Examination of the role of subunits and lipids in cytochrome c oxidase based on x-ray crystallographic structure

细胞色素c氧化酶是呼吸链的末端酶，根据生物体的进化阶段，它含有数量可变的亚基。脱氮副球藻的酶含有三个亚基，它们与真核酶的三个线粒体编码的亚基同源。酵母酶包含另外8个核编码亚基，哺乳动物酶包含10个亚基。牛心细胞色素c氧化酶在2.8A分辨率下的晶体结构表明该酶含有8种磷脂。尽管组成不同，但所有这些酶催化的反应基本相同。本研究的目的是从结构和功能两个方面考察附加的亚基、脂质和金属的作用。对脱氮假单胞菌的2个亚基组成的酶进行纯化和结晶。但是，由于对X射线辐射的敏感性，数据的收集并没有成功。对牛心酶的结晶条件进行了改进，得到了X射线衍射分辨率高达1.9A的晶体，并在2.3A分辨率下测定了该酶的晶体结构。该结构除了含有两个亚铁血红素a和三个铜以外，还含有钠/钠位、镁位和锌位。用质谱仪测定了结晶制剂中磷脂的组成和结构。从色谱图和质谱图中鉴定了PC、RE、PG和CL。MAS-MAS分析表明，每种磷脂的脂肪酸结构都比较独特。

Cytochrome c oxidase, the terminal enzyme of the respiratoly chain, contains a variable number of the subunits depending on the evolutionry stage of the organism. The Paracoccus denitrificans enzymes contain three subunits, which are homologous to three mitochondrial-encoded subunits of the eukaryotic enzyme. The yeast enzymes contain additional eight nuclear-encoded subunits, the mammalian enzymes contain ten subunits. The crystal structure of bovine heart cytochrome c oxidase at 2.8 A resolution reveals eight phospholipids in this enzyme. All of thses enzyme catalize essentially same reaction in spite of the differences of the compornents. the aim of the present study is examination of the role of the additional subunits, lipids and metals from both the structures and functions. The p.denitrificas enzyme, composed of 2 subunits, was purified and crystalized. But, the collection of data was not succeeded because of the sensitivity to X-ray irradiation. Crystallization conditions of bovine heart enzyme have been improved to provide crystals which diffract x-rays up to 1.9A resolution, and the crystal structures of this enzyme were determined at 2.3A resolution. This structure reveled Na/Na site, Mg site and Zn site in addition to two hemes a and three cuppers. The composition and Structure of the phosholipids in the crystalline preparation were determined with mass spectrometry. PC,RE,PG and CL were assingned from the chromatographic pattern as well as the mass spectrometry. The MAS-MAS analysis indicate that structure of fatty acids of each phospholipids are fairly unique.