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The study on the structure and function of the transcriptionalregulator containing a heme as a signal sensor

含血红素信号传感器的转录调节因子的结构与功能研究

基本信息

批准号：
12680631
负责人：
AONO Shigetoshi
金额：
$ 2.37万
依托单位：
Japan Advanced Institute of Science and Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

CooA is a heme-containing and CO-sensing transcriptional activator whose activity is regulated by CO. A protoheme acts as a CO sensor in CooA. Mutagenesis and spectroscopic studies revealed the unique coordination structure of the heme in CooA, the Pro^2/Cys^<75>, Pro^2/His^<77>, and CO/His^<77> coordinations for the ferric, ferrous, and CO-bound hemes, respectively. The ligand exchange between Cys^<75> and His^<77> took place during the change in the oxidation state of the heme. The ligand-switching process induced by the reduction of the heme was investigated by the technique of pulse radiolysis. Hydrated electron reduced the heme iron in ferric CooA within 1 μs to form the first intermediate with the Soret peak at 440 nm, suggesting that a six-coordinated ferrous heme with a thiolate axial ligand was formed initially.The first intermediate was converted into the second intermediate with the time constantjof 40 μs (k = 2.5 x 10^4 s^<-1>). In the second intermediate, the thiolate from Cys^<75> was thought to be protonated and/or the Fe-S bond was thought to be elongated. The second intermediate was converted into the final reduced form with the time constant of 2.9 ms (k = 3.5 x 102 s^<-1>) for wild-type CooA. The ligand exchange between Cys^<75> and His^<77> took place during the conversion of the second intermediate into the final reduced form. We constructed a truncated mutant, CooAΔN5, in which the first five residues in the N-terminal were deleted, to examine the functional role of Pro^2 coordination. CooAΔN5 showed a similar spectroscopic and functional properties to those of wild type, indicating the flexibility of the polypeptide chain of CooA in its N-terminal region.

CooA是一种含血红素的CO敏感转录激活剂，其活性受CO的调节。原血红素在CooA中起着CO感受器的作用。诱变和光谱研究揭示了CooA、Pro^2/Cys^&lt；75&gt；、Pro^2/His^&lt；77&gt；和CO/His^&lt；77&gt；；CO/His^&lt；77&gt；；Cys^&lt；75&gt；与His^&lt；77&gt；之间的配体交换发生在血红素氧化态的变化过程中。用脉冲辐解技术研究了血红素还原引起的配基转换过程。水合电子在1μS作用下还原CoOA铁中的血红素铁，生成第一中间体，Soret峰位于440 nm，初步表明生成了六配位含硫键轴向配体的亚铁血红素，转化成第二中间体的时间常数为40μS(k=2.5x10^4 S^-1&gt；)。在第二个中间体中，Cys^&lt；75&gt；的硫酸盐被认为是质子化的和/或Fe-S键被拉长了。对于野生型CooA，第二个中间体被转化为最终的还原形式，时间常数为2.9ms(k=3.5x102CooA)。Cys^&lt；75&gt；和他的^&lt；77&gt；之间的配体交换发生在第二中间体转化为最终还原形式的过程中。我们构建了一个缺失N端前5个残基的截短突变体CooAΔN5，以研究Pro^2配位的功能作用。CooAΔN5显示出与野生型相似的光谱和功能性质，表明CooA的多肽链在其N-末端区域具有灵活性。