REPLACEMENT OF LEUCINE-93 BY ALANINE OR THREONINE SLOWS DOWN THE DECAY OF THE N-INTERMEDIATE AND O-INTERMEDIATE 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 almost-equal-to 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.