A redirected proton pathway in the bacteriorhodopsin mutant Tyr-57-->Asp. Evidence for proton translocation without Schiff base deprotonation.
Light-driven proton pumping in bacteriorhodopsin involves deprotonation of the retinylidene Schiff base during M formation and reprotonation during N formation as key steps. This study reports on the spectroscopic characterization of the bacteriorhodopsin mutant Tyr-57-->Asp (Y57D). The results reveal that although formation of the M intermediate and Schiff base deprotonation is blocked, the mutant still exhibits a significant level of light-driven proton translocation. The photocycle of Y57D involves formation of K and L intermediates accompanied by the normal chromophore isomerization and changes in the hydrogen bonding of Asp-96 and Asp-115. However, an additional Asp residue deprotonates during formation of the L intermediate along with a transmembrane alpha-helical structural change that normally occurs upon N formation. We postulate that proton transport in Y57D occurs through a redirected pathway that does not involve the deprotonation of the Schiff base. Chromophore isomerization, which normally results in the transfer of a proton from the Schiff base to Asp-85, instead causes the deprotonation of Asp-57 in Y57D, most likely through an interaction involving Asp-212. This deprotonation of Asp-57 causes the release of a proton into the extracellular medium. Reprotonation of Asp-57 occurs through the Schiff base reprotonation pathway, which consists of a hydrogen-bonded network of residues spanning from Asp-96 to Asp-212. The results also indicate that the transmembrane alpha-helical structural changes observed during N formation (Rothschild, K.J., Marti, T., Sonar, S., He, Y.W., Rath, P., Fischer, W., Bousche, O., and Khorana, H. G. (1993) J. Biol. Chem. 268, 27046-27052) do not require deprotonation of Asp-96 or of the Schiff base.
Sonar
S
S
Physics Department, Boston University, Massachusetts 02215.
Marti
T
T
Rath
P
P
Fischer
W
W
Coleman
M
M
Nilsson
A
A
Khorana
H G
HG
Rothschild
K J
KJ
D001224:Aspartic Acid / Q000378:metabolism; D001436:Bacteriorhodopsins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D009154:Mutation; D011522:Protons; D012545:Schiff Bases / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman; D013696:Temperature
D011522:Protons; D012545:Schiff Bases; D001224:Aspartic Acid; D001436:Bacteriorhodopsins