Vibrational spectroscopy of bacteriorhodopsin mutants: Evidence that Asp-96 deprotonates during the M → N transition

The role of Asp-96 in the bacteriorhodopsin (bR) photocycle has been investigated by time-resolved and static low-temperature Fourier transform infrared difference spectroscopy. Bands in the time-resolved difference spectra of bR were assigned by obtaining analogous time-resolved spectra from the site-directed mutants Asp-96 → Ala and Asp-96 → Glu. As concluded previously (Braiman, M. S., Mogi, T., Marti, T., Stern, L. J., Khorana, H. G., and Rothschild, K. J. (1988) Biochemistry 27, 8516-8520) Asp-96 is predominantly in a protonated state in the M intermediate. Upon formation of the N intermediate, deprotonation of Asp-96 occurs. This is consistent with its postulated role as a key residue in the reprotonation pathway leading from the cytoplasm to the Schiff base. A broad band centered at 1400 cm^-1, which increases in intensity upon N formation is assigned to the Asp-96 symmetric COO^- vibration. The Asp-96 → Ala mutation also causes a delay in the Asp-212 protonation which normally occurs during the L → M transition. It is concluded that Asp-96 donates a proton into the Schiff base reprotonation pathway during N formation and that it accepts a proton from the cytoplasm during the N → O or O → bR transition.