The retinal Schiff base-counterion complex of bacteriorhodopsin: changed geometry during the photocycle is a cause of proton transfer to aspartate 85.
Bacteriorhodopsin contains all-trans-retinal linked via a protonated Schiff base to K216. The proton transport in this pump is initiated by all-trans to 13-cis photoisomerization of the retinal and the ensuing transfer of the Schiff base proton to D85. Changed geometrical relationship of the Schiff base and D85 after the photoisomerization is a possible reason for the proton transfer. We introduced small volume/shape changes with site-specific mutagenesis of residues V49 and A53 that contact the side chain of K216, in order to force the Schiff base into somewhat different positions relative to D85. Earlier [Zimányi, L., Váró, G., Chang, M., Ni, B., Needleman, R., & Lanyi, J. K. (1992) Biochemistry 31, 8535-8543] we had described the kinetics of absorbance changes in the microsecond to millisecond time range after photoexcitation with the scheme L<-->M1<-->M2 + H+ (where the first equilibrium is the internal proton transfer and the second is proton release on the extracellular surface). Testing it at various pH values with mutants, where selected rate constants are changed, now confirms the validity of this scheme. The kinetics of the M state thus allowed examination of the transient equilibrium that develops in the L<-->M1 reaction and represents the redistribution of the proton between the Schiff base and D85. From the structure of the protein, the V49A and V49M residue replacements were both predicted to cause decreased alignment of the Schiff base and D85, and indeed we found that they both changed the equilibrium toward the protonated Schiff base. In contrast, the residue replacements A53V and A53G were predicted to move the Schiff base in opposite directions, away from and closer to alignment with D85, respectively. The former indeed changed the equilibrium toward the protonated Schiff base and the latter toward the deprotonated Schiff base. In addition, the hydroxyl stretch band of a bound water in the L state was affected by all mutations that disfavor proton transfer to D85. We conclude that the geometry of the proton donor and acceptor in the Schiff base-D85 pair, mediated by bound water, is a determinant of the proton transfer equilibrium.
Brown
L S
LS
Department of Physiology and Biophysics, University of California, Irvine 92717.
Gat
Y
Y
Sheves
M
M
Yamazaki
Y
Y
Maeda
A
A
Needleman
R
R
Lanyi
J K
JK
D001224:Aspartic Acid / Q000737:chemistry* / Q000235:genetics; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D001692:Biological Transport; D019617:Halobacterium salinarum / Q000737:chemistry / Q000235:genetics / Q000254:growth & development; D006863:Hydrogen-Ion Concentration; D016014:Linear Models; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D017440:Photoperiod; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics; D017494:Proton Pumps; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012160:Retina / Q000378:metabolism; D012545:Schiff Bases / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared; D013237:Stereoisomerism
D045322:Photosynthetic Reaction Center Complex Proteins; D017494:Proton Pumps; D011994:Recombinant Proteins; D012545:Schiff Bases; D001224:Aspartic Acid; D001436:Bacteriorhodopsins