Effects of individual genetic substitutions of arginine residues on the deprotonation and reprotonation kinetics of the Schiff base during the bacteriorhodopsin photocycle.
The rates are determined for the deprotonation and reprotonation of the protonated Schiff base (PSB) as well as of formation and decay of the UV transient in the photocycle of seven bacteriorhodopsin (bR) mutants in which Arg-7, 82, 164, 175, 225, or 227 are replaced by glutamine and Arg-134 by cysteine. The results show that all these mutations increase the rate of deprotonation of the PSB compared to ebR, (wild-type bacteriorhodopsin expressed in Escherichia coli) greatly increase the rate of the reprotonation of the SB (Schiff base) in the case of the Arg-164 and Arg-175 mutations and dramatically decrease this rate in the case of the Arg-227 mutation. Temperature studies on the latter mutant suggest that the observed change in its rate of reprotonation is due to large decrease in the energy and entropy of activation, similar to those observed for Asp-96 mutations (Miller, A. and D. Orsterhelt. 1990. Biochim. Biophys. Acta. 1020:57-64). These results suggest that the reprotonation process is changed to a proton diffusion-controlled mechanism in the Arg-227 mutant due to a change in the structure of the proton channel. The absorption intensity ratio (AUV/AMslow) of each arginine mutant relative to that of ebR is found to be similar to that for native purple membrane (PM) except for the Arg-227 mutant where it is greatly reduced, and for the Arg-82 mutant where it is not observed, suggesting that both Arg-227 and Arg-82 residues somehow play roles in inducing the UV transient absorption. All the above results are discussed in terms of the model for the structure of bR proposed by Henderson, R., J.M. Baldwin, T.A. Ceska, F. Zemlin, E. Beckmann, and K.H. Downing. (1990. J. Mol. Biol. 213:899-929).
Lin
G C
GC
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
el-Sayed
M A
MA
Marti
T
T
Stern
L J
LJ
Mogi
T
T
Khorana
H G
HG
D000595:Amino Acid Sequence; D001120:Arginine; D001436:Bacteriorhodopsins / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006217:Halobacterium / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry; D011994:Recombinant Proteins / Q000378:metabolism / Q000528:radiation effects; D013816:Thermodynamics
D011994:Recombinant Proteins; D001436:Bacteriorhodopsins; D001120:Arginine