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) photo cycle has been investigated by Fourier transform infrared difference s pectroscopy. L-Threonine labeled at the hydroxyl group with O-18 (L-[3 -O-18]threonine) was incorporated into bR and the bR --> M FTIR differ ence spectra measured. Bands are assigned to threonine vibrational mod es on the basis of O-18 induced isotope frequency shifts and normal mo de calculations. In the 3500 cm(-1) region, a negative band is assigne d to the OH stretch of threonine. In the 1125 cm(-1) region, a negativ e 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 bo nded threonine hydroxyl group in light adapted bR which undergoes a ch ange 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 Th r-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 th at its interaction with the retinylidene Schiff base and Asp-85 may pl ay an important role in regulating the color of bacteriorhodopsin and the transfer of a proton to the Schiff base. (C) 1998 Elsevier Science B.V. All rights reserved.