A new spectral window on retinal protein photochemistry

A VIS pump/hyperspectral NIR probe study of all-trans-retinal protonated Schiff base (RPSB) in ethanol is presented. Upon irradiation, a short-lived absorption band covers the recorded range of λ = 1-2 μm. It decays to reveal the tail of S(1) emission at λ < 1.3 μm, along with a residual absorption at longer wavelengths, both of which decay with the known kinetics of internal conversion to S(0). The existence of this hitherto unrecorded excited-state absorption deep in the NIR will require a revision of current models for RPSB electronic structure. The phenomenological similarity of these observations with ultrafast NIR studies of carotenoids raises the question of whether three, and not two, electronic states participate in RPSB photochemistry as well. The relevance of these observations to retinal protein photochemistry is discussed.     

Excitation energies of retinal chromophores: critical role of the structural model

We employ a variety of highly-correlated approaches including quantum Monte Carlo (QMC) and the n-electron valence state perturbation theory (NEVPT2) to compute the vertical excitation energies of retinal protonated Schiff base (RPSB) models in the gas phase. We find that the NEVPT2 excitation energies are in good agreement with the QMC values and confirm our previous findings that the complete-active-space perturbation (CASPT2) approach yields accurate excitations for RPSB models only when the more recent zero-order IPEA Hamiltonian is employed. The excitations computed with the original zero-order formulation of CASPT2 are instead systematically red-shifted by more than 0.3 eV. We then focus on the full 11-cis retinal chromophore and show that the M06-2X and MP2 approaches provide reliable ground-state equilibrium structures for this system while the complete-active-space self-consistent field (CASSCF) geometry is characterized by significantly higher ground-state energies at the NEVPT2 and CASPT2 level. Our calibration of the structural model together with the general agreement of all highly-correlated excited-state methods allows us to reliably assign a value of about 2.3 eV to the vertical excitation of 11-cis RPSB in the gas-phase.     

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.     

Absorption studies of neutral retinal Schiff base chromophores

The neutral retinal Schiff base is connected to opsin in UV sensing pigments and in the blue-shifted meta-II signaling state of the rhodopsin photocycle. We have designed and synthesized two model systems for this neutral chromophore and have measured their gas-phase absorption spectra in the electrostatic storage ring ELISA with a photofragmentation technique. By comparison to the absorption spectrum of the protonated retinal Schiff base in vacuo, we found that the blue shift caused by deprotonation of the Schiff base is more than 200 nm. The absorption properties of the UV absorbing proteins are thus largely determined by the intrinsic properties of the chromophore. The effect of approaching a positive charge to the Schiff base was also studied, as well as the susceptibility of the protonated and unprotonated chromophores to experience spectral shifts in different solvents.     

Absorption of schiff-base retinal chromophores in vacuo

The absorption spectrum of the all-trans retinal chromophore in the protonated Schiff-base form, that is, the biologically relevant form, has been measured in vacuo, and a maximum is found at 610 nm. The absorption of retinal proteins has hitherto been compared to that of protonated retinal in methanol, where the absorption maximum is at 440 nm. In contrast, the new gas-phase absorption data constitute a well-defined reference for spectral tuning in rhodopsins in an environment devoid of charges and dipoles. They replace the misleading comparison with absorption properties in solvents and lay the basis for reconsidering the molecular mechanisms of color tuning in the large family of retinal proteins. Indeed, our measurement directly shows that protein environments in rhodopsins are blue- rather than red shifting the absorption. The absorption of a retinal model chromophore with a neutral Schiff base is also studied. The data explain the significant blue shift that occurs when metharhodopsin I becomes deprotonated as well as the purple-to-blue transition of bacteriorhodopsin upon acidification.     

The opsin shift and mechanism of spectral tuning in rhodopsin

Molecular dynamics simulations and combined quantum mechanical and molecular mechanical calculations have been performed to investigate the mechanism of the opsin shift and spectral tuning in rhodopsin. A red shift of -980 cm(-1) was estimated in the transfer of the chromophore from methanol solution environment to the protonated Schiff base (PSB)-binding site of the opsin. The conformational change from a 6-s-cis-all-trans configuration in solution to the 6-s-cis-11-cis conformer contributes additional -200 cm(-1), and the remaining effects were attributed to dispersion interactions with the aromatic residues in the binding site. An opsin shift of 2100 cm(-1) was obtained, in reasonable accord with experiment (2730 cm(-1)). Dynamics simulations revealed that the 6-s-cis bond can occupy two main conformations for the β-ionone ring, resulting in a weighted average dihedral angle of about -50°, which may be compared with the experimental estimate of -28° from solid-state NMR and Raman data. We investigated a series of four single mutations, including E113D, A292S, T118A, and A269T, which are located near the PSB, along the polyene chain of retinal and close to the ionone ring. The computational results on absorption energy shift provided insights into the mechanism of spectral tuning, which involves all means of electronic structural effects, including the stabilization or destabilization of either the ground or the electronically excited state of the retinal PSB.     

Butyl conformational reorganization as a possible explanation for the longitudinal flexibility of the binding site of bacteriorhodopsin. The azulene and C-22 retinoid analogs.

The UV-VIS absorption data of four bacteriorhodopsin (BR) analogs formed from azulene-retinals of varying polyene chain length show that the one-bond-shortened to one-bond-lengthened analogs possess comparable opsin shift values to that of BR. A two-bond-shortened analog exhibited a much smaller opsin shift. These data, combined with those reported for the C-22 retinal analog (Tokunaga et al., 1977, Biophys. J. 19, 191-198) were analyzed by molecular modelling and computer graphics in terms of a model where conformational flexibility of the appended butyl is the controlling factor in determining ease of pigment formation and protein/substrate interaction.     

Following photoinduced dynamics in bacteriorhodopsin with 7-fs impulsive vibrational spectroscopy

Sub-10-fs laser pulses are used to impulsively photoexcite bacteriorhodopsin (BR) suspensions and probe the evolution of the resulting vibrational wave packets. Fourier analysis of the spectral modulations induced by transform-limited as well as linearly chirped excitation pulses allows the delineation of excited- and ground-state contributions to the data. On the basis of amplitude and phase variations of the modulations as a function of the dispersed probe wavelength, periodic modulations in absorption above 540 nm are assigned to ground-state vibrational coherences induced by resonance impulsive Raman spectral activity (RISRS). Probing at wavelengths below 540 nm-the red edge of the intense excited-state absorption band-uncovers new vibrational features which are accordingly assigned to wave packet motions along bound coordinates on the short-lived reactive electronic surface. They consist of high- and low-frequency shoulders adjacent to the strong C=C stretching and methyl rock modes, respectively, which have ground-state frequencies of 1008 and 1530 cm-1. Brief activity centered at approximately 900 cm-1, which is characteristic of ground-state HOOP modes, and strong modulations in the torsional frequency range appear as well. Possible assignments of the bands and their implication to photoinduced reaction dynamics in BR are discussed. Reasons for the absence of similar signatures in the pump-probe spectral modulations at longer probing wavelengths are considered as well.     

Ultrafast dynamics of 2E state formation in Cr(acac)3

Femtosecond time-resolved absorption spectroscopy has been used to elucidate the excited-state dynamics associated with formation of the (2)E excited state in a Cr(III) transition metal complex. Cr(acac)(3) (where acac is the deprotonated monoanion of acetylacetone) exhibits monophasic decay kinetics with tau = 1.1 +/- 0.1 ps following excitation into the lowest-energy ligand-field absorption band; the time constant is found to be independent of both excitation and probe wavelength across the entire (4)A(2) --> (4)T(2) absorption envelope. The lack of a significant shift in the excited-state absorption spectrum combined with the observed spectral narrowing is consistent with an assignment of this process as vibrational cooling (k(vib)) in the (2)E state. The data on Cr(acac)(3) indicate that intersystem crossing associated with the (4)T(2) --> (2)E conversion occurs at a rate k(ISC) > 10(13) s(-)(1) and furthermore competes effectively with vibrational relaxation in the initially formed (4)T(2) state. Excitation into the higher energy (4)LMCT state (lambda(ex) = 336 nm) gives rise to biphasic kinetics with tau( 1) = 50 +/- 20 fs and tau( 2) = 1.2 +/- 0.2 ps. The slower component is again assigned to vibrational cooling in the (2)E state, whereas the subpicosecond process is attributed to conversion from the charge-transfer to the ligand-field manifold. In addition to detailing a process central to the photophysics of Cr(III), these results reinforce the notion that the conventional picture of excited-state dynamics in which k(vib) > k(IC) > k(ISC) does not generally apply when describing excited-state formation in transition metal complexes.     

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.     

RESONANCE RAMAN SPECTRA OF BACTERIORHODOPSIN MUTANTS WITH SUBSTITUTIONS AT ASP-85, ASP-96, AND ARG-82

Detergent solubilized bacteriorhodopsin (BR) proteins which contain alterations made by site-directed mutagenesis (Asp-96----Asn, D96N; Asp-85----Asn, D85N; and Arg-82----Gln, R82Q) have been studied with resonance Raman spectroscopy. Raman spectra of the light-adapted (BRLA) and M species in D96N are identical to those of native BR, indicating that this residue is not located near the chromophore. The BRLA states of D85N and especially R82Q contain more of the 13-cis, C = N syn (BR555) species under ambient illumination compared to solubilized native BR. Replacement of Asp-85 with Asn causes a 25 nm red-shift of the absorption maximum and a frequency decrease in both the ethylenic (-7 cm-1) and the Schiff base C = NH+ (-3 cm-1) stretching modes of BRLA. These changes indicate that Asp-85 is located close to the protonated retinal Schiff base. The BRLA spectrum of R82Q exhibits a slight perturbation of the C = NH+ band, but its M spectrum is unperturbed. The Raman spectra and the absorption properties of D85N and R82Q suggest that the protein counterion environment involves the residues Asp-85-, Arg-82+ and presumably Asp-212-. These data are consistent with a model where the strength of the protein-chromophore interaction and hence the absorption maximum depends on the overall charge of the Schiff base counterion environment.     

THE 'OPSIN SHIFT' IN BACTERIORHODOPSIN: STUDIES WITH ARTIFICIAL BACTERIORHODOPSINS

Abstract— The difference (in cm⁻¹) in absorption maxima between the protonated Schiff base of retinals and the pigment derived therefrom has been defined as the opsin shift. It represents the influence of the opsin binding site on the chromophore. The analysis of the opsin shifts of a series of dihydrobacteriorhodopsins has led to the external point-charge model, which in addition to a counter anion near the Schiff base ammonium, carries another negative charge in the vicinity of the β-ionone ring. This is in striking contrast to the external point-charge model proposed earlier for the bovine visual pigment. The absorption maxima of rhodopsins formed from bromo- and phenyl retinals support the two models. A retinal carrying a photoaffinity label has yielded a nonbleachable bacteriorhodopsin.     

Decisive role of electronic polarization of the protein environment in determining the absorption maximum of halorhodopsin

It is known that the absorption maximum of halorhodopsin is red shifted by 10 nm with the uptake of a chloride ion Cl(-). According to the X-ray structure, the ion is located at the position of the counterion of the chromophore, protonated retinal Schiff base. Thus, the direction of the observed spectral change is opposite to that expected from the pi-electron redistribution (an increase in the bond alternation) induced by the counterion. The physical origin of this abnormal shift is never explained in terms of any simple chemical analogues. We successfully explain this phenomenon by a QM/MM type of excitation energy calculation. The three-dimensional structure of the protein is explicitly taken into account using the X-ray structure. We reveal that the electronic polarization of the protein environment plays an essential role in tuning the absorption maximum of halorhodopsin.     

Excited-state dynamics of spiropyran-derived merocyanine isomers

Merocyanine (MC) isomers that are formed after absorption of a UV photon by 1',3'-dihydro-1',3'-3'-trimethyl-6-nitrospiro[2H-1-benzopyran-2',2'-(2H)-indole] were studied. Several, predominantly TTC and TTT, merocyanine isomers are present in toluene solution ("T" and "C" indicate trans and cis conformations of the C-C bonds in the methine bridge). Excitation in the MC visible absorption band (at 490, 550, and 630 nm) with 100 fs laser pulses was used to study MC excited-state dynamics. Internal conversion on the picosecond time scale was found to be the dominant relaxation pathway. Excited-state isomerization reactions were also observed. Excitation at 630 nm (assigned to TTC isomer excitation) leads to formation of a third isomer (either CTC or CTT). Excitation at 490 nm (assigned to TTT isomer excitation) leads to more complex excited-state relaxation, including formation of two isomers: TTC (absorption at 600 nm) and CTC or CTT (absorption at 650 nm).     

Chromophore interaction in xanthorhodopsin--retinal dependence of salinixanthin binding

Xanthorhodopsin is a light-driven proton pump in the extremely halophilic bacterium Salinibacter ruber. Its unique feature is that besides retinal it has a carotenoid, salinixanthin, with a light harvesting function. Tight and specific binding of the carotenoid antenna is controlled by binding of the retinal. Addition of all-trans retinal to xanthorhodopsin bleached with hydroxylamine restores not only the retinal chromophore absorption band, but causes sharpening of the salinixanthin bands reflecting its rigid binding by the protein. In this report we examine the correlation of the changes in the two chromophores during bleaching and reconstitution with native all-trans retinal, artificial retinal analogs and retinol. Bleaching and reconstitution both appear to be multistage processes. The carotenoid absorption changes during bleaching occurred not only upon hydrolysis of the Schiff base but continued while the retinal was leaving its binding site. In the case of reconstitution, the 13-desmethyl analog formed the protonated Schiff base slower than retinal, and provided the opportunity to observe changes in carotenoid binding at various stages. The characteristic sharpening of the carotenoid bands, indicative of its reduced conformational heterogeneity in the binding site, occurs when the retinal occupies the binding site but the covalent bond to Lys-240 via a Schiff base is not yet formed. This is confirmed by the results for retinol reconstitution, where the Schiff base does not form but the carotenoid exhibits its characteristic spectral change from the binding.     

Influence of environment on the electronic structure of Cob(III)alamins: time-resolved absorption studies of the S(1) state spectrum and dynamics

Transient absorption spectroscopy has been used to elucidate the nature of the S1 intermediate state populated following excitation of cob(III)alamin (Cbl(III)) compounds. This state is sensitive both to axial ligation and to solvent polarity. The excited-state lifetime as a function of temperature and solvent environment is used to separate the dynamic and electrostatic influence of the solvent. Two distinct types of excited states are identified, both assigned to pi3d configurations. The spectra of both types of excited states are characterized by a red absorption band (ca. 600 nm) assigned to Co 3d --> 3d or Co 3d --> corrin pi* transitions and by visible absorption bands similar to the corrin pi-->pi* transitions observed for ground state Cbl(III) compounds. The excited state observed following excitation of nonalkyl Cbl(III) compounds has an excited-state spectrum characteristic of Cbl(III) molecules with a weakened bond to the axial ligand (Type I). A similar excited-state spectrum is observed for adenosylcobalamin (AdoCbl) in water and ethylene glycol. The excited-state spectrum of methyl, ethyl, and n-propylcobalamin is characteristic of a Cbl(III) species with a sigma-donating alkyl anion ligand (Type II). This Type II excited-state spectrum is also observed for AdoCbl bound to glutamate mutase. The results are discussed in the context of theoretical calculations of Cbl(III) species reported in the literature and highlight the need for additional calculations exploring the influence of the alkyl ligand on the electronic structure of cobalamins.     

A molecular spring for vision

Light absorption by the visual pigment rhodopsin leads to vision via a complex signal transduction pathway that is initiated by the ultrafast and highly efficient photoreaction of its chromophore, the retinal protonated Schiff base (RPSB). Here, we investigate this reaction in real time by means of unrestrained molecular dynamics simulations of the protein in a membrane mimetic environment, treating the chromophore at the density functional theory level. We demonstrate that a highly strained all-trans RPSB is formed starting from the 11-cis configuration (dark state) within approximately 100 fs by a minor rearrangement of the nuclei under preservation of the saltbridge with Glu113 and virtually no deformation of the binding pocket. Hence, the initial step of vision can be understood as the compression of a molecular spring by a minor change of the nuclear coordinates. This spring can then release its strain by altering the protein environment.     

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.     

Vibrational analysis of excited and ground electronic states of all-trans retinal protonated Schiff-bases

We report on vibrational coherence dynamics in excited and ground electronic states of all-trans retinal protonated Schiff-bases (RPSB), investigated by time-resolved Degenerate Four-Wave-Mixing (DFWM). The results show that wave packet dynamics in the excited state of RPSB consist of only low-frequency (<800 cm(-1)) modes. Such low-frequency wave packet motion is observed over a broad range of detection wavelengths ranging from excited state absorption (～500 nm) to stimulated emission (>600 nm). Our results indicate that low-frequency coherences in the excited state are not activated directly by laser excitation but rather by internal vibrational energy redistribution. This is supported by the observation that similar coherence dynamics are not observed in the electronic ground state. Challenging previous experimental results, we show that the formation of low-frequency coherence dynamics in RPSB does not require significant excess vibrational energy deposition in the excited state vibrational manifolds. Concerning ground state wave packet dynamics, we observe a set of high-frequency (>800 cm(-1)) modes, reflecting mainly single and double bond stretching motion in the retinal polyene-chain. Dephasing of these high-frequency coherences is mode-dependent and partially differs from analogous vibrational dephasing of the all-trans retinal chromophore in a protein environment (bacteriorhodopsin).     

The excited-state chemistry of phycocyanobilin: a semiempirical study.

Based on previous time-resolved absorption studies, phycocyanobilin undergoes a photoreaction from an A- into a B- and C-form, with the latter two photoproducts showing absorption spectra red-shifted from A. To identify the molecular mechanism involved in the excited-state reactions, the structural origin of the red shift in the absorption spectra is investigated. Using semiempirical AM1 calculations that include configuration interaction by pair doubles excitation configuration interaction, the absorption spectra of different conformers as well as different protonation states were calculated. The results clearly indicate a pronounced red shift in the spectra of structures either protonated or deprotonated at the basic/acidic centres of the tetrapyrrole chromophore whereas, in contrast, conformational changes alone result in a blue shift. Furthermore, it is shown by quantum chemical calculations that the basicity of phycocyanobilin is much higher in the excited than in the ground state, with a decrease in the excited-state pK(B)* of approximately 9.5 units. The acidity is only slightly enhanced with a drop in pK(A)* of only approximately 1.6 units. From these findings, a reaction model for the excited-state processes in phycocyanobilin is proposed. According to this model, photoexcitation of phycocyanobilin triggers an excited-state proton transfer giving rise to the formation of a protonated species. In parallel, the local increase in the medium pH associated with protonation then forwards a deprotonation at an acidic NH-group so that in effect both protonated and deprotonated phycocyanobilin would arise from the initial photoreaction and account for the observed red shift in the spectra of the B- and C-forms.