The nature of the delocalized cations in azulenic bacteriorhodopsin analogs.

Depending on the size and shape of their azulenic chromophores, azulenic bacteriorhodopsin (bR) pigment analogs can exist as either an initial pigment P1, a more red-shifted final pigment P2 or an equilibrium mixture of both. The absorption spectra of red-shifted bR analogs exhibit characteristic narrow-band shapes similar to charge fully delocalized cyanine-like dyes. Therefore, all such red-shifted pigments are believed to be highly delocalized, bond-equalized carbocations. We have determined structural requirements that facilitate their formation. To describe fully the red-shift potentials of these retinal analogs, we have introduced a new parameter-percent red-shift (PRS). A large PRS value not only reflects the extent of red-shift, but is also suggestive of extensive delocalization of the positive charge. Relevance of these findings in consideration of the possibility of forming stable O-intermediates is presented. The postulated resonance hybrid-like structures for different cations of the positively charged protonated Schiff base chromophores are in fact structurally distinct species, equilibrating in response to local perturbations within the supramolecular protein environment.     

Structure, spectroscopy, and spectral tuning of the gas-phase retinal chromophore: the beta-ionone "handle" and alkyl group effect

The low-lying singlet states (i.e. S0, S1, and S2) of the chromophore of rhodopsin, the protonated Schiff base of 11-cis-retinal (PSB11), and of its all-trans photoproduct have been studied in isolated conditions by using ab initio multiconfigurational second-order perturbation theory. The computed spectroscopic features include the vertical excitation, the band origin, and the fluorescence maximum of both isomers. On the basis of the S0-->S1 vertical excitation, the gas-phase absorption maximum of PSB11 is predicted to be 545 nm (2.28 eV). Thus, the predicted absorption maximum appears to be closer to that of the rhodopsin pigment (2.48 eV) and considerably red-shifted with respect to that measured in solution (2.82 eV in methanol). In addition, the absorption maxima associated with the blue, green, and red cone visual pigments are tentatively rationalized in terms of the spectral changes computed for PSB11 structures featuring differently twisted beta-ionone rings. More specifically, a blue-shifted absorption maximum is explained in terms of a large twisting of the beta-ionone ring (with respect to the main conjugated chain) in the visual S-cone (blue) pigment chromophore. In contrast, the chromophore of the visual L-cone (red) pigment is expected to have a nearly coplanar beta-ionone ring yielding a six double bond fully conjugated framework. Finally, the M-cone (green) chromophore is expected to feature a twisting angle between 10 and 60 degrees. The spectroscopic effects of the alkyl substituents on the PSB11 spectroscopic properties have also been investigated. It is found that they have a not negligible stabilizing effect on the S1-S0 energy gap (and, thus, cause a red shift of the absorption maximum) only when the double bond of the beta-ionone ring conjugates significantly with the rest of the conjugated chain.     

COMPARISON OF THE PATHWAYS AND QUANTUM YIELDS OF DIRECT PHOTOISOMERIZATION OF UNPROTONATED AND PROTONATED N-BUTYLAMINE SCHIFF BASES OF ISOMERIC RETINYLIDENEACETALDEHYDE WITH THOSE OF SCHIFF BASES OF ISOMERIC RETINAL: RATIONALIZATION OF THE SELECTION OF THE RETINYLIDENE CHROMOPHORE BY RETINOCHROME

Abstract— The pathways and quantum yields of direct photoisomerization of unprotonated and pro-tonated n-butylamine Schiff bases (SB and PSB) of isomeric retinylideneacetaldehyde (C22 aldehyde) were determined in n-hexane, acetonitrile and methanol for the former and in acetonitrile and methanol for the latter. The results are compared with those of the Schiff bases of isomeric retinal (C20 SB and C20 PSB) reported previously (Koyama et al., Photochem. Photobiol. 54 , 433–443, 1991). The isomerization pathways and quantum yields of C22 SB are more or less similar to those of C20 SB, but conspicuous differences in the isomerization pathways are found between C22 PSB and C20 PSB. The homogeneous (exclusive) isomerization of the retinylidene chromophore from all-trum to 11-cis in retinochrome is rationalized not by C22 PSB but by C20 PSB.
Almost complete one-way isomerization from cis to trans of C22 SB (in n-hexane) is ascribed to isomerization via the T₁ state, while mutual isomeritation between cis and tram of C22 PSB is ascribed to isomerization via the S₁ (B_u) state. The T_I potential of C22 SB and the S₁ potential of C22 PSB are discussed based on photostationary state compositions.     

Combined QM/MM study of the opsin shift in bacteriorhodopsin

Combined quantum mechanical and molecular mechanical (QM/MM) calculations and molecular dynamics simulations of bacteriorhodopsin (bR) in the membrane matrix have been carried out to determine the factors that make significant contributions to the opsin shift. We found that both solvation and interactions with the protein significantly shifts the absorption maximum of the retinal protonated Schiff base, but the effects are much more pronounced in polar solvents such as methanol, acetonitrile, and water than in the protein environment. The differential solvatochromic shifts of PSB in methanol and in bR leads to a bathochromic shift of about 1800 cm(-1). Because the combined QM/MM configuration interaction calculation is essentially a point charge model, this contribution is attributed to the extended point-charge model of Honig and Nakanishi. The incorporation of retinal in bR is accompanied by a change in retinal conformation from the 6-s-cis form in solution to the 6-s-trans configuration in bR. The extension of the pi-conjugated system further increases the red-shift by 2400 cm(-1). The remaining factors are due to the change in dispersion interactions. Using an estimate of about 1000 cm(-1) in the dispersion contribution by Houjou et al., we obtained a theoretical opsin shift of 5200 cm(-1) in bR, which is in excellent agreement with the experimental value of 5100 cm(-1). Structural analysis of the PSB binding site revealed the specific interactions that make contributions to the observed opsin shift. The combined QM/MM method used in the present study provides an opportunity to accurately model the photoisomerization and proton transfer reactions in bR.     

ISOMER COMPOSITION and SPECTRA OF THE DARK and LIGHT ADAPTED FORMS OF ARTIFICIAL BACTERIORHODOPSINS*

Abstract– The isomer composition and spectral properties of 15 artificial bacteriorhodopsin (bR) pigments, based on a series of retinal analogs with polyene residue modified below C₉ are determined for both dark-adapted (DA) and light-adapted (LA) forms. Similarly to native bR, in all cases only two isomers, C₁₃=C₁₄cis (13-cis) and M-trans, are observed. However, the artificial DA pigments have a lower 13-d.s content than native DA bR (? 66%) while the corresponding LA pigments have a much higher 13-cis content (11-69%) than native LA bR (<2%). Thus, in variance with the native pigment, in all of the artificial systems light also induced the reversed all-trans13-cis process. The data are accounted for in terms of specific steric interactions between the polyene and the protein binding site which allow a (C₁₅-anti)(C_ls-syn) isomerization during the photocycle of the artificial pigments, but not in the case of native bR. This accounts for the high proton pumping efficiency of the natural pigment. The nature of a highly red shifted light-adapted form of two of the artificial pigments is investigated and discussed. It is also shown that, in variance with native bR, several artificial pigments exhibit identical absorption spectra for their 13-cis and all-trans isomers. It is concluded that the spectral data for the above species of artificial pigments do not lead to a clear molecular model for the origin of the spectral shift between 13-cis and all-trans bR.     

TIME-RESOLVED RESONANCE RAMAN SPECTRA OF THE PHOTOCYCLE INTERMEDIATES OF ACID AND DEIONIZED BACTERIORHODOPSIN

Abstract— The photocycle of bacteriorhodopsin (bR) and its perturbed forms are investigated by a time-resolved resonance Raman study. These experiments were performed in the C=C stretching and in the fingerprint spectral regions for the acid blue, acid purple and deionized forms of bR.
The main observations are as follows: (1) isomerization of the retinal, from all- trans to 13- cis , occurs in native bR and in all of the acid and deionized perturbed bR species; (2) formation of the early intermediates (the K₆₁₀ and L₅₅₀ analogues) also occur in native bR and in all of the perturbed species; and (3) deprotonation of the protonated Schiff base (PSB), to give the M₄₁₂ type intermediate, occurs in native bR, but is inhibited in all of the perturbed bR species on the time-scale of the native bR photocycle.
The results show that isomerization alone is not a prerequisite for the PSB deprotonation process. The observed photocycle, initiated with retinal isomerization, is found to occur from all- trans to 13- cis in all of the perturbed forms of bR. In addition, the results imply that removal of the cations, of an increase in the hydrogen ion concentration, prevent only the PSB deprotonation process and not the formation of earlier cycle intermediates. Some attention is focused on the two blue forms of bR (acid and deionized) due to the fact that their ground-state absorption maximum, unphotolyzed Raman spectra, and Raman spectra changes during the photocycle are all very similar. The similarities between the acid blue and deionized blue forms in the fingerprint region support previous suggestions that both blue species have nearly the same retinal active site.     

EFFECTS OF MUTAGENETIC SUBSTITUTION OF PROLINES ON THE RATE OF DEPROTONATION and REPROTONATION OF THE SCHIFF BASE DURING THE PHOTOCYCLE OF BACTERIORHODOPSIN

Abstract— Membrane-buried proline residues are found in many transport proteins. To study their roles in the structure and function of bacteriorhodopsin (bR), effects of the individual substitutions ofPro–50,Pro–91 andPro–186 on the deprotonation and reprotonation kinetics of the Schiffbase (SB) were determined by flash photolysis. The obtained rate constants and the amplitudes of the slow and fast components were compared with those of ebR (wild-type bR, the native protein that is expressed in Escherichia coli). The deprotonation rates of PSB were found to be 10 times faster than that of ebR for P50A, P91A and P91G mutants, and 4 times faster for the P50G mutant. These mutations also increased the initial reprotonation rate of the SB, although the overall change in the reprotonation rate is not as significant as that in the deprotonation rate. Our results indicate thatPro–50 andPro–91, as well asPro–186, are important for the proton-pumping function of bR.     

Coupling of protonation switches during rhodopsin activation

Recent studies of the activation mechanism of rhodopsin involving Fourier-transform infrared spectroscopy and a combination of chromophore modifications and site-directed mutagenesis reveal an allosteric coupling between two protonation switches. In particular, the ring and the 9-methyl group of the all-trans retinal chromophore serve to couple two proton-dependent activation steps: proton uptake by a cytoplasmic network between transmembrane (TM) helices 3 and 6 around the conserved ERY (Glu-Arg-Tyr) motif and disruption of a salt bridge between the retinal protonated Schiff base (PSB) and a protein counterion in the TM core of the receptor. Retinal analogs lacking the ring or 9-methyl group are only partial agonists--the conformational equilibrium between inactive Meta I and active Meta II photoproduct states is shifted to Meta I. An artificial pigment was engineered, in which the ring of retinal was removed and the PSB salt bridge was weakened by fluorination of C14 of the retinal polyene. These modifications abolished allosteric coupling of the proton switches and resulted in a stabilized Meta I state with a deprotonated Schiff base (Meta I(SB)). This state had a partial Meta II-like conformation due to disruption of the PSB salt bridge, but still lacked the cytoplasmic proton uptake reaction characteristic of the final transition to Meta II. As activation of native rhodopsin is known to involve deprotonation of the retinal Schiff base prior to formation of Meta II, this Meta I(SB) state may serve as a model for the structural characterization of a key transient species in the activation pathway of a prototypical G protein-coupled receptor.     

RESONANCE RAMAN STUDIES OF BACTERIORHODOPSIN ANALOGUES

Abstract— We present the results of resonance Raman measurements on a series of bacteriorhodopsin (bR) analogues formed from synthetic retinals which have replaced the native chromophore in the active site. Specifically, 5,6-dihydro-bR, 13-desmethyl-bR, 10-methyl-bR, 14-methyl-bR, and 10.14-dimethyl-bR have been studied. All five analogues bind and form Schiff base retinal-apoprotein linkages. While the Schiff base linkages of 5,6-dihydro-bR, 13-desmethyl-bR, and 10-methyl-bR are protonated, like the native chromophore, the 14-methyl-bR, and 10,14-dimethyl-bR Schiff bases are unprotonated. These results suggest that the binding site of bacteriorhodopsin near the Schiff base moiety is different from that of rhodopsin. The protonated Schiff base -C=NH- stretching frequency of 5.6-dihydro-bR lies at 1660 cm^-1 which is unusually high for a bacteriorhodopsin based pigment. The downward shift upon deuteration is 16 cm^-1, essentially identical to that measured for bacteriorhodopsin. This and the other analogue results strongly reinforce our previous arguments that the Schiff base stretching frequency is determined in large part by two factors, the C=N force constant and the stretch interaction with C=N-H bend. On the other hand, the deuterium isotope effect is determined primarily by the stretch-bend interaction.     

The protonated Schiff base of halorhodopsin from Natronobacterium pharaonis is hydrolyzed at elevated temperatures

Halorhodopsin from Natronobacterium pharaonis (pHR) is a light-driven chloride pump in which photoisomerzation of a retinal chromophore triggers a photocycle which leads to a chloride anion transport across the plasma membrane. Similarly to other retinal proteins the protonated Schiff base (PSB), which covalently links the retinal to the protein, does not experience hydrolysis reaction at room temperature even though several water molecules are located in the protonated Schiff base (PSB) vicinity. In the present studies we have revealed that in contrast to other studied archaeal rhodopsins, temperature increase to about 70 degrees C hydrolyses the PSB linkage of pHR. The rate of the reaction is affected by Cl-concentration and reveals an anion binding site (in addition to the Cl- in the SB vicinity) with a binding constant of 100mM (measured at 70 degrees C). We suggest that this binding site is located on the extracellular side and its possible role in the Cl-pumping mechanism is discussed. The rate of the hydrolysis reaction is affected by the nature of the anion bound to pHR. Substitution of the Cl- anion by Br-, I- and SCN- exhibits similar behavior to that of CI- in the region of 100mM but higher concentrations are needed for N3-, HCOO- and NO2-to achieve similar behavior. Steady state pigment illumination accelerates the reaction and reduces the energy of activation and the frequency factor. Adjusting the sample temperature to 25 degrees C following the hydrolysis reaction led to about 80% pigment recovery. However, the newly reformed pigment is different from the mother pigment and has different characteristics. It is concluded that the apo-membrane adopts a modified conformation and/or aggregated state which rebinds the retinal to give a new conformation of the pHR pigment.     

THE ORIGIN OF THE RED-SHIFTED ABSORPTION MAXIMUM OF THE M412 INTERMEDIATE IN THE BACTERIORHODOPSIN PHOTOCYCLE

The factors that red shift the absorption maximum of the retinal Schiff base chromophore in the M₄₁₂ intermediate of bacteriorhodopsin photocycle relative to absorption in solution were investigated using a series of artificial pigments and studies of model compounds in solution. The artificial pigments derived from retinal analogs that perturb chromophore-protein interactions in the vicinity of the ring moiety indicate that a considerable part of the red shift may originate from interactions in the vicinity of the Schiff base linkage. Studies with model compounds revealed that hydrogen bonding to the Schiff base moiety can significantly red shift the absorption maximum. Furthermore, it was demonstrated that although s-trans ring-chain planarity prevails in the M₄₁₂ intermediate it does not contribute significantly (only ca 750 cm⁻¹) to the opsin shift observed in M₄₁₂. It is suggested that in M₄₁₂, the Schiff base linkage is hydrogen bonded to bound water and/or protein residues inducing a considerable red shift in the absorption maximum of the retinal chromophore.     

SPECTROSCOPY OF POLYENES–III. ABSORPTION AND EMISSION SPECTRAL INVESTIGATION OF POLYENE SCHIFF BASES AND PROTONATED SCHIFF BASES RELATED TO VISUAL PIGMENTS

Abstract— A number of n -butylamine Schiff bases of polyenals related to retinals as homologues and analogues, and their protonated forms, have been studied for absorption and emission spectral properties. The polyene Schiff bases exhibit the same general features in their absorption spectra as those of the parallel polyenals except that the ^lB_u←¹A_g and π*← n singlet transitions are at substantially higher energy in the Schiff bases (the shift being larger for the π *← n transition). The Schiff bases with short polyene chainlength ( n = 2, 3 where n is the number of double bonds including C=N) do not fluoresce or phosphoresce in 3-methylpentane in the temperature range 298–77 K. The Schiff bases with intermediate chainlength ( n = 4, 5) show fluorescence at 77 K with intensity strongly dependent on the nature of solvent. The Schiff bases with relatively long chainlength ( n = 5–7) show strong or moderately strong fluorescence at 77 K and very weak fluorescence at 298 K ( n = 7) with intrinsic radiative lifetimes much longer than those estimated from the oscillator strength of the low-energy, strong absorption band (¹B_u←¹ A_g ). A discussion on the possible state order and nature of the fluorescing state of the various polyene Schiff base systems is presented.     

Ring oxidized retinals form unusual bacteriorhodopsin analogue pigments.

Three ring oxidized retinal analogues have been isolated from the exhaustive oxidation of all-trans retinal. All-trans 4-oxoretinal and 2,3-dehydro-4-oxoretinal have similar absorption maxima to that of all-trans retinal and have been shown to be in the 6-s-cis conformation in solution. Pigments formed with bacterioopsin exhibit absorption maxima (520 nm) blue-shifted from that of bacteriorhodopsin (bR), indicating a disturbance of the external point charge by the electronegative carbonyl moiety at the 4 position. The third analogue contains a ring contracted to a cyclopentenyl-alpha,beta-dione. Unlike the majority of retinals, this analogue displays a 6-s-trans conformation in solution and has a red-shifted absorption maximum at 435 nm. The resulting bR analogue pigment (515 nm) is formed five times faster than the other oxoretinal pigments. All three oxoretinal pigments show an irreversible 20 nm blue shift upon exposure to white light. The 4-oxo and 2,3-dehydro-4-oxoretinal pigments, after irradiation, undergo a small reversible blue shift (4-8 nm) on dark adaptation. These two pigments pump protons, although with slowed photocycle kinetics, demonstrating that these structural changes (addition of the carbonyl at the C-4 and insertion of a double bond in the ring) do not block the function of the pigment. Extraction of the C-15 tritiated analogue retinals from illuminated and non-illuminated pigments of all three oxoretinals yield identical results. Therefore, any crosslinking of these oxoretinals to the protein is by linkages which are unstable to the extraction procedures.     

Lanthanons(III) Complexes of Bifunctional Tridentate Schiff's Bases

A systematic study of the reactions of isopropoxides of praseodymium, neodymium and samarium with bifunctional tridentate Schiff bases (acetylacetone-O-aminophenol and benzoylacetone-O-aminophenol) in equimolar ratios resulting in the formation of products with formula M(OPrⁱ) (SB), M(SB) (SBH) and M₂(SB)3 (where SB is Schiff base anion, SBH is Schiff base and M is metal). The reactions have been found to be quite facile even at the room temperature as observed by immediate change of colour. The intensity of the colour of acetylacetone-O-aminophenol derivatives of rare earths decreased with the increasing stoichiometric ratio of the ligand i.e. from yellow to light yellow.     

Probing for the Threshold Energy for Visual Transduction: Red-Shifted Visual Pigment Analogs from 3-Methoxy-3-Dehydroretinal and Related Compounds

While azulenic retinal analogs failed to yield a red-shifted visual pigment analog, the 9-cis isomers of the push-pull polyenals 3-methoxy-3-dehydroretinal and 14F-3-methoxy-3-dehydroretinal yielded iodopsin pigment analogs with absorption maxima at, respectively, 663 and 720 nm. The former gave a relatively stable batho product (700 nm) and was able to activate transducin. A lower activity was observed for the latter. One possible explanation for the combined results is that the excitation energies of these red-shifted pigments are approaching the threshold energy for visual transduction (although at this time we cannot rigorously exclude a role of the added F-atom in reducing the transducin activity).     

SHAPE OF THE CHROMOPHOIW BINDING SITE IN pharaonis PHOBORHODOPSIN FROM A STUDY USING RETINAL ANALOGS

Abstract To investigate the shape of the chromophore binding site of pharaonis phoborhodopsin (ppR), ppR-opsin was incubated with five ring-modified retinal analogs: an acyclic retinal, phenylretinal, α-retinal, cyclohexylretinal and 5-isopropyl-α-retinal. The experimental results were compared with those obtained from bacteriorhodopsin-opsin (bR-opsin) and the same retinal analogs. It was suggested that ring chain conformation is important in affecting the spectral shoulder unique for the absorption spectrum of ppR. The rate of pigment formation depended greatly on the analogs used with the planar analogs showing rapid formation. Thus, we concluded that the space of the retinal binding site of ppR is restricted to the plane of the cyclohexenyl ring of the chromophore, whereas that of bR is less restricted.     

The effect of the protein environment on the structure and charge distribution of the retinal Schiff base in bacteriorhodopsin

The effects of the amino acid side chains of the binding pocket of bacteriorhodopsin (bR) and of a water molecule on the structure of the retinal Schiff base have been studied using Becke3LYP/6-31G* level of density functional theory. A model protonated Schiff base structure including six conjugated double bonds and methyl substituents was optimized in the presence of several amino acid side chains and of a water molecule, separately. The Schiff base structure was also calculated in the form of a neutral species. At each optimized complex geometry the atomic charges of the model Schiff base were calculated using Mulliken population analysis. In agreement with previously proposed counterion(s) of the protonated retinal Schiff base in bR, the results show that Asp₈₅ and Asp₂₁₂, which are present in the form of negatively charged groups, have significantly large effects on the structure and electronic configuration of both unprotonated and protonated model Schiff bases. The presence of a water molecule in the vicinity of the Schiff base demonstrates significant effects which are comparable to those of aspartate groups. Other side chains studied did not show any significant effect in this direction. Apart from the aspartate groups and the water molecule, in none of the other complexes studied are the atomic charges and the bond alternation of the model Schiff base significantly influenced by the presence of the neighboring amino acids. Received: 24 March 1998 / Accepted: 3 September 1998 / Published online: 10 December 1998     

KINETIC MODEL OF BACTERIORHODOPSIN PHOTOCYCLE: PATHWAY FROM M STATE TO bR

A model of the last parts of the bacteriorhodopsin (bR) photocycle is proposed on the basis of experimental data for the kinetic behavior of the 'O' intermediate during a temperature pulse in distilled water suspension. The model includes the previously proposed (but not well characterized) intermediate 'N' between the 'M' and 'O' states of bR. This intermediate exists in fast temperature-dependent quasi-stationary equilibrium with the red-shifted intermediate 'O' and has a maximum of absorption close to the bR spectrum.     

INVESTIGATION INTO THE SPECTROSCOPY AND PHOTOISOMERIZATION OF A SERIES OF POLY (ETHYLENE GLYCOL) PEPTIDE SCHIFF BASES OF 11-cis RETINAL

Abstract— The 11-cis and all-trans isomers of a series of poly(ethylene glycol)-oligopeptide - Schiff bases as models for rhodopsin were synthesized and studied. Absorption data for certain of the PEG-peptide Schiff bases demonstrated that no intramolecular hydrogen-bonding (or protonation) occurs between the Schiff base and an acidic amino acid residue, as was previously thought. Photoisomerization of the 11-cis protonated and unprotonated Schiff bases were examined using both steady state and laser flash techniques. Also with 355 nm excitation (and additionally 532 nm in one case), an approximate 40% increase in quantum yield of isomerization (φ) occurred for all protonated PEG-peptide Schiff bases compared to the H⁺-n-butylamine counterparts (in methanol). In one case, a > 100% increase in φ was found in dichloromethane. These data show that PEG-oligopeptide Schiff bases are still further improved models for rhodopsin compared to their n-butylamine analogs.     

Exploring the molecular mechanism for color distinction in humans

We examine here the role of the red, green, and blue human opsin structures in modulating the absorption properties of 11-cis-retinal bonded to the protein via a protonated Schiff base (PSB). We built the three-dimensional structures of the human red, green, and blue opsins using homology modeling techniques with the crystal structure of bovine rhodopsin as the template. We then used quantum mechanics (QM) combined with molecular mechanics (MM) (denoted as QM/MM) techniques in conjunction with molecular dynamics to determine how the room temperature molecular structures of the three human color opsin proteins modulate the absorption frequency of the same bound 11-cis-retinal chromophore to account for the differences in the observed absorption spectra. We find that the conformational twisting of the 11-cis-retinal PSB plays an important role in the green to blue opsin shift, whereas the dipolar side chains in the binding pocket play a surprising role of red-shifting the blue opsin with respect to the green opsin, as a fine adjustment to the opsin shift. The dipolar side chains play a large role in the opsin shift from red to green.