Fourier transform infrared spectra of a late intermediate of the bacteriorhodopsin photocycle suggest transient protonation of Asp-212.
We measured time-resolved difference spectra, in the visible and the infrared, for the Glu-194 and Glu-204 mutants of bacteriorhodopsin and detected an anomalous O state, labeled O', in addition to the authentic O intermediate, before recovery of the initial state in the photocycle. The O' intermediate exhibits prominent bands at 1712 cm(-1) (positive) and 1387 cm(-1) (negative). These bands arise with the same time constant as the deprotonation of Asp-85. Both bands are shifted to lower frequency upon labeling of the protein with [4-(13)C]aspartic acid. The former band, but not the latter, is shifted in D2O. These shifts identify the two bands as the carboxyl stretch of a protonated aspartic acid and the symmetric carbonyl stretch of an unprotonated aspartate, respectively, and suggest that in O' an initially anionic aspartate enters into protonation equilibrium with Asp-85. Elimination of the few other candidates, on various grounds, identifies Asp-212 as the unknown residue. It is possible, therefore, that in the last step of the photocycle of the mutants studied the proton released from Asp-85 is conducted to the extracellular surface via Asp-212. An earlier report of a weak band at 1712 cm(-1) late in the wild-type photocycle [Zscherp and Heberle (1997) J. Phys. Chem. B 101, 10542-10547] suggests that Asp-212 might play this role in the wild-type protein also.
Dioumaev
A K
AK
Department of Physiology and Biophysics, University of California, Irvine 92697, USA.
Brown
L S
LS
Needleman
R
R
Lanyi
J K
JK
D001224:Aspartic Acid / Q000737:chemistry* / Q000378:metabolism; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid / Q000235:genetics; D005973:Glutamine / Q000235:genetics; D019617:Halobacterium salinarum; D007700:Kinetics; D010782:Photolysis; D017440:Photoperiod; D011522:Protons; D017550:Spectroscopy, Fourier Transform Infrared; D013997:Time Factors
D011522:Protons; D005973:Glutamine; D001224:Aspartic Acid; D018698:Glutamic Acid; D001436:Bacteriorhodopsins