Puettner I, Carafoli E, Malatesta F
J Biol Chem. 1985 Mar 25;260(6):3719-23.
Beef heart cytochrome c oxidase has been depleted of subunit III by treatment with chymotrypsin. The removal of subunit III has been evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel fluorography of preparations of the oxidase labeled with [14C]dicyclohexylcarbodiimide prior to proteolysis. Removal of subunit III resulted in a perturbation of the visible spectrum of reduced cytochrome oxidase. Subunit III-depleted oxidase is spectroscopically very similar to the oxidase from Paracoccus denitrificans. When reconstituted into liposomes, the depleted enzyme still pumped protons in response to a pulse of reduced cytochrome c. The H+/e- stoichiometry averaged 0.5. Redox-linked proton translocation could be observed only when respiratory control ratios were higher than 3 and the reductant pulse was of a magnitude that allowed for no more than 5 turnovers of the oxidase.
用胰凝乳蛋白酶处理牛肉心细胞色素c氧化酶，可使其亚基III缺失。在蛋白水解之前，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和对用[14C]二环己基碳二亚胺标记的氧化酶制剂进行凝胶荧光显影，评估亚基III的去除情况。亚基III的去除导致还原型细胞色素氧化酶可见光谱发生扰动。缺失亚基III的氧化酶在光谱上与反硝化副球菌的氧化酶非常相似。当重新组装到脂质体中时，缺失的酶仍能响应还原型细胞色素c的脉冲而泵出质子。H+/e-化学计量平均为0.5。只有当呼吸控制率高于3且还原剂脉冲的幅度允许氧化酶周转不超过5次时，才能观察到氧化还原相关的质子转运。
