title: Infrared study of the L, M, and N intermediates of bacteriorhodopsin using the photoreaction of M.
abstract: Infrared spectroscopy is used to characterize the transitions in the photocycle of bR involving the M intermediate. It has been shown previously that in this part of the photocycle a large protein conformational change takes place that is important for proton pumping. In this work we separate the spectra of the L, M, and N intermediates in order to better describe the timing of the molecular changes. We use the photoreaction of the M intermediate to separate its spectrum from those of L and N. At temperatures between 220 and 270 K a mixture of M and L or N is produced by illumination with green light. Subsequent blue illumination selectively drives M back into the ground state and the difference between the spectra before and after blue excitation yields the spectrum of M. Below about 250 K and L/M mixture is separated; at higher temperatures an M/N mixture is seen. We find that the spectrum of M is identical in the two temperature regions. The large protein conformational change is seen to occur during the M to N transition. Our results confirm that Asp-96 is transiently deprotonated in the L state. The only aspartic protonation changes between M and bR are the protonation of Asp-85 and Asp-212 that occur simultaneously during the L to M transition. Blue-light excitation of M results in deprotonation of both. The results suggest a quadrupolelike interaction of the Schiff base, Asp-85, Asp-212, and an additional positive charge in bR.
authors.affiliation: Institute of Biophysics, Hungarian Academy of Sciences, Szeged.
authors.forename: P
authors.initials: P
authors.lastname: Ormos
authors.forename: K
authors.initials: K
authors.lastname: Chu
authors.forename: J
authors.initials: J
authors.lastname: Mourant
mesh_terms: D001436:Bacteriorhodopsins / Q000737:chemistry*; D019617:Halobacterium salinarum / Q000737:chemistry*; D010777:Photochemistry; D013055:Spectrophotometry, Infrared
chemical_list: D001436:Bacteriorhodopsins