Coupling of the reisomerization of the retinal, proton uptake, and reprotonation of Asp-96 in the N photointermediate of bacteriorhodopsin.
In the N to O reaction of the bacteriorhodopsin photocycle, Asp-96 is protonated from the cytoplasmic surface, and coupled to this, the retinal isomerizes from 13-cis,15-anti back to the initial all-trans configuration. To dissect the two steps, and to better understand how and why they occur, we describe the properties of two groups of site-specific mutants in which the N intermediate has greatly increased lifetime. In the first group, with the mutations near the retinal, an unusual N state is produced in which the retinal is 13-cis,15-anti but Asp-96 has a protonated carboxyl group. The apparent pK(a) for the protonation is 7.5, as in the wild-type. It is likely that here the interference with N decay is the result of steric conflict of side-chains with the retinal or with the side-chain of Lys-216 connected to the retinal, which delays the reisomerization after protonation of Asp-96. In the second group, with the mutations located near Asp-96 or between Asp-96 and the cytoplasmic surface, reprotonation of Asp-96 is strongly perturbed. The reisomerization of the retinal occurs only after recovery from a long-living protein conformation in which reprotonation of Asp-96 is either entirely blocked or blocked at low pH.
Dioumaev
A K
AK
Department of Physiology & Biophysics, University of California, Irvine, California 92697, USA.
Brown
L S
LS
Needleman
R
R
Lanyi
J K
JK
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001224:Aspartic Acid; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism* / Q000528:radiation effects; D001665:Binding Sites; D018360:Crystallography, X-Ray; D019617:Halobacterium salinarum / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012172:Retinaldehyde / Q000737:chemistry* / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared; D013237:Stereoisomerism
D011994:Recombinant Proteins; D001224:Aspartic Acid; D001436:Bacteriorhodopsins; D012172:Retinaldehyde