Question

Question 1 (25 points).

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.

Part 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)

Part 2

The following graph (Fig. 2) shows the changes of oxygen concentration in a solution containing the patient’s mitochondria when the following reactants were sequentially added:

1. S’ = succinate + rotenone 2. ADP
3. ADP
4. ADP

a. Explain the effect of each reactant on oxygen consumption (4 pts)
b. Does this experiment confirm your previous conclusion regarding the defect affecting the

patient’s respiratory chain? Explain. (2 pts)

c. One of your co-worker pointed out that the graphs for the patient and the control are the same and suggested you used the wrong graph for the control. Did you make a mistake? Briefly explain (2 pts)

Part 3

Further tests show an important elevation of the concentration of lactate and of some intermediates of the citric acid cycle in the blood plasma and urine of this patient. Explain this observation (4 pts).

Answer 1

Part 1:

· Pyruvate + Malate: they’re substrates (pyruvate after transforming to acetyl-CoA) of the tricarboxylic acid cycle (TCAc), and their oxidation produces NADH and FADH₂, which are used to pump protons to the intermembrane mitochondrial space (ITS).

· ADP: is the substrate of the final complex the F₀ of the electron transport chain (ETC) also called ATPsynthase.

· DNP: it’s full name is 2,4-dinitrophenol, it’s a uncoupler of the ETC because it binds to the internal mitochondrial membrane allowing the flow of protons from the ITS to stroma.

· Succinate: is another substrate of the TCAc like pyruvate or malate.

a) The addition of ADP changes the equilibrium of ATP/ADP/AMP concentrations, therefore, affecting the neat velocity of respiration.

b) It probably is the ATPsynthase complex (F1 part) and the complex I, but I think both complexes have at least 1 mutation in their protein components for the following reasons:

a. ADP addition doesn’t drop O2 consumption as it should (meaning that something (i.e F1 complex) that binds to ADP could be different).

b. DNP also doesn’t drop the O2 consumption as it should (meaning that the proton gradient in-between mitochondrial membranes is maintained).

c. The addition of succinate to the TCAc only produces NADH (and CO2).

Part 2:

a) The same as part 1a, but, rotenone is an inhibitor of the pumping activity of the NADH-ubiquinone-reductase (complex I) of the CTE, therefore it decreases the O2 consumption since no electrons can travel to the oxygen.

b) Yes, it does, since this shows that the inhibition of a specific element of the ETC in the control mitochondria has the same results as in the patient mitochondria (whose are inhibited per se).

c) No, I haven’t made a mistake, the graphs are the same because they’re showing the same thing: “o2 consumption when NADH produced by TCAc can’t be used”.