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+ H₂O tion? 1/202 Q₁H₂ NADH- NAD+ Complex I Complex III ADP Complex IV Complex II ATP ADP ATP In the diagram protein complexes colored gray indicate that they are inhibited while complexes colored red, green, etc. are catalytically active. (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. (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- 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.) (d) For the system in Panel B, particles were prepared as in Question (b) to measure the stoichiometry of protons pumped only by Complex I /ATP synthesized. As seen in the diagram, CoQ was present to receive electrons from Complex I. While Complex III was inhibited, it was found neces- sary to also inhibit Complex IV with cyanide. In order to measure the stoichiometry of protons pumped by only Complex I/ ATP synthesized, explain why was it necessary to inhibit Complex IV? To which cytochrome does cyanide bind?

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+ H₂O tion? 1/202 Q₁H₂ NADH- NAD+ Complex I Complex III ADP Complex IV Complex II ATP ADP ATP In the diagram protein complexes colored gray indicate that they are inhibited while complexes colored red, green, etc. are catalytically active. (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. (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- 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.) (d) For the system in Panel B, particles were prepared as in Question (b) to measure the stoichiometry of protons pumped only by Complex I /ATP synthesized. As seen in the diagram, CoQ was present to receive electrons from Complex I. While Complex III was inhibited, it was found neces- sary to also inhibit Complex IV with cyanide. In order to measure the stoichiometry of protons pumped by only Complex I/ ATP synthesized, explain why was it necessary to inhibit Complex IV? To which cytochrome does cyanide bind?

Biochemistry

6th Edition

ISBN:9781305577206

Author:Reginald H. Garrett, Charles M. Grisham

Publisher:Reginald H. Garrett, Charles M. Grisham

Chapter21: Photosynthesis

Section: Chapter Questions

Problem 5P: The Relative Efficiency of ATP Synthesis in Noncyclic versus Cyclic Photophosphorylation If...

See similar textbooks