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.     

Interaction of rhodopsin with the G-protein,transducin

Rhodopsin, upon activation by light, transduces the photon signal by activation of the G-protein, transducin. The well-studied rhodopsin/transducin system serves as a model for the understanding of signal transduction by the large class of G-protein-coupled receptors. The interactive form of rhodopsin, R*, is conformationally similar or identical to rhodopsin's photolysis intermediate Metarhodopsin II (MII). Formation of MII requires deprotonation of rhodopsin's protonated Schiff base which appears to facilitate some opening of the rhodopsin structure. This allows a change in conformation at rhodopsin's cytoplasmic surface that provides binding sites for transducin. Rhodopsin's 2nd, 3rd and putative 4th cytoplasmic loops bind transducin at sites including transducin's 5 kDa carboxyl-terminal region. Site-specific mutagenesis of rhodopsin is being used to distinguish sites on rhodopsin's surface that are important in binding transducin from those that function in activating transducin. These observations are consistent with and extend studies on the action of other G-protein-coupled receptors and their interactions with their respective G proteins.     

Conformational similarities in the beta-ionone ring region of the rhodopsin chromophore in its ground state and after photoactivation to the metarhodopsin-I intermediate

High-resolution solid-state NMR methods have been used to analyze the conformation of the chromophore in the late photointermediate metarhodopsin-I, from observation of (13)C nuclei introduced into the beta-ionone ring (at the C16, C17, and C18 methyl groups) and into the adjoining segment of the polyene chain (at C8). Bovine rhodopsin in its native membrane was also regenerated with retinal that was (13)C-labeled close to the 11-Z bond (C20 methyl group) to provide a reporter for optimizing and quantifying the photoconversion to metarhodopsin-I. Indirect photoconversion via the primary intermediate, bathorhodopin, was adopted as the preferred method since approximately 44% conversion to the metarhodopsin-I component could be achieved, with only low levels (approximately 18%) of ground-state rhodopsin remaining. The additional photoproduct, isorhodopsin, was resolved in (13)C spectra from C8 in the chain, at levels of approximately 38%, and was shown using rotational resonance NMR to adopt the 6-s-cis conformation between the ring and the polyene chain. The C8 resonance was not shifted in the metarhodopsin-I spectral component but was strongly broadened, revealing that the local conformation had become less well defined in this segment of the chain. This line broadening slowed rotational resonance exchange with the C17 and C18 ring methyl groups but was accounted for to show that, despite the chain being more relaxed in metarhodopsin-I, its average conformation with respect to the ring was similar to that in the ground state protein. Conformational restraints are also retained for the C16 and C17 methyl groups on photoactivation, which, together with the largely preserved conformation in the chain, argues convincingly that the ring remains with strong contacts in its binding pocket prior to activation of the receptor. Previous conclusions based on photocrosslinking studies are considered in view of the current findings.     

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.     

Light-stable rhodopsin. I. A rhodopsin analog reconstituted with a nonisomerizable 11-cis retinal derivative.

With the aim of preparing a light-stable rhodopsin-like pigment, an analog, II, of 11-cis retinal was synthesized in which isomerization of the C11-C12 cis-double bond is blocked by a cyclohexene ring built around the C10 to C13-methyl. The analog II formed a rhodopsin-like pigment (rhodopsin-II) with opsin expressed in COS-1 cells and with opsin from rod outer segments. The rate of rhodopsin-II formation from II and opsin was approximately 10 times slower than that of rhodopsin from 11-cis retinal and opsin. After solubilization in dodecyl maltoside and immunoaffinity purification, rhodopsin-II displayed an absorbance ratio (A280nm/A512nm) of 1.6, virtually identical with that of rhodopsin. Acid denaturation of rhodopsin-II formed a chromophore with lambda max, 452 nm, characteristic of protonated retinyl Schiff base. The ground state properties of rhodopsin-II were similar to those of rhodopsin in extinction coefficient (41,200 M-1 cm-1) and opsin-shift (2600 cm-1). Rhodopsin-II was stable to hydroxylamine in the dark, while light-dependent bleaching by hydroxylamine was slowed by approximately 2 orders of magnitude relative to rhodopsin. Illumination of rhodopsin-II for 10 s caused approximately 3 nm blue-shift and 3% loss of visible absorbance. Prolonged illumination caused a maximal blue-shift up to approximately 20 nm and approximately 40% loss of visible absorbance. An apparent photochemical steady state was reached after 12 min of illumination. Subsequent acid denaturation indicated that the retinyl Schiff base linkage was intact. A red-shift (approximately 12 nm) in lambda max and a 45% recovery of visible absorbance was observed after returning the 12-min illuminated pigment to darkness. Rhodopsin-II showed marginal light-dependent transducin activation and phosphorylation by rhodopsin kinase.     

Constitutively active mutants of rhodopsin.

Two critical amino acids in the visual pigment rhodopsin are Lys-296, the site of attachment of retinal to the protein through a protonated Schiff base linkage, and Glu-113, the Schiff base counterion. Mutation of Lys-296 or Glu-113 results in constitutive activation of opsin, as assayed by its ability to activate transducin in the absence of added chromophore. We conclude that opsin is constrained to an inactive conformation by a salt bridge between Lys-296 and Glu-113. Recently, one of the mutants, K296E, was found in a family with retinitis pigmentosa, suggesting that degeneration of the photoreceptor cells in individuals with this mutation may result from persistent stimulation of the phototransduction pathway.     

Photoreactions of metarhodopsin III

Meta III is an inactive intermediate thermally formed following light activation of the visual pigment rhodopsin. It is produced from the Meta I/Meta II photoproduct equilibrium of rhodopsin by a thermal isomerization of the protonated Schiff base C=N bond of Meta I, and its chromophore configuration is therefore all-trans 15-syn. In contrast to the dark state of rhodopsin, which catalyzes exclusively the cis to trans isomerization of the C11=C12 bond of its 11-cis 15-anti chromophore, Meta III does not acquire this photoreaction specificity. Instead, it allows for light-dependent syn to anti isomerization of the C15=N bond of the protonated Schiff base, yielding Meta II, and for trans to cis isomerizations of C11=C12 and C9=C10 of the retinal polyene, as shown by FTIR spectroscopy. The 11-cis and 9-cis 15-syn isomers produced by the latter two reactions are not stable, decaying on the time scale of few seconds to dark state rhodopsin and isorhodopsin by thermal C15=N isomerization, as indicated by time-resolved FTIR methods. Flash photolysis of Meta III produces therefore Meta II, dark state rhodopsin, and isorhodopsin. Under continuous illumination, the latter two (or its unstable precursors) are converted as well to Meta II by presumably two different mechanisms.     

Localization of the retinal protonated Schiff base counterion in rhodopsin.

Semiempirical molecular orbital calculations are combined with 13C NMR chemical shifts to localize the counterion in the retinal binding site of vertebrate rhodopsin. Charge densities along the polyene chain are calculated for an 11-cis-retinylidene protonated Schiff base (11-cis-RPSB) chromophore with 1) a chloride counterion at various distances from the Schiff base nitrogen, 2) one or two chloride counterions at different positions along the retinal chain from C10 to C15 and at the Schiff base nitrogen, and 3) a carboxylate counterion out of the retinal plane near C12. Increasing the distance of the negative counterion from the Schiff base results in an enhancement of alternating negative and positive partial charge on the even- and odd-numbered carbons, respectively, when compared to the 11-cis-RPSB chloride model compound. In contrast, the observed 13C NMR data of rhodopsin exhibit downfield chemical shifts from C8 to C13 relative to the 11-cis-RPSB.Cl corresponding to a net increase of partial positive or decrease of partial negative charge at these positions (Smith, S. O., I. Palings, M. E. Miley, J. Courtin, H. de Groot, J. Lugtenburg, R. A. Mathies, and R. G. Griffin. 1990. Biochemistry. 29:8158-8164). The anomalous changes in charge density reflected in the rhodopsin NMR chemical shifts can be qualitatively modeled by placing a single negative charge above C12. The calculated fit improves when a carboxylate counterion is used to model the retinal binding site. Inclusion of water in the model does not alter the fit to the NMR data, although it is consistent with observations based on other methods.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultra-high-field MAS NMR assay of a multispin labeled ligand bound to its G-protein receptor target in the natural membrane environment: electronic structure of the retinylidene chromophore in rhodopsin

11-Z-[8,9,10,11,12,13,14,15,19,20-(13)C10]Retinal prepared by total synthesis is reconstituted with opsin to form rhodopsin in the natural lipid membrane environment. The 13C shifts are assigned with magic angle spinning NMR dipolar correlation spectroscopy in a single experiment and compared with data of singly labeled retinylidene ligands in detergent-solubilized rhodopsin. The use of multispin labeling in combination with 2-D correlation spectroscopy improves the relative accuracy of the shift measurements. We have used the chemical shift data to analyze the electronic structure of the retinylidene ligand at three levels of understanding: (i) by specifying interactions between the 13C-labeled ligand and the G-protein-coupled receptor target, (ii) by making a charge assessment of the protonation of the Schiff base in rhodopsin, and (iii) by evaluating the total charge on the carbons of the retinylidene chromophore. In this way it is shown that a conjugation defect is the predominant ground-state property governing the molecular electronics of the retinylidene chromophore in rhodopsin. The cumulative chemical shifts at the odd-numbered carbons (Delta(sigma)odd) of 11-Z-protonated Schiff base models relative to the unprotonated Schiff base can be used to measure the extent of delocalization of positive charge into the polyene. For a series of 11-Z-protonated Schiff base models and rhodopsin, Delta(sigma)odd appears to correlate linearly with the frequency of maximum visible absorption. Since rhodopsin has the largest value of Delta(sigma)odd, the data contribute to existing and converging spectroscopic evidence for a complex counterion stabilizing the protonated Schiff base in the binding pocket.     

Equilibrium between metarhodopsin-I and metarhodopsin-II is dependent on the conformation of the third cytoplasmic loop

Rhodopsin is a G-protein-coupled receptor (GPCR) that is the light detector in the rod cells of the eye. Rhodopsin is the best understood member of the large GPCR superfamily and is the only GPCR for which atomic resolution structures have been determined. However, these structures are for the inactive, dark-adapted form. Characterization of the conformational changes in rhodopsin caused by light-induced activation is of wide importance, because the metarhodopsin-II photoproduct is analogous to the agonist-occupied conformation of other GPCRs, and metarhodopsin-I may be similar to antagonist-occupied GPCR conformations. In this work we characterize the interaction of antibody K42-41L with the metarhodopsin photoproducts. K42-41L is shown to inhibit formation of metarhodopsin-II while it stabilizes the metarhodopsin-I state. Thus, K42-41L recognizes an epitope accessible in dark-adapted rhodopsin and metarhodopsin-I that is lost upon formation of metarhodopsin-II. Previous work has shown that the peptide TGALQERSK is able to mimic the K42-41L epitope, and we have now determined the structure of the K42-41L-peptide complex. The structure demonstrates a central role for elements of the rhodopsin C3 loop, particularly Gln238 and Glu239, in the interaction with K42-41L. Geometric constraints taken from the antibody-bound peptide were used to model the epitope on the rhodopsin surface. The resulting model suggests that K42-41L locks the C3 loop into an extended conformation that is intermediate between two compact conformations seen in crystal structures of dark-adapted rhodopsin. Together, the structural and functional data strongly suggest that the equilibrium between metarhodopsin-I and metarhodopsin-II is dependent upon the conformation of the C3 loop. The biological implications of this model and its possible relations to dimeric and multimeric complexes of rhodopsin are discussed.     

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.     

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.     

Low-temperature solid-state 13C NMR studies of the retinal chromophore in rhodopsin

Magic angle sample spinning (MASS) 13C NMR spectra have been obtained of bovine rhodopsin regenerated with retinal prosthetic groups isotopically enriched with 13C at C-5 and C-14. In order to observe the 13C retinal chromophore resonances, it was necessary to employ low temperatures (-15-----35 degrees C) to restrict rotational diffusion of the protein. The isotropic chemical shift and principal values of the chemical shift tensor of the 13C-5 label indicate that the retinal chromophore is in the twisted 6-s-cis conformation in rhodopsin, in contrast to the planar 6-s-trans conformation found in bacteriorhodopsin. The 13C-14 isotropic shift and shift tensor principal values show that the Schiff base C = N bond is anti. Furthermore, the 13C-14 chemical shift (121.2 ppm) is within the range of values (120-123 ppm) exhibited by protonated (C = N anti) Schiff base model compounds, indicating that the C = N linkage is protonated. Our results are discussed with regard to the mechanism of wavelength regulation in rhodopsin.     

High-resolution solid-state 13C-NMR study of carbons C-5 and C-12 of the chromophore of bovine rhodopsin. Evidence for a 6-S-cis conformation with negative-charge perturbation near C-12

Solid-state 13C magic-angle spinning NMR spectroscopy has been employed to study the conformation of the 11-cis-retinylidene Schiff base chromophore in bovine rhodopsin. Spectra were obtained from lyophilized samples of bovine rhodopsin selectively 13C-labeled at position C-5 or C-12 of the retinyl moiety, and reconstituted in the fully saturated branched-chain phospholipid diphytanoyl glycerophosphocholine. Comparison of the NMR parameters for carbon C-5 presented in this paper with those published for retinyl Schiff base model compounds and bacteriorhodopsin by Harbison and coworkers [Harbison et al. (1985) Biochemistry 24, 6955-6962], indicate that in bovine rhodopsin the C-6-C-7 single bond has the unperturbed cis conformation. This is in contrast to the 6-S-trans conformation found in bacteriorhodopsin. The NMR parameters for bovine [12-13C]rhodopsin present evidence for the presence of a negative charge interacting with the retinyl moiety near C-12, in agreement with the model for the opsin shift presented by Honig and Nakanishi and coworkers [Kakitani et al. (1985) Photochem. Photobiol. 41, 471-479].     

Rhodopsin with 11-cis-locked chromophore is capable of forming an active state photoproduct

The visual pigment rhodopsin is characterized by an 11-cis retinal chromophore bound to Lys-296 via a protonated Schiff base. Following light absorption the C(11)=C(12) double bond isomerizes to trans configuration and triggers protein conformational alterations. These alterations lead to the formation of an active intermediate (Meta II), which binds and activates the visual G protein, transducin. We have examined by UV-visible and Fourier transform IR spectroscopy the photochemistry of a rhodopsin analogue with an 11-cis-locked chromophore, where cis to trans isomerization around the C(11)=C(12) double bond is prevented by a 6-member ring structure (Rh(6.10)). Despite this lock, the pigment was found capable of forming an active photoproduct with a characteristic protein conformation similar to that of native Meta II. This intermediate is further characterized by a protonated Schiff base and protonated Glu-113, as well as by its ability to bind a transducin-derived peptide previously shown to interact efficiently with native Meta II. The yield of this active photointermediate is pH-dependent and decreases with increasing pH. This study shows that with the C(11)=C(12) double bond being locked, isomerization around the C(9)=C(10) or the C(13)=C(14) double bonds may well lead to an activation of the receptor. Additionally, prolonged illumination at pH 7.5 produces a new photoproduct absorbing at 385 nm, which, however, does not exhibit the characteristic active protein conformation.     

Crystallographic Study of the LUMI Intermediate of Squid Rhodopsin

Upon absorption of light, the retinal chromophore in rhodopsin isomerizes from the 11-cis to the trans configuration, initiating a photoreaction cycle. The primary photoreaction state, bathorhodopsin (BATHO), relaxes thermally through lumirhodopsin (LUMI) into a photoactive state, metarhodopsin (META), which stimulates the conjugated G-protein. Previous crystallographic studies of squid and bovine rhodopsins have shown that the structural change in the primary photoreaction of squid rhodopsin is considerably different from that observed in bovine rhodopsin. It would be expected that there is a fundamental difference in the subsequent thermal relaxation process between vertebrate and invertebrate rhodopsins. In this work, we performed crystallographic analyses of the LUMI state of squid rhodopsin using the P62 crystal. When the crystal was illuminated at 100 K with blue light, a half fraction of the protein was converted into BATHO. This reaction state relaxed into LUMI when the illuminated crystal was warmed in the dark to 170 K. It was found that, whereas trans retinal is largely twisted in BATHO, it takes on a more planar configuration in LUMI. This relaxation of retinal is accompanied by reorientation of the Schiff base NH bond, the hydrogen-bonding partner of which is switched to Asn185 in LUMI. Unlike bovine rhodopsin, the BATHO-to-LUMI transition in squid rhodopsin was accompanied by no significant change in the position/orientation of the beta-ionone ring of retinal.     

Rhodopsin's carboxyl-terminal threonines are required for wild-type arrestin-mediated quench of transducin activation in vitro

Many recent reports have demonstrated that rhodopsin's carboxyl-terminal serine residues are the main targets for phosphorylation by rhodopsin kinase. Phosphorylation at the serines would therefore be expected to promote high-affinity arrestin binding. We have examined the roles of the carboxyl serine and threonine residues during arrestin-mediated deactivation of rhodopsin using an in vitro transducin activation assay. Mutations were introduced into a synthetic bovine rhodopsin gene and expressed in COS-7 cells. Individual serine and threonine residues were substituted with neutral amino acids. The ability of the mutants to act as substrates for rhodopsin kinase was analyzed. The effect of arrestin on the activities of the phosphorylated mutant rhodopsins was measured in a GTPgammaS binding assay involving purified bovine arrestin, rhodopsin kinase, and transducin. A rhodopsin mutant lacking the carboxyl serine and threonine residues was not phosphorylated by rhodopsin kinase, demonstrating that phosphorylation is restricted to the seven putative phosphorylation sites. A rhodopsin mutant possessing a single phosphorylatable serine at 338 demonstrated no phosphorylation-dependent quench by arrestin. These results suggest that singly phosphorylated rhodopsin is deactivated through a mechanism that does not involve arrestin. Analysis of additional mutants revealed that the presence of threonine in the carboxyl tail of rhodopsin provides for greater arrestin-mediated quench than does serine. These results suggest that phosphorylation site selection could serve as a mechanism to modulate the ability of arrestin to quench rhodopsin.     

Investigations of the rhodopsin/Meta I and rhodopsin/Meta II transitions of bovine rod outer segments by means of kinetic infrared spectroscopy

We have applied our recently developed technique of flash induced kinetic infrared spectroscopy to the rhodopsin/Meta I and rhodopsin/Meta II transitions. Features of the infrared spectrum reflecting the C=C-vibration and the isomeric form of the chromophore are in agreement with resonant Raman experiments. Different results are obtained for the C=N-vibration of the Schiff base retinal opsin link. They are interpreted in terms of a Schiff base protonated via an hydrogen bond. A proton transfer in the excited state is suggested to explain the deviating results. In addition we have obtained spectral changes which cannot be attributed to molecular changes in the chromophore. We assume that these spectral features reflect molecular events in the protein part of rhodopsin.     

Signaling states of rhodopsin. Retinal provides a scaffold for activating proton transfer switches

The G-protein-coupled receptor rhodopsin is activated by photoconversion of its covalently bound ligand 11-cis-retinal to the agonist all-trans-retinal. After light-induced isomerization and early photointermediates, the receptor reaches a G-protein-dependent equilibrium between active and inactive conformations distinguished by the protonation of key opsin residues. In this report, we study the role of the 9-methyl group of retinal, one of the crucial steric determinants of light activation. We find that when this group is removed, the protonation equilibrium is strongly shifted to the inactive conformation. The residually formed active species is very similar to the active form of normal rhodopsin, metarhodopsin II. It has a deprotonated Schiff base, binds to the retinal G-protein transducin, and is favored at acidic pH. Our data show that the normal proton transfer reactions are inhibited in 9-demethyl rhodopsin but are still mandatory for receptor activation. We propose that retinal and its 9-methyl group act as a scaffold for opsin to adjust key proton donor and acceptor side chains for the proton transfer reactions that stabilize the active conformation. The mechanism may also be applicable to related receptors and may thus explain the partial agonism of certain ligands.     

Conformational changes in the photocycle of Anabaena sensory rhodopsin: absence of the Schiff base counterion protonation signal

Anabaena sensory rhodopsin (ASR) is a novel microbial rhodopsin recently discovered in the freshwater cyanobacterium Anabaena sp. PCC7120. This protein most likely functions as a photosensory receptor as do the related haloarchaeal sensory rhodopsins. However, unlike the archaeal pigments, which are tightly bound to their cognate membrane-embedded transducers, ASR interacts with a soluble cytoplasmic protein analogous to transducers of animal vertebrate rhodopsins. In this study, infrared spectroscopy was used to examine the molecular mechanism of photoactivation in ASR. Light adaptation of the pigment leads to a phototransformation of an all-trans/15-anti to 13-cis/15-syn retinylidene-containing species very similar in chromophore structural changes to those caused by dark adaptation in bacteriorhodopsin. Following 532 nm laser-pulsed excitation, the protein exhibits predominantly an all-trans retinylidene photocycle containing a deprotonated Schiff base species similar to those of other microbial rhodopsins such as bacteriorhodopsin, sensory rhodopsin II, and Neurospora rhodopsin. However, no changes are observed in the Schiff base counterion Asp-75, which remains unprotonated throughout the photocycle. This result along with other evidence indicates that the Schiff base proton release mechanism differs significantly from that of other known microbial rhodopsins, possibly because of the absence of a second carboxylate group at the ASR photoactive site. Several conformational changes are detected during the ASR photocycle including in the transmembrane helices E and G as indicated by hydrogen-bonding alterations of their native cysteine residues. In addition, similarly to animal vertebrate rhodopsin, perturbations of the polar head groups of lipid molecules are detected.