Biochemistry: The Chemical Reactions of Living Cells, Volume 2 - D. Metzler 1980

On how electrons meet oxygen, how ATR is generated in the process, and some related phenomena
Electron transport chain and oxidative phosphorylation
Reconstitution of phosphorylating particles

Many attempts have been made to resolve and reconstitute submitochondrial phosphorylating particles. The first breakthrough was the removal of several "coupling factors," the most famous of which is coupling factor F₁(Sec. D,1). Removal of factor F₁ from submitochondrial particles invariably leads to the loss of their ability to synthesize ATP, while electron transport remains unaffected. Phosphorylating activity can be restored by adding F₁ back to the membrane preparations. Consequently, F₁ is intimately associated with ATP synthesis. This important protein has been isolated from Cell/35.html">Mitochondria and METABOLISM/14.html">Chloroplasts as homogeneous particles with a Molecular Weight of ~285 000. It apparently consists of five Different types of polypeptide chains with molecular weights of ~60 000, 56 000, 36 000, 17 000, and 13 000 [78, 79]

Solubilization of The inner mitochondrial membrane can be achieved using detergents. For example, Ragan and Racker [80] homogenized mitochondria in a sucrose solution and added the steroid detergent sodium cholate (Fig. 12-16), along with Other Reagents. After gently stirring the mixture and centrifuging, the supernatant was fractionated with ammonium sulfate. A hydrophobic protein fraction was obtained, which was devoid of Phospholipids, cytochrome oxidase, and soluble components such as cytochrome c. Upon adding an appropriate mixture of phospholipids (including phosphatidylethanolamine and phosphatidylcholine) to the hydrophobic protein complex, The ability to phosphorylate ADP during The oxidation of NADH by coenzyme Q₁ was restored. The reconstituted system was sensitive to inhibition by rotenone (Table 10-2), uncoupling agents, and oligomycin. Up to 0.5 mole of ATP was synthesized per mole of NADH oxidized. There are also reports of similar success in reconstituting phosphorylation sites 2 and 3 [81].

Attempts to resolve and reconstitute the Oxidative Phosphorylation system are of paramount importance for designing future experiments. However, significant difficulties arise in interpreting the results. Only a few of the components have been isolated in a completely homogeneous state. These components must be further purified and their properties investigated, while learning to handle each protein in a way that avoids Denaturation. We can probably still hope that the time will come when it will be possible, by mixing many highly purified components of mitochondrial membranes, to reconstitute a functionally active Electron Transport and phosphorylation system. Such experiments will also help answer the question: is a membrane absolutely required for oxidative phosphorylation? Although some researchers believe that past experiments have already shown that an intact membrane may not be necessary, no one has yet succeeded in achieving phosphorylation using truly soluble enzyme preparations. ATP synthesis has been observed only when the appropriate Proteins were incorporated into phospholipid "vesicles".