LOCALIZATION OF THE RETINAL PROTONATED SCHIFF-BASE COUNTERION IN RHODOPSIN

Semiempirical molecular orbital calculations are combined with C-13 NMR chemical shifts to localize the counterion in the retinal binding site of vertebrate rhodopsin. Charge densities along the polyene chain are calculated for an 11-cis-retinylidene protonated Schiff base (11-cis-RPSB) chromophore with 1) a chloride counterion at various distances from the Schiff base nitrogen, 2) one or two chloride counterions at different positions along the retinal chain from C-10 to C-15 and at the Schiff base nitrogen, and 3) a carboxylate counterion out of the retinal plane near C-12. Increasing the distance of the negative counterion from the Schiff base results in an enhancement of alternating negative and positive partial charge on the even- and odd-numbered carbons, respectively, when compared to the 11-cis-RPSB chloride model compound. In contrast, the observed C-13 NMR data of rhodopsin exhibit downfield chemical shifts from C-8 to C-13 relative to the 11-cis-RPSB.Cl corresponding to a net increase of partial positive or decrease of partial negative charge at these positions (Smith, S. O., 1. Palings, M. E. Miley, J. Courtin, H. de Groot, J. Lugtenburg, R. A. Mathies, and R. G. Griffin. 1990. Biochemistry. 29:8158-8164). The anomalous changes in charge density reflected in the rhodopsin NMR chemical shifts can be qualitatively modeled by placing a single negative charge above C-12. The calculated fit improves when a carboxylate counterion is used to model the retinal binding site. Inclusion of water in the model does not alter the fit to the NMR data, although it is consistent with observations based on other methods. These data constrain the location and the orientation of the Glu113 side chain, which is known to be the counterion in rhodopsin, and argue for a strong interaction centered at C-12 of the retinylidene chain.