Matsushita K, Patel L, Kaback H R
Biochemistry. 1984 Sep 25;23(20):4703-14. doi: 10.1021/bi00315a028.
Cytochrome o type oxidase purified from the membrane of Escherichia coli consists of four polypeptides (Mr 66000, 35000, 22000, and 17000), and the monomeric form predominates in octyl beta-D-glucopyranoside. The oxidase complex contains two b-type cytochromes (b-558 and b-563) and 2 mol of heme/mol of enzyme. Cytochrome o utilizes ubiquinol-1 and a number of other artificial electron donors as substrates but does not oxidize reduced cytochrome c or ferrocyanide. Activity is highly dependent upon exogenous phospholipids and/or Tween 20, and the quinone analogues 2-heptyl-4-hydroxyquinoline N-oxide and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole are potent inhibitors. Proteoliposomes were formed by detergent dilution or dialysis in the presence of the oxidase and phospholipids, followed by freeze-thaw/sonication. Vesicles formed by this means are unilamellar and contain a random distribution of 85-90-A intramembranous particles on the convex and concave fracture surfaces. During oxidase turnover, the reconstituted system generates a proton electrochemical gradient (interior negative and alkaline) of -115 to -140 mV; however, respiratory control is minimal (i.e., respiratory control ratios of about 1.5 are observed). By using a glass electrode to measure changes in external pH and the fluorescence of entrapped 8-hydroxy-1,3,6-pyrenetrisulfonate to measure changes in internal pH, it is apparent that during ubiquinol oxidation, protons are released on the external surface of the membrane and consumed on the internal surface. In contrast, with N,N,N',-N'-tetramethyl-p-phenylenediamine, an electron donor that carries few protons at neutral pH, little change in external pH is observed until the protonophore carbonyl cyanide m-chlorophenylhydrazone is added, at which point the medium becomes alkaline. The results taken as a whole are consistent with the concept that oxidase turnover generates an electrical potential (interior negative) due to vectorial electron flow from the outer to the inner surface of the membrane. The pH gradient (interior alkaline), on the other hand, appears to result from scalar (i.e., nonvectorial) reactions that consume and release protons at the inner and/or outer surfaces of the membrane, respectively. In other words, cytochrome o oxidase from Escherichia coli does not appear to catalyze vectorial proton translocation.
从大肠杆菌细胞膜中纯化得到的细胞色素 o 型氧化酶由四种多肽组成（分子量分别为 66000、35000、22000 和 17000），在辛基 β - D - 吡喃葡萄糖苷中单体形式占主导。该氧化酶复合物含有两种 b 型细胞色素（b - 558 和 b - 563），每摩尔酶含有 2 摩尔血红素。细胞色素 o 利用泛醇 - 1 和许多其他人工电子供体作为底物，但不氧化还原型细胞色素 c 或亚铁氰化物。其活性高度依赖于外源磷脂和/或吐温 20，醌类似物 2 - 庚基 - 4 - 羟基喹啉 N - 氧化物和 5 - N - 十一烷基 - 6 - 羟基 - 4,7 - 二氧苯并噻唑是有效的抑制剂。通过在氧化酶和磷脂存在下用去污剂稀释或透析，然后进行冻融/超声处理来形成蛋白脂质体。通过这种方式形成的囊泡是单层的，在凸面和凹面断裂面上含有随机分布的 85 - 90 Å 膜内颗粒。在氧化酶周转过程中，重构系统产生 -115 至 -140 mV 的质子电化学梯度（内部为负且呈碱性）；然而，呼吸控制最小（即观察到的呼吸控制率约为 1.5）。通过使用玻璃电极测量外部 pH 的变化以及用包封的 8 - 羟基 - 1,3,6 - 芘三磺酸盐的荧光测量内部 pH 的变化，很明显在泛醇氧化过程中，质子在膜的外表面释放并在内表面消耗。相比之下，对于 N，N，N'， - N' - 四甲基 - p - 苯二胺，一种在中性 pH 下携带很少质子的电子供体，在加入质子载体羰基氰化物间氯苯腙之前，观察到外部 pH 几乎没有变化，此时介质变为碱性。总体而言，这些结果与以下概念一致，即氧化酶周转由于从膜的外表面到内表面的矢量电子流而产生电势（内部为负）。另一方面，pH 梯度（内部呈碱性）似乎是由分别在膜的内表面和/或外表面消耗和释放质子的标量（即非矢量）反应导致的。换句话说，来自大肠杆菌的细胞色素 o 氧化酶似乎不催化矢量质子转运。
