Structure and function of the heme-based gas sensor proteins

基于血红素的气体传感器蛋白的结构和功能

基本信息

批准号：
14380303
负责人：
AONO Shigetoshi
金额：
$ 9.73万
依托单位：
Okazaki National Research Institutes
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14380303/
关键词：
Sensor Protein Aerotaxis Control System Hemoprotein Oxygen Sensor protein

项目摘要

HemAT-Bs is a heme-containing signal transducer protein responsible for aerotaxis of Bacillus subtilis. The recombinant HemAT-Bs expressed in Escherichia coli was purified as the oxy form in which oxygen was bound to the ferrous heme. Oxygen binding and dissociation rate constants were determined to be kon = 32 μM^<-1> s^<-1> and koff = 23^<-1>, respectively, revealing that HemAT-Bs has a moderate oxygen affinity similar to that of sperm whale myoglobin (Mb). The rate constant for autoxidation at 37 ℃ was 0.06 h^<-1>, which is also close to that of Mb. Although the electronic absorption spectra of HemAT-Bs were similar to those of Mb, HemAT-Bs showed some unique characteristics in its resonance Raman spectra. Oxygen-bound HemAT-Bs gave the Fe-0_2 band at a noticeably low frequency (560 cm^<-1>), which suggests a unique hydrogen bonding between a distal amino acid residue and the proximal atom of the bound oxygen molecule. Deoxy HemAT-Bs gave the Fe-His band at a higher frequency (225 cm<-1>) than those of ordinary His-coordinated deoxy heme proteins. CO-bound HemAT-Bs gave the Fe-CO and C-O bands at 494 and 1964 cm1, respectively, which fall on the same C-O versus Fe-CO correlation line as that of Mb. Based on these results, the structural and functional properties of HemAT-Bs has been elucidated.In this work, a new CooA homologue from C.hydrogenoformans (Ch-CooA) was also studied. Ch-CooA also contains a protoheme as the active site for sensing CO. The heme in Ch-CooA is six-coordinate, low spin state in the ferric, ferrous and CO-bound forms. The heme environmental structure of Ch-CooA was elucidated by mutagenesis and spectroscopic studies.

HemAT-Bs 是一种含血红素的信号转导蛋白，负责枯草芽孢杆菌的趋气性。在大肠杆菌中表达的重组 HemAT-Bs 被纯化为氧形式，其中氧与亚铁血红素结合。确定为 kon = 32 μM^<-1> s^<-1> 且 koff = 23^<-1>，分别揭示HemAT-Bs具有与抹香鲸肌红蛋白(Mb)相似的中等氧亲和力，37℃下的自氧化速率常数为0.06h^<-1>，这也是尽管HemAT-Bs的电子吸收光谱与Mb相似，但HemAT-Bs的共振拉曼光谱显示出一些独特的特征。氧结合的HemAT-B以明显低的频率(560cm^-1)给出Fe-0_2带，这表明结合的氧分子的远端氨基酸残基和近端原子之间存在独特的氢键。 HemAT-Bs以比普通His配位的CO结合的HemAT-Bs更高的频率(225cm -1 )给出Fe-His带。分别给出了 494 和 1964 cm1 处的 Fe-CO 和 C-O 谱带，它们与 Mb 落在相同的 C-O 与 Fe-CO 相关线上。基于这些结果，HemAT-B 的结构和功能特性已得到阐明。在这项工作中，还研究了来自 C.hydrogenoformans 的新 CooA 同源物 (Ch-CooA)，Ch-CooA 也含有原血红素作为传感活性位点。 Ch-CooA 中的血红素是六配位、低自旋态，呈三价铁、二价铁和 CO 结合形式。Ch-CooA 的血红素环境结构通过诱变和光谱研究得以阐明。