Fourier transform Raman spectroscopy of the bacteriorhodopsin mutant Tyr-185-->Phe: formation of a stable O-like species during light adaptation and detection of its transient N-like photoproduct.

Near-infrared FT-Raman spectroscopy can be used to measure the vibrations of the bacteriorhodopsin (bR) chromophore without the disadvantage of conventional visible resonance Raman spectroscopy, where the visible excitation drives the bR photoreactions. We utilized this technique to investigate the light-dark adaptation of bacteriorhodopsin and the mutant Tyr-185-->Phe (Y185F) at room temperature in solution. Compared to wild-type bR, both the FT-Raman and resonance Raman spectra of the light-adapted Y185F displayed new features characteristic of the vibrations of the O intermediate. Light adaptation of Y185F was found to involve a 13-cis, C=N syn-->all-trans isomerization of the retinal chromophore which produces a species similar to bR570 and a second O-like species. Dark adaptation, which was much slower in Y185F compared to wild-type bR, involved a parallel decay of the bR570 and O-like species and resulted in a decreased all-trans:13-cis ratio compared to wild type. Further evidence for the existence of an O-like species in Y185F comes from pump-probe Raman difference spectroscopy, where a red pump beam is found to produce a species very similar to the N intermediate in the photocycle. This species is shown by stroboscopic Raman measurements to exist transiently even at high pH. We postulate that when the Y185F chromophore has an all-trans structure the effective pKa of Asp-85 and Asp-212 is elevated in Y185F due to the disruption of the Asp-212/Tyr-185 hydrogen bond, thereby accounting for the increased protonation of these residues in the O-like species.