a) NADH, Q, Complexes I, III, and IV.
b) NADH, Q, Cytochrome c, Complexes II and III.
c) succinate, Q, cytochrome c, Complexes II, III and  IV.
d) succinate, Q, cytochrome c, Complexes II and III.

2)  In electron transfer only the quinone portion of the ubiquinone undergoes oxidation/reduction;  the isoprenoid chain remains unchanged.  What is the function of this chain?

3)  All the dehydrogenases of glycolysis and the citric acid cycle use NAD⁺ (E^o'= -0.32 v) as electron acceptor except succinate dehydrogenase, which uses covalently bound FAD (E^o'= 0.05v for enzyme bound FAD/FADH₂), Suggest why FAD is a more appropriate electron acceptor  than NAD⁺ in the dehydrogenation of succinate, based on the Eo' values of fumarate/succinate (E^o'= 0.03v), NAD⁺/NADH, and the succinate dehydrogenase FAD/FADH₂.

4)  The degree of reduction of each carrier in the respiratory chain is determined  by the conditions in the mitochondrion. For example,  when NADH and O₂ are abundant, the steady state degree of reduction of the carriers decreases as the electrons pass from substrate to oxygen. When electron transfer is blocked, the carriers before the block become more reduced and the carriers after the block become relatively more oxidized. For each of the conditions below, predict the state of oxidation of ubiquinone and cytochromes b, c₁, c, and a+a₃.

a) Abundant NADH and O₂, but cyanide added.
b) Abundant NADH but O₂ exhausted.
c) Abundant O₂, but NADH exhausted.
d) Abundant NADH and O₂.

5)  A gene has been identified in humans that appears to play a role in the efficacy with which calories are utilized, and  anti-obesity drugs have been proposed to regulate the amount of uncoupling protein (UCP-2) produced by this gene. The UCP-2 protein is present in many human tissues and has been shown to be a proton translocator in mitochondrial membranes. Explain how increasing the presence of UCP-2  protein might lead to weight loss in humans.

6) a) When widely prescribed pain-killer, Demerol (meperidine), is added to a suspension of respiring mitochondria, the ratios of NADH/NAD⁺ and Q/QH₂ increase. Which electron transport complex is inhibited by demerol?
b) When the antibiotic myoxythiazole  is added to respiring bacteria, the ratios of cytochrome c1(Fe3+)/cytochrome c1(Fe2+), and cytochrome b₅₆₆(Fe³⁺)/cytochrome b566(Fe²⁺) increase. Where does myoxythiazole inhibit the electron transport chain?

7)  a)  the toxicity of cyanide (CN^-) results from its binding to the iron atoms of cytochrome a,a3 complex and subsequent inhibition of mitochondrial electron transport. How does the CN-/iron complex prevent oxygen from accepting electrons from the electron transport chain?
b) Patients who have been exposed to cyanide can be given nitrites that convert Fe³⁺ in oxyhemoglobin to Fe³⁺ met hemoglobin.  Given the affinity of CN^- for Fe³⁺, suggest how this nitrite treatment might function to decrease the effects of cyanide on the electron transport chain.

8) Under aerobic conditions, extra mitochondrial NADH must be oxidized by the mitochondrial electron-transport chain.  Consider a preparation of rat hepatocytes containing  mitochondria and all the cytosolic enzymes.  If [4-³H]NADH is introduced, radioactivity soon appears in the in the mitochondrial matrix. However if [7-¹⁴C] NADH is introduced, no radioactivity appears in the matrix. What do these observations reveal about the oxidation of extramitochondrial NADH by the electron transport chain?

9) Plant chloroplasts contain thylokoid vesicles that contain an electron transfer chain and an oxidative phosphorylation  system analogous to those found in mitochondria.  In thylokoid vesicles protons are pumped from the outside (stroma) to the inside (lumen).  If the mechanism of ATP synthesis in thylokoid vesicles is similar to that in mitochondria, draw a diagram showing how the CF_oCF₁ ATP synthase of chloroplasts is oriented in the membrane.   In what compartment will ATP synthesis take place?

10) a) Calculate the proton motive force across the the inner mitochondrial membrane at 25^oC, when the electrical difference is -0.18v (inside negative), the pH outside is 6.7, and the pH inside is 7.5.  (Hint:  the proton motive force, Dp,  is defined as Dp=DG/nF)
b) What percentage of energy is from the chemical (pH) gradient, and what percentage is from the charge gradient?
c)  What is the total free energy available for the phosphorylation of ADP?

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