Two-photon spectroscopy of locked-11-cis-rhodopsin: evidence for a protonated Schiff base in a neutral protein binding site.

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Abstract
The nature of the protein binding site of rhodopsin was studied using 2-photon spectroscopy to assign the location of the low-lying covalent 1Ag*--like .pi..pi.* state in a model rhodopsin containing a locked-11-cis chromophore. The 2-proton thermal lens maximum is observed at 22,800 cm-1, .apprxeq. 2000 cm-1 above the 1 photon absorption maximum, indicating that the protein environment has induced a level ordering reversal of the low-lying .pi..pi.* states relative to that observed in retinyl Schiff bases in solution. The spectroscopic results clearly indicate that the chromophore is protonated and that the binding site is unchanged. Electrostatic energy contour maps of the binding site are calculated, showing possible locations for the external counterion(s). Two models of the binding site are proposed that accommodate the available spectroscopic data. One model involves a protonated Schiff base chromophore stabilized by a single negatively charged Asp or Glu residue. A more complicated model involving 2 residues (one charged, the other neutral) is also proposed. The latter model is interesting because it also accomodates the observed deuterium isotope effect in the form of a proton translocation between the 2 residues. The translocation is assumed to be a ground state process, initiated subsequent to the photoisomerization of the chromophore and energetically driven via destabilization of the counterion environment as a result of isomerization-induced charge separation.