Question

28 You want to study the biochemical properties of the respiratory enzyme complexes required for oxidative phosphorylation. Your initial goal is to measure proton pumping by the NADH dehydrogenase complex. You mix purified NADH dehydrogenase complex with phospholipids and a pH indicator dye that changes color at acidic pH, and you follow a protocol for making liposomes. After verifying that the protein complex was incorporated into liposomes, you add NADH and incubate at 37°C. You are dismayed to observe no color change in the pH indicator dye. a. One labmate says the result is not surprising, because you forgot to add a key component. What is this key component? Explain why it is important. (6 pts) b. Another labmate suggests that the failure to observe a pH change occurred because the NADH dehydrogenase protein complexes are inserted in the lipid bilayer in both possible orientations. Do you agree that this might prevent the establishment of a pH gradient in your experiment? Explain your reasoning. (6 pts)

Answer 1

NADH dehydrogenase complex catalyzes the transfer of electrons from NADH to coenzyme Q10 (CoQ10) and translocates protons across the inner mitochondrial membrane in eukaryotes or the plasma membrane of bacteria. The key component that was needed for the setup to work was an electron acceptor. With proton translocation, the mechanism will not work.

Again we also see that the correct orientation of NADH dehydrogenase complex is necessary. H+ will only flow from the matrix to IMS as the matrix that has high pH or low H+ concentration and the intermembrane space that contains low pH or high H+ concentration. Therefore, without a proper pH gradient, the complex fails to work. If the complex is in reverse orientation, it would fail to work.

P.S. Please rate this answer (Thumbs Up) if you like it. Thanks.