Studies on the Signal Transduction Mechanism by Gas Sensor Proteins

本研究制备了DOS和CooA两种气体传感器蛋白的不同突变体，以揭示氧和CO等气体分子触发的信号转导机制。首先，由于发现该蛋白含有六配位低自旋血红素，因此确定了DOS的轴向配体。根据与Dos同源的FixL的结构信息，用Gln取代Dos的Met 95，测定其吸收光谱和共振拉曼光谱。该突变体含有五配位的高自旋血红素，Met 95被确定为Dos的轴向配体之一。另一方面，为了揭示CooA的DNA结合机制，在血红素辅因子附近的氨基酸残基中引入突变，CooA是一种转录因子，可感知CO分压。His 77残基被具有不同极性的Asn、Ser和Arg取代，并且研究了被CO取代的轴向配体的身份。发现这些突变导致五配位血红素的形成，但结合v（Fe-CO）伸缩拉曼谱带对突变不敏感。因此，建议CO替代His 77配体的Pro2残基反式。此外，还对位于CooA中心螺旋上的突变体Cys 105、Leu 112、Ile 113、Ala 114、Leu 116、Gly 117、Arg 118、Leu 120、Tnr 121、Arg 125和Met 131进行了共振拉曼光谱测定，推测这些突变体与信号转导有关。发现L116 Q、G117 N和L120 Q的v（Fe-CO）伸缩频率发生了很大的偏移，推测这些残基的位置与CO气体的结合位置很接近。此外，足迹分析显示，DNA结合活性在这些突变体中丢失。

In this research, various mutants of two gas sensor proteins, DOS and CooA, were prepared in order to reveal signal transduction mechanism triggered by gas molecules such as oxygen and CO. Firstly, axial ligands of Dos were identified since this protein was found to contain six-coordinate low-spin heme. On the basis of structural information of FixL which is homologous to Dos, Met 95 of Dos was replaced by Gln, and absorption and resonance Raman spectra were measured. This mutant was found to contain five-coordinate high-spin heme, and the Met95 was established to be one of the axial ligand of Dos. On the other hand, mutation was introduced to amino acid residues in close proximity of the heme cofactor in order to reveal the DNA binding mechanism of CooA, which is a transcription factor and senses CO partial pressure. His77 residue was replaced by Asn, Ser, and Arg which have different polarity, and identity of the axial ligand replaced by CO was investigated. These mutations were found to result in the formation of five-coordinate heme, but bound v(Fe-CO) stretching Raman band was insensitive to the mutations. Thus, CO was suggested to replace Pro2 residue trans to the His77 ligand. In addition, resonance Raman spectra were measured for the mutants Cys105, Leu112, Ile113, Ala114, Leu116, Gly117, Arg118, Leu120, Tnr121, Arg125, and Met131, which reside on the center helix of CooA and are supposed to concern in the signal transduction. The v(Fe-CO) stretching fiequency were found to shift greatly for L116Q, G117N, and L120Q, and these residues were suggested to locate closely to the bound CO gas. Furthermore, footprint analysis revealed that DNA binding activity was lost in these mutants.