Effects of chloride on chicken iodopsin and the chromophore transfer reactions from iodopsin to scotopsin and B-photopsin

Abstract

Spectroscopic properties of chicken iodopsin were investigated in correlation with the concentration of chloride in digitonin extracts. When chloride in the extract was depleted by extensive dialysis, chloride-depleted iodopsin (absorption maximum, 512 nm) was formed. It was converted to chloride-bound iodopsin (absorption maximum, 562 nm) by the addition of chloride in the extract. There existed an equilibrium between two forms of iodopsin with a dissociation constant of 0.8 mM chloride. The chromophore-transfer reaction from iodopsin to scotopsin or B-photopsin, the protein moiety of chicken rhodopsin or chicken blue-sensitive cone pigment, respectively, in digitonin extract was also investigated in correlation with the concentrations of chloride, other monovalent and divalent anions, and detergent. The chromophore of chloride-depleted iodopsin was easily transferred to scotopsin in the extract, resulting in formation of rhodopsin. On the other hand, chloride-bound iodopsin was fairly stable even in the presence of scotopsin, indicating that the reaction is inhibited by binding of chloride to iodopsin. The chromophore-transfer reaction to B-photopsin was also observed from chloride-depleted iodopsin but not from chloride-bound iodopsin. The reaction was observable in the 10% digitonin extract as well as in the 2% digitonin extract. The reaction was also observed when 25 mM Na2SO4 was present in the mixture instead of NaCl, but was not when 67 mM NaNO3 was present. All these facts suggest that the chloride binding site of iodopsin does not accept a divalent anion such as SO4(2+), but does accept a monovalent anion such as Cl- or NO3-, which causes inhibition of the chromophore transfer.

Anion sensitivity and spectral tuning of cone visual pigments in situ.

“…Previous work on detergentextracted pigments provided evidence for anion sensitivity of the green-absorbing pigment P521 in gecko (10-12) and the red-absorbing cone pigment iodopsin in chicken (13,14). The anion selectivity and affinity of the fish and amphibian pigments studied here are very similar to the corresponding results obtained on gecko and chicken pigments.…”

“…For example, when the gecko pigment was studied in a Cl--free environment, its Amax was found to be '500 nm; when Clwas added, Amax shifted to the native value of 521 nm (10,11). A similar Cl--induced shift from 520 to 565 nm was found in chicken iodopsin (13,14). In this paper, we provide evidence that suggests that Cl--mediated spectral tuning of longwavelength-absorbing cone pigments is a widespread phenomenon and may indeed be universal among vertebrates.…”

“…Hence, Cl-binding constitutes an important component of spectral tuning in these pigments. Moreover, in the gecko P521 pigment (39) and in chicken iodopsin (14), 11-cis-retinal continually exchanges in the dark, and Cl-reduces the rate of this dark exchange. Crescitelli's (40) studies on the effect of raising the pH on the gecko pigment suggest that binding of Cl-shifts the pKa of the chromophore Schiff base in the alkaline direction.…”

“…Evidence for this possibility was first provided for the gecko P521 pigment (10)(11)(12), then for chicken iodopsin (13,14), and recently for the red-absorbing cone pigment in frog (15). For example, when the gecko pigment was studied in a Cl--free environment, its Amax was found to be '500 nm; when Clwas added, Amax shifted to the native value of 521 nm (10,11).…”

Measurement of Slow Spontaneous Release of 11-cis-Retinal from Rhodopsin

“…Chromophore exchange of 11CR by 9CR has been demonstrated for cone pigments (21)(22)(23). The exchange in gecko cone pigment P521 reconstituted in digitonin was shown to be~30% in 15 h at 15 C, corresponding to a k off of 6.6 Â 10 À6 s À1 (22).…”