COMPLETE IDENTIFICATION OF C=O STRETCHING VIBRATIONAL BANDS OF PROTONATED ASPARTIC-ACID RESIDUES IN THE DIFFERENCE INFRARED-SPECTRA OF M-INTERMEDIATE AND N-INTERMEDIATE VERSUS BACTERIORHODOPSIN

Fourier transform infrared difference spectra were obtained for the M and N intermediates versus light-adapted bacteriorhodopsin (BR) with site-directed mutant proteins in which aspartic acid residues at positions 96 and 115 were replaced by asparagine. The positive and negative bands at 1740 and 1732 cm(-1) in the M/BR spectrum are shown to be the superposition of bands due to C=O stretching vibrations of Asp-96 and Asp-115 (a positive band at 1736 cm(-1) and a negative band at 1742 cm(-1) of Asp-96, and a positive band at 1742 cm(-1) and a negative band at 1734 cm(-1) of Asp-115). The positive band at 1738 cm(-1) and the negative band at 1734 cm(-1) in the N/BR spectrum are attributed to Asp-115. On the basis of these results, Asp-115 is protonated in M and N as well as in the ground state. On the other hand, no bands corresponding to Asp-212 were found in the region of protonated carboxylic acid vibration, indicating that Asp-212 remains unprotonated in M and N. The frequencies of the C=O stretching modes of protonated Asp-96 and Asp-115 change in the opposite direction in the BR-to-M conversion relative to the shifts in the BR-to-L conversion, indicating different environmental changes for these residues in L and M.