Molecular Construction of Cytochrome bc_1 and b_6/f Complexes

1.建立了从牛心线粒体中分离细胞色素BC_1复合体的新方法。该方法缩短了实际工作周期，提高了成品率。然而，在X-射线衍射分析中，几次使所制备的络合物结晶的尝试都没有成功。Euglena细胞色素BC_1复合体是由其亚线粒体颗粒制备的。该复合体的亚基组成与其他来源的相似，含有细胞色素b-和c1、铁硫蛋白以及其他几个亚基。在553&gt；553&gt；中发现细胞色素c-1的非典型的血红素结合模式。C-1多肽的N端序列研究表明，第一个半胱氨酸残基被苯丙氨酸取代，血红素-C只通过一个硫醚键与半胱氨酸结合。分离了一个与细胞色素c&lt；@D5-@&gt；D5&lt；@D21@&gt；d1多肽对应的基因，并测定了其全序列，推导出Euglena细胞色素&lt；@D5-@&gt；D5&lt；@D21@&gt；D2。该成熟蛋白由243个氨基酸组成，并证实了上述替换。此外，该序列与其他序列的比较也揭示了在细胞色素c&lt；@D5-@&gt；D5&lt；@D21@&gt；D2.4中常见的功能重要区域。利用基因工程技术，确定了酵母细胞色素c-1功能和组装的重要区域。(继续到另一页)

1. A new method was developed for the isolation of cytochrome bc_1 complex from beef heart mitochondria. This method shortens the practical working period and raises the yield of the complex. However, several trials to crystallize the complex thus prepared were unsuccessful in X-ray diffraction analysis.2. Euglena cytochrome bc_1 complex was prepared from its submitochondrial particles. The subunit composition of this complex was similar to those from other sources containing cytochromes b__- and c__-_1 and iron-sulfur protein with other several subunits. However, the -peak of cytochrome c__-_1 was found at 561nm and that of pyridine ferror=hemechrome at 553nm, indicating atypical heme-binding mode of euglena cytochrome c__-_1 as found in Euglena cytochrome c_<553>. The N-terminal sequence studies of the c__-_1 polypeptide showed that the first cysteine residue was replace by phenylalanine and the heme-c was bound to a cysteine through only a single thioether bond.3. A cDNA corresponding to cytochrome c<@D5-@>D5<@D21@>D1 polypeptide was isolated and its total seq=uence was determined to deduce the amino acid sequence of Euglena cytochrome <@D5-@>D5<@D21@>D2. The mature protein was composed of 243 amino acids and the replacement mentioned above was confirmed. Also the comparison of this sequence with others revealed the functionally important regions commonly found in cytochromes c<@D5-@>D5<@D21@>D2.4. Using gene engineering technique the important regions for function and assembly of yeast cytochrome c__-_1 were identified. (continues to a separate page)