Exploring the function of Tyr83 in bacteriorhodopsin: features of the Y83F and Y83N mutants.
Tyrosine-83, a residue which is conserved in all halobacterial retinal proteins, is located at the extracellular side in helix C of bacteriorhodopsin. Structural studies indicate that its hydroxyl group is hydrogen bonded to Trp189 and possibly to Glu194, a residue which is part of the proton release complex (PRC) in bacteriorhodopsin. To elucidate the role of Tyr83 in proton transport, we studied the Y83F and Y83N mutants. The Y83F mutation causes an 11 nm blue shift of the absorption spectrum and decreases the size of the absorption changes seen upon dark adaptation. The light-induced fast proton release, which accompanies formation of the M intermediate, is observed only at pH above 7 in Y83F. The pK(a) of the PRC in M is elevated in Y83F to about 7.3 (compared to 5.8 in WT). The rate of the recovery of the initial state (the rate of the O --> BR transition) and light-induced proton release at pH below 7 is very slow in Y83F (ca. 30 ms at pH 6). The amount of the O intermediate is decreased in Y83F despite the longer lifetime of O. The Y83N mutant shows a similar phenotype in respect to proton release. As in Y83F, the recovery of the initial state is slowed several fold in Y83N. The O intermediate is not seen in this mutant. The data indicate that the PRC is functional in Y83F and Y83N but its pK(a) in M is increased by about 1.5 pK units compared to the WT. This suggests that Tyr83 is not the main source for the proton released upon M formation in the WT; however, Tyr83 is involved in the proton release affecting the pK(a) of the PRC in M and the rate of proton transport from Asp85 to PRC during the O --> bR transition. Both the Y83F and the Y83N mutations lead to a greatly decreased functionality of the pigment at high pH because most of the pigment is converted into the inactive P480 species, with a pK(a) 8-9.
Imasheva
E S
ES
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Lu
M
M
Balashov
S P
SP
Ebrey
T G
TG
Chen
Y
Y
Ablonczy
Z
Z
Menick
D R
DR
Crouch
R K
RK
D001436:Bacteriorhodopsins / Q000235:genetics / Q000378:metabolism*; D019617:Halobacterium salinarum / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010782:Photolysis; D010957:Plasmids; D011522:Protons; D014443:Tyrosine / Q000737:chemistry / Q000502:physiology*
D011522:Protons; D014443:Tyrosine; D001436:Bacteriorhodopsins