Regulation of photoactivation in vertebrate short wavelength visual pigments: protonation of the retinylidene Schiff base and a counterion switch

Xenopus violet cone opsin (VCOP) and its counterion variant (VCOP-D108A) are expressed in mammalian COS1 cells and regenerated with 11-cis-retinal. The phototransduction process in VCOP-D108A is investigated via cryogenic electronic spectroscopy, homology modeling, molecular dynamics, and molecular orbital theory. The VCOP-D108A variant is a UV-like pigment that displays less efficient photoactivation than the mouse short wavelength sensitive visual pigment (MUV) and photobleaching properties that are significantly different. Theoretical calculations trace the difference to the protonation state of the nearby glutamic acid residue E176, which is the homology equivalent of E181 in rhodopsin. We find that E176 is negatively charged in MUV but neutral (protonated) in VCOP-D108A. In the dark state, VCOP-D108A has an unprotonated Schiff base (SB) chromophore (lambdamax = 357 nm). Photolysis of VCOP-D108A at 70 K generates a bathochromic photostationary state (lambdamax = 380 nm). We identify two lumi intermediates, wherein the transitions from batho to the lumi intermediates are temperature- and pH-dependent. The batho intermediate decays to a more red-shifted intermediate called lumi I. The SB becomes protonated during the lumi I to lumi II transition. Decay of lumi II forms meta I, followed by the formation of meta II. We conclude that even in the absence of a primary counterion in VCOP-D108A, the SB becomes protonated during the photoactivation cascade. We examine the relevance of this observation to the counterion switch mechanism of visual pigment activation.     

Phototransduction by vertebrate ultraviolet visual pigments: protonation of the retinylidene Schiff base following photobleaching

The photochemical and subsequent thermal reactions of the mouse short-wavelength visual pigment (MUV) were studied by using cryogenic UV-visible and FTIR difference spectroscopy. Upon illumination at 75 K, MUV forms a batho intermediate (lambda(max) approximately 380 nm). The batho intermediate thermally decays to the lumi intermediate (lambda(max) approximately 440 nm) via a slightly blue-shifted intermediate not observed in other photobleaching pathways, BL (lambda(max) approximately 375 nm), at temperatures greater than 180 K. The lumi intermediate has a significantly red-shifted absorption maximum at 440 nm, suggesting that the retinylidene Schiff base in this intermediate is protonated. The lumi intermediate decays to an even more red-shifted meta I intermediate (lambda(max) approximately 480 nm) which in turn decays to meta II (lambda(max) approximately 380 nm) at 248 K and above. Differential FTIR analysis of the 1100-1500 cm(-1) region reveals an integral absorptivity that is more than 3 times smaller than observed in rhodopsin and VCOP. These results are consistent with an unprotonated Schiff base chromophore. We conclude that the MUV-visual pigment possesses an unprotonated retinylidene Schiff base in the dark state, and undergoes a protonation event during the photobleaching cascade.     

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.     

Differences in the photobleaching process between 7-cis- and 11-cis-rhodopsins: a unique interaction change between the chromophore and the protein during the lumi-meta I transition.

The photochemical and subsequent thermal reactions of 7-cis-rhodopsin prepared from cattle opsin and 7-cis-retinal were investigated by low-temperature spectrophotometry and laser photolysis, and compared with those of 11-cis-rhodopsin prepared from cattle opsin and 11-cis-retinal. Low-temperature experiments revealed that the absorption maxima of batho and lumi intermediates from 7-cis-rhodopsin were at slightly shorter wavelengths than those of 11-cis-rhodopsin while the meta I intermediates of both rhodopsin isomers showed the same absorption maxima. Kinetic experiments of the photobleaching process of 7-cis-rhodopsin using picosecond and nanosecond laser pulses revealed the formation of intermediates corresponding to the batho, lumi, meta I, and meta II intermediates from 11-cis-rhodopsin. An intermediate of 7-cis-rhodopsin corresponding to photorhodopsin (a precursor of bathorhodopsin), however, was not detected. Batho and lumi intermediates from 7-cis-rhodopsin had shorter lifetimes (approximately 40 ns and 300 microseconds) than those of 11-cis-rhodopsin (250 ns and 800 microseconds), but the lifetime of the meta I intermediate from 7-cis-rhodopsin was identical with that from 11-cis-rhodopsin (12 ms). These results indicate that the difference in configuration of the original chromophore between 7-cis- and 11-cis-rhodopsins is a cause of different chromophore-opsin interactions in the batho and lumi stages, while in the meta I stage the difference has disappeared by the relaxation of the protein near the chromophores. A possible interaction change between the 9-methyl group of the chromophore and its neighboring protein during the lumi-meta I transition will be discussed.     

Electrostatic interaction between retinylidene chromophore and opsin in rhodopsin studied by fluorinated rhodopsin analogues

Photochemical reactions of fluorinated rhodopsin analogues (F-rhodopsins) prepared from 10- or 12-fluorinated retinals (10- or 12-F-retinals) and cattle opsin were investigated by means of low-temperature spectrophotometry. On irradiation with blue light at liquid nitrogen temperature (-191 degrees C), the F-rhodopsins were converted to their respective batho intermediates. On warming, they decomposed to their respective fluororetinals and cattle opsin through lumi and meta intermediates. There was a difference in photochemical behavior between batho-12-F-rhodopsin and batho-10-F-rhodopsin. Upon irradiation with red light at -191 degrees C, batho-12-F-rhodopsin was converted to a mixture of 12-F-rhodopsin and 9-cis-12-F-rhodopsin like that of the natural bathorhodopsin, whereas batho-10-F-rhodopsin was not converted to 9-cis-10-F-rhodopsin but only to 10-F-rhodopsin. This fact suggests that the fluorine substituent at the C10 position (i.e., 10-fluoro) of the retinylidene chromophore may interact with the protein moiety during the process of isomerization of the chromophore or in the state of the batho intermediate. On irradiation with blue light at -191 degrees C, 9-cis-10-F-rhodopsin was converted to another bathochromic intermediate that was different in absorption spectrum from batho-10-F-rhodopsin. 9-cis-10-F-rhodopsin was practically "photoinsensitive" at liquid helium temperature (-265 degrees C), whereas 10-F-rhodopsin was converted to a photo-steady-state mixture of 10-F-rhodopsin and batho-10-F-rhodopsin. The specific interaction between the fluorine atom at the C10 position of the retinylidene chromophore and the opsin was discussed in terms of electrostatic interactions.     

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.     

Characterization of the primary photointermediates of Drosophila rhodopsin

Invertebrate opsins are unique among the visual pigments because the light-activated conformation, metarhodopsin, is stable following exposure to light in vivo. Recovery of the light-activated pigment to the dark conformation (or resting state) occurs either thermally or photochemically. There is no evidence to suggest that the chromophore becomes detached from the protein during any stage in the formation or recovery processes. Biochemical and structural studies of invertebrate opsins have been limited by the inability to express and purify rhodopsins for structure-function studies. In this study, we used Drosophila to produce an epitope-tagged opsin, Rh1-1D4, in quantities suitable for spectroscopic and photochemical characterization. When expressed in Drosophila, Rh1-1D4 is localized to the rhabdomere membranes, has the same spectral properties in vivo as wild-type Rh1, and activates the phototransduction cascade in a normal manner. Purified Rh1-1D4 visual pigment has an absorption maximum of the dark-adapted state of 474 nm, while the metarhodopsin absorption maximum is 572 nm. However, the metarhodopsin state is not stable as purified in dodecyl maltoside but decays with kinetics that require a double-exponential fit having lifetimes of 280 and 2700 s. We investigated the primary properties of the pigment at low temperature. At 70 K, the pigment undergoes a temperature-induced red shift to 486 nm. Upon illumination with 435 nm light, a photostationary state mixture is formed consisting of bathorhodopsin (lambda(max) = 545 nm) and isorhodopsin (lambda(max) = 462 nm). We also compared the spectroscopic and photochemical properties of this pigment with other vertebrate pigments. We conclude that the binding site of Drosophila rhodopsin is similar to that of bovine rhodopsin and is characterized by a protonated Schiff base chromophore stabilized via a single negatively charged counterion.     

Divergent mechanisms for the tuning of shortwave sensitive visual pigments in vertebrates.

Of the four classes of vertebrate cone visual pigments, the shortwave-sensitive SWS1 class shows the shortest lambda(max) values with peaks in different species in either the violet (390-435 nm) or ultraviolet (around 365 nm) regions of the spectrum. Phylogenetic evidence indicates that the ancestral pigment was probably UV-sensitive (UVS) and that the shifts between violet and UV have occurred many times during evolution. This is supported by the different mechanisms for these shifts in different species. All visual pigments possess a chromophore linked via a Schiff base to a Lys residue in opsin protein. In violet-sensitive (VS) pigments, the Schiff base is protonated whereas in UVS pigments, it is almost certainly unprotonated. The generation of VS from ancestral UVS pigments most likely involved amino acid substitutions in the opsin protein that serve to stabilise protonation. The key residues in the opsin protein for this are at sites 86 and 90 that are adjacent to the Schiff base and the counterion at Glu113. In this review, the different molecular mechanisms for the UV or violet shifts are presented and discussed in the context of the structural model of bovine rhodopsin.     

Rhodopsin photoproducts in 2D crystals

The published electron microscope and X-ray structures of rhodopsin have made available a detailed picture of the inactive dark state of rhodopsin. Yet, the photointermediates of rhodopsin that ultimately lead to the activated receptor species still await a similar analysis. Such an analysis first requires the generation and characterization of the photoproducts that can be obtained in crystals of rhodopsin. We therefore studied with Fourier-transform infrared (FTIR) difference spectroscopy the photoproducts in 2D crystals of bovine rhodopsin in a p22(1)2(1) crystal form. The spectra obtained by cryotrapping revealed that in this crystal form the still inactive early intermediates batho, lumi, and meta I are similar to those obtained from rhodopsin in native disk membranes, although the transition from lumi to meta I is shifted to a higher temperature. However, at room temperature, the formation of the active state, meta II, is blocked in the crystalline environment. Instead, an intermediate state is formed that bears some features of meta II but lacks the specific conformational changes required for activity. Despite being unable to activate its cognate G protein, transducin, to a significant extent, this intermediate state is capable of interacting with functional transducin-derived peptides to a limited extent. Therefore, while unable to support formation of rhodopsin's active state meta II, 2D p22(1)2(1) crystals proved to be very suitable for determining 3D structures of its still inactive precursors, batho, lumi, and meta I. In future studies, FTIR spectroscopy may serve as a sensitive assay to screen crystals grown under altered conditions for potential formation of the active state, meta II.     

Photochemical reaction of 9-cis-retro-gamma-rhodopsin at low temperatures.

9-cis-Retro-gamma-rhodopsin (lambda max = 420 nm) was prepared from 9-cis-retro-gamma-retinal and cattle opsin. After cooling to liquid nitrogen temperature (77 K), the pigment was irradiated with light at 380 nm. The spectrum shifted to the longer wavelengths, owing to formation of a batho product. This fact indicates that the conjugated double bond system from C-5 to C-8 of the chromophoric retinal in rhodopsin was not necessary for formation of bathorhodopsin. Reirradiation of the batho product with light at wavelengths longer than 520 nm yielded a mixture composed of presumably 9- or 11-cis forms of retro-gamma-rhodopsin. These three isomers are interconvertible by light at liquid nitrogen temperature. Thus the retro-gamma-rhodopsin system is similar in photochemical reaction at 77 K to cattle rhodopsin system. Each system has its own batho product. Based on these results, it was infered that the formation of batho-rhodopsin is due to photoisomerization of the chromophoric retinal of rhodopsin and is not due to translocation of a proton on the ring or on the side chain from C-6 to C-8 of the chromophoric retinal to the Schiff-base nitrogen.     

Chimeric nature of pinopsin between rod and cone visual pigments.

Chicken pineal pinopsin is the first example of extra-retinal opsins, but little is known about its molecular properties as compared with retinal rod and cone opsins. For characterization of extra-retinal photon signaling, we have developed an overexpression system providing a sufficient amount of purified pinopsin. The recombinant pinopsin, together with similarly prepared chicken rhodopsin and green-sensitive cone pigment, was subjected to photochemical and biochemical analyses by using low-temperature spectroscopy and the transducin activation assay. At liquid nitrogen temperature (-196 degrees C), we detected two kinds of photoproducts, bathopinopsin and isopinopsin, having their absorption maxima (lambda(max)) at 527 and approximately 440 nm, respectively, and we observed complete photoreversibility among pinopsin, bathopinopsin, and isopinopsin. A close parallel of the photoreversibility to the rhodopsin system strongly suggests that light absorbed by pinopsin triggers the initial event of cis-trans isomerization of the 11-cis-retinylidene chromophore. Upon warming, bathopinopsin decayed through a series of photobleaching intermediates: lumipinopsin (lambda(max) 461 nm), metapinopsin I (460 nm), metapinopsin II (385 nm), and metapinopsin III (460 nm). Biochemical and kinetic analyses showed that metapinopsin II is a physiologically important photoproduct activating transducin. Detailed kinetic analyses revealed that the formation of metapinopsin II is as fast as that of a chicken cone pigment, green, but that the decay process of metapinopsin II is as slow as that of the rod pigment, rhodopsin. These results indicate that pinopsin is a new type of pigment with a chimeric nature between rod and cone visual pigments in terms of the thermal behaviors of the meta II intermediate. Such a long-lived active state of pinopsin may play a role in the pineal-specific phototransduction process.     

Spectroscopic characterization of the photocycle intermediates of photoactive yellow protein.

The absorption spectra of photocycle intermediates of photoactive yellow protein mutants were compared with those of the corresponding intermediates of wild type to probe which amino acid residues interact with the chromophore in the intermediate states. B and H intermediates were produced by irradiation and trapped at 80 K, and L intermediates at 193 K. The absorption spectra of these intermediates produced from R52Q were identical to those from wild type, whereas those from E46Q and T50V were 7-15 nm red-shifted as those in the dark states. The absorption spectra of M intermediates were measured by flash photolysis at room temperature. Those of Y42F, T50V, and R52Q were identical to that of wild type, whereas that of E46Q was 11 nm red-shifted. Assuming that the intermediates of mutants have a structure comparable to that of wild type, these findings suggest the following: Glu46 interacts with the chromophore throughout the photocycle, interaction between the chromophore and Thr50 as well as Tyr42 is lost upon the formation of M intermediate, and Arg52 never interacts with the chromophore directly. The hydrogen-bonding network around the phenolic oxygen of the chromophore would be thus maintained until L intermediate decays, and the global conformational change would take place by the loss of the hydrogen bond between the chromophore and Tyr42. This model conflicts with some of the results of previous crystallographic studies, suggesting that the reaction mechanism in the crystal may be different from that in solution.     

Spectroscopic study of the batho-to-lumi transition during the photobleaching of rhodopsin using ring-modified retinal analogues

Photochemical and subsequent thermal reactions of rhodopsin containing 9-cis-retinal [Rh(9)] or one of four analogues with 9-cis geometries formed from ring-modified retinals, alpha-retinal [alpha Rh(9)], acyclic retinal [AcRh(9)], acyclic alpha-retinal [Ac alpha Rh(9)], and 5-isopropyl-alpha-retinal [P alpha Rh(9)] were investigated by low-temperature spectrophotometry and nanosecond laser photolysis. Irradiation of each pigment at -180 degrees C produced a photosteady-state mixture containing the original 9-cis pigment, its 11-cis pigment, and a photoproduct, indicating that the primary process of each pigment is a photoisomerization of its chromophore. The photoproduct produced by the irradiation of AcRh(9) had an absorption spectrum red shifted from the original AcRh(9) and was identified as the batho intermediate of AcRh(9). It was converted to the lumi intermediate through a metastable species, the BL intermediate, which has never been detected in Rh(9) at low temperature and whose absorption maximum was at shorter wavelengths than that of the batho intermediate. In contrast, the absorption maxima of the photoproducts produced from the other analogue pigments were at shorter wavelengths than those of the original pigments. They were identified as BL intermediates on the basis of their absorption maxima and thermal stabilities. The formation time constant of the lumi intermediate at room temperature was found to be dependent on the extent of modification of the ring portion of the chromophore, decreasing with the complete truncation of the cyclohexenyl ring [Ac alpha Rh(9)] and increasing with the attachment of the isopropyl group to the ring [P alpha Rh(9)].(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative studies on the late bleaching processes of four kinds of cone visual pigments and rod visual pigment

Visual pigments in rod and cone photoreceptor cells of vertebrate retinas are highly diversified photoreceptive proteins that consist of a protein moiety opsin and a light-absorbing chromophore 11-cis-retinal. There are four types of cone visual pigments and a single type of rod visual pigment. The reaction process of the rod visual pigment, rhodopsin, has been extensively investigated, whereas there have been few studies of cone visual pigments. Here we comprehensively investigated the reaction processes of cone visual pigments on a time scale of milliseconds to minutes, using flash photolysis equipment optimized for cone visual pigment photochemistry. We used chicken violet (L-group), chicken blue (M1-group), chicken green (M2-group), and monkey green (L-group) visual pigments as representatives of the respective groups of the phylogenetic tree of cone pigments. The S, M1, and M2 pigments showed the formation of a pH-dependent mixture of meta intermediates, similar to that formed from rhodopsin. Although monkey green (L-group) also formed a mixture of meta intermediates, pH dependency of meta intermediates was not observed. However, meta intermediates of monkey green became pH dependent when the chloride ion bound to the monkey green was replaced with a nitrate ion. These results strongly suggest that rhodopsin and S, M1, and M2 cone visual pigments share a molecular mechanism for activation, whereas the L-group pigment may have a special reaction mechanism involving the chloride-binding site.     

Dual photoactive species in Glu46Asp and Glu46Ala mutants of photoactive yellow protein: a pH-driven color transition.

Photoactive yellow protein (PYP) is a blue light sensor present in the purple photosynthetic bacterium Ectothiorhodospira halophila, which undergoes a cyclic series of absorbance changes upon illumination at its lambda(max) of 446 nm. The anionic p-hydroxycinnamoyl chromophore of PYP is covalently bound as a thiol ester to Cys69, buried in a hydrophobic pocket, and hydrogen-bonded via its phenolate oxygen to Glu46 and Tyr42. The chromophore becomes protonated in the photobleached state (I(2)) after it undergoes trans-cis isomerization, which results in breaking of the H-bond between Glu46 and the chromophore and partial exposure of the phenolic ring to the solvent. In previous mutagenesis studies of a Glu46Gln mutant, we have shown that a key factor in controlling the color and photocycle kinetics of PYP is this H-bonding system. To further investigate this, we have now characterized Glu46Asp and Glu46Ala mutants. The ground-state absorption spectrum of the Glu46Asp mutant shows a pH-dependent equilibrium (pK = 8.6) between two species: a protonated (acidic) form (lambda(max) = 345 nm), and a slightly blue-shifted deprotonated (basic) form (lambda(max) = 444 nm). Both of these species are photoactive. A similar transition was also observed for the Glu46Ala mutant (pK = 7.9), resulting in two photoactive red-shifted forms: a basic species (lambda(max) = 465 nm) and a protonated species (lambda(max) = 365 nm). We attribute these spectral transitions to protonation/deprotonation of the phenolate oxygen of the chromophore. This is demonstrated by FT Raman spectra. Dark recovery kinetics (return to the unphotolyzed state) were found to vary appreciably between these various photoactive species. These spectral and kinetic properties indicate that the hydrogen bond between Glu46 and the chromophore hydroxyl group is a dominant factor in controlling the pK values of the chromophore and the glutamate carboxyl.     

Bathoproducts of rhodopsin, isorhodopsin I, and isorhodopsin II.

Bathorhodopsins were prepared by partially (10--15%) photoconverting bovine rhodopsin (11-cis chromophore) or isorhodopsin I (9-cis chromophore) at 77 degrees K; care was taken to avoid establishing photostationary states. The absorption spectra calculated for the bathorhodopsins derived from the two parent pigments are identical in their lambda max 'S, bandwidths, and extinction coefficients. This result provides further support for the hypothesis that bathorhodopsin is a common intermediate between an 11-cis pigment (rhodopsin) and a 9-cis one (isorhodopsin I) and thus probably has an all-trans chromophore. This in turn is strong evidence for the cis-trans isomerization model of the primary event in vision. The spectrum of the bathoproduct of isorhodopsin II (9,13-dicis chromophore) is different from the other pigments' bathoproducts.     

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)     

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.     

Light-induced hydrolysis and rebinding of nonisomerizable bacteriorhodopsin pigment

Bacteriorhodopsin (bR) is characterized by a retinal-protein protonated Schiff base covalent bond, which is stable for light absorption. We have revealed a light-induced protonated Schiff base hydrolysis reaction in a 13-cis locked bR pigment (bR5.13; lambda(max) = 550 nm) in which isomerization around the critical C13==C14 double bond is prevented by a rigid ring structure. The photohydrolysis reaction takes place without isomerization around any of the double bonds along the polyene chain and is indicative of protein conformational alterations probably due to light-induced polarization of the retinal chromophore. Two photointermediates are formed during the hydrolysis reaction, H450 (lambda(max) = 450 nm) and H430 (lambda(max) = 430 nm), which are characterized by a 13-cis configuration as analyzed by high-performance liquid chromatography. Upon blue light irradiation after the hydrolysis reaction, these intermediates rebind to the apomembrane to reform bR5.13. Irradiation of the H450 intermediate forms the original pigment, whereas irradiation of H430 at neutral pH results in a red shifted species (P580), which thermally decays back to bR5.13. Electron paramagnetic resonance (EPR) spectroscopy indicates that the cytoplasmic side of bR5.13 resembles the conformation of the N photointermediate of native bR. Furthermore, using osmotically active solutes, we have observed that the hydrolysis rate is dependent on water activity on the cytoplasmic side. Finally, we suggest that the hydrolysis reaction proceeds via the reversed pathway of the binding process and allows trapping a new intermediate, which is not accumulated in the binding process.     

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.