Sato Maki, Kikukawa Takashi, Araiso Tsunehisa, Okita Hirotaka, Shimono Kazumi, Kamo Naoki, Demura Makoto, Nitta Katsutoshi
Division of Biological Sciences, Graduate School of Science, Hokkaido University, 060-0810.
J Biochem. 2003 Jul;134(1):151-8. doi: 10.1093/jb/mvg123.
Pharaonis halorhodopsin (phR) is an inward light-driven chloride ion pump in Natronobacterium pharaonis. In order to clarify the roles of the Ser130(phR) and Thr126(phR) residues, which correspond to Ser115(shR) and Thr111(shR) of salinarum hR (shR), with regard to their Cl(-)binding affinity and the photocycle, the wild-type phR, and S130 and T126 mutants were expressed in Escherichia coli cells. The photocycles of the wild-type phR, and S130 and T126 mutants were investigated in the presence of 1 M NaCl. Based on results of kinetic analysis involving singular value decomposition and global fitting, typical photointermediates K, L and O were identified, and the kinetic constants of decay or formation varied depending on the mutant. The photocycle scheme was linear for the wild-type phR, and S130C, S130T and T126V mutants. On the other hand, the S130A mutant showed a branched pathway between the L-hR and L-O steps. The present study revealed the following two facts with respect to the Ser130(phR) residue: 1) The OH group of this residue is important for Cl(-) ion binding next to the Schiff base nitrogen, and 2) replacement of an Ala residue, which is unable to form a hydrogen bond, results in a branched photocycle. The implication of this branching was discussed.
法老嗜盐菌视紫红质（phR）是法老嗜盐菌中一种内向光驱动的氯离子泵。为了阐明与盐沼嗜盐菌视紫红质（shR）的Ser115（shR）和Thr111（shR）相对应的Ser130（phR）和Thr126（phR）残基在氯离子结合亲和力和光循环方面的作用，在大肠杆菌细胞中表达了野生型phR以及S130和T126突变体。在1 M NaCl存在的情况下研究了野生型phR以及S130和T126突变体的光循环。基于涉及奇异值分解和全局拟合的动力学分析结果，鉴定出了典型的光中间体K、L和O，并且衰变或形成的动力学常数因突变体而异。野生型phR以及S130C、S130T和T126V突变体的光循环方案是线性的。另一方面，S130A突变体在L-hR和L-O步骤之间显示出分支途径。本研究揭示了关于Ser130（phR）残基的以下两个事实：1）该残基的OH基团对于席夫碱氮旁边的Cl(-)离子结合很重要，2）替换不能形成氢键的Ala残基会导致分支光循环。讨论了这种分支的含义。
