HYDROGEN-BONDS OF WATER AND C=O GROUPS COORDINATE LONG-RANGE STRUCTURAL IN THE L-PHOTOINTERMEDIATE OF BACTERIORHODOPSIN

Fourier transform infrared spectra of light-adapted bacteriorhodopsin exhibit a band at 1618 cm(-1) that shifts to 1625 cm(-1) upon formatio n of the L intermediate. It is assigned to the peptide C=O of Va149 fr om the fact that it shifts in [1-C-13]valine-labeled bacteriorhodopsin and appears perturbed in the Val49-->Met mutant. The intensity of the BR-->L difference band is reduced in the Thr46-->Val mutant but resto red by the additional mutation of Asp96-->Asn. These intensity changes are closely correlated with the H-bonding change of water molecules, suggesting that the peptide C=O of Va149 Is hydrated, This could arise In the Thr46-->Val mutant because of perturbation of the C=O of Val46 , which points toward Val49. The Val49-->Ala mutation influences a pep tide N-H, presumably of Va149, and the carboxylic C=O of Asp96, as wel l as water molecules proximal to Asp85. Conversely, the water molecule assumed to be in the cavity that arises from the missing two methyl g roups in V49A could be affected in the mutant of Asp96-->Asn. We propo se that the perturbation exerted on Asp85 by the Schiff base in the L intermediate is transmitted to Asp96 through I-I-bonding of water mole cules in the Asp85-Val49 region,the C=O of Val49, H-bonding between Va 149 and Thr46, and H-bonding between Thr46 and Asp96.