Roles of Ser130 and Thr126 in chloride binding and photocycle of pharaonis halorhodopsin.
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.
Sato
Maki
M
Division of Biological Sciences, Graduate School of Science, Hokkaido University, 060-0810.
Kikukawa
Takashi
T
Araiso
Tsunehisa
T
Okita
Hirotaka
H
Shimono
Kazumi
K
Kamo
Naoki
N
Demura
Makoto
M
Nitta
Katsutoshi
K
D000042:Absorption; D019943:Amino Acid Substitution; D002712:Chlorides / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D025602:Halorhodopsins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D019633:Natronobacterium / Q000737:chemistry*; D010507:Periodicity; D010777:Photochemistry; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D013053:Spectrophotometry / Q000379:methods; D013912:Threonine / Q000235:genetics / Q000378:metabolism*
D002712:Chlorides; D025602:Halorhodopsins; D011994:Recombinant Proteins; D013912:Threonine; D012694:Serine