B/B 09 · Electron transport and oxidative phosphorylation
Mitochondria and Their Inhibitors
Use an oxygen electrode experiment to work out how inhibitors and uncouplers change respiration and ATP synthesis.
Passage
In mitochondria, electrons from NADH and FADH₂ pass through a series of protein complexes in the inner membrane to oxygen. The energy released is used to pump protons from the matrix into the intermembrane space, which builds an electrochemical proton gradient. Protons flow back into the matrix through ATP synthase, and the energy of this flow drives the synthesis of ATP from ADP and inorganic phosphate. NADH gives electrons to Complex I, succinate gives electrons through Complex II, and the last step is the transfer of electrons from Complex IV to O₂, which forms water.
Oxidative phosphorylation is regulated by the supply of ADP. When ATP use is low, ADP is scarce, the gradient is high, and the electron transport chain runs slowly. When ATP use rises, ADP builds up and the chain speeds up to meet the demand. In some tissues, such as brown fat, a protein called an uncoupling protein allows protons to leak back across the membrane on purpose, so that the energy of the gradient is released as heat and not used to make ATP.
A researcher studies isolated mitochondria in a closed chamber fitted with an oxygen electrode that measures the rate of O₂ consumption. The mitochondria are suspended in a buffer that contains phosphate and the substrates glutamate and malate, which feed electrons to Complex I through NADH. Additions are made in the order shown in Table 1, and the following measurements are made after each addition: the rate of oxygen consumption, the rate of ATP synthesis, and the size of the proton gradient, which is expressed as a percentage of the largest value.
Table 1. Oxygen consumption, ATP synthesis and proton gradient after each addition.
| Step | Addition | O₂ consumption (nmol/min) | ATP synthesis (nmol/min) | Proton gradient (% of maximum) |
|---|---|---|---|---|
| 1 | Mitochondria + glutamate + malate (no ADP) | 20 | 0 | 100 |
| 2 | ADP | 100 | 300 | 80 |
| 3 | Oligomycin | 25 | 0 | 100 |
| 4 | FCCP | 140 | 0 | 5 |
The researcher then runs a second series with fresh mitochondria in the presence of ADP, glutamate and malate, and measures the same variables after adding inhibitors of different complexes. Table 2 shows the results.
Table 2. Effects of inhibitors on mitochondria that are actively making ATP.
| Condition | O₂ consumption (nmol/min) | ATP synthesis (nmol/min) | Proton gradient (% of maximum) |
|---|---|---|---|
| No inhibitor | 100 | 300 | 80 |
| Rotenone (inhibits Complex I) | 5 | 0 | 10 |
| Rotenone, then succinate added | 80 | 150 | 60 |
| Cyanide (inhibits Complex IV) | 0 | 0 | 0 |
Oligomycin binds to ATP synthase and blocks the channel through which protons flow. FCCP is a lipid-soluble weak acid that can carry protons across the inner mitochondrial membrane. The researcher notes that the amount of mitochondria, the temperature and the volume were the same in all of the experiments. ATP synthesis is measured by following the formation of ATP from a limited amount of added ADP with a light-emitting enzyme assay, and the chamber is sealed so that the only source of oxygen is the dissolved oxygen in the buffer, which is enough for several minutes at these rates.
Questions
Question 1 of 6
In Table 1, oxygen consumption rises from 20 to 100 nmol/min when ADP is added (Step 2). What is the best explanation?
The electrons come from NADH produced from glutamate and malate. ADP does not donate electrons.
ADP acts on ATP synthase, which uses the gradient. It does not control oxygen entry at Complex IV.
The proton gradient fell from 100% to 80% after ADP. A gradient does not pump electrons.
Why the answer is what it is
Without ADP, protons cannot flow back through ATP synthase, so the high gradient slows the chain. When ADP is available, ATP synthase uses the gradient, which falls slightly (100% to 80%), and the chain speeds up. This coupling is called respiratory control.
Review: Electron Transport Chain · Oxidative Phosphorylation · Redox Chemistry & Electron Carriers · The TCA Cycle · Metabolic Regulation & Integration
Question 2 of 6
After oligomycin is added (Step 3), O₂ consumption falls from 100 to 25 nmol/min and ATP synthesis stops. What is the best explanation?
The explanation would not account for the rise in the proton gradient to 100%.
If Complex IV were inhibited, O₂ consumption would fall to zero, as it does with cyanide. Here, the gradient is high (100%).
An uncoupler would collapse the gradient and raise O₂ consumption. Here, the gradient rises and O₂ consumption falls.
Why the answer is what it is
By blocking the proton channel of ATP synthase, oligomycin stops ATP synthesis and the return of protons, so the gradient returns to its maximum. The large gradient makes it harder to pump more protons, and electron flow and O₂ consumption fall back to a low rate.
Review: Electron Transport Chain · Oxidative Phosphorylation · Redox Chemistry & Electron Carriers · The TCA Cycle · Metabolic Regulation & Integration
Question 3 of 6
How does FCCP (Step 4) cause O₂ consumption to rise to 140 nmol/min while ATP synthesis remains zero?
Blocking Complex I would lower O₂ consumption, as with rotenone in Table 2.
The gradient fell to 5%, not rose.
The result in the table is explained by the loss of the gradient, and no donor is needed.
Why the answer is what it is
An uncoupler makes the membrane leaky to protons. The gradient collapses (5%), so nothing restrains the chain and it runs at its maximum rate, but ATP synthase has no gradient to use. The energy released appears as heat.
Review: Electron Transport Chain · Oxidative Phosphorylation · Redox Chemistry & Electron Carriers · The TCA Cycle · Metabolic Regulation & Integration
Question 4 of 6
In Table 2, rotenone lowers O₂ consumption to 5 nmol/min, and adding succinate restores most of it (80). Which conclusion is best supported?
ATP synthesis rose to 150 nmol/min, so the coupling remained intact.
If NADH entered Complex I, rotenone would still block it.
Rotenone blocks Complex I only, and the chain continues to work if electrons enter downstream.
Why the answer is what it is
Succinate is oxidized by Complex II, which passes electrons to the ubiquinone pool, downstream of Complex I. The chain resumes and ATP is made again, so Complex I is the site of the block.
Review: Electron Transport Chain · Oxidative Phosphorylation · Redox Chemistry & Electron Carriers · The TCA Cycle · Metabolic Regulation & Integration
Question 5 of 6
The ATP synthesis rate with succinate after rotenone (150 nmol/min) is lower than the uninhibited rate (300 nmol/min), even though O₂ consumption is similar (80 compared with 100). What is the most likely reason?
ATP synthase does not use succinate.
Rotenone acts on Complex I. Complex IV is not blocked, as shown by the O₂ consumption.
A rise in matrix pH would reduce the gradient and would not raise ATP synthesis.
Why the answer is what it is
Electrons from NADH pass through Complexes I, III and IV, which each pump protons. Electrons from succinate (FADH₂) enter at Complex II, which does not pump protons, so each pair of electrons builds a smaller gradient and makes less ATP.
Review: Electron Transport Chain · Oxidative Phosphorylation · Redox Chemistry & Electron Carriers · The TCA Cycle · Metabolic Regulation & Integration
Question 6 of 6
What is the effect of cyanide on mitochondria that are actively making ATP?
The proton gradient falls to zero with cyanide, which means that proton pumping stopped.
Cyanide gives zero O₂ consumption in Table 2. An uncoupler would increase it.
All of the complexes in the chain are needed for continuous electron flow to oxygen.
Why the answer is what it is
Cyanide blocks the transfer of electrons to O₂ at Complex IV. All of the upstream carriers stay reduced, so electron flow stops, no protons are pumped, the gradient disappears (0%), and ATP synthesis stops.
Review: Electron Transport Chain · Oxidative Phosphorylation · Redox Chemistry & Electron Carriers · The TCA Cycle · Metabolic Regulation & Integration