J. Biochem, 2003, Vol. 134, No. 1 151-158
© 2003 Japanese Biochemical Society
BIOCHEMISTRY |
Roles of Ser130 and Thr126 in Chloride Binding and Photocycle of pharaonis Halorhodopsin
1 Division of Biological Sciences, Graduate School of Science, Hokkaido University, 060-0810; 2 Center for Advanced Science and Technology, Hokkaido University, 001-0021; and 3 Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812
Pharaonis halorhodopsin (phR) is an inward light-driven chloride ion pump in Natronobacterium pharaonis. In order to clarify the roles of the Ser130phR and Thr126phR residues, which correspond to Ser115shR and Thr111shR 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 Ser130phR 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.
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