The molecular evolution of avian ultraviolet- and violet-sensitive visual pigments

The shortwave-sensitive SWS1 class of vertebrate visual pigments range in lambda(max) from the violet (385-445 nm) to the ultraviolet (UV) (365-355 nm), with UV-sensitivity almost certainly ancestral. In birds, however, the UV-sensitive pigments present in a number of species have evolved secondarily from an avian violet-sensitive (VS) pigment. All avian VS pigments expressed in vitro to date encode Ser86 whereas Phe86 is present in all non-avian ultraviolet sensitive (UVS) pigments. In this paper, we show by site directed mutagenesis of avian VS pigments that Ser86 is required in an avian VS pigment to maintain violet-sensitivity and therefore underlies the evolution of avian VS pigments. The major mechanism for the evolution of avian UVS pigments from an ancestral avian VS pigment is undoubtedly a Ser90Cys substitution. However, Phe86, as found in the Blue-crowned trogon, will also short-wave shift the pigeon VS pigment into the UV whereas Ala86 and Cys86 which are also found in natural avian pigments do not generate short-wave shifts when substituted into the pigeon pigment. From available data on avian SWS1 pigments, it would appear that UVS pigments have evolved on at least 5 separate occasions and utilize 2 different mechanisms for the short-wave shift.     

Spectral tuning and evolution of primate short-wavelength-sensitive visual pigments

The peak sensitivities (λ(max)) of the short-wavelength-sensitive-1 (SWS1) pigments in mammals range from the ultraviolet (UV) (360-400 nm) to the violet (400-450 nm) regions of the spectrum. In most cases, a UV or violet peak is determined by the residue present at site 86, with Phe conferring UV sensitivity (UVS) and either Ser, Tyr or Val causing a shift to violet wavelengths. In primates, however, the tuning mechanism of violet-sensitive (VS) pigments would appear to differ. In this study, we examine the tuning mechanisms of prosimian SWS1 pigments. One species, the aye-aye, possesses a pigment with Phe86 but in vitro spectral analysis reveals a VS rather than a UVS pigment. Other residues (Cys, Ser and Val) at site 86 in prosimians also gave VS pigments. Substitution at site 86 is not, therefore, the primary mechanism for the tuning of VS pigments in primates, and phylogenetic analysis indicates that substitutions at site 86 have occurred at least five times in primate evolution. The sole potential tuning site that is conserved in all primate VS pigments is Pro93, which when substituted by Thr (as found in mammalian UVS pigments) in the aye-aye pigment shifted the peak absorbance into the UV region with a λ(max) value at 371 nm. We, therefore, conclude that the tuning of VS pigments in primates depends on Pro93, not Tyr86 as in other mammals. However, it remains uncertain whether the initial event that gave rise to the VS pigment in the ancestral primate was achieved by a Thr93Pro or a Phe86Tyr substitution.     

Ultraviolet vision in birds: the importance of transparent eye media

Ultraviolet (UV)-sensitive visual pigments are widespread in the animal kingdom but many animals, for example primates, block UV light from reaching their retina by pigmented lenses. Birds have UV-sensitive (UVS) visual pigments with sensitivity maxima around 360–373 nm (UVS) or 402–426 nm (violet-sensitive, VS). We describe how these pigments are matched by the ocular media transmittance in 38 bird species. Birds with UVS pigments have ocular media that transmit more UV light (wavelength of 50% transmittance, λ_T0.5, 323 nm) than birds with VS pigments (λ_T0.5, 358 nm). Yet, visual models predict that colour discrimination in bright light is mostly dependent on the visual pigment (UVS or VS) and little on the ocular media. We hypothesize that the precise spectral tuning of the ocular media is mostly relevant for detecting weak UV signals, e.g. in dim hollow-nests of passerines and parrots. The correlation between eye size and UV transparency of the ocular media suggests little or no lens pigmentation. Therefore, only small birds gain the full advantage from shifting pigment sensitivity from VS to UVS. On the other hand, some birds with VS pigments have unexpectedly low UV transmission of the ocular media, probably because of UV blocking lens pigmentation.     

Ultraviolet visual sensitivity in three avian lineages: paleognaths, parrots, and passerines

Ultraviolet (UV) light-transmitted signals play a major role in avian foraging and communication, subserving functional roles in feeding, mate choice, egg recognition, and nestling discrimination. Sequencing functionally relevant regions of the short wavelength sensitive type 1 (SWS1) opsin gene that is responsible for modulating the extent of SWS1 UV sensitivity in birds allows predictions to be made about the visual system's UV sensitivity in species where direct physiological or behavioral measures would be impractical or unethical. Here, we present SWS1 segment sequence data from representative species of three avian lineages for which visually based cues for foraging and communication have been investigated to varying extents. We also present a preliminary phylogenetic analysis and ancestral character state reconstructions of key spectral tuning sites along the SWS1 opsin based on our sequence data. The results suggest ubiquitous ultraviolet SWS1 sensitivity (UVS) in both paleognaths, including extinct moa (Emeidae), and parrots, including the nocturnal and flightless kakapo (Strigops habroptilus), and in most, but not all, songbird (oscine) lineages, and confirmed violet sensitivity (VS) in two suboscine families. Passerine hosts of avian brood parasites were included both UVS and VS taxa, but sensitivity did not co-vary with egg rejection behaviors. The results should stimulate future research into the functional parallels between the roles of visual signals and the genetic basis of visual sensitivity in birds and other taxa.     

Photochemistry of the primary event in short-wavelength visual opsins at low temperature.

Two short-wavelength cone opsins, frog (Xenopus laevis) violet and mouse UV, were expressed in mammalian COS1 cells, purified in delipidated form, and studied using cryogenic UV-vis spectrophotometry. At room temperature, the X. laevis violet opsin has an absorption maximum at 426 nm when generated with 11-cis-retinal and an absorption maximum of 415 nm when generated with 9-cis-retinal. The frog short-wavelength opsin has two different batho intermediates, one stable at 30 K (lambda(max) approximately 446 nm) and the other at 70 K (lambda(max) approximately 475 nm). Chloride ions do not affect the absorption maximum of the violet opsin. At room temperature, mouse UV opsin has an absorption maximum of 357 nm, while at 70 K, the pigment exhibits a bathochromic shift to 403 nm with distinct vibronic structure and a strong secondary vibronic band at 380 nm. We have observed linear relationships when analyzing the energy difference between the initial and bathochromic intermediates and the normalized difference spectra of the batho-shifted intermediates of rod and cone opsins. We conclude that the binding sites of these pigments change from red to green to violet via systematic shifts in the position of the primary counterion relative to the protonated Schiff base. The mouse UV cone opsin does not fit this trend, and we conclude that wavelength selection in this pigment must operate via a different molecular mechanism. We discuss the possibility that the mouse UV chromophore is initially unprotonated.     

A novel amino acid substitution is responsible for spectral tuning in a rodent violet-sensitive visual pigment

Cone short-wave (SWS1) visual pigments can be divided into two categories that correlate with spectral sensitivity, violet sensitive above 390 nm and ultraviolet sensitive below that wavelength. The evolution and mechanism of spectral tuning of SWS1 opsins are proving more complex than those of other opsin classes. Violet-sensitive pigments probably evolved from an ancestral ultraviolet-sensitive opsin, although in birds ultraviolet sensitivity has re-evolved from violet-sensitive pigments. In certain mammals, a single substitution involving the gain of a polar residue can switch sensitivity from ultraviolet to violet sensitivity, but where such a change is not involved, several substitutions may be required to effect the switch. The guinea pig, Cavia porcellus, is a hystricognathous rodent, a distinct suborder from the Sciurognathi, such as rats and mice. It has been shown by microspectrophotometry to have two cone visual pigments at 530 and 400 nm. We have ascertained the sequence of the short-wave pigment and confirmed its violet sensitivity by expression and reconstitution of the pigment in vitro. Moreover, we have shown by site-directed mutagenesis that a single residue is responsible for wavelength tuning of spectral sensitivity, a Val86Phe causing a 60 nm short-wave shift into the ultraviolet and a Val86Tyr substitution shifting the pigment 8 nm long wave. The convergent evolution of this mammalian VS pigment provides insight into the mechanism of tuning between the violet and UV.     

Concerted Shifts in Absorption Maxima of Yellow and Red Plumage Carotenoids Support Specialized Tuning of Chromatic Signals to Different Visual Systems in Near-Passerine Birds

Recent evidence that absorption maxima (λR_min) expressed by colorful plumage pigments align to diagnostic cone sensitivities of affiliated visual systems suggests that birds employ specialized signals in relation to their color vision. However, these studies compared different pigments and clades for the violet (porphyrins in non-passerines) and ultraviolet (carotenoids in passerines) sensitive system, which confounds chemistry and phylogeny with tuning patterns. To test whether signal alignments to violet (VS) and ultraviolet (UVS) systems transcend confounding factors, parallel analyses were conducted for a diversity of near-passerines, a group in which plumage carotenoids occur in taxa with either visual system. Conventional and phylogenetically informed analyses confirmed earlier findings: short wavelength absorbing (yellow carotenoid) pigments aligned λR_min with the violet-sensitive (V) cone of VS species but with the short wavelength-sensitive (S) cone of UVS species, whereas long wavelength-absorbing (red carotenoid) pigments aligned only with the S cone of VS species. More extensive variation among VS yellow carotenoids produced λR_min alignments to cone sensitivities that differed at shorter (peaks) versus longer (overlaps) wavelengths. Ancestral trait reconstructions indicated that signals evolved to match pre-existing VS systems, but did not resolve scenarios for UVS systems. Regardless of historical details, alignments expressed a higher-level pattern in which λR_min values were blue-shifted for yellow and red carotenoids in VS compared to UVS species, a pattern opposite that expressed by receptor sensitivities between systems. Thus, generalized functional designs attributed to avian color vision allow for specialized visual communication through the development of chromatic signals suited to each perceptual system.     

Visual modelling suggests a weak relationship between the evolution of ultraviolet vision and plumage coloration in birds

Birds have sophisticated colour vision mediated by four cone types that cover a wide visual spectrum including ultraviolet (UV) wavelengths. Many birds have modest UV sensitivity provided by violet‐sensitive (VS) cones with sensitivity maxima between 400 and 425 nm. However, some birds have evolved higher UV sensitivity and a larger visual spectrum given by UV‐sensitive (UVS) cones maximally sensitive at 360–370 nm. The reasons for VS–UVS transitions and their relationship to visual ecology remain unclear. It has been hypothesized that the evolution of UVS‐cone vision is linked to plumage colours so that visual sensitivity and feather coloration are ‘matched’. This leads to the specific prediction that UVS‐cone vision enhances the discrimination of plumage colours of UVS birds while such an advantage is absent or less pronounced for VS‐bird coloration. We test this hypothesis using knowledge of the complex distribution of UVS cones among birds combined with mathematical modelling of colour discrimination during different viewing conditions. We find no support for the hypothesis, which, combined with previous studies, suggests only a weak relationship between UVS‐cone vision and plumage colour evolution. Instead, we suggest that UVS‐cone vision generally favours colour discrimination, which creates a nonspecific selection pressure for the evolution of UVS cones.     

Photoisomerization efficiency in UV-absorbing visual pigments: protein-directed isomerization of an unprotonated retinal Schiff base

A visual pigment consists of an opsin protein and a chromophore, 11-cis-retinal, which binds to a specific lysine residue of opsin via a Schiff base linkage. The Schiff base chromophore is protonated in pigments that absorb visible light, whereas it is unprotonated in ultraviolet-absorbing visual pigments (UV pigments). To investigate whether an unprotonated Schiff base can undergo photoisomerization as efficiently as a protonated Schiff base in the opsin environment, we measured the quantum yields of the bovine rhodopsin E113Q mutant, in which the Schiff base is unprotonated at alkaline pH, and the mouse UV pigment (mouse UV). Photosensitivities of UV pigments were measured by irradiation of the pigments followed by chromophore extraction and HPLC analysis. Extinction coefficients were estimated by comparing the maximum absorbances of the original pigments and their acid-denatured states. The quantum yield of the bovine rhodopsin E113Q mutant at pH 8.2, where the Schiff base is unprotonated, was significantly lower than that of wild-type rhodopsin, whereas the mutant gave a quantum yield almost identical to that of the wild type at pH 5.5, where the Schiff base is protonated. These results suggest that Schiff base protonation plays a role in increasing quantum yield. The quantum yield of mouse UV, which has an unprotonated Schiff base chromophore, was significantly higher than that of the unprotonated form of the rhodopsin E113Q mutant, although it was still lower than the visible-absorbing pigments. These results suggest that the mouse UV pigment has a specific mechanism for the efficient photoisomerization of its unprotonated Schiff base chromophore.     

Ultraviolet-sensitive vision in long-lived birds

Long-term exposure to ultraviolet (UV) light generates substantial damage, and in mammals, visual sensitivity to UV is restricted to short-lived diurnal rodents and certain marsupials. In humans, the cornea and lens absorb all UV-A and most of the terrestrial UV-B radiation, preventing the reactive and damaging shorter wavelengths from reaching the retina. This is not the case in certain species of long-lived diurnal birds, which possess UV-sensitive (UVS) visual pigments, maximally sensitive below 400 nm. The Order Psittaciformes contains some of the longest lived bird species, and the two species examined so far have been shown to possess UVS pigments. The objective of this study was to investigate the prevalence of UVS pigments across long-lived parrots, macaws and cockatoos, and therefore assess whether they need to cope with the accumulated effects of exposure to UV-A and UV-B over a long period of time. Sequences from the SWS1 opsin gene revealed that all 14 species investigated possess a key substitution that has been shown to determine a UVS pigment. Furthermore, in vitro regeneration data, and lens transparency, corroborate the molecular findings of UV sensitivity. Our findings thus support the claim that the Psittaciformes are the only avian Order in which UVS pigments are ubiquitous, and indicate that these long-lived birds have UV sensitivity, despite the risks of photodamage.     

Regulation of phototransduction in short-wavelength cone visual pigments via the retinylidene Schiff base counterion.

Short-wavelength visual pigments (SWS1) have lambda(max) values that range from the ultraviolet to the blue. Like all visual pigments, this class has an 11-cis-retinal chromophore attached through a Schiff base linkage to a lysine residue of opsin apoprotein. We have characterized a series of site-specific mutants at a conserved acidic residue in transmembrane helix 3 in the Xenopus short-wavelength sensitive cone opsin (VCOP, lambda(max) approximately 427 nm). We report the identification of D108 as the counterion to the protonated retinylidene Schiff base. This residue regulates the pK(a) of the Schiff base and, neutralizing this charge, converts the violet sensitive pigment into one that absorbs maximally in the ultraviolet region. Changes to this position cause the pigment to exhibit two chromophore absorbance bands, a major band with a lambda(max) of approximately 352-372 nm and a minor, broad shoulder centered around 480 nm. The behavior of these two absorbance bands suggests that these represent unprotonated and protonated Schiff base forms of the pigment. The D108A mutant does not activate bovine rod transducin in the dark but has a significantly prolonged lifetime of the active MetaII state. The data suggest that in short-wavelength sensitive cone visual pigments, the counterion is necessary for the characteristic rapid production and decay of the active MetaII state.     

Evolution of ultraviolet vision in shorebirds (Charadriiformes)

Diurnal birds belong to one of two classes of colour vision. These are distinguished by the maximum absorbance wavelengths of the SWS1 visual pigment sensitive to violet (VS) and ultraviolet (UVS). Shifts between the classes have been rare events during avian evolution. Gulls (Laridae) are the only shorebirds (Charadriiformes) previously reported to have the UVS type of opsin, but too few species have been sampled to infer that gulls are unique among shorebirds or that Laridae is monomorphic for this trait. We have sequenced the SWS1 opsin gene in a broader sample of species. We confirm that cysteine in the key amino acid position 90, characteristic of the UVS class, has been conserved throughout gull evolution but also that the terns Anous minutus, A. tenuirostris and Gygis alba, and the skimmer Rynchops niger carry this trait. Terns, excluding Anous and Gygis, share the VS conferring serine in position 90 with other shorebirds but it is translated from a codon more similar to that found in UVS shorebirds. The most parsimonious interpretation of these findings, based on a molecular gene tree, is a single VS to UVS shift and a subsequent reversal in one lineage.     

Complex distribution of avian color vision systems revealed by sequencing the SWS1 opsin from total DNA

To gain insights into the evolution and ecology of visually acute animals such as birds, biologists often need to understand how these animals perceive colors. This poses a problem, since the human eye is of a different design than that of most other animals. The standard solution is to examine the spectral sensitivity properties of animal retinas through microspectophotometry-a procedure that is rather complicated and therefore only has allowed examinations of a limited number of species to date. We have developed a faster and simpler molecular method, which can be used to estimate the color sensitivities of a bird by sequencing a part of the gene coding for the ultraviolet or violet absorbing opsin in the avian retina. With our method, there is no need to sacrifice the animal, and it thereby facilitates large screenings, including rare and endangered species beyond the reach of microspectrophotometry. Color vision in birds may be categorized into two classes: one with a short-wavelength sensitivity biased toward violet (VS) and the other biased toward ultraviolet (UVS). Using our method on 45 species from 35 families, we demonstrate that the distribution of avian color vision is more complex than has previously been shown. Our data support VS as the ancestral state in birds and show that UVS has evolved independently at least four times. We found species with the UVS type of color vision in the orders Psittaciformes and Passeriformes, in agreement with previous findings. However, species within the families Corvidae and Tyrannidae did not share this character with other passeriforms. We also found UVS type species within the Laridae and Struthionidae families. Raptors (Accipitridae and Falconidae) are of the violet type, giving them a vision system different from their passeriform prey. Intriguing effects on the evolution of color signals can be expected from interactions between predators and prey. Such interactions may explain the presence of UVS in Laridae and Passeriformes.     

E113 is required for the efficient photoisomerization of the unprotonated chromophore in a UV-absorbing visual pigment

Protonation of the retinal Schiff base chromophore is responsible for the absorption of visible light and is stabilized by the counterion residue E113 in vertebrate visual pigments. However, this residue is also conserved in vertebrate UV-absorbing visual pigments (UV pigments) which have an unprotonated Schiff base chromophore. To elucidate the role played by this residue in the photoisomerization of the unprotonated chromophore in UV pigments, we measured the quantum yield of the E113Q mutant of the mouse UV cone pigment (mouse UV). The quantum yield of the mutant was much lower than that of the wild type, indicating that E113 is required for the efficient photoisomerization of the unprotonated chromophore in mouse UV. Introduction of the E113Q mutation into the chicken violet cone pigment (chicken violet), which has a protonated chromophore, caused deprotonation of the chromophore and a reduction in the quantum yield. On the other hand, the S90C mutation in chicken violet, which deprotonated the chromophore with E113 remaining intact, did not significantly affect the quantum yield. These results suggest that E113 facilitates photoisomerization in both UV-absorbing and visible light-absorbing visual pigments and provide a possible explanation for the complete conservation of E113 among vertebrate UV pigments.     

Multiple shifts between violet and ultraviolet vision in a family of passerine birds with associated changes in plumage coloration

Colour vision in diurnal birds falls into two discrete classes, signified by the spectral sensitivity of the violet- (VS) or ultraviolet-sensitive (UVS) short wavelength-sensitive type 1 (SWS1) single cone. Shifts between sensitivity classes are rare; three or four are believed to have happened in the course of avian evolution, one forming UVS higher passerines. Such shifts probably affect the expression of shortwave-dominated plumage signals. We have used genomic DNA sequencing to determine VS or UVS affinity in fairy-wrens and allies, Maluridae, a large passerine family basal to the known UVS taxa. We have also spectrophotometrically analysed male plumage coloration as perceived by the VS and UVS vision systems. Contrary to any other investigated avian genus, Malurus (fairy-wrens) contains species with amino acid residues typical of either VS or UVS cone opsins. Three bowerbird species (Ptilonorhynchidae) sequenced for outgroup comparison carry VS opsin genes. Phylogenetic reconstructions render one UVS gain followed by one or more losses as the most plausible evolutionary scenario. The evolution of avian ultraviolet sensitivity is hence more complex, as a single shift no longer explains its distribution in Passeriformes. Character correlation analysis proposes that UVS vision is associated with shortwave-reflecting plumage, which is widespread in Maluridae.     

pH dependence of photolysis intermediates in the photoactivation of rhodopsin mutant E113Q

Glutamic acid at position 113 in bovine rhodopsin ionizes to form the counterion to the protonated Schiff base (PSB), which links the 11-cis-retinylidene chromophore to opsin. Photoactivation of rhodopsin requires both Schiff base deprotonation and neutralization of Glu-113. To better understand the role of electrostatic interactions in receptor photoactivation, absorbance difference spectra were collected at time delays from 30 ns to 690 ms after photolysis of rhodopsin mutant E113Q solubilized in dodecyl maltoside at different pH values at 20 degrees C. The PSB form (pH 5. 5, lambda(max) = 496 nm) and the unprotonated Schiff base form (pH 8. 2, lambda(max) = 384 nm) of E113Q rhodopsin were excited using 477 nm or 355 nm light, respectively. Early photointermediates of both forms of E113Q were qualitatively similar to those of wild-type rhodopsin. In particular, early photoproducts with spectral shifts to longer wavelengths analogous to wild-type bathorhodopsin were seen. In the case of the basic form of E113Q, the absorption maximum of this intermediate was at 408 nm. These results suggest that steric interaction between the retinylidene chromophore and opsin, rather than charge separation, plays the dominant role in energy storage in bathorhodopsin. After lumirhodopsin, instead of deprotonating to form metarhodopsin I(380) on the submillisecond time scale as is the case for wild type, the acidic form of E113Q produced metarhodopsin I(480), which decayed very slowly (exponential lifetime = 12 ms). These results show that Glu-113 must be present for efficient deprotonation of the Schiff base and rapid visual transduction in vertebrate visual pigments.     

Wavelength regulation in iodopsin, a cone pigment.

The opsin shift, the difference in wavenumber between the absorption peak of a visual pigment and the protonated Schiff base of the chromophore, represents the influence of the opsin binding site on the chromophore. The opsin shift for the chicken cone pigment iodopsin is much larger than that for rhodopsin. To understand the origin of this opsin shift and the mechanism of wavelength regulation in iodopsin, a series of synthetic 9-cis and 11-cis dehydro- and dihydro-retinals was used to regenerate iodopsin-based pigments. The opsin shifts of these pigments are quite similar to those found in bacteriorhodopsin-based artificial pigments. On the basis of these studies, a tentative model of wavelength regulation in iodopsin is proposed.     

Regeneration of bovine and octopus opsins in situ with natural and artificial retinals

We consider the problem of color regulation in visual pigments for both bovine rhodopsin (lambda max = 500 nm) and octopus rhodopsin (lambda max = 475 nm). Both pigments have 11-cis-retinal (lambda max = 379 nm, in ethanol) as their chromophore. These rhodopsins were bleached in their native membranes, and the opsins were regenerated with natural and artificial chromophores. Both bovine and octopus opsins were regenerated with the 9-cis- and 11-cis-retinal isomers, but the octopus opsin was additionally regenerated with the 13-cis and all-trans isomers. Titration of the octopus opsin with 11-cis-retinal gave an extinction coefficient for octopus rhodopsin of 27,000 +/- 3000 M-1 cm-1 at 475 nm. The absorption maxima of bovine artificial pigments formed by regenerating opsin with the 11-cis dihydro series of chromophores support a color regulation model for bovine rhodopsin in which the chromophore-binding site of the protein has two negative charges: one directly hydrogen bonded to the Schiff base nitrogen and another near carbon-13. Formation of octopus artificial pigments with both all-trans and 11-cis dihydro chromophores leads to a similar model for octopus rhodopsin and metarhodopsin: there are two negative charges in the chromophore-binding site, one directly hydrogen bonded to the Schiff base nitrogen and a second near carbon-13. The interaction of this second charge with the chromophore in octopus rhodopsin is weaker than in bovine, while in metarhodopsin it is as strong as in bovine.     

Opsin shift in an aldolase antibody.

An antibody-retinal assembly that mimics the opsin shift (OS) of the naturally occurring visual pigments is reported. Both experiments and calculations show that the aldolase antibody 33F12 covalently binds all-trans retinal via a protonated Schiff base with a lysine residue. This chromophore, which exhibits a remarkable opsin red shift (140 nm), represents a useful model system for studying the factors that contribute to the OS.     

Specific reaction of 9-cis-retinoyl fluoride with bovine opsin

Opsin readily undergoes Schiff base formation between an active site lysine and 9-cis- or 11-cis-retinaldehyde to form the visual pigments isorhodopsin (lambda max = 487 nm) and rhodopsin (lambda max = 500 nm), respectively (Dratz, 1977). It would be predicted that 9-cis-retinoyl fluoride (1), an isostere of 9-cis-retinal, should be an active site directed, mechanism-based labeling agent of opsin, since a stable peptide bond should be formed instead of a Schiff base. It is shown here that 9-cis-retinoyl fluoride (1) reacts with opsin in a time-dependent fashion (t1/2 = 9 min at 25 microM 1) to form a new, nonbleachable pigment with a lambda max of approximately 365 nm. beta-Ionone competitively slows down the rate of the reaction. The absorbance of the new pigment at approximately 365 nm is similar to that of model amide compounds. This result is consistent in a general and qualitative way with the Nakanishi-Honig point-charge model for visual pigments which requires that the chromophore be charged, a situation not possible when the retinoid is linked to opsin via a peptide bond rather than a protonated Schiff base [Honig, B., Dinur, U., Nakanishi, K., Balogh-Nair, V., Gawinowicz, M.A., Arnabaldi, M., & Motto, M.G. (1979) J. Am. Chem. Soc. 101, 7084-7086]. 9-cis-Retinoyl fluoride (1) is approximately 4-fold more potent than all-trans-retinoyl fluoride (2) as an inactivator of bovine opsin. Importantly, 13-cis-retinoyl fluoride (3) is inactive, and no new absorption band at 365 nm is observed.(ABSTRACT TRUNCATED AT 250 WORDS)