WATER STRUCTURAL-CHANGES AT THE PROTON UPTAKE SITE (THE THR46-ASP96 DOMAIN) IN THE L-INTERMEDIATE OF BACTERIORHODOPSIN

Fourier transform infrared spectra of the L intermediate of light-adap ted bacteriorhodopsin were examined for recombinant proteins with amin o acid substitutions at Thr46 and Asp96. Two O-H stretching vibrationa l bands of water, at 3607 and 3577 cm(-1), change into stronger H-bond ing states in L of the wild type. Thr46-->Val substitution abolished t hese bands in spite of the fact that [3-O-18]threonine-labeling did no t shift them, indicating that they correspond to coordination of the w ater with Thr46. The two water bands were restored, although with chan ged frequencies, by an additional Asp96-->Asn substitution in Thr46--> Val/Asp96-->Asn, A single Asp96-->Asn substitution abolished the 3607 cm(-1) band. Thus, Asp96 also takes part in structural changes in wate r. The perturbations of these water molecules in the L intermediate di splayed a weak correlation with the ratio of intensity change in the t wo vibrational bands of the Schiff base mode at 1312 and 1301 cm(-1) a nd the rate for the deprotonation of the Schiff base at the L-to-M rea ction of the photocycle. We find, therefore, that the water molecules in the cytoplasmic Asp96-Thr46 domain, which comprises the site of pro ton uptake after formation of the M intermediate, undergo structural c hanges in the L intermediate already. These changes are transmitted to the extracellular domain and affect interaction of the Schiff base wi th Asp85, that is far removed from this region.