Water and Peptide Backbone Structure in the Active Center of Bovine Rhodopsin

Abstract

Difference FTIR spectra in the conversion of rhodopsin or isorhodopsin to bathorhodopsin were recorded for recombinant wild-type and E113Q bovine rhodopsins. Differences in various vibrational modes between E113Q and the wild-type proteins whose Schiff bases interact with chloride and Glu113, respectively, were analyzed. Water molecules in rhodopsin that change upon formation of bathorhodopsin are detected by a change in frequency of the O−H stretching vibration from 3538 to 3525 cm^-1. This change in the wild-type protein is absent in E113Q. One or a few water molecules are therefore suggested to be located in the proximity of Glu113, the counterion of the Schiff base. Another water vibration at 3564 cm^-1, which is shifted to 3542 cm^-1 in bathorhodopsin in the wild type, persists in E113Q but with ∼5-cm^-1 shift toward higher frequency. This is due to water molecules that may be located at a site somewhat more remote from Glu113. Structural changes of some peptide carbonyls and amides are also absent in E113Q. On the other hand, the E113Q protein shows shifts of the N−H⁺ stretching vibrational band, that is probably due to the protonated Schiff base, upon conversion of rhodopsin to bathorhodopsin. No corresponding changes were observed in the wild type. We propose a model in which a water molecule interacts with Glu113, the protonated Schiff base, and peptide carbonyls, and amides. These residues undergo structural changes upon formation of bathorhodopsin.