The residues Leu 93 and Asp 96 act independently in the bacteriorhodopsin photocycle: studies with the leu 93-->Ala, Asp 96-->Asn double mutant.
Previous mutagenesis studies with bacteriorhodopsin have shown that reprotonation of the Schiff's base is the rate-limiting step in the photocycle of the D96N mutant, whereas retinal re-isomerization and return of the protein to the initial state constitute the rate-limiting events in the photocycle of the L93A mutant. Thus, in the D96N mutant, decay of the M intermediate is slowed down by more than 100-fold at pH 7. In the L93A mutant, decay of the O intermediate is slowed down by 250-fold. We report here that in the L93A, D96N double mutant, decay of the M intermediate, as well as the formation and decay of the O intermediate, are slowed down dramatically. The photocycle is completed by the decay of a long-lived O intermediate, as in the L93A mutant. The decay of the M and O intermediates in the double mutant parallels the behavior seen in the single mutants over a wide temperature and pH range, arguing that the observed independence is an intrinsic property of the mutant. The slow decay of the M and O intermediates can be selectively and independently reversed under conditions identical to those used for the corresponding intermediates in the D96N and L93A single mutants. Because the effects of the two individual mutations are preserved in the double mutant and can be independently reversed, we conclude that residues Asp 96 and Leu 93 act independently and at different stages of the bacteriorhodopsin photocycle. These results also show that formation of the O intermediate only requires protonation of the Schiff's base and is independent of the protonation of Asp 96 from the aqueous medium.
Delaney
J K
JK
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Subramaniam
S
S
D000409:Alanine; D001216:Asparagine; D001224:Aspartic Acid; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism*; D006217:Halobacterium / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007930:Leucine; D008027:Light; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012545:Schiff Bases; D013053:Spectrophotometry; D013997:Time Factors
D011994:Recombinant Proteins; D012545:Schiff Bases; D001224:Aspartic Acid; D001436:Bacteriorhodopsins; D001216:Asparagine; D007930:Leucine; D000409:Alanine