Chloride Effect on Iodopsin Studied by Low-Temperature Visible and Infrared Spectroscopies

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Abstract
To investigate the chloride effect on the spectral properties of iodopsin, we have prepared an anion-free iodopsin (iodopsin·free) by extensive dialysis of an iodopsin sample against a buffer containing no chloride, and visible and infrared difference spectra between iodopsin·free and its photoproduct at 77 K were recorded. The absorption maximum of iodopsin·free in l-α-phosphatidylcholine liposomes was 528 nm, which was almost identical with that of the nitrate-bound form of iodopsin (526 nm, iodopsin·NO3), but 43 nm blue-shifted from that of the chloride-bound form of iodopsin (iodopsin·Cl). The iod/batho visible difference spectrum obtained from iodopsin·free was similar in shape to that from iodopsin·NO3, but not to that from iodopsin·Cl. FTIR spectroscopy revealed that the chromophore vibrational bands and the peptide bonds of the original state in iodopsin·free were identical with those in iodopsin·NO3 and were also similar to those in iodopsin·Cl except for the ethylenic vibrations of the chromophore. In contrast, those of the batho state in iodopsin·free were similar to those in iodopsin·NO3 but considerably different from those in iodopsin·Cl. These results suggested that the binding of chloride but not nitrate induces a conformational change in the protein and that the chloride binding site is situated in a position where it directly interacts with the chromophore when the chromophore is photoisomerized. FTIR spectroscopy also revealed that one of the four water bands observed in the batho/iod spectrum of iodospin·Cl is absent in the spectra of iodopsin·free and iodopsin·NO3. Thus, in contrast to nitrate, a lyotropic anion, chloride would bind to the binding site with water molecule(s) which could form a hydrogen-bonding network with amino acid residue(s) near the chromophore, thereby resulting in the red shift of the absorption maximum of iodopsin.

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