FTIR Difference Spectroscopy of the Bacteriorhodopsin Mutant Tyr-185→Phe: Detection of a Stable O-like Species and Characterization of Its Photocycle at Low Temperature

Fourier transform infrared difference spectroscopy has been used to study the photocycle of the mutant Tyr-185→Phe expressed in native Halobacterium halobium and isolated as intact purple membrane fragments. We find several changes in the low-temperature bR→K, bR→L, and bR→M FTIR difference spectra of Y185F relative to wild-type bR which are not directly related to the absorption bands associated with Tyr-185. We show that these features arise from the photoreaction of a stable red-shifted species (O^Y185F) with a vibrational spectrum similar to the O intermediate. By using photoselection and FTIR spectroscopy, we have been able to characterize the photoproducts of this O^Y185F species. A K-like photoproduct is formed at 80 K which has a 13-cis structure. However, it differs from K₆₃₀, exhibiting an intense band at 990 cm⁻¹ most likely due to a hydrogen-out-of-plane vibrational mode of the chromophore. At 170 and 250 K, photoexcitation of O^Y185F produces an intermediate with vibrational features similar to the N intermediate in the wild-type bR photocycle. However, no evidence for an M-like intermediate is found. Although Asp-96 undergoes a change in its environment/protonation state during the O^Y185F photocycle, no protonation changes involving Asp-85 and Asp-212 were detected. These results provide strong evidence that light adaptation of Y185F produces two species similar to bR₅₇₀ and the O intermediate. Differences in their respective photocycles can be explained on the basis of differences in the protonation states of the residues Asp-85 and Asp-212 which are ionized in bR₅₇₀ and undergo net protonation upon Q^Y185F formation.