Effects of substitution of tyrosine 57 with asparagine and phenylalanine on the properties of bacteriorhodopsin.
Tyrosine 57 is one of the residues present in the retinal binding pocket and is conserved in all the halophilic retinal proteins. We have studied mutants of bacteriorhodopsin, expressed in Halobacterium salinarium, in which tyrosine 57 is replaced by an asparagine (Y57N) or phenylalanine (Y57F). In Y57N the photocycle proceeds only up to the L intermediate; no M is formed at neutral pH. The lifetime of L intermediate is extremely long, ca. 500 ms. Proton release is severely affected in both the mutants which suggests that Y57 is associated with the proton release pathway. By comparing the pH-induced absorption changes in the UV in Y57N and Y57F with those in the wild-type (WT), we determined that the pKa of Y57 is 10.2. In Y57F, which shows M formation, the rate constant of the L-->M transition is pH dependent (pKa 8.7) suggesting that Y57 is probably not the residue that normally controls the transition into the alkaline photocycle. Y57 is either part of the counterion complex or in close proximity to D85 since its mutation influences the pKa of Asp85. In Y57F the pKa of D85 is approximately 4.9 (compared to approximately 2.9 in the WT). The Y57N mutant shows two pKa's in the purple to blue transition, approximately 3.8 and < 1. In the presence of hydroxylamine, at neutral pH, Y57N is stable in the dark but bleaches very rapidly upon illumination compared to the WT. Since the lifetime of L intermediate is long in Y57N, we suggest that the Schiff base becomes accessible to hydroxylamine in this state.
Govindjec
R
R
Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign 61801, USA.
Kono
M
M
Balashov
S P
SP
Imasheva
E
E
Sheves
M
M
Ebrey
T G
TG
D001216:Asparagine / Q000378:metabolism*; D001436:Bacteriorhodopsins / Q000737:chemistry / Q000378:metabolism*; D003080:Cold Temperature; D003624:Darkness; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000737:chemistry; D007700:Kinetics; D008027:Light; D010649:Phenylalanine / Q000378:metabolism*; D010777:Photochemistry; D011522:Protons; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine / Q000378:metabolism*
D006898:Hydroxylamines; D011522:Protons; D019811:Hydroxylamine; D014443:Tyrosine; D010649:Phenylalanine; D001436:Bacteriorhodopsins; D001216:Asparagine