Replacement of leucine-93 by alanine or threonine slows down the decay of the N and O intermediates in the photocycle of bacteriorhodopsin: implications for proton uptake and 13-cis-retinal----all-trans-retinal reisomerization.
We report that the replacement of Leu-93 in bacteriorhodopsin by Ala (L93A) or Thr (L93T) slows down the photocycle by approximately 100-fold relative to wild-type bacteriorhodopsin. Time-resolved visible absorption spectroscopy and resonance Raman experiments, respectively, show the presence of long-lived O-like and N-like intermediates in the photocycles of the above mutants. We infer the existence of an equilibrium between the N and O intermediates in the photocycles of these mutants. The L93A and L93T mutants exhibit normal proton pumping under continuous illumination, suggesting that the decay of the N and/or O intermediate, and consequently, proton translocation, can be accelerated by the absorption of a second photon. Since the 13-cis----all-trans reisomerization of retinal is completed during the decay of the N and O intermediates, we conclude that the interaction of Leu-93 with retinal is important in this phase of the photocycle. This conclusion is supported by a recent structural model of bacteriorhodopsin that suggests that Leu-93 is near the C-13 methyl group of retinal.
Subramaniam
S
S
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Greenhalgh
D A
DA
Rath
P
P
Rothschild
K J
KJ
Khorana
H G
HG
D000409:Alanine; D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003624:Darkness; D004926:Escherichia coli / Q000235:genetics; D007536:Isomerism; D007700:Kinetics; D007930:Leucine; D008027:Light; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011522:Protons; D011994:Recombinant Proteins / Q000378:metabolism; D012172:Retinaldehyde / Q000378:metabolism*; D012243:Rhodopsin / Q000528:radiation effects; D013053:Spectrophotometry; D013912:Threonine
D011522:Protons; D011994:Recombinant Proteins; D013912:Threonine; D001436:Bacteriorhodopsins; D012243:Rhodopsin; D007930:Leucine; D000409:Alanine; D012172:Retinaldehyde