The pKa of the protonated Schiff bases of gecko cone and octopus visual pigments.

A visual pigment is composed of retinal bound to its apoprotein by a protonated Schiff base linkage. Light isomerizes the chromophore and eventually causes the deprotonation of this Schiff base linkage at the meta II stage of the bleaching cycle. The meta II intermediate of the visual pigment is the active form of the pigment that binds to and activates the G protein transducin, starting the visual cascade. The deprotonation of the Schiff base is mandatory for the formation of meta II intermediate. We studied the proton binding affinity, pKa, of the Schiff base of both octopus rhodopsin and the gecko cone pigment P521 by spectral titration. Several fluorinated retinal analogs have strong electron withdrawing character around the Schiff base region and lower the Schiff base pKa in model compounds. We regenerated octopus and gecko visual pigments with these fluorinated and other retinal analogs. Experiments on these artificial pigments showed that the spectral changes seen upon raising the pH indeed reflected the pKa of the Schiff base and not the denaturation of the pigment or the deprotonation of some other group in the pigment. The Schiff base pKa is 10.4 for octopus rhodopsin and 9.9 for the gecko cone pigment. We also showed that although the removal of Cl- ions causes considerable blue-shift in the gecko cone pigment P521, it affects the Schiff base pKa very little, indicating that the lambda max of visual pigment and its Schiff base pKa are not tightly coupled.     

Evidence for a bound water molecule next to the retinal Schiff base in bacteriorhodopsin and rhodopsin: a resonance Raman study of the Schiff base hydrogen/deuterium exchange.

The retinal chromophores of both rhodopsin and bacteriorhodopsin are bound to their apoproteins via a protonated Schiff base. We have employed continuous-flow resonance Raman experiments on both pigments to determine that the exchange of a deuteron on the Schiff base with a proton is very fast, with half-times of 6.9 +/- 0.9 and 1.3 +/- 0.3 ms for rhodopsin and bacteriorhodopsin, respectively. When these results are analyzed using standard hydrogen-deuteron exchange mechanisms, i.e., acid-, base-, or water-catalyzed schemes, it is found that none of these can explain the experimental results. Because the exchange rates are found to be independent of pH, the deuterium-hydrogen exchange can not be hydroxyl (or acid-)-catalyzed. Moreover, the deuterium-hydrogen exchange of the retinal Schiff base cannot be catalyzed by water acting as a base because in that case the estimated exchange rate is predicted to be orders of magnitude slower than that observed. The relatively slow calculated exchange rates are essentially due to the high pKa values of the Schiff base in both rhodopsin (pKa > 17) and bacteriorhodopsin (pKa approximately 13.5). We have also measured the deuterium-hydrogen exchange of a protonated Schiff base model compound in aqueous solution. Its exchange characteristics, in contrast to the Schiff bases of the pigments, is pH-dependent and consistent with the standard base-catalyzed schemes. Remarkably, the water-catalyzed exchange, which has a half-time of 16 +/- 2 ms and which dominates at pH 3.0 and below, is slower than the exchange rate of the Schiff base in rhodopsin and bacteriorhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Octopus photoreceptor membranes. Surface charge density and pK of the Schiff base of the pigments.

The chromophore of octopus rhodopsin is 11-cis retinal, linked via a protonated Schiff base to the protein backbone. Its stable photoproduct, metarhodopsin, has all-trans retinal as its chromphore. The Schiff base of acid metarhodopsin (lambda max = 510 nm) is protonated, whereas that of alkaline metarhodopsin (lambda max = 376 nm) is unprotonated. Metarhodopsin in photoreceptor membranes was titrated and the apparent pK of the Schiff base was measured at different ionic strengths. From these salt-dependent pKs the surface charge density of the octopus photoreceptor membranes and the intrinsic Schiff base pK of metarhodopsin were obtained. The surface charge density is sigma = -1.6 +/- 0.1 electronic charges per 1,000 A2. Comparison of the measured surface charge density with values from octopus rhodopsin model structures suggests that the measured value is for the extracellular surface and so the Schiff base in metarhodopsin is freely accessible to protons from the extracellular side of the membrane. The intrinsic Schiff base pK of metarhodopsin is 8.44 +/- 0.12, whereas that of rhodopsin is found to be 10.65 +/- 0.10 in 4.0 M KCl. These pK values are significantly higher than the pK value around 7.0 for a retinal Schiff base in a polar solvent; we suggest that a plausible mechanism to increase the pK of the retinal pigments is the preorganization of their chromophore-binding sites. The preorganized site stabilizes the protonated Schiff base with respect to the unprotonated one. The difference in the pK for the octopus rhodopsin compared with metarhodopsin is attributed to the relative freedom of the latter's chromophore-binding site to rearrange itself after deprotonation of the Schiff base.     

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.     

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.     

A study of the Schiff base mode in bovine rhodopsin and bathorhodopsin

We have obtained the resonance Raman spectra of bovine rhodopsin, bathorhodopsin, and isorhodopsin for a series of isotopically labeled retinal chromophores. The specific substitutions are at retinal's protonated Schiff base moiety and include -HC = NH+-, -HC = ND+-, -H13C = NH+-, and -H13C = ND+-. Apart from the doubly labeled retinal, we find that the protonated Schiff base frequency is the same, within experimental error, for both rhodopsin and bathorhodopsin for all the substitutions measured here and elsewhere. We develop a force field that accurately fits the observed ethylenic (C = C) and protonated Schiff base stretching frequencies of rhodopsin and labeled derivatives. Using MINDO/3 quantum mechanical procedures, we investigate the response of this force field, and the ethylenic and Schiff base stretching frequencies, to the placement of charges close to retinal's Schiff base moiety. Specifically, we find that the Schiff base frequency should be measurably affected by a 3.0-4.5-A movement of a negatively charged counterion from the positively charged protonated Schiff base moiety. That there is no experimentally discernible difference in the Schiff base frequency between rhodopsin and bathorhodopsin suggests that models for the efficient conversion of light to chemical energy in the rhodopsin to bathorhodopsin photoconversion based solely on salt bridge separation of the protonated Schiff base and its counterion are probably incorrect. We discuss various alternative models and the role of electrostatics in the rhodopsin to bathorhodopsin primary process.     

Rapid release of retinal from a cone visual pigment following photoactivation

As part of the visual cycle, the retinal chromophore in both rod and cone visual pigments undergoes reversible Schiff base hydrolysis and dissociation following photobleaching. We characterized light-activated release of retinal from a short-wavelength-sensitive cone pigment (VCOP) in 0.1% dodecyl maltoside using fluorescence spectroscopy. The half-time (t(1/2)) of release of retinal from VCOP was 7.1 s, 250-fold faster than that of rhodopsin. VCOP exhibited pH-dependent release kinetics, with the t(1/2) decreasing from 23 to 4 s with the pH decreasing from 4.1 to 8, respectively. However, the Arrhenius activation energy (E(a)) for VCOP derived from kinetic measurements between 4 and 20 °C was 17.4 kcal/mol, similar to the value of 18.5 kcal/mol for rhodopsin. There was a small kinetic isotope (D(2)O) effect in VCOP, but this effect was smaller than that observed in rhodopsin. Mutation of the primary Schiff base counterion (VCOP(D108A)) produced a pigment with an unprotonated chromophore (λ(max) = 360 nm) and dramatically slowed (t(1/2) ~ 6.8 min) light-dependent retinal release. Using homology modeling, a VCOP mutant with two substitutions (S85D and D108A) was designed to move the counterion one α-helical turn into the transmembrane region from the native position. This double mutant had a UV-visible absorption spectrum consistent with a protonated Schiff base (λ(max) = 420 nm). Moreover, the VCOP(S85D/D108A) mutant had retinal release kinetics (t(1/2) = 7 s) and an E(a) (18 kcal/mol) similar to those of the native pigment exhibiting no pH dependence. By contrast, the single mutant VCOP(S85D) had an ~3-fold decreased retinal release rate compared to that of the native pigment. Photoactivated VCOP(D108A) had kinetics comparable to those of a rhodopsin counterion mutant, Rho(E113Q), both requiring hydroxylamine to fully release retinal. These results demonstrate that the primary counterion of cone visual pigments is necessary for efficient Schiff base hydrolysis. We discuss how the large differences in retinal release rates between rod and cone visual pigments arise, not from inherent differences in the rate of Schiff base hydrolysis but rather from differences in the properties of noncovalent binding of the retinal chromophore to the protein.     

Structural changes in the Schiff base region of squid rhodopsin upon photoisomerization studied by low-temperature FTIR spectroscopy

Low-temperature Fourier transform infrared (FTIR) spectroscopy is used to study squid rhodopsin at 77 K in investigating structural changes in the Schiff base region upon photoisomerization. The analysis of O-D stretching vibrations in D(2)O revealed that there are more internal water molecules near the retinal chromophore in squid rhodopsin than in bovine rhodopsin. Among nine O-D stretching vibrations of water in squid rhodopsin, eight peaks are identical between rhodopsin and 9-cis-rhodopsin (Iso). On the other hand, the isomer-specific O-D stretch of water was observed for rhodopsin (2451 cm(-)(1)) and Iso (2382 cm(-)(1)). Low frequencies of these bands suggest that the water forms a strong hydrogen bond with a negatively charged counterion. In addition, it was suggested that the hydrogen bond of the Schiff base is weaker in squid rhodopsin than in bacteriorhodopsin and bovine rhodopsin, and squid rhodopsin possessed similar hydrogen bonding strength for the Schiff base among rhodopsin, Iso, and bathorhodopsin. Most vibrational bands in the X-D stretch region originate from water O-D or the Schiff base N-D stretches, suggesting that the hydrogen bonding network in the Schiff base region of squid rhodopsin is composed of only water molecules. On the basis of these results, we propose that squid rhodopsin possesses a "bridge" water between the Schiff base and its counterion as well as squid retinochrome [Furutani, Y., Terakita, A., Shichida, Y., and Kandori, H. (2005) Biochemistry 44, 7988-7997], which is absent in vertebrate rhodopsin [Furutani, Y., Shichida, Y., and Kandori, H. (2003) Biochemistry 42, 9619-9625].     

Energetics of primary processes in visula escitation: photocalorimetry of rhodopsin in rod outer segment membranes.

A sensitive technique for the direct calorimetric determination of the energetics of photochemical reactions under low levels of illumination, and its application to the study of primary processes in visula excitation, are described. Enthlpies are reported for various steps in the bleaching of rhodopsin in intact rod outer segment membranes, together with the heats of appropriate model reactions. Protonation changes are also determined calorimetrically by use of buffers with differing heats of proton ionization. Bleaching of rhodopsin is accompanied by significant uptake of heat energy, vastly in excess of the energy required for simple isomerization of the retinal chromophore. Metarhodopsin I formation involves the uptake of about 17 kcal/mol and no net change in proton ionization of the system. Formation of metarhodopsin II requires an additional energy of about 10 kcal/mol and involves the uptake on one hydrogen ion from solution. The energetics of the overall photolysis reaction, rhodopsin leads to opsin + all-trans-retinal, are pH dependent and involve the exposure of an additional titrating group on opsin. This group has a heat of proton ionization of about 12 kcal/mal, characteristic of a primary amine, but a pKa in the region of neutrality. We suggest that this group is the Schiff base lysine of the chromophore binding site of rhodopsin which becomes exposed on photolysis. The low pKa for this active lysine would result in a more stable retinal-opsin linkage, and might be induced by a nearby positively charged group on the protein (either arginine or a second lysine residue). This leads to a model involving intramolecular protonation of the Schiff base nitrogen in the retinal-opsin linkage of rhodopsin, which is consistent with the thermodynamic and spectroscopic properties of the system. We further propose that the metarhodopsin I leads to metarhodopsin II step in the bleaching sequence involves reversible hydrolysis of the Schiff base linkage in the chromophore binding site, and that subsequent steps are the result of migration of the chromophore from this site.     

Crystallographic analysis of the primary photochemical reaction of squid rhodopsin

Visual signal transduction is initiated by the photoisomerization of 11-cis retinal upon rhodopsin ligation. Unlike vertebrate rhodopsin, which interacts with Gt-type G-protein to stimulate the cyclic GMP signaling pathway, invertebrate rhodopsin interacts with Gq-type G-protein to stimulate a signaling pathway that is based on inositol 1,4,5-triphosphate. Since the inositol 1,4,5-triphosphate signaling pathway is utilized by mammalian nonvisual pigments and a large number of G-protein-coupled receptors, it is important to elucidate how the activation mechanism of invertebrate rhodopsin differs from that of vertebrate rhodopsin. Previous crystallographic studies of squid and bovine rhodopsins have shown that there is a profound difference in the structures of the retinal-binding pockets of these photoreceptors. Here, we report the crystal structures of all-trans bathorhodopsin (Batho; the first photoreaction intermediate) and the artificial 9-cis isorhodopsin (Iso) of squid rhodopsin. Upon the formation of Batho, the central moiety of the retinal was observed to move largely towards the cytoplasmic side, while the Schiff base and the ionone ring underwent limited movements (i.e., the all-trans retinal in Batho took on a right-handed screwed configuration). Conversely, the 9-cis retinal in Iso took on a planar configuration. Our results suggest that the light energy absorbed by squid rhodopsin is mostly converted into the distortion energy of the retinal polyene chain and surrounding residues.     

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.     

Photosensitivities of rhodopsin mutants with a displaced counterion

Visual pigments consist of a protein moiety opsin and an 11-cis-retinal chromophore that is covalently bound to the opsin via a Schiff base linkage. They have a high photosensitivity, which can be attributed to the high probability of photon absorption and the high photoisomerization quantum yield of the retinal chromophore. Both of these parameters are regulated by the opsin, though the precise mechanism is unknown. We previously found that counterion residue E113, which stabilizes the proton on the Schiff base, is involved in the efficient photoisomerization in vertebrate visual pigments. To test the positional effect of the counterion on the photon absorption and the photoisomerization, we measured the photosensitivities of a set of mutants of bovine rhodopsin in which the counterion was displaced to position 90, 94, 117, or 292. The molar extinction coefficient was reduced in many of the mutants, leading to reductions in the photosensitivity for monochromatic lights. However, the oscillator strength, the probability of photon absorption integrated over the entire wavenumber range of the absorption band, was relatively similar among the mutants and the wild type. In addition, the quantum yields of the mutants were not markedly different from that of the wild type. These results indicate that the counterion does not need to be located at position 113 for a high photosensitivity for natural light. Interestingly, all of the mutants exhibited greatly increased hydroxylamine sensitivity. This result suggests that the counterion in vertebrate visual pigments is optimally located for the stability of the Schiff base linkage.     

Resonance Raman spectroscopy of octopus rhodopsin and its photoproducts

We report here the resonance Raman spectra of octopus rhodopsin and its photoproducts, bathorhodopsin and acid metarhodopsin. These studies were undertaken in order to make comparisons with the well-studied bovine pigments, so as to understand the similarities and the differences in pigment structure and photochemical processes between vertebrates and invertebrates. The flow method was used to obtain the Raman spectrum of rhodopsin at 13 degrees C. The bathorhodopsin spectrum was obtained by computer subtraction of the spectra containing different photostationary mixtures of rhodopsin, isorhodopsin, hypsorhodopsin, and bathorhodopsin, obtained at 12 K using the pump-probe technique and from measurements at 80 K. Like their bovine counterparts, the Schiff base vibrational mode appears at approximately 1660 cm-1 in octopus rhodopsin and the photoproducts, bathorhodopsin and acid metarhodopsin, suggesting a protonated Schiff base linkage between the chromophore and the protein. Differences between the Raman spectra of octopus rhodopsin and bathorhodopsin indicate that the formation of bathorhodopsin is associated with chromophore isomerization. This inference is substantiated by the chromophore chemical extraction data which show that, like the bovine system, octopus rhodopsin is an 11-cis pigment, while the photoproducts contain an all-trans pigment, in agreement with previous work. The octopus rhodopsin and bathorhodopsin spectra show marked differences from their bovine counterparts in other respects, however. The differences are most dramatic in the structure-sensitive fingerprint and the HOOP regions. Thus, it appears that although the two species differ in the specific nature of the chromophore-protein interactions, the general process of visual transduction is the same.     

Resonance Raman microprobe spectroscopy of rhodopsin mutants: effect of substitutions in the third transmembrane helix.

A microprobe system has been developed that can record Raman spectra from as little as 2 microL of solution containing only micrograms of biological pigments. The apparatus consists of a liquid nitrogen (l-N2)-cooled cold stage, an epi-illumination microscope, and a substractive-dispersion, double spectrograph coupled to a l-N2-cooled CCD detector. Experiments were performed on native bovine rhodopsin, rhodopsin expressed in COS cells, and four rhodopsin mutants: Glu134 replaced by Gln (E134Q), Glu122 replaced by Gln (E122Q), and Glu113 replaced by Gln (E113Q) or Ala (E113A). Resonance Raman spectra of photostationary steady-state mixtures of 11-cis-rhodopsin, 9-cis-isorhodopsin, and all-trans-bathorhodopsin at 77 K were recorded. The Raman spectra of E134Q and the wild-type are the same, indicating that Glu134 is not located near the chromophore. Substitution at Glu122 also does not affect the C = NH stretching vibration of the chromophore. The fingerprint and Schiff base regions of the Raman spectra of the 380-nm, pH 7 forms of E113Q and E113A are characteristic of unprotonated retinal Schiff bases. The C = NH modes of the approximately 500-nm, pH 5 forms of E113Q and E113A in H2O (D2O) are found at 1648 (1629) and 1645 (1630) cm-1, respectively. These frequencies indicate that the protonated Schiff base interacts more weakly with its protein counterion in the Glu113 mutants than it does in the native pigment. Furthermore, perturbations of the unique bathorhodopsin hydrogen out-of-plane (HOOP) vibrations in E113Q and E113A indicate that the strength of the protein perturbation near C12 is weakened compared to that in native bathorhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resonance raman spectroscopy of an ultraviolet-sensitive insect rhodopsin

We present the first visual pigment resonance Raman spectra from the UV-sensitive eyes of an insect, Ascalaphus macaronius (owlfly). This pigment contains 11-cis-retinal as the chromophore. Raman data have been obtained for the acid metarhodopsin at 10 degrees C in both H2O and D2O. The C = N stretching mode at 1660 cm-1 in H2O shifts to 1631 cm-1 upon deuteriation of the sample, clearly showing a protonated Schiff base linkage between the chromophore and the protein. The structure-sensitive fingerprint region shows similarities to the all-trans-protonated Schiff base of model retinal chromophores, as well as to the octopus acid metarhodopsin and bovine metarhodopsin I. Although spectra measured at -100 degrees C with 406.7-nm excitation, to enhance scattering from rhodopsin (lambda max 345 nm), contain a significant contribution from a small amount of contaminants [cytochrome(s) and/or accessory pigment] in the sample, the C = N stretch at 1664 cm-1 suggests a protonated Schiff base linkage between the chromophore and the protein in rhodopsin as well. For comparison, this mode also appears at approximately 1660 cm-1 in both the vertebrate (bovine) and the invertebrate (octopus) rhodopsins. These data are particularly interesting since the absorption maximum of 345 nm for rhodopsin might be expected to originate from an unprotonated Schiff base linkage. That the Schiff base linkage in the owlfly rhodopsin, like in bovine and in octopus, is protonated suggests that a charged chromophore is essential to visual transduction.     

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.     

Constitutive activity of a UV cone opsin

Vertebrate visual pigment proteins contain a conserved carboxylic acid residue in the third transmembrane helix. In rhodopsin, Glu113 serves as a counterion to the positively charged protonated Schiff base formed by 11-cis retinal attached to Lys296. Activation involves breaking of this ion pair. In UV cone pigments, the retinyl Schiff base is unprotonated, and hence such a salt bridge is not present; yet the pigment is inactive in the dark. Mutation of Glu108, which corresponds to rhodopsin's Glu113, to Gln yields a pigment that remains inactive in the dark. The apoproteins of both the wild-type and mutant, however, are constitutively active with the mutant being of significantly higher activity. Thus, one important role for preserving the negatively charged glutamate in the third helix of UV pigments is to maintain a less active opsin in a manner similar to rhodopsin. Ligand binding itself in the absence of a salt bridge is sufficient for deactivation.     

Role of the retinal hydrogen bond network in rhodopsin Schiff base stability and hydrolysis

Little is known about the molecular mechanism of Schiff base hydrolysis in rhodopsin. We report here our investigation into this process focusing on the role of amino acids involved in a hydrogen bond network around the retinal Schiff base. We find conservative mutations in this network (T94I, E113Q, S186A, E181Q, Y192F, and Y268F) increase the activation energy (E(a)) and abolish the concave Arrhenius plot normally seen for Schiff base hydrolysis in dark state rhodopsin. Interestingly, two mutants (T94I and E113Q) show dramatically faster rates of Schiff base hydrolysis in dark state rhodopsin, yet slower hydrolysis rates in the active MII form. We find deuterium affects the hydrolysis process in wild-type rhodopsin, exhibiting a specific isotope effect of approximately 2.5, and proton inventory studies indicate that multiple proton transfer events occur during the process of Schiff base hydrolysis for both dark state and MII forms. Taken together, our study demonstrates the importance of the retinal hydrogen bond network both in maintaining Schiff base integrity in dark state rhodopsin, as well as in catalyzing the hydrolysis and release of retinal from the MII form. Finally, we note that the dramatic alteration of Schiff base stability caused by mutation T94I may play a causative role in congenital night blindness as has been suggested by the Oprian and Garriga laboratories.     

Photochemical reaction of 7,8-dihydrorhodopsin at low temperatures

The photoreaction of 9-cis-7,8-dihydrorhodopsin was examined at liquid nitrogen temperatures (-180 degrees C) in order to elucidate the photochemical events in visual pigments. This rhodopsin analog was prepared by incubating 9-cis-7,8-dihydroretinal with bovine opsin in the dark. 9-cis-7,8-Dihydrorhodopsin (lambda max = 427 nm) was cooled to -180 degrees C, and then irradiated at -180 degrees C with a 390 nm light, resulting in formation of its bathochromic product (lambda max = 465 nm). This result indicates that the presence of four double-bonds adjacent to the Schiff base nitrogen is sufficient to allow formation of a bathochromic product. Thus, the mechanism of formation of bathorhodopsin (in bovine rhodopsin system) may be considered as some change of the interaction between the conjugated double-bond system from C-9 to the Schiff base nitrogen and its surrounding charges in opsin, caused by rotation of 11-12 double-bond.     

Function of extracellular loop 2 in rhodopsin: glutamic acid 181 modulates stability and absorption wavelength of metarhodopsin II

The second extracellular loop of rhodopsin folds back into the membrane-embedded domain of the receptor to form part of the binding pocket for the 11-cis-retinylidene chromophore. A carboxylic acid side chain from this loop, Glu181, points toward the center of the retinal polyene chain. We studied the role of Glu181 in bovine rhodopsin by characterizing a set of site-directed mutants. Sixteen of the 19 single-site mutants expressed and bound 11-cis-retinal to form pigments. The lambda(max) value of mutant pigment E181Q showed a significant spectral red shift to 508 nm only in the absence of NaCl. Other substitutions did not significantly affect the spectral features of the mutant pigments in the dark. Thus, Glu181 does not contribute significantly to spectral tuning of the ground state of rhodopsin. The most likely interpretation of these data is that Glu181 is protonated and uncharged in the dark state of rhodopsin. The Glu181 mutants displayed significantly increased reactivity toward hydroxylamine in the dark. The mutants formed metarhodopsin II-like photoproducts upon illumination but many of the photoproducts displayed shifted lambda(max) values. In addition, the metarhodopsin II-like photoproducts of the mutant pigments had significant alterations in their decay rates. The increased reactivity of the mutants to hydroxylamine supports the notion that the second extracellular loop prevents solvent access to the chromophore-binding pocket. In addition, Glu181 strongly affects the environment of the retinylidene Schiff base in the active metarhodopsin II photoproduct.