EFFECT OF PROTONATION ON THE ISOMERIZATION PROPERTIES OF n-BUTYLAMINE SCHIFF BASE OF ISOMERIC RETINAL AS REVEALED BY DIRECT HPLC ANALYSES: SELECTION OF ISOMERIZATION PATHWAYS BY RETINAL PROTEINS

Alumina adsorption chromatography and ion-pair reversed-phase chromatography were developed to analyze the isomers of unprotonated and protonated n-butylamine Schiff base of retinal (RSB and PRSB), respectively. Photoisomerization starting from the all-trans, 11-cis and 13-cis isomers was traced for RSB in n-hexane, acetonitrile, methanol and 1-butanol, and for PRSB in methanol, acetonitrile and 1-butanol. The quantum yields of photoisomerization for the all-trans, 9-cis, 11-cis and 13-cis isomers were determined for RSB and PRSB in the above solvents except 1-butanol. On the other hand, photoisomerization of isomeric retinal bound (through Schiff base linkage) to bovine serum albumin (RBSA) in aqueous solution (pH 3, 7 and 12) as well as thermal isomerization of RSB (in n-hexane), PRSB (in methanol) and RBSA (in aqueous solution, pH 7) were traced starting from the all-trans, 11-cis, and 13-cis isomers. Protonation of RSB drastically changes the pathway of photoisomerization and increases the quantum yields of isomeric RSB. The solvent polarity increases the quantum yields of RSB differently depending on the configuration. Protonation enhances thermal isomerization also. The results of the above model systems are compared with those of retinal proteins to rationalize their selection of the particular isomerization pathways.     

Activity switches of rhodopsin

Rhodopsin, the visual pigment of the rod photoreceptor cell contains as its light-sensitive cofactor 11-cis retinal, which is bound by a protonated Schiff base between its aldehyde group and the Lys296 side chain of the apoprotein. Light activation is achieved by 11-cis to all-trans isomerization and subsequent thermal relaxation into the active, G protein-binding metarhodopsin II state. Metarhodopsin II decays via two parallel pathways, which both involve hydrolysis of the Schiff base eventually to opsin and released all-trans retinal. Subsequently, rhodopsin's dark state is regenerated by a complicated retinal metabolism, termed the retinoid cycle. Unlike other retinal proteins, such as bacteriorhodopsin, this regeneration cycle cannot be short cut by light, because blue illumination of active metarhodopsin II does not lead back to the ground state but to the formation of largely inactive metarhodopsin III. In this review, mechanistic details of activating and deactivating pathways of rhodopsin, particularly concerning the roles of the retinal, are compared. Based on static and time-resolved UV/Vis and FTIR spectroscopic data, we discuss a model of the light-induced deactivation. We describe properties and photoreactions of metarhodopsin III and suggest potential roles of this intermediate for vision.     

Characterization and photochemistry of 13-desmethyl bacteriorhodopsin

The photochemistry of the 13-desmethyl (DM) analogue of bacteriorhodopsin (BR) is examined by using spectroscopy, molecular orbital theory, and chromophore extraction followed by conformational analysis. The removal of the 13-methyl group permits the direct photochemical formation of a thermally stable, photochemically reversible state, P1(DM) (lambda(max) = 525 nm), which can be generated efficiently by exciting the resting state, bR(DM) with yellow or red light (lambda > 590 nm). Chromophore extraction analysis reveals that the retinal configuration in P1(DM) is 9-cis, identical to that of the retinal configuration in the native BR P1 state. Fourier transform infrared and Raman experiments on P1(DM) indicate an anti configuration around the C15=N bond, as would be expected of an O-state photoproduct. However, low-temperature spectroscopy and ambient, time-resolved studies indicate that the P1(DM) state forms primarily via thermal relaxation from the L(D)(DM) state. Theoretical studies on the BR binding site show that 13-dm retinal is capable of isomerizing into a 9-cis configuration with minimal steric hindrance from surrounding residues, in contrast to the native chromophore in which surrounding residues significantly obstruct the corresponding motion. Analysis of the photokinetic experiments indicates that the Arrhenius activation energy of the bR(DM) --> P1(DM) transition in 13-dm-BR is less than 0.6 kcal/mol (vs 22 +/-5 kcal/mol measured for the bR --> P (P1 and P2) reaction in 85:15 glycerol:water suspensions of wild type). Consequently, the P1(DM) state in 13-dm-BR can form directly from all-trans, 15-anti intermediates (bR(DM) and O(DM)) or all-trans, 15-syn (K(D)(DM)/L(D)(DM)) intermediates. This study demonstrates that the 13-methyl group, and its interactions with nearby binding site residues, is primarily responsible for channeling one-photon photochemical and thermal reactions and is limited to the all-trans and 13-cis species interconversions in the native protein.     

Reaction path analysis of the "tunable" photoisomerization selectivity of free and locked retinal chromophores

Multiconfigurational second-order perturbation theory computations and reaction path mapping for the retinal protonated Schiff base models all-trans-nona-2,4,6,8-tetraeniminium and 2-cis-nona-2,4,6,8-tetraeniminium cation demonstrate that, in isolated conditions, retinal chromophores exhibit at least three competing excited-state double bond isomerization paths. These paths are associated with the photoisomerization of the double bonds in positions 9, 11, and 13, respectively, and are controlled by barriers that favor the position 11. The computations provide a basis for the understanding of the observed excited-state lifetime in both naturally occurring and synthetic chromophores in solution and, tentatively, in the protein environment. In particular, we provide a rationalization of the excited-state lifetimes observed for a group of locked retinal chromophores which suggests that photoisomerization in bacteriorhodopsin is the result of simultaneous specific "catalysis" (all-trans --> 13-cis path) accompanied by specific "inhibition" (all-trans --> 11-cis path). The nature of the S(1) --> S(0) decay channel associated with the three paths has also been investigated at the CASSCF level of theory. It is shown that the energy surfaces in the vicinity of the conical intersection for the photoisomerization about the central double bond of retinal (position 11) and the two corresponding lateral double bonds (positions 9 and 13) are structurally different.     

Constraints of opsin structure on the ligand-binding site: studies with ring-fused retinals

Ring-fused retinal analogs were designed to examine the hula-twist mode of the photoisomerization of the 9-cis retinylidene chromophore. Two 9-cis retinal analogs, the C11-C13 five-membered ring-fused and the C12-C14 five-membered ring-fused retinal derivatives, formed the pigments with opsin. The C11-C13 ring-fused analog was isomerized to a relaxed all-trans chromophore (lambda(max) > 400 nm) at even -269 degrees C and the Schiff base was kept protonated at 0 degrees C. The C12-C14 ring-fused analog was converted photochemically to a bathorhodopsin-like chromophore (lambda(max) = 583 nm) at -196 degrees C, which was further converted to the deprotonated Schiff base at 0 degrees C. The model-building study suggested that the analogs do not form pigments in the retinal-binding site of rhodopsin but form pigments with opsin structures, which have larger binding space generated by the movement of transmembrane helices. The molecular dynamics simulation of the isomerization of the analog chromophores provided a twisted C11-C12 double bond for the C12-C14 ring-fused analog and all relaxed double bonds with a highly twisted C10-C11 bond for the C11-C13 ring-fused analog. The structural model of the C11-C13 ring-fused analog chromophore showed a characteristic flip of the cyclohexenyl moiety toward transmembrane segments 3 and 4. The structural models suggested that hula twist is a primary process for the photoisomerization of the analog chromophores.     

Key role of electrostatic interactions in bacteriorhodopsin proton transfer

The first proton transport step following photon absorption in bacteriorhodopsin is from the 13-cis retinal Schiff base to Asp85. Configurational and energetic determinants of this step are investigated here by performing quantum mechanical/molecular mechanical minimum-energy reaction-path calculations. The results suggest that retinal can pump protons when in the 13-cis, 15-anti conformation but not when 13-cis, 15-syn. Decomposition of the proton transfer energy profiles for various possible pathways reveals a conflict between the effect of the intrinsic proton affinities of the Schiff base and Asp85, which favors the neutral, product state (i.e., with Asp85 protonated), with the mainly electrostatic interaction between the protein environment with the reacting partners, which favors the ion pair reactant state (i.e., with retinal protonated). The rate-limiting proton-transfer barrier depends both on the relative orientations of the proton donor and acceptor groups and on the pathway followed by the proton; depending on these factors, the barrier may arise from breaking and forming of hydrogen bonds involving the Schiff base, Asp85, Asp212, and water w402, and from nonbonded interactions involving protein groups that respond to the charge rearrangements in the Schiff base region.     

Participation of the BC Loop in the Correct Folding of Bacteriorhodopsin as Revealed by Solid‐state NMR†

Structural changes in bacteriorhodopsin (bR) in two different processes of retinal reconstitutions were investigated by observing the ¹³C and ¹⁵N solid‐state NMR spectra of [1‐¹³C]Val‐ and [¹⁵N]Pro‐labeled bR. We found that NMR signals of the BC loop were sensitive to changes in protein structure and dynamics, from wild‐type (WT) bR to bacterio‐opsin (bO), regenerated bR and E1001 bR. Regenerated bR was prepared following the addition of retinal into bO obtained from photobleached WT‐bR. E1001 bR was cultured from a retinal‐deficient strain termed E1001 following the addition of retinal to growing cells. ¹⁵N NMR signal at Pro70 in the BC loop in WT‐bR was observed at 122.4 p.p.m., whereas signals were not apparent or partly suppressed in bO and regenerated bR, respectively. Similarly, the ¹³C NMR signal at Val69 in the BC loop at 172.0 p.p.m. that was observed in WT‐bR was significantly decreased in both regenerated bR and bO. These results suggest that the dynamic structure of the BC loop in bO was substantially altered following the removal of retinal. As a consequence, the correct protein structure failed to be recovered via the regenerating process of retinal to bO. On the other hand, ¹³C and ¹⁵N NMR signals at the BC loop in E1001 bR appeared at positions identical to those of WT‐bR. The results of the current study indicate that the BC loop may not always fold correctly in the regenerated bR, which leads to different properties in the regenerated bR compared to that of WT‐bR.     

Rhodopsin regeneration is accelerated via noncovalent 11-cis retinal-opsin complex--a role of retinal binding pocket of opsin

The regeneration of bovine rhodopsin from its apoprotein opsin and the prosthetic group 11-cis retinal involves the formation of a retinylidene Schiff base with the epsilon-amino group of the active lysine residue of opsin. The pH dependence of a Schiff base formation in solution follows a typical bell-shaped profile because of the pH dependence of the formation and the following dehydration of a 1-aminoethanol intermediate. Unexpectedly, however, we find that the formation of rhodopsin from 11-cis retinal and opsin does not depend on pH over a wide pH range. These results are interpreted by the Matsumoto and Yoshizawa (Nature 258 [1975] 523) model of rhodopsin regeneration in which the 11-cis retinal chromophore binds first to opsin through the beta-ionone ring, followed by the slow formation of the retinylidene Schiff base in a restricted space. We find the second-order rate constant of the rhodopsin formation is 6100+/-300 mol(-1) s(-1) at 25 degrees C over the pH range 5-10. The second-order rate constant is much greater than that of a model Schiff base in solution by a factor of more than 10(7). A previous report by Pajares and Rando (J Biol Chem 264 [1989] 6804) suggests that the lysyl epsilon-NH(2) group of opsin is protonated when the beta-ionone ring binding site is unoccupied. The acceleration of the Schiff base formation in rhodopsin is explained by stabilization of the deprotonated form of the lysyl epsilon-NH(2) group which might be induced when the beta-ionone ring binding site is occupied through the noncovalent binding of 11-cis retinal to opsin at the initial stage of rhodopsin regeneration, followed by the proximity and orientation effect rendered by the formation of noncovalent 11-cis retinal-opsin complex.     

1H-13C/1H-15N heteronuclear dipolar recoupling by R-symmetry sequences under fast magic angle spinning for dynamics analysis of biological and organic solids

Fast magic angle spinning (MAS) NMR spectroscopy is becoming increasingly important in structural and dynamics studies of biological systems and inorganic materials. Superior spectral resolution due to the efficient averaging of the dipolar couplings can be attained at MAS frequencies of 40 kHz and higher with appropriate decoupling techniques, while proton detection gives rise to significant sensitivity gains, therefore making fast MAS conditions advantageous across the board compared with the conventional slow- and moderate-MAS approaches. At the same time, many of the dipolar recoupling approaches that currently constitute the basis for structural and dynamics studies of solid materials and that are designed for MAS frequencies of 20 kHz and below, fail above 30 kHz. In this report, we present an approach for (1)H-(13)C/(1)H-(15)N heteronuclear dipolar recoupling under fast MAS conditions using R-type symmetry sequences, which is suitable even for fully protonated systems. A series of rotor-synchronized R-type symmetry pulse schemes are explored for the determination of structure and dynamics in biological and organic systems. The investigations of the performance of the various RN(n)(v)-symmetry sequences at the MAS frequency of 40 kHz experimentally and by numerical simulations on [U-(13)C,(15)N]-alanine and [U-(13)C,(15)N]-N-acetyl-valine, revealed excellent performance for sequences with high symmetry number ratio (N/2n > 2.5). Further applications of this approach are presented for two proteins, sparsely (13)C/uniformly (15)N-enriched CAP-Gly domain of dynactin and U-(13)C,(15)N-Tyr enriched C-terminal domain of HIV-1 CA protein. Two-dimensional (2D) and 3D R16(3)(2)-based DIPSHIFT experiments carried out at the MAS frequency of 40 kHz, yielded site-specific (1)H-(13)C/(1)H-(15)N heteronuclear dipolar coupling constants for CAP-Gly and CTD CA, reporting on the dynamic behavior of these proteins on time scales of nano- to microseconds. The R-symmetry-based dipolar recoupling under fast MAS is expected to find numerous applications in studies of protein assemblies and organic solids by MAS NMR spectroscopy.     

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.     

Asymmetric toggling of a natural photoswitch: ultrafast spectroscopy of Anabaena sensory rhodopsin

Photochemistry in retinal proteins (RPs) is determined both by the properties of the retinal chromophore and by its interactions with the surrounding protein. The initial retinal configuration, and the isomerization coordinates active in any specific protein, must be important factors influencing the course of photochemistry. This is illustrated by the vast differences between the photoisomerization dynamics in visual pigments which start 11-cis and end all-trans, and those observed in microbial ion pumps and sensory rhodopsins which start all-trans and end in a 13-cis configuration. However, isolating these factors is difficult since most RPs accommodate only one active stable ground-state configuration. Anabaena sensory rhodopsin, allegedly functioning in cyanobacteria as a wavelength sensor, exists in two stable photoswitchable forms, containing all-trans and 13-cis retinal isomers, at a wavelength-dependent ratio. Using femtosecond spectroscopy, and aided by extraction of coherent vibrational signatures, we show that cis-to-trans photoisomerization, as in visual pigments, is ballistic and over in a fraction of a picosecond, while the reverse is nearly 10 times slower and kinetically reminiscent of other microbial rhodopsins. This provides a new test case for appreciating medium effects on primary events in RPs.     

The mechanism of bacteriorhodopsin functioning. I. The light-induced proton throw-over by retinal

The path of proton motion in protein bacteriorhodopsin is constructed: Tyr 43 → Tyr 26 → Asp 212 → Tyr 83 → Tyr 57 → Tyr 79 → Glu 204 → Tyr 64 → Glu 74. From the position of quantum theory the light-induced isomerization of retinal, the process carrying out the proton and throw-over from the inlet H-bond chain (Tyr 43 → Tyr 26 → Lys 216) to the outlet one (Lys 216 → Asp 212 → … → Glu 74) are investigated. The kinetics of the transition of the excitation from the π-electron subsystem to the vibrational one is studied; the function of the nonequilibrial excitation distribution of the collective intramolecular retinal vibrations is found. The mechanism of deprotonation of Schiff base (transmission of proton to the outlet channel) is considered. The correspondence between the theoretically estimated states and spectroscopically identified forms of bacteriorhodosin (initial form bR, intermediates J, K) is proposed.     

TD-DFT calculations of the potential energy curves for the trans-cis photo-isomerization of protonated Schiff base of retinal

One-dimensional potential energy curves for the isomerization of protonated Schiff base of retinal (PSBR) in bacteriorhodopsin (bR), i.e., isomerization from all-trans- to 13-cis-forms, have been calculated by means of time-dependent density functional theory (TD-DFT) calculations, in order to elucidate the mechanism of initial step in photo-absorption. The transition state of the isomerization in the first excited state is located at theta(13-14)=58 degrees , where theta(13-14) means twist angle around the C(13)=C(14) double bond of PSBR The potential barrier is formed by the avoided crossing between S(1) (B(u)-like) and S(2) (A(g)-like) states. The mechanism of the isomerization was discussed on the basis of theoretical results.     

A semiempirical study of the optimized ground and excited state potential energy surfaces of retinal and its protonated Schiff base.

The electronic ground and first excited states of retinal and its Schiff base are optimized for the first time using the semiempirical AM1 Hamiltonian. The barrier for rotation about the C(11)-C(12) double bond is characterized by variation of both the twist angle delta(C(10)-C(11)-C(12)-C(13)) and the bond length d(C(11)-C(12)). The potential energy surface is obtained by varying these two parameters. The calculated ground state rotational barrier is equal to 15.6 kcal/mol for retinal and 20.5 kcal/mol for its Schiff base. The all-trans conformation is more stable by 3.7 kcal/mol than the 11-cis geometry. For the first excited state, S(1,) the 90 degrees twisted geometry represents a saddle point for retinal with the rotational barrier of 14.6 kcal/mol. In contrast, this conformation is an energy minimum for the Schiff base. It can be easily reached at room temperature from the planar minima since it is separated from them by a barrier of only 0.6 kcal/mol. The 90 degrees minimum conformation is more stable than the all-trans by 8.6 kcal/mol. We are thus able to present a reaction path on the S(1) surface of the retinal Schiff base with an almost barrier-less geometrical relaxation into a twisted minimum geometry, as observed experimentally. The character of the ground and first excited singlet states underscores the need for the inclusion of double excitations in the calculations.     

A resonance Raman study of octopus bathorhodopsin with deuterium labeled retinal chromophores.

The resonance Raman spectrum of octopus bathorhodopsin in the fingerprint region and in the ethylenic-Schiff base region have been obtained at 80 K using the "pump-probe" technique as have its deuterated chromophore analogues at the C7D; C8D; C8,C7D2; C10D; C11D; C11, C12D2; C14D; C15D; C14, C15D2; and N16D positions. While these data are not sufficient to make definitive band assignments, many tentative assignments can be made. Because of the close spectral similarity between the octopus bathorhodopsin spectrum and that of bovine bathorhodopsin, we conclude that the essential configuration of octopus bathorhodopsin's chromophore is all-trans like. The data suggest that the Schiff base, C = N, configuration is trans (anti). The observed conformationally sensitive fingerprint bands show pronounced isotope shifts upon chromophore deuteration. The size of the shifts differ, in certain cases, from those found for bovine bathorhodopsin. Thus, the internal mode composition of the fingerprint bands differs somewhat from bovine bathorhodopsin, suggesting a somewhat different in situ chromophore conformation. An analysis of the NH bend frequency, the Schiff base C = N stretch frequency, and its shift upon Schiff base deuteration suggests that the hydrogen bonding between the protonated Schiff base with its protein binding pocket is weaker in octopus bathorhodopsin than in bovine bathorhodopsin but stronger than that found in bacteriorhodopsin's bR568 pigment.     

MAGIC ANGLE SPINNING NMR STUDIES ON THE METARHODOPSIN II INTERMEDIATE OF BOVINE RHODOPSIN: EVIDENCE FOR AN UNPROTONATED SCHIFF BASE

Magic angle spinning (MAS)13C-NMR spectra of the metarhodopsin II intermediate have been obtained using bovine rhodopsin regenerated with retinal 13C-labeled at the C-13 and C-15 positions to investigate the protonation state of the retinal Schiff base linkage. The 13C-labeled rhodopsin was reconstituted into 1,2-dipalmitoleoylphosphatidylcholine bilayers to increase the amount of meta II trapped at low temperature. Both the 13C-15 (159.2 ppm) and 13C-13 (144.0 ppm) isotropic chemical shifts are characteristic of an unprotonated Schiff base, while the 13C-15 shift is significantly different from that of retinal (191 ppm) or a tetrahedral carbinolamine group (70-90 ppm) previously proposed as an intermediate in the hydrolysis of the Schiff base at the meta II stage. This rules out the possibility that meta II non-covalently binds retinal or is a carbinolamine intermediate and provides convincing evidence that Schiff base deprotonation occurs in the meta I-meta II transition, an event that is likely to be important in triggering the activation of transducin.     

Novel retinylidene iminium salts for defining opsin shifts: synthesis and intrinsic chromophoric properties

Retinal Schiff bases serve as chromophores in many photoactive proteins that carry out functions such as signalling and light-induced ion translocation. The retinal Schiff base can be found as neutral or protonated, as all-trans, 11-cis or 13-cis isomers and can adopt different conformations in the protein binding pocket. Here we present the synthesis and characterisation of isomeric retinylidene iminium salts as mimics blocked towards isomerisation at the C11 position and conformationally restrained. The intrinsic chromophoric properties are elucidated by gas phase absorption studies. These studies reveal a small blue-shift in the S0-->S1 absorption for the 11-locked derivative as compared to the unlocked one. The gas phase absorption spectra of all the cationic mimics so far investigated show almost no absorption in the blue region. This observation stresses the importance of protein interactions for colour tuning, which allows the human eye to perceive blue light.     

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.     

全反式球形烯的光敏异构化及异构体的光谱

从Rhodobacter sphaeroides 2.4.1提取了全反式球形烯, 用高压液相色谱(HPLC)分离了它的I2增感异构化产物: 9,13'-cis(峰A), 5,13-cis+13,9'-cis(峰B), 13-cis(峰C), 5,13'-cis(峰E1), 9-cis(峰E2), 13'-cis(峰F),5,9'-cis(峰G), 9'-cis(峰H), all-trans(峰I), 研究了异构体的结构对其电子吸收光谱及HPLC保留时间的影响, 从而确认峰E1为5,13'-cis。对全反式球形烯的光敏异构化机理也作了初步探讨。     

Photoreduction of bacteriorhodopsin Schiff base at low humidity. A study with C13=C14 nonisomerizable artificial pigments

The retinal protonated Schiff base of bacteriorhodopsin is photoreactive to reducing agents such as NaBH4. In the present work we have studied the effect of different protein hydration levels on the photoreductive reaction, as well as the consequences of preventing isomerization around the critical C13=C14 retinal double bond. It was revealed that the rate of light-induced NaBH4 reaction can be fitted to three phases, between 100 and 87%, from 87 to 35% and below 35% relative humidities (r.h.). The three phases are attributed to three protein regions characterized by different water affinities. Furthermore, it is shown that the PSB reduction reaction is light catalyzed even in artificial pigments derived from retinal analogs, in which isomerization around the C13=C14 double bond is prevented. It is suggested that the protein experiences light-induced conformational alterations that are not associated with C13=C14 double bond isomerization. In the 13-cis locked pigment the rate of reduction reaction is affected by r.h. levels only below 35%. The relatively low r.h. required for withdrawing water from the protein is attributed to the increased protein-water affinity in this specific pigment.