To determine which segments of the electron transport system (ETS) are responsible for proton pumping and hence for ATP synthesis, investigators usually incubate isolated mitochondria under conditions such that only a portion of the ETS is functional. One approach is to supply the mitochondria with an electron donor and an electron acceptor that are known to tap into the ETS at specific points. In addition, inhibitors of known specificity are often added. In one such experiment, mitochondria were incubated with hydroxybutyrate, oxidized cytochrome c, ADP, Pi, and cyanide. (Mitochondria have an NAD+-dependent dehydrogenase that is capable of oxidizing hydroxybutyrate to ketobutyrate.)

a. What is the electron donor in this system? What is the electron acceptor?

b. Based on what you already know about the ETS, how many moles of ATP would you expect to be formed per mole of ?-hydroxybutyrate oxidized? Write a balanced equation for the reaction that occurs in this system.

c. Cyanide is the only reagent added to the system that is not a part of the balanced equation for the reaction. What was the purpose of adding cyanide to the system? What result would expect if the cyanide had not been added?

d. Would you expect the enzymes of the TCA cycle to be active in this assay system? Explain your reasoning.

e. Why is it important that ?-ketobutyrate cannot be further metabolized in this system? Lactate is quite similar in structure to ?-ketobutyrate; what effect would it have had if the investigators had used lactate as the oxidizable substrate instead of hydroxybutyrate?