Studies on the Signal Transduction Mechanism by Gas Sensor Proteins

本研究制备了两种气体敏感蛋白DOS和CooA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，确定了Dos的轴向配体，因为该蛋白含有六配位的低自旋血红素。根据与Dos同源的FixL的结构信息，用Gln取代了Dos的Met 95，并测定了其吸收光谱和共振拉曼光谱。该突变体含有5个配位的高自旋血红素，Met95被确定为DOS的轴向配体之一。另一方面，对血红素辅因子附近的氨基酸残基进行突变，以揭示转录因子CooA的DNA结合机制。CooA是一种感受CO分压的转录因子。用不同极性的AsN、Ser和Arg取代His77残基，并考察了CO取代的轴向配体的同一性。这些突变导致了五配位的血红素的形成，但结合的v(Fe-CO)拉曼光谱对突变不敏感。因此，建议用CO取代反式为His77配体的Pro2残基。此外，还测量了位于CooA中心螺旋上的突变体Cys105、Leu112、Ile113、Ala114、Leu116、Gly117、Arg118、Leu120、Tnr121、Arg125和Met131的共振拉曼光谱，推测它们与信号转导有关。L116Q、G117N和L120Q的v(Fe-CO)伸缩频率发生了较大的移动，推测这些残基位于束缚CO气体附近。此外，足迹分析表明，这些突变体的DNA结合活性丢失。

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.