Uncoupling and the Proton Circuit in a Respiring Vesicle
An investigator reconstitutes a bacterial respiratory chain into lipid vesicles: a NADH dehydrogenase, a quinone pool, and a terminal oxidase that reduces O2. NADH is added outside the vesicles. The vesicles accumulate protons internally, and a membrane potential (inside negative) develops. When the protonophore FCCP is added, O2 consumption rises about 3-fold and the proton gradient collapses. When valinomycin (K+ ionophore) is added in the presence of external K+, the membrane potential collapses but the pH gradient increases and O2 consumption rises modestly. Adding both FCCP and valinomycin does not raise respiration further than FCCP alone.
1. Explain why FCCP stimulates O2 consumption while valinomycin produces only a modest increase.
Read the worked answer
Respiration is limited by the back-pressure of the protonmotive force (pmf = membrane potential plus pH gradient). FCCP carries protons across the membrane, dissipating both components of pmf, so the respiratory chain runs faster-hence the ~3-fold rise. Valinomycin is a K+ carrier, not a proton carrier: it collapses the electrical component (membrane potential) by allowing K+ to move down its gradient. But because the chain continues to pump protons, the chemical component (delta-pH) builds up; the pmf is redistributed rather than fully dissipated. With only one component removed, back-pressure remains, so the respiratory stimulation is modest. The pmf is conserved in the pH term, which is why valinomycin cannot match FCCP.
2. The observation that FCCP plus valinomycin gives no more respiration than FCCP alone is used to argue that the two agents act on different components of pmf. Evaluate this argument.
Read the worked answer
The argument is consistent but not uniquely diagnostic. FCCP alone already collapses the proton gradient, which in turn eliminates the membrane potential (the two components are interconvertible through the proton circuit: if delta-pH is zero, continued pumping cannot sustain a potential because protons equilibrate). Once pmf is fully dissipated by FCCP, there is no remaining back-pressure for valinomycin to relieve, so no additional stimulation is expected-regardless of whether valinomycin's target is distinct. The result therefore shows that FCCP is sufficient to remove all back-pressure, which is what you would predict if pmf has two components and FCCP collapses both. To prove the components are separable you need the valinomycin-alone result (potential down, delta-pH up, modest respiration), which is the informative arm.