Question

Question 1

The mitochondria from a patient suffering from muscle pain during physical exercises were analyzed to detect any defect in one of the components of the respiratory chain. Isolated mitochondria were incubated in an isotonic buffer, and changes in the oxygen consumption were measured as various components were sequentially added to the reaction.

1) The following graph (Fig. 1) shows the changes of oxygen concentration in a solution containing the patient’s mitochondria or mitochondria from a healthy donor (control). The following reactants were sequentially added:

1. S = Pyruvate + malate

2. ADP

3. DNP (uncoupling agent)

4. Succinate

S DNP S ADP Succinate Succinate ADP DNP Patient Control Figure 1

a. Explain the effect of each reactant on oxygen consumption. (8 pts)

b. In the control, why does the oxygen consumption slow after the addition of ADP (change in slope). (2 pts)

c. Predict which protein complex from the respiratory chain is affected in the patient. Briefly explain your reasoning. (3 pts)

Answer 1

a) Let us address each one:

S: Both pyruvate and malate are krebs cycle fuel molecules, one produces acetyl CoA to enter the cycle and the other one oxaloacetate to also start the cycle.

ADP: This one is crucial for the final goal that is ATP production, it does not matter how many NADH you produce in Krebs cycle, without ADP oxygen won't be consumed and ATP won't be produced

DNP: This uncouples the electron transport chain, that means the energy is not focused on taking ADP to create ATP but is released as heat instead

Succinate: This is a intermediate molecule in Krebs cycle, its addition is not normally required if you have your acetyl CoA and oxaloacetate beggining the cycle... unless the enzyme that turns succinyl CoA into succinate is not working

b) This question is wrongly made, because ADP is not lowering the oxygen consumption, it is actually increasing it. The curve goes down because the line represents the oxygen concentration, not the oxygen consumption.

The addition of ADP changes the slope because now the production of ATP is possible (ADP is the substrate), and this is the particular step that consumes oxygen, taking it as the final electron receptor to turn it into water

c) Note how in the patient oxygen is not being consumed even when ADP is added, but when succinate is added everything starts to work. This means that an enzyme between acetyl CoA and Succinate is missing, it's non functional. This could be either citrate synthase, aconitase, isocitrate dehydrogenase, alpha ketoglutarate dehydrogenase or succinyl CoA synthase, or maybe all of them. The first part of the krebs cycle is the problem