TIME‐RESOLVED RESONANCE RAMAN SPECTRA OF THE PHOTOCYCLE INTERMEDIATES OF ACID AND DEIONIZED BACTERIORHODOPSIN

Abstract

Abstract— The photocycle of bacteriorhodopsin (bR) and its perturbed forms are investigated by a time‐resolved resonance Raman study. These experiments were performed in the C=C stretching and in the fingerprint spectral regions for the acid blue, acid purple and deionized forms of bR.The main observations are as follows: (1) isomerization of the retinal, from all‐trans to 13‐cis, occurs in native bR and in all of the acid and deionized perturbed bR species; (2) formation of the early intermediates (the K610 and L550 analogues) also occur in native bR and in all of the perturbed species; and (3) deprotonation of the protonated Schiff base (PSB), to give the M412 type intermediate, occurs in native bR, but is inhibited in all of the perturbed bR species on the time‐scale of the native bR photocycle.The results show that isomerization alone is not a prerequisite for the PSB deprotonation process. The observed photocycle, initiated with retinal isomerization, is found to occur from all‐trans to 13‐cis in all of the perturbed forms of bR. In addition, the results imply that removal of the cations, of an increase in the hydrogen ion concentration, prevent only the PSB deprotonation process and not the formation of earlier cycle intermediates. Some attention is focused on the two blue forms of bR (acid and deionized) due to the fact that their ground‐state absorption maximum, unphotolyzed Raman spectra, and Raman spectra changes during the photocycle are all very similar. The similarities between the acid blue and deionized blue forms in the fingerprint region support previous suggestions that both blue species have nearly the same retinal active site.