MCAT practice · Biological and Biochemical Foundations · 1D Bioenergetics and metabolism
Respiratory control in fresh and stored mitochondria
An original practice passage written for this site, not an AAMC item. Exam pace is about 8 minutes for a passage like this.
Isolated mitochondria allow electron transport and ATP synthesis to be studied separately. In a sealed chamber fitted with an oxygen electrode, the rate at which O₂ disappears mainly reports the rate at which electrons reach Complex IV. By adding reagents in a fixed order, investigators can probe one part of the coupled system at a time. One group used this approach to ask how cold storage affects rat liver mitochondria. They compared mitochondria tested within an hour of isolation (fresh) with mitochondria from the same isolation that had been held on ice for 24 hours (stored).
Each run contained 1.0 mg of mitochondrial protein in 2.0 mL of buffer with phosphate and Mg²⁺ at 30 °C. The investigators made five additions in sequence. First came the NADH-linked substrates glutamate and malate (state 2). Next came 200 nmol of ADP (state 3); the fast rate lasted until all of the added ADP had been phosphorylated, and then the rate fell (state 4). Third came oligomycin, an inhibitor of the Fo portion of ATP synthase. Fourth, the protonophore FCCP was titrated to the concentration giving the highest rate. Last came antimycin A, which blocks Complex III. In parallel runs under identical conditions, the membrane potential (ΔΨ) was estimated from the distribution of a fluorescent cationic dye that accumulates in the matrix to an extent set by ΔΨ.
Oxygen consumption rates are shown in Table 1 and membrane potentials in Figure 1. In fresh mitochondria, 40 nmol of O₂ was consumed during the state 3 interval in which the 200 nmol of ADP was phosphorylated. The respiratory control ratio, defined as the state 3 rate divided by the state 4 rate, was 5.7 for fresh mitochondria and 1.8 for stored mitochondria. The investigators concluded that storage had impaired the coupling of respiration to phosphorylation, and they set out to identify which component of the system was responsible.
| Condition | Fresh | Stored |
|---|---|---|
| State 2 (glutamate + malate) | 12 | 30 |
| State 3 (+ 200 nmol ADP) | 85 | 70 |
| State 4 (ADP used up) | 15 | 38 |
| + Oligomycin | 9 | 33 |
| + FCCP (optimal) | 110 | 95 |
| + Antimycin A | 2 | 2 |
- Fresh
- Stored
Data are illustrative, constructed for practice.
1. Based on the passage, what is the ADP/O ratio (ADP phosphorylated per oxygen atom reduced) of the fresh mitochondria oxidizing glutamate and malate?
- A. Wrong: 0.4 is 80 nmol O ÷ 200 nmol ADP, the ratio inverted (oxygen atoms per ADP rather than ADP per oxygen atom).
- B. Right: 40 nmol O₂ is 80 nmol of oxygen atoms, and 200 nmol ADP ÷ 80 nmol O = 2.5, close to the modern value for NADH-linked substrates.
- C. Wrong: 3.0 is the classic textbook P/O for NADH, recalled rather than calculated from the passage's numbers.
- D. Wrong: 5.0 is 200 ÷ 40, which divides by molecules of O₂ rather than by oxygen atoms.
2. According to Table 1, about how much of the oxygen consumption of fresh mitochondria after oligomycin addition is due to electron transport through the respiratory chain?
- A. Wrong: 2 nmol O₂/min/mg is the rate that survives antimycin A, which is O₂ use that does not pass through the electron transport chain.
- B. Right: subtracting the antimycin-insensitive rate, which does not pass through the chain, gives 9 − 2 = 7 nmol O₂/min/mg.
- C. Wrong: 9 nmol O₂/min/mg is the whole oligomycin rate, which still includes the 2 nmol O₂/min/mg that does not pass through the chain.
- D. Wrong: 13 is the state 4 rate minus the antimycin rate; it describes the system before oligomycin was added, not after.
3. A reviewer proposes that storage lowered the respiratory control ratio by making the inner membrane leakier to protons, not by reducing ATP synthase activity. Which comparison in Table 1 tests this proposal most directly?
- A. Right: With Fo blocked, respiration can only replace protons that leak back across the membrane. A rise from 9 to 33 nmol O₂/min/mg points to more leak, and less ATP synthase activity could not raise this rate.
- B. Wrong: State 3 rates depend on the chain, the leak and ATP synthase together, so this comparison cannot tell the two explanations apart.
- C. Wrong: The antimycin rates are identical in both preparations and measure O₂ use that bypasses the chain, which bears on neither explanation.
- D. Wrong: FCCP rates measure how fast the chain can run with no gradient. They are similar in the two preparations and say nothing about membrane leak versus synthase activity.
4. If the fresh mitochondria were given succinate plus rotenone instead of glutamate and malate, how would the measured ADP/O ratio most likely change?
- A. Wrong: Succinate electrons enter through Complex II, which pumps no protons, and so they pass only Complexes III and IV, not all three pumps.
- B. Wrong: The protons needed per ATP stay the same, but fewer protons are pumped per oxygen atom reduced, so ATP made per O falls.
- C. Wrong: Succinate dehydrogenase does not consume ATP. The direction is right, but the reason is wrong: the ratio falls because Complex II pumps no protons.
- D. Right: Complex II feeds electrons to ubiquinone below Complex I, so each pair pumps about 6 rather than 10 protons and the ADP/O falls from about 2.5 to about 1.5.
5. In fresh mitochondria, adding ADP raised O₂ consumption (Table 1) while slightly lowering ΔΨ (Figure 1). Which explanation best accounts for both observations?
- A. Wrong: ΔΨ is smaller in state 3 than in state 2 or state 4, and a larger gradient would slow the pumps rather than speed them up. This reverses the relationship.
- B. Wrong: Phosphorylation of ADP neither donates nor releases electrons. Electrons come from NADH made by oxidizing the substrates.
- C. Right: ATP synthase lets protons flow back into the matrix, which partly drains the gradient. With less opposing ΔΨ, the pumps and electron flow to O₂ speed up. This is respiratory control.
- D. Wrong: An FCCP-like effect would collapse ΔΨ to about 25 mV and could not be stopped by oligomycin. ADP lowers ΔΨ only a little, and its effect ends once ADP is used up.
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