ATP Synthase

1. ATP synthase is an F-type ATPase. It was originally identified as a proton pump that used primary active transport to move H⁺ across the membrane against its gradient.
a. Which part of ATP synthase–F_o or F₁– binds either ADP or ATP?
b. Which part of ATP synthase–F_o or F₁– binds H⁺?
c. Primary active transporters always have a conformation change associated with converting ATP to ADP. Which two parts of ATP synthase change conformation?
d. Does the ring of c subunits rotate clockwise or counterclockwise when ADP + P_i → ATP?
e. Does the ring of c subunits rotate clockwise or counterclockwise when ATP → ADP + P_i?

2. Start with an active site of ATP synthase that is in the loose conformation, which has a high affinity for ADP + P_i. What happens to that active site and to ADP when H⁺'s move from the P side to the N side?   Describe each conformation change and the change to ADP that is the result of the conformation change.

3. Start with 1 H⁺ on the P side of the membrane. What happens to it as it moves through ATP synthase to the N side? Name each subunit involved and each conformation change that occurs.

4. Synthesis of 1 ATP requires the transport of 3 H⁺ through ATP synthase + 1 H⁺ that accompanies P_i into the matrix using a symport.
a. How many H⁺'s are transported from N to P as a result of the oxidation of 1 NADH?
b. How many H⁺'s are transported from N to P as a result of the oxidation of 1 FADH₂?
c. How many ATP's can be synthesized for each NADH oxidized? for each FADH₂?

5. What happens to the proton gradient in the presence of an uncoupler? How does this affect the synthesis of ATP?

6. Histidine has a pK_a closer to pH 7 than aspartate, which means that it readily donates and accepts H⁺ near neutral pH. Why isn't histidine, rather than aspartate, used to move H⁺ across the membrane?

7. Calculate the total number of ATP's produced as a result of degrading one glucose molecule in the presence of O₂ by filling in Table 1. Assume that NADH produced in glycolysis is equivalent to NADH produced in the mitochondrial matrix.

Shuttles

1. Figure 8-38 shows the structure of NADH.
a. Draw the structures of malate, α-ketoglutarate, aspartate, and glutamate.
      Compare the structure of NADH to the molecules you have drawn.
b. Which is more easily transported across a membrane by a transport protein?
c. What is the net charge on each of the molecules you drew?
d. Separate them into two pairs based so that each pair has the same net charge.

2. Look at Figure 19-29 in the text. Locate the oxaloacetate in the cytosol.
a. How is that oxaloacetate converted to malate? (Enzyme & source of NADH)
b. How is the malate that is produced moved into the mitochondrial matrix?
c. What happens to malate in the mitochondrial matrix? (Enzyme & source of NAD⁺)
d. How is oxaloacetate moved back to the cytosol in order to shuttle more NADH
      into the matrix?

3. The process of moving NADH into the mitochondrial matrix by the malate/aspartate shuttle is one of diffusion rather than active transport. Explain why this is the case, using the definitions of diffusion and active transport.

4. Glycerol-3-phosphate dehydrogenase and the malate-aspartate shuttle system are methods for virtually (not actually) moving NADH produced in glycolysis from the cytosol into the matrix. Fill in Table 2 to compare and contrast the two methods.

5. Assume that [H₂PO₄⁻] matrix and [H₂PO₄⁻] cytosol are the same, and that [H⁺] is also the same on both sides of the inner mitochondrial membrane. Use ΔV = − 0.2 V (measured from the cytosol to the matrix).
a. What is ΔG_T for moving H₂PO₄⁻ into the matrix?
b. What is ΔG_T for moving H⁺ into the matrix?
c. Given that   [H⁺] is lower on the N side, is ΔG_T for   H⁺–H₂PO₄⁻   transport into
       the matrix > 0, = 0, or < 0?

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