Asp83, Glu113 and Glu134 are not specifically involved in Schiff base protonation or wavelength regulation in bovine rhodopsin

Site-specific mutagenesis was employed to investigate the proposed contribution of proton-donating residues (Glu, Asp) in the membrane domains of bovine rhodopsin to protonation of the Schiff base-linking protein and chromophore or to wavelength modulation of this visual pigment. Three point-mutations were introduced to replace the highly conserved residues Asp83 by Asn (D83N), Glu113 by Gln (E113 Q) or Glu134 by Asp (E134D), respectively. All 3 substitutions had only marginal effects on the spectral properties of the final pigment (less than or equal to 3 nm blue-shift relative to native rhodopsin). Hence, none of these residues by itself is specifically involved in Schiff base protonation or wavelength modulation of bovine rhodopsin.     

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.     

Proton movement and photointermediate kinetics in rhodopsin mutants

The role of ionizable amino acid side chains in the bovine rhodopsin activation mechanism was studied in mutants E134Q, E134R/R135E, H211F, and E122Q. All mutants exhibited bathorhodopsin stability on the 30 ns to 1 micros time scale similar to that of the wild type. Lumirhodopsin decay was also similar to that of the wild type except for the H211F mutant where early decay (20 micros) to a second form of lumirhodopsin was seen, followed by formation of an extremely long-lived Meta I(480) product (34 ms), an intermediate which forms to a much reduced extent, if at all, in dodecyl maltoside suspensions of wild-type rhodopsin. A smaller amount of a similar long-lived Meta I(480) product was seen after photolysis of E122Q, but E134Q and E134R/R135Q displayed kinetics much more similar to those of the wild type under these conditions (i.e., no Meta I(480) product). These results support the idea that specific interaction of His211 and Glu122 plays a significant role in deprotonation of the retinylidene Schiff base and receptor activation. Proton uptake measurements using bromcresol purple showed that E122Q was qualitatively similar to wild-type rhodopsin, with at least one proton being released during lumirhodopsin decay per Meta I(380) intermediate formed, followed by uptake of at least two protons per rhodopsin bleached on a time scale of tens of milliseconds. Different results were obtained for H211F, E134Q, and E134R/R135E, which all released approximately two protons per rhodopsin bleached. These results show that several ionizable groups besides the Schiff base imine are affected by the structural changes involved in rhodopsin activation. At least two proton uptake groups and probably at least one proton release group in addition to the Schiff base are present in rhodopsin.     

Chromophore structure in lumirhodopsin and metarhodopsin I by time-resolved resonance Raman microchip spectroscopy.

Time-resolved resonance Raman microchip flow experiments have been performed on the lumirhodopsin (Lumi) and metarhodopsin I (Meta I) photointermediates of rhodopsin at room temperature to elucidate the structure of the chromophore in each species as well as changes in protein-chromophore interactions. Transient Raman spectra of Lumi and Meta I with delay times of 16 micros and 1 ms, respectively, are obtained by using a microprobe system to focus displaced pump and probe laser beams in a microfabricated flow channel and to detect the scattering. The fingerprint modes of both species are very similar and characteristic of an all-trans chromophore. Lumi exhibits a relatively normal hydrogen-out-of-plane (HOOP) doublet at 951/959 cm(-1), while Meta I has a single HOOP band at 957 cm(-1). These results suggest that the transitions from bathorhodopsin to Lumi and Meta I involve a relaxation of the chromophore to a more planar all-trans conformation and the elimination of the structural perturbation that uncouples the 11H and 12H wags in bathorhodopsin. Surprisingly, the protonated Schiff base C=N stretching mode in Lumi (1638 cm(-1)) is unusually low compared to those in rhodopsin and bathorhodopsin, and the C=ND stretching mode shifts down by only 7 cm(-1) in D2O buffer. This indicates that the Schiff base hydrogen bonding is dramatically weakened in the bathorhodopsin to Lumi transition. However, the C=N stretching mode in Meta I is found at 1654 cm(-1) and exhibits a normal deuteration-induced downshift of 24 cm(-1), identical to that of the all-trans protonated Schiff base. The structural relaxation of the chromophore-protein complex in the bathorhodopsin to Lumi transition thus appears to drive the Schiff base group out of its hydrogen-bonded environment near Glu113, and the hydrogen bonding recovers to a normal solvated PSB value but presumably a different hydrogen bond acceptor with the formation of Meta I.     

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.     

The role of Glu181 in the photoactivation of rhodopsin

The visual pigment rhodopsin is a prototypical seven transmembrane helical G protein-coupled receptor. Photoisomerization of its protonated Schiff base (PSB) retinylidene chromophore initiates a progression of metastable intermediates. We studied the structural dynamics of receptor activation by FTIR spectroscopy of recombinant pigments. Formation of the active state, Meta II, is characterized by neutralization of the PSB and its counterion Glu113. We focused on testing the hypothesis of a PSB counterion switch from Glu113 to Glu181 during the transition of rhodopsin to the still inactive Meta I photointermediate. Our results, especially from studies of the E181Q mutant, support the view that both Glu113 and Glu181 are deprotonated, forming a complex counterion to the PSB in rhodopsin, and that the function of the primary counterion shifts from Glu113 to Glu181 during the transition to Meta I. The Meta I conformation in the E181Q mutant is less constrained compared with that of wild-type Meta I. In particular, the hydrogen bonded network linking transmembrane helices 1, 2, and 7, adopts a conformation that is already Meta II-like, while other parts of the receptor appear to be in a Meta I-like conformation similar to wild-type. We conclude that Glu181 is responsible, in part, for controlling the extraordinary high pK(a) of the chromophore PSB in the dark state, which very likely decreases upon transition to Meta I in a stepwise weakening of the interaction between PSB and its complex counterion during the course of receptor activation. A model for the specific role in coupling chromophore isomerization to protein conformational changes concomitant with receptor activation is presented.     

Fourier-transform infrared difference spectroscopy of rhodopsin and its photoproducts at low temperature

Fourier-transform infrared difference spectroscopy has been used to detect the vibrational modes in the chromophore and protein that change in position or intensity between rhodopsin and the photoproducts formed at low temperature (70 K), bathorhodopsin and isorhodopsin. A method has been developed to obtain infrared difference spectra between rhodopsin and bathorhodopsin, bathorhodopsin and isorhodopsin, and rhodopsin and isorhodopsin. To aid in the identification of the vibrational modes, we performed experiments on deuterated and hydrated films of native rod outer segments and rod outer segments regenerated with either retinal containing 13C at carbon 15 or 15-deuterioretinal. Our infrared measurements provide independent verification of the resonance Raman result that the retinal in bathorhodopsin is distorted all-trans. The positions of the C = N stretch in the deuterated pigment and the deuterated pigments regenerated with 11-cis-15-deuterioretinal or 11-cis-retinal containing 13C at carbon 15 are indicative that the Schiff-base linkage is protonated in rhodopsin, bathorhodopsin, and isorhodopsin. Furthermore, the C = N stretching frequency occurs at the same position in all three species. The data indicate that the protonated Schiff base has a C = N trans conformation in all three species. Finally, we present evidence that, even in these early stages of the rhodopsin photosequence, changes are occurring in the opsin and perhaps the associated lipids.     

Resonance Raman analysis of the mechanism of energy storage and chromophore distortion in the primary visual photoproduct

The vibrational structure of the chromophore in the primary photoproduct of vision, bathorhodopsin, is examined to determine the cause of the anomalously decoupled and intense C(11)=C(12) hydrogen-out-of-plane (HOOP) wagging modes and their relation to energy storage in the primary photoproduct. Low-temperature (77 K) resonance Raman spectra of Glu181 and Ser186 mutants of bovine rhodopsin reveal only mild mutagenic perturbations of the photoproduct spectrum suggesting that dipolar, electrostatic, or steric interactions with these residues do not cause the HOOP mode frequencies and intensities. Density functional theory calculations are performed to investigate the effect of geometric distortion on the HOOP coupling. The decoupled HOOP modes can be simulated by imposing approximately 40 degrees twists in the same direction about the C(11)=C(12) and C(12)-C(13) bonds. Sequence comparison and examination of the binding site suggests that these distortions are caused by three constraints consisting of an electrostatic anchor between the protonated Schiff base and the Glu113 counterion, as well as steric interactions of the 9- and 13-methyl groups with surrounding residues. This distortion stores light energy that is used to drive the subsequent protein conformational changes that activate rhodopsin.     

Changing the location of the Schiff base counterion in rhodopsin.

Rhodopsin and all of the vertebrate visual pigments have a carboxylic acid residue, Glu113, in the third transmembrane segment that serves as a counterion to the protonated Schiff base nitrogen of the chromophore. We show here that the counterion in bovine rhodopsin can be moved from position 113 to 117 without significantly changing the wild-type spectral properties of the protein. A series of double mutants were constructed where the Glu113 counterion was changed to Gln and an Asp residue was substituted for amino acid residues from position 111 to 121 in the third transmembrane segment of the protein. Only at position 117 can an Asp fully substitute for the counterion at position 113. The double mutant E113Q,-A117D has an absorption maximum at 493 nm which is independent of pH in the range 5.6-8.4 and independent of the presence of external chloride anions. An Asp at no other position tested in the third transmembrane segment can fully substitute for the Glu counterion at position 113. Partial substitution is observed for an Asp at position 120. Residues 113, 117, and 120 are expected to lie along the same face of an alpha-helix. These results suggest that the Schiff base nitrogen in rhodopsin is located between residues 113 and 117 but there is enough flexibility in the protein to allow partial interaction with an Asp at position 120. Position 117 is the same location of the counterion in the related biogenic amine receptors.     

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.     

Resonance Raman studies of the primary photochemical event in visual pigments.

Resonance Raman multicomponent spectra of bovine rhodopsin, isorhodopsin, and bathorhodopsin have been obtained at low temperature. Application of the double beam "pump-probe" technique allows us to extract a complete bathorhodopsin spectrum from the mixture in both protonated and deuterated media. Our results show that the Schiff base of bathorhodopsin is fully protonated and that the extent of protonation is unaffected by its photochemical formation from either rhodopsin or isorhodopsin. The Raman spectrum of bathorhodopsin is significantly different than that of either parent pigment, thus supporting the notion that a geometric change in the chromophore is an important component of the primary photochemical event in vision. A normal mode analysis is carried out with particular attention devoted to the factors that determine the frequency of the C=N stretching vibration. We find that the increased frequency of this mode in protonated relative to unprotonated Schiff bases is due to coupling between C=N stretching and C=N-H bending motions, and the shift observed upon deuteration of the Schiff base can also be understood in these terms. Various models for the primary event are discussed in light of our experimental and theoretical results.     

Transducin-dependent protonation of glutamic acid 134 in rhodopsin

A highly conserved carboxylic acid residue in rhodopsin, Glu(134), modulates transducin (G(t)) interaction. It has been postulated that Glu(134) becomes protonated upon receptor activation. We studied the interaction between rhodopsin and G(t) using Fourier transform infrared (FTIR) difference spectroscopy combined with attenuated total reflection (ATR). Formation of the complex between G(t) and photoactivated rhodopsin reconstituted into phosphatidylcholine vesicles caused prominent infrared absorption increases at 1641, 1550, and 1517 cm(-)(1). The rhodopsin mutant E134Q was also studied. When measured in the presence of G(t), replacement of Glu(134) by glutamine abolished the low-frequency part of a broad absorption band at 1735 cm(-)(1) that is normally superimposed on the light-induced absorption changes of Asp(83) and Glu(122) of rhodopsin. In addition, a negative absorption band at 1400 cm(-)(1) that is evoked by interaction of native metarhodopsin II (MII) with G(t) was not observed in the difference spectrum of the E134Q mutant. Thus, Glu(134) is ionized in the dark and exhibits a symmetrical COO(-) stretching vibration at 1400 cm(-)(1). Glu(134) becomes protonated in the G(t)-MII complex and displays a C=O stretching mode near 1730 cm(-)(1). The E134Q mutation also affects absorption changes attributable to lipids, suggesting that the protonation of Glu(134) is linked to transfer of the carboxylic acid side chain from a polar to a nonpolar environment by becoming exposed to the lipid phase when G(t) binds. These results show directly that Glu(134) becomes protonated in MII upon G(t) binding and suggest that changes in receptor conformation affect lipid-protein interactions.     

A comparative study of the infrared difference spectra for octopus and bovine rhodopsins and their bathorhodopsin photointermediates

Fourier-transform infrared difference spectroscopy has been used to detect the vibrational modes in the chromophore and protein that change in position and intensity between octopus rhodopsin and its photoproducts formed at low temperature (85 K), bathorhodopsin and isorhodopsin. The infrared difference spectra between octopus rhodopsin and octopus bathorhodopsin, octopus bathorhodopsin and octopus isorhodopsin, and octopus isorhodopsin and octopus rhodopsin are compared to analogous difference spectra for the well-studied bovine pigments, in order to understand the similarities in pigment structure and photochemical processes between the vertebrate and invertebrate systems. The structure-sensitive fingerprint region of the infrared spectra for octopus bathorhodopsin shows strong similarities to spectra of both all-trans-retinal and bovine bathorhodopsin, thus confirming chemical extraction data that suggest that octopus bathorhodopsin contains an all-trans-retinal chromophore. In contrast, we find dramatic differences in the hydrogen out-of-plane modes of the two bathorhodopsins, and in the fingerprint lines of the rhodopsins and isorhodopsins for the two pigments. These observations suggest that while the primary effect of light in the octopus rhodopsin system, as in the bovine rhodopsin system, is 11-cis/11-trans isomerization, the protein-chromophore interactions for the two systems are quite different. Finally, striking similarities and differences in infrared lines attributable to changes in amino acid residues in the opsin are found between the two pigment systems. They suggest that no carboxylic acid or tyrosine residues are affected in the initial changes of light-energy transduction in octopus rhodopsin. Comparing the amino acid sequences for octopus and bovine pigments also allows us to suggest that the carboxylic acid residues altered in the bovine transitions are Glu-122 and/or Glu-134.     

Mechanism of activation and inactivation of opsin: role of Glu113 and Lys296.

In previous studies, mutation of either Lys296 or Glu113 in bovine rhodopsin has been shown to result in constitutive activation of the apoprotein form, opsin [Robinson et al. (1992) Neuron 9, 719-725]. In this report, pH-rate profiles for the rhodopsin-catalyzed exchange of GTPgS for GDP on transducin are established for the constitutively active opsin mutants. All of the mutants, including the double-mutant E113Q,K296G, show a bell-shaped pH-rate profile. Therefore, it is evident that at least two ionizable groups in addition to Lys296 and Glu113 control the formation of the active opsin state. The sole effect of mutation at position 113 or 296 is to alter the ionization constant of the group with the higher pKa, called pka2. pKa2 decreases in the following order: rhodopsin/light (9.0) > K296E = K296G = E113Q,K296G (8.0) > E113Q (6.8) > K296H (6.6) > wild-type opsin (< 5.0). These results are consistent with a model where activation of opsin involves (i) breaking of the salt bridge between Lys296 and Glu113, (ii) deprotonation of Lys296, and (iii) the net uptake of a proton from the solvent. Furthermore, exogenous addition of the chromophore all-trans-retinal shifts the wild-type and E113Q opsin equilibrium to favor the active state. In all these respects, the light-independent activation of the opsin mutants appears to proceed by a mechanism similar to that of light-activated rhodopsin.     

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.     

Structural impact of the E113Q counterion mutation on the activation and deactivation pathways of the G protein-coupled receptor rhodopsin

Disruption of an interhelical salt bridge between the retinal protonated Schiff base linked to H7 and Glu113 on H3 is one of the decisive steps during activation of rhodopsin. Using previously established stabilization strategies, we engineered a stabilized E113Q counterion mutant that converted rhodopsin to a UV-absorbing photoreceptor with deprotonated Schiff base and allowed reconstitution into native-like lipid membranes. Fourier-transform infrared difference spectroscopy reveals a deprotonated Schiff base in the photoproducts of the mutant up to the active state Meta II, the absence of the classical pH-dependent Meta I/Meta II conformational equilibrium in favor of Meta II, and an anticipation of active state features under conditions that stabilize inactive photoproduct states in wildtype rhodopsin. Glu181 on extracellular loop 2, is found to be unable to maintain a counterion function to the Schiff base on the activation pathway of rhodopsin in the absence of the primary counterion, Glu113. The Schiff base becomes protonated in the transition to Meta III. This protonation is, however, not associated with a deactivation of the receptor, in contrast to wildtype rhodopsin. Glu181 is suggested to be the counterion in the Meta III state of the mutant and appears to be capable of stabilizing a protonated Schiff base in Meta III, but not of constraining the receptor in an inactive conformation.     

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.     

Nanosecond photolysis of rhodopsin: evidence for a new, blue-shifted intermediate

Early photolysis intermediates of native bovine rhodopsin (RHO) are investigated by nanosecond laser photolysis near physiological temperature. Absorption difference spectra are collected after excitation with 477-, 532-, and 560-nm laser pulses of various energies and with 477-nm laser excitation at 5, 12, 17, 21, and 32 degrees C. The data are analyzed by using singular-value decomposition (SVD) and a global exponential fitting routine. Two rate constants associated with distinct spectral changes are observed during the time normally associated with the decay of bathorhodopsin to lumirhodopsin. Various models consistent with this observation are considered. A sequential model in which there is a reversible step between a bathorhodopsin intermediate and a new intermediate (BSI), which is blue-shifted relative to lumirhodopsin, is shown to best fit the data. The temperature dependence of the observed and calculated rate constants leads to linear Arrhenius plots. Extrapolation of the temperature dependence suggests that BSI should not be observable after rhodopsin photolysis at temperatures below -100 degrees C. The results are discussed with regard to the artificial visual pigments cis-5,6-dihydroisorhodopsin and 13-demethylrhodopsin. It is proposed that the rate of the BATHO to BSI transition is limited by the relaxation of the strained all-trans-retinal chromophore within a tight protein environment. The transition to LUMI involves chromophore relaxation concurrent with protein relaxation. While the first process is strongly affected by changes in the chromophore, the second transition seems to be determined more by protein relaxation.     

Time-resolved photointermediate changes in rhodopsin glutamic acid 181 mutants

The role of glutamic acid 181 in the bovine rhodopsin retinylidene chromophore pocket was studied by expressing E181 mutants in COS cells and measuring, as a function of time, the absorbance changes produced after excitation of lauryl maltoside pigment suspensions with 7 ns laser pulses. All mutants studied except E181D showed accelerated decay of bathorhodopsin compared to wild type. Even for E181D, an anomalously large blue shift was observed in the absorption spectrum of the bathorhodopsin decay product, BSI. These observations support the idea that E181 plays a significant role in the earliest stages of receptor activation. E181 mutations have a pronounced effect on the decay of the lumirhodopsin photointermediate, primarily affecting the size of the red shift that occurs in the lumirhodopsin I to lumirhodopsin II transition that takes place on the 10 micros time scale after wild-type photoexcitation. While the spectral change that occurs in the lumirhodopsin I to lumirhodopsin II transition in wild-type rhodopsin is very small ( approximately 2 nm), making it difficult to detect, it is larger in E181D ( approximately 6 nm), making it evident even in the lower signal-to-noise ratio measurements possible with rhodopsin mutants. The change seen is even larger for the E181F mutant where significant amounts of a deprotonated Schiff base intermediate are produced with the 10 micros time constant of lumirhodopsin II formation. The E181Q mutant shows lumirhodopsin decay more similar to wild-type behavior, and no lumirhodopsin I to lumirhodopsin II transition can be resolved. The addition of chloride ion to E181Q increases the lumirhodopsin I-lumirhodopsin II spectral shift and slows the deprotonation of the Schiff base. The latter result is consistent with the idea that a negative charge at position 181 contributes to protonated Schiff base stability in the later intermediates.     

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.