2. Mitochondria isolated from bovine cardiac muscle, when subjected to sonication, form closed mem- brane vesicles containing the electron transport catalysts and the ATP synthase enzyme located in the inner mitochondrial membrane. A diagrammatic representation of these electron transport particles retaining the capacity for oxidative phosphorylation is given below. NADH NAD+ tion? ATP H₂O BIOS 20200 ADP 1/202 NADH Q₁H₂ NAD+ Complex I Complex III ADP ATP In the diagram protein complexes colored gray indicate that they are inhibited while complexes colored red, green, etc. are catalytically active. Complex IV (a) In Panel A indicate which side of the membrane vesicle faced the matrix and which side faced the intermembrane space prior to sonication. During coupled substrate oxidation which compart- ment will be of higher pH and which of lower pH. Problem Set #3 Fall Quarter 2023 Complex II (b) For the system in Panel A, sonication of mitochondria was carried out in the presence of an excess of ferricytochrome c (i.e., the heme iron is in the ferric state) and Coenzyme Q (CoQ or ubiquinone) enabling both to be trapped into the vesicular cavity upon closing of membrane fragments into spherical vesicles. For oxidation of NADH by O2 coupled to phosphorylation of ADP, in addition to NADH and ADP, what other reagent must be added to the suspension of electron transport particles to observe oxidative phosphorylation and how does this differ from the corresponding process in intact mitochondria?. Why would addition of oligomycin not alter the coupling of oxidation and phosphoryla- Page 2 Question #2, continued: (c) For the system in Panel A, if sonication had been carried out in the absence of ferricyto- chrome c, why would addition of cytochrome c to the suspension of electron transport particles not restore oxidative phosphorylation assuming that all other reagents were present? (Hint: The answer is not simply because of the absence of a critical electron transfer catalyst.)