Cytochrome oxidase in thermophilic bacterium PS3 -Crystalization, First and Higher order structure, and H^+ pump activity-

1. 从嗜热细菌PS3的EMBL-3文库中克隆细胞色素氧化酶亚基基因并进行测序。这些基因按亚基II、I、III和IV的顺序形成了一个操纵子。将每个亚基的氨基酸序列与线粒体和反硝化副球菌的氨基酸序列进行比较，我们可以得到重要氨基酸残基的位置和三维结构的粗略图像。对酶的结晶进行了试验。但在母液中加入辛基葡萄糖苷和庚烷三醇时，只出现小结晶，不适合结晶学分析。测定了PS3细胞色素氧化酶靶大小与氧化酶和质子泵活性的关系。二聚体结构可能是质子泵送所必需的，而单体可以介导氧化。该酶以单体形式存在于几种非离子洗涤剂溶液中。二聚体酶可以在磷脂床上形成。在细胞色素氧化酶水平上测量质子泵活性的几种方法已经开发出来，并应用于各种细菌的几种制剂。在查阅了几篇有关这一主题的论文后，对细菌中产生电化学质子梯度的呼吸复合物进行了综述。细胞色素氧化酶表达系统的建立将使细胞色素氧化酶结构与功能之间的关系得到进一步的证实。

1. The genes for subunits of cytochrome oxidase were cloned from a library in EMBL-3 of the thermophilic bacterium PS3 and sequenced. The genes formed an operon in the order of subunits II,I,III and IV. Comparison of the amino acid sequences of each subunit with those from mitochondria and paracoccus denitrificans tells us places of important amino acid residues and a rough image of three-dimensional structure.2. Crystalization of the enzyme was tried. However, only small crystals seemed to appear, when octylglucoside and heptane triol were added to the mother liquor, which was not suitable for crystalography.3. Target size of PS3 cytochrome oxidase in respect to both oxidase and proton pump activities were measured. Dimeric structure may be necessary for proton pumping, whereas the monomer can mediate oxidation. The enzyme exist as monomer in several non-ionic detergent solution. Dimeric enzyme may be vormed in the bed of phospholipids.4. Several methods to measure proton pump activity at the cytochrome oxidase-level have been developed, and applied on several preparations from various bacteria. After consulting several papers on this topics, a review on respiratory complexes in bacteria generating electrochemical proton gradients have been written,5. Introduction of an expression system will make it possible to confirm putative relationship between structure and function in cytochrome oxidase.