Detection of threonine structural changes upon formation of the M-intermediate of bacteriorhodopsin: Evidence for assignment to Thr-89

The behavior of threonine residues in the bacteriorhodopsin (bR) photocycle has been investigated by Fourier transform infrared difference spectroscopy. l-Threonine labeled at the hydroxyl group with ¹⁸O (l-[3-¹⁸O]threonine) was incorporated into bR and the bR→M FTIR difference spectra measured. Bands are assigned to threonine vibrational modes on the basis of ¹⁸O induced isotope frequency shifts and normal mode calculations. In the 3500 cm^-1 region, a negative band is assigned to the OH stretch of threonine. In the 1125 cm^-1 region, a negative band is assigned to a mixed CH₃ rock/CO stretch mode. The frequency of both these bands indicates the presence of at least one hydrogen bonded threonine hydroxyl group in light adapted bR which undergoes a change in structure by formation of the M intermediate. Spectral changes induced by the substitution Thr-89→Asn but not Thr-46→Asn or Asp-96→Asn are consistent with the assignment of these bands to Thr-89. These results along with another related study on the mutant Thr-89→Asn indicate that the active site of bR includes Thr-89 and that its interaction with the retinylidene Schiff base and Asp-85 may play an important role in regulating the color of bacteriorhodopsin and the transfer of a proton to the Schiff base. Copyright (C) 1998 Elsevier Science B.V.