Structure and function of two novel respiratory chain complex from Gram-positive thermophilic bacteria

基因编码细胞色素bd-型quinol oxidase完全克隆自Gram-positive thermophilic bacteria Bacillus stearothermophilus。与其他细胞色素的序列进行比较和对齐，因为这些氧化物可以被分成两个亚家族，而B。stearothermophilus enzyme是第二群体的第一成员，它已被孤立和表征。关于细胞色素Bd的前一个结构模型的液压分析指标，主要基于E.的分析。coli enzyme needs to be revised.The genes for cytochrome bo D23-type cytochrome c oxidase were ligated to the shuttle vector pSTE12 and introduced in the wild B. stearothermophilus cells to be over-expressed。产生的氧化酶是纯化的，重新构建成脂质体，并显示出H-D1 + Y-D1泵入活动，而H-D1 + Y-D1/O比率低于细胞色素Caa Y-D23 Y-D2型氧化酶。一根突变细胞在哪一个细胞色素bo中的一根菌株D23-D-2型氧化酶主要操作是孤立的。The Hy D1+ D1 pumping activity and steady-state growth extent of the cells were higher than those of the mutant K17， in which cytochromes bd operates as the main terminal oxidase， and lower than those of the wild cells， in which cytochrome caa D23 mainly operates。以下是关于细胞能量代谢的有效性取决于任何Respiratory Enzyme实际起作用的建议：Furthermore，ctaA基因，定位于细胞色素Caa的上游，D23和D2操作器，从B克隆。stearothermophilus和over-expressed in E.大肠杆菌宿主细胞。CtaA产品在膜准备转换成Heme O到Heme A体外。这一发现是一个直接的证据，因为CtaA是一个同义词。

The genes encoding cytochrome bd-type quinol oxidase were completely cloned from the Gram-positive thermophilic bacteria Bacillus stearothermophilus. Comparison and alignment with sequences reported for other cytochromes bd show that these oxidases can be divided into two subfamilies and that the B. stearothermophilus enzyme is the first member of the second group which has been isolated and characterized. Hydropathic analysis indicates that the previous structural model for cytochrome bd mainly based on the analysis of the E. coli enzyme needs to be revised.The genes for cytochrome boィイD23ィエD2-type cytochrome c oxidase were ligated to the shuttle vector pSTE12 and introduced in the wild B. stearothermophilus cells to be over-expressed. The produced oxidase was purified, reconstituted into liposomes, and showed HィイD1+ィエD1 pumping activity, although the HィイD1+ィエD1/O ratio was lower than that of cytochrome caaィイD23ィエD2-type oxidase. A strain of mutant cells in which cytochrome boィイD23ィエD2-type oxidase mainly operates was isolated. The HィイD1+ィエD1 pumping activity and steady-state growth extent of the cells were higher than those of the mutant K17, in which cytochromes bd operates as the main terminal oxidase, and lower than those of the wild cells, in which cytochrome caaィイD23ィエD2 mainly operates. This finding suggests that the efficiency of cellular energy metabolism depends on which respiratory enzyme actually works.Furthermore, the ctaA gene, located upstream of the cytochrome caaィイD23ィエD2 operon, was cloned from B. stearothermophilus and over-expressed in E. coli host cells. The CtaA product in the membrane preparation converts heme O into heme A in vitro. This finding is a direct evidence to show that CtaA is the heme A synthase.