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 是一种含有血红素的 CO 感应转录激活剂，其活性受 CO 调节。原血红素在 CooA 中充当 CO 传感器。诱变和光谱研究揭示了 CooA 中血红素的独特配位结构，即 Pro^2/Cys。 ^<75>、Pro^2/His^<77> 和 CO/His^<77> 配位，用于铁、亚铁和CO结合的血红素之间的配体交换分别发生在血红素氧化态的变化期间。由血红素还原诱导的配体转换过程被研究。脉冲放射分解技术在 1 μs 内还原了 CooA 铁中的血红素铁，形成第一个中间体，索雷峰位于 440 nm，表明六配位亚铁最初形成带有硫醇盐轴向配体的血红素。第一个中间体以40 μs (k = 2.5 x 10^4 s^-1>)的时间常数转化为第二个中间体。在第二个中间体中，来自Cys的硫醇盐。 ^<75>被认为是质子化的和/或Fe-S键被认为是延长的。第二中间体被转化为时间常数为2.9的最终还原形式。 ms (k = 3.5 x 102 s^<-1>) 对于野生型 CooA。 Cys^<75> 和 His^<77> 之间的配体交换发生在第二个中间体转化为最终还原形式的过程中。我们构建了一个截短的突变体 CooAΔN5，其中 N 端的前 5 个残基被删除，以检查 Pro^2 配位的功能作用，CooAΔN5 显示出类似的光谱。以及与野生型相比的功能特性，表明 CooA 的多肽链在其 N 末端区域具有灵活性。