Explain.Why does mitochondrial NADH yields more ATPs than cytosolic NADH?The enzyme dihydrofolate reductase (DHFR) normally resides in the cytosol, andDHFR can be imported into mitochondria by appending a mitochondrial signalsequence. However, when this modified DHFR is incubated with methotrexate, whichis a substrate analog that binds tightly to the active site, the modified DHFR is nolonger imported.a) Propose an explanation for why methotrexate prevents import of DHFR intomitochondria.b) Suppose that DHFR were modified instead by appending a nuclear localizationsignal. Would you expect methotrexate to prevent transport of this modified DHFRinto the nucleus? Why or why not?