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

细胞色素c氧化酶是细胞色素C链的末端酶，根据生物体的进化阶段，其亚基的数目是可变的。副球菌的酶含有三个亚基，这是同源的真核酶的三个蛋白质编码的亚基。酵母酶含有另外八个核编码亚基，哺乳动物酶含有十个亚基。牛心细胞色素c氧化酶在2.8 A分辨率的晶体结构揭示了这种酶中的八种磷脂。尽管这些酶的组分不同，但它们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检查额外的亚基、脂质和金属的作用。对由2个亚基组成的P. acetificas酶进行了纯化和结晶。但是，由于对X射线照射的敏感性，数据的收集没有成功。本文改进了牛心酶的结晶条件，使其晶体对X射线的分辨力达到1.9A，并在2.3A的分辨力下测定了该酶的晶体结构。该结构除含有两个血红素a和三个铜外，还含有Na/Na位、Mg位和Zn位。用质谱法测定了晶体制剂中磷脂的组成和结构。从色谱图和质谱图中鉴定出PC、RE、PG和CL。MAS-MAS分析表明，每种磷脂的脂肪酸结构都相当独特。

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.