Fourier Transform Infrared Spectra of a Late Intermediate of the Bacteriorhodopsin Photocycle Suggest Transient Protonation of Asp-212†

摘要: We measured time-resolved difference spectra, in the visible and infrared, for Glu-194 Glu-204 mutants of bacteriorhodopsin detected an anomalous O state, labeled O', addition to authentic intermediate, before recovery initial state photocycle. The O' intermediate exhibits prominent bands at 1712 cm(-1) (positive) 1387 (negative). These arise with same time constant as deprotonation Asp-85. Both are shifted lower frequency upon labeling protein [4-(13)C]aspartic acid. former band, but not latter, is D2O. shifts identify two carboxyl stretch a protonated aspartic acid symmetric carbonyl unprotonated aspartate, respectively, suggest that initially anionic aspartate enters into protonation equilibrium Elimination few other candidates, on various grounds, identifies Asp-212 unknown residue. It possible, therefore, last step photocycle studied proton released from Asp-85 conducted extracellular surface via Asp-212. An earlier report weak band late wild-type [Zscherp Heberle (1997) J. Phys. Chem. B 101, 10542-10547] suggests might play this role also.