EVIDENCE FOR A LONG-LIVED 13-CIS-CONTAINING INTERMEDIATE IN THE PHOTOCYCLE OF THE LEU93-]ALA BACTERIORHODOPSIN MUTANT

Previous studies have shown that replacement of Leu 93 by amino acids such as Ala or Thr dramatically slows the rate of completion of the ba cteriorhodopsin photocycle through the formation of a long-lived inter mediate assigned to the O intermediate on the basis of its red-shifted absorption spectrum. Spectroscopic and biochemical studies have impli cated this kinetic defect with a slowing of the reisomerization of ret inal from the 13-cis to the initial all-trans state in the Leu 93 muta nts. These results have suggested that the van der Waals interaction o f Leu 93 with retinal is essential for the rapid thermal reisomerizati on of retinal in the photocycle. Here, this hypothesis is independentl y tested by carrying out time-resolved resonance Raman spectroscopic s tudies of the long-lived intermediate in the photocycle of the Leu 93 --> Ala mutant. Raman difference spectra obtained with a 631 nm probe show a single band in the fingerprint region at 1192 cm(-1) confirming the previous observation of a 13-cis configuration for retinal during the lifetime of the long-lived intermediate. We conclude that a 13-ci s-containing intermediate is accumulated in the late stages of the pho tocycle of the Leu 93 --> Ala mutant, supporting our hypothesis that t he interaction of Leu 93 with retinal is critical for the rapid therma l reisomerization of retinal in the bacteriorhodopsin photocycle.