Anions stabilize a metarhodopsin II-like photoproduct with a protonated Schiff base

In rhodopsin, the retinal chromophore is covalently bound to the apoprotein by a protonated Schiff base, which is stabilized by the negatively charged counterion Glu(113), conferring upon it a pK(a) of presumably > 16. Upon p hotoexcitation and conformational relaxation of the initial photoproducts, the Schiff base proton neutralizes the counterion, a step that is considere d a prerequisite for formation of the active state of the receptor, metarho dopsin II (MII). We show that the pK(a) of the Schiff base drops below 2.5 in MII In the presence of solute anions, however, it may be increased consi derably, thereby leading to the formation of a MII photoproduct with a prot onated Schiff base (PSB) absorbing at 480 nm. This PSB is not stabilized by Glu(113), which is shown to be neutral, but by stoichiometric binding of a n anion near the Schiff base. Protonation of the Schiff base in MII changes neither coupling to G protein, as assessed by binding to a transducin-deri ved peptide, nor the conformation of the protein, as judged by FTIR and UV spectroscopy. A PSB and an active state conformation are therefore compatib le, as suggested previously by mutants of rhodopsin. The anion specificity of the stabilization of the PSB follows the series thiocyanate > iodide > n itrate > bromide > chloride > sulfate in order of increasing efficiency. Th is specificity correlates inversely with the strength of hydration of the r espective anion species in solution and seems therefore to be determined ma inly by its partitioning into the considerably less polar protein interior.