Visible‐Light‐Induced Photodimerization of a Photoactive Yellow Protein (PYP) Chromophore Model in a Single Crystal

The chromophore of the photoactive yellow protein (PYP), the photoreceptor in the photomotility of the bacterium Halorhodospira halophila, is a deprotonated para‐coumaric thioester linked to the side residue of a cysteine residue. The photophysics of the PYP chromophore is conveniently modeled with para‐hydroxycinnamic thiophenyl esters. Herein, we report the first direct evidence, obtained with X‐ray diffraction, of photodimerization of a para‐hydroxycinnamic thiophenyl ester in single crystalline state. This result represents the first direct observation of [2+2] dimerization of a model PYP chromophore, and demonstrates that even very weak light in the visible region is capable of inducing parallel radical reactions in PYP from the excited state of the chromophore, in addition to the main reaction pathway (trans–cis isomerization). This PYP model system adds an interesting example to the known solid‐state photodimerizations, because unlike the anhydrous crystal (which is not capable of sustaining the stress and disintegrates in the course of photodimerization), a single water molecule “dilutes” the structure to the extent sufficient for single‐crystal‐to‐single‐crystal reaction.     

Solvent effect on the excited-state dynamics of analogues of the photoactive yellow protein chromophore

We previously reported that two analogues of the Photoactive Yellow Protein chromophore, trans-p-hydroxycinnamic acid (pCA(2-)) and its amide derivative (pCM-) in their deprotonated forms, undergo a trans-cis photoisomerization whereas the thioester derivative, trans-p-hydroxythiophenyl cinnamate (pCT-), does not. pCT- is also the only one to exhibit a short-lived intermediate on its excited-state deactivation pathway. We here further stress the existence of two different relaxation mechanisms for these molecules and examine the reaction coordinates involved. We looked at the effect of the solvent properties (viscosity, polarity, solvation dynamics) on their excited-state relaxation dynamics, probed by ultrafast transient absorption spectroscopy. Sensitivity to the solvent properties is found to be larger for pCT- than for pCA(2-) and pCM-. This difference is considered to reveal that either the relaxation pathway or the reaction coordinate is different for these two classes of analogues. It is also found to be correlated to the electron donor-acceptor character of the molecule. We attribute the excited-state deactivation of analogues bearing a weaker acceptor group, pCA(2-) and pCM-, to a stilbene-like photoisomerization mechanism with the concerted rotation of the ethylenic bond and one adjacent single bond. For pCT-, which contains a stronger acceptor group, we consider a photoisomerization mechanism mainly involving the single torsion of the ethylenic bond. The excited-state deactivation of pCT- would lead to the formation of a ground-state intermediate at the "perp" geometry, which would return to the initial trans conformation without net isomerization.     

Subpicosecond Excited-State Proton Transfer Preceding Isomerization During the Photorecovery of Photoactive Yellow Protein

The ultrafast excited-state dynamics underlying the receptor state photorecovery is resolved in the M100A mutant of the photoactive yellow protein (PYP) from Halorhodospira halophila. The M100A PYP mutant, with its distinctly slower photocycle than wt PYP, allows isolation of the pB signaling state for study of the photodynamics of the protonated chromophore cis-p-coumaric acid. Transient absorption signals indicate a subpicosecond excited-state proton-transfer reaction in the pB state that results in chromophore deprotonation prior to the cis-trans isomerization required in the photorecovery dynamics of the pG state. Two terminal photoproducts are observed, a blue-absorbing species presumed to be deprotonated trans-p-coumaric acid and an ultraviolet-absorbing protonated photoproduct. These two photoproducts are hypothesized to originate from an equilibrium of open and closed folded forms of the signaling state, I(2) and I(2)'.     

Structure and photoreaction of photoactive yellow protein, a structural prototype of the PAS domain superfamily

Photoactive yellow protein (PYP) is a water-soluble photosensor protein found in purple photosynthetic bacteria. Unlike bacterial rhodopsins, photosensor proteins composed of seven transmembrane helices and a retinal chromophore in halophilic archaebacteria, PYP is a highly soluble globular protein. The alpha/beta fold structure of PYP is a structural prototype of the PAS domain superfamily, many members of which function as sensors for various kinds of stimuli. To absorb a photon in the visible region, PYP has a p-coumaric acid chromophore binding to the cysteine residue via a thioester bond. It exists in a deprotonated trans form in the dark. The primary photochemical event is photo-isomerization of the chromophore from trans to cis form. The twisted cis chromophore in early intermediates is relaxed and finally protonated. Consequently, the chromophore becomes electrostatically neutral and rearrangement of the hydrogen-bonding network triggers overall structural change of the protein moiety, in which local conformational change around the chromophore is propagated to the N-terminal region. Thus, it is an ideal model for protein conformational changes that result in functional change, responding to stimuli and expressing physiological activity. In this paper, recent progress in investigation of the photoresponse of PYP is reviewed.     

Ultrafast dynamics of isolated model photoactive yellow protein chromophores: "Chemical perturbation theory" in the laboratory

Pump-probe and pump-dump probe experiments have been performed on several isolated model chromophores of the photoactive yellow protein (PYP). The observed transient absorption spectra are discussed in terms of the spectral signatures ascribed to solvation, excited-state twisting, and vibrational relaxation. It is observed that the protonation state has a profound effect on the excited-state lifetime of p-coumaric acid. Pigments with ester groups on the coumaryl tail end and charged phenolic moieties show dynamics that are significantly different from those of other pigments. Here, an unrelaxed ground-state intermediate could be observed in pump-probe signals. A similar intermediate could be identified in the sinapinic acid and in isomerization-locked chromophores by means of pump-dump probe spectroscopy; however, in these compounds it is less pronounced and could be due to ground-state solvation and/or vibrational relaxation. Because of strong protonation-state dependencies and the effect of electron donor groups, it is argued that charge redistribution upon excitation determines the twisting reaction pathway, possibly through interaction with the environment. It is suggested that the same pathway may be responsible for the initiation of the photocycle in native PYP.     

Ultrafast photoisomerization of photoactive yellow protein chromophore analogues in solution: influence of the protonation state.

We investigate solvent viscosity and polarity effects on the photoisomerization of the protonated and deprotonated forms of two analogues of the photoactive yellow protein (PYP) chromophore. These are trans-p-hydroxybenzylidene acetone and trans-p-hydroxyphenyl cinnamate, studied in solutions of different polarity and viscosity at room temperature, by means of femtosecond fluorescence up-conversion. The fluorescence lifetimes of the protonated forms are found to be barely sensitive to solvent viscosity, and to increase with increasing solvent polarity. In contrast, the fluorescence decays of the deprotonated forms are significantly slowed down in viscous media and accelerated in polar solvents. These results elucidate the dramatic influence of the protonation state of the PYP chromophore analogues on their photoinduced dynamics. The viscosity and polarity effects are, respectively, interpreted in terms of different isomerization coordinates and charge redistribution in S(1). A trans-to-cis isomerization mechanism involving mainly the ethylenic double-bond torsion and/or solvation is proposed for the anionic forms, whereas "concerted" intramolecular motions are proposed for the neutral forms.     

Photoisomerization and proton transfer in photoactive yellow protein

The photoactive yellow protein (PYP) is a bacterial photosensor containing a para-coumaryl thioester chromophore that absorbs blue light, initiating a photocycle involving a series of conformational changes. Here, we present computational studies to resolve uncertainties and controversies concerning the correspondence between atomic structures and spectroscopic measurements on early photocycle intermediates. The initial nanoseconds of the PYP photocycle are examined using time-dependent density functional theory (TDDFT) to calculate the energy profiles for chromophore photoisomerization and proton transfer, and to calculate excitation energies to identify photocycle intermediates. The calculated potential energy surface for photoisomerization matches key, experimentally determined, spectral parameters. The calculated excitation energy of the photocycle intermediate cryogenically trapped in a crystal structure by Genick et al. [Genick, U. K.; Soltis, S. M.; Kuhn, P.; Canestrelli, I. L.; Getzoff, E. D. Nature 1998, 392, 206-209] supports its assignment to the PYP(B) (I(0)) intermediate. Differences between the time-resolved room temperature (298 K) spectrum of the PYP(B) intermediate and its low temperature (77 K) absorbance are attributed to a predominantly deprotonated chromophore in the former and protonated chromophore in the latter. This contrasts with the widely held belief that chromophore protonation does not occur until after the PYP(L) (I(1) or pR) intermediate. The structure of the chromophore in the PYP(L) intermediate is determined computationally and shown to be deprotonated, in agreement with experiment. Calculations based on our PYP(B) and PYP(L) models lead to insights concerning the PYP(BL) intermediate, observed only at low temperature. The results suggest that the proton is more mobile between Glu46 and the chromophore than previously realized. The findings presented here provide an example of the insights that theoretical studies can contribute to a unified analysis of experimental structures and spectra.     

Electronic structure of the PYP chromophore in its native protein environment

We report on supermolecular ab initio calculations which clarify the role of the local amino acid environment in determining the unique electronic structure properties of the photoactive yellow protein (PYP) chromophore. The extensive ab initio calculations, at the level of the CC2 and EOM-CCSD methods, allow us to explicitly address how the interactions between the deprotonated p-coumaric thio-methyl ester (pCTM-) chromophore and the surrounding amino acids act together to create a specifically stabilized pCTM- species. Particularly noteworthy is the role of the Arg52 amino acid in stabilizing the chromophore against autoionization, and the role of the Tyr42 and Glu46 amino acids in determining the hydrogen-bonding properties that carry the dominant energetic effects.     

Spatial confinement alters the ultrafast photoisomerization dynamics of azobenzenes

Ultrafast transient absorption spectroscopy reveals new excited-state dynamics following excitation of trans-azobenzene (t-Az) and several alkyl-substituted t-Az derivatives encapsulated in a water-soluble supramolecular host–guest complex. Encapsulation increases the excited-state lifetimes and alters the yields of the trans → cis photoisomerization reaction compared with solution. Kinetic modeling of the transient spectra for unsubstituted t-Az following nπ* and ππ* excitation reveals steric trapping of excited-state species, as well as an adiabatic excited-state trans → cis isomerization pathway for confined molecules that is not observed in solution. Analysis of the transient spectra following ππ* excitation for a series of 4-alkyl and 4,4′-dialkyl substituted t-Az molecules suggests that additional crowding due to lengthening of the alkyl tails results in deeper trapping of the excited-state species, including distorted trans and cis structures. The variation of the dynamics due to crowding in the confined environment provides new evidence to explain the violation of Kasha''s rule for nπ* and ππ* excitation of azobenzenes based on competition between in-plane inversion and out-of-plane rotation channels.

Ultrafast transient absorption spectroscopy reveals new excited-state dynamics following excitation of trans-azobenzene (t-Az) and several alkyl-substituted t-Az derivatives encapsulated in a water-soluble supramolecular host–guest complex.     

Unraveling the similarity of the photoabsorption of deprotonated p-coumaric acid in the gas phase and within the photoactive yellow protein

Using advanced QM/MM methods, the surprisingly negligible shift of the lowest-lying bright electronic excitation of the deprotonated p-coumaric acid (pCA(-)) within the photoactive yellow protein (PYP) is shown to stem from a subtle balance between hypsochromic and bathochromic effects. More specifically, it is found that the change in the excitation energy as a consequence of the disruption of the planarity of pCA(-) inside PYP is nearly canceled out by the shift induced by the intermolecular interactions of the chromophore and the protein as a whole. These results provide important insights about the primary absorption and the tuning of the chromophore by the protein environment in PYP.     

Probing the primary event in the photocycle of photoactive yellow protein using photochemical hole-burning technique

Photochemical hole-burning spectroscopy was used to study the excited-state electronic structure of the 4-hydroxycinnamyl chromophore in photoactive yellow protein (PYP). This system is known to undergo a trans-to-cis isomerization process on a femtosecond-to-picosecond time scale, similar to membrane-bound rhodopsins, and is characterized by a broad featureless absorbance at 446 nm. Resolved vibronic structure was observed for the hole-burned spectra obtained when PYP in phosphate buffer at pH 7 was frozen at low temperature and irradiated with narrow bandwidth laser light at 431 nm. The approximate homogeneous width of 752 cm-1 could be calculated from the deconvolution of the hole-burned spectra leading to an estimated dephasing time of approximately 14 fs for the PYP excited-state structure. The resolved vibronic structure also enabled us to obtain an estimated change in the C=C stretching frequency, from 1663 cm-1 in the ground state to approximately 1429 cm-1 upon photoexcitation. The results obtained allowed us to speculate about the excited-state structure of PYP. We discuss the data for PYP in relation to the excited-state model proposed for the photosynthetic membrane protein bacteriorhodopsin, and use it to explain the primary event in the function of photoactive biological protein systems. Photoexcitation was also carried out at 475 nm. The vibronic structure obtained was quite different both in terms of the frequencies and Franck-Condon envelope. The origin of this spectrum was tentatively assigned.     

Low-temperature spectroscopy of Met100Ala mutant of photoactive yellow protein

The trans-to-cis photoisomerization of the p-coumaroyl chromophore of photoactive yellow protein (PYP) triggers the photocycle. Met100, which is located in the vicinity of the chromophore, is a key residue for the cis-to-trans back-isomerization of the chromophore, which is a rate-determining reaction of the PYP photocycle. Here we characterized the photocycle of the Met100Ala mutant of PYP (M100A) by low temperature UV-visible spectroscopy. Irradiation of M100A at 80 K yielded a 380 nm species (M100A(BL)), while the corresponding intermediate of wild type (WT; PYP(BL)) is formed above 90 K. The amounts of redshifted intermediates produced from M100A (M100A(B') and M100A(L)) were substantially less than those from WT. While the near-UV intermediate (PYP(M)) is not formed from WT in glycerol samples at low temperature, M100A(M) was clearly observed above 190 K. These alterations of the photocycle of M100A were explained by the shift in the equilibrium between the intermediates. The carbonyl oxygen of the thioester linkage of the cis-chromophore in the photocycle intermediates is close to the phenyl ring of Phe96 (<3.5 A), which would be displaced by the mutation of Met100. These findings imply that the interaction between chromophore and amino acid residues near Met100 is altered during the early stage of the PYP photocycle.     

Structural evolution of the chromophore in the primary stages of trans/cis isomerization in photoactive yellow protein

We have studied the structural changes induced by optical excitation of the chromophore in wild-type photoactive yellow protein (PYP) in liquid solution with a combined approach of polarization-sensitive ultrafast infrared spectroscopy and density functional theory calculations. We identify the nuC8-C9 marker modes for solution phase PYP in the P and I0 states, from which we derive that the first intermediate state I0 that appears with a 3 ps time constant can be characterized to have a cis geometry. This is the first unequivocal demonstration that the formation of I0 correlates with the conversion from the trans to the cis state. For the P and I0 states we compare the experimentally measured vibrational band patterns and anisotropies with calculations and find that for both trans and cis configurations the planarity of the chromophore has a strong influence. The C7=C8-(C9=O)-S moiety of the chromophore in the dark P state has a trans geometry with the C=O group slightly tilted out-of-plane, in accordance with the earlier reported structure obtained in an X-ray diffraction study of PYP crystals. In the case of I0, experiment and theory are only in agreement when the C7=C8-(C9=O)-S moiety has a planar configuration. We find that the carboxylic side group of Glu46 that is hydrogen-bonded to the chromophore phenolate oxygen does not alter its orientation on going from the electronic ground P state, via the electronic excited P state to the intermediate I0 state, providing conclusive experimental evidence that the primary stages of PYP photoisomerization involve flipping of the enone thioester linkage without significant relocation of the phenolate moiety.     

Regio- and stereoselective synthesis of the enantiomers of monoterpene-based β-amino acid derivatives

The regio- and stereospecific addition of chlorosulfonyl isocyanate to cis-δ-pinene enantiomers has furnished monoterpene-fused β-lactams. The observed regioselectivity can be explained by ab initio DFT modeling of transition state structures. In contrast with the less reactive α-pinane-fused β-lactam 4, the resulting β-lactams 5 and 13 containing an amino group connected to a secondary carbon possess similar reactivity to the cycloalkane-fused analogues and can be easily converted to the β-amino acid and its protected derivatives. The base-catalyzed isomerization of the cis-amino ester afforded the corresponding trans-amino ester in moderate yield.     

Analogue chromophore study of the influence of electronic perturbation on color regulation of photoactive yellow protein

We report a unique lambdamax shift of the absorption maximum of a photoactive yellow protein (PYP) analogue reconstituted with a fluorinated chromophore (F-PYP). The difference in lambdamax between the free chromophore and the protein was significantly larger than that with the native chromophore. We concluded that the unusual lambdamax shift is caused by the electronegative character of the fluorine atom and not by steric hindrance. This result suggests that formation of a hydrogen bond between the fluorine atom and one or more amino acid residues could neutralize its electron-withdrawing character. The properties of analogues of PYP with brominated and methylated chromophore could be explained as an effect of steric hindrance.     

Controlling Light-Induced Proton Transfer from the GFP Chromophore

Green Fluorescent Protein (GFP) is known to undergo excited-state proton transfer (ESPT). Formation of a short H-bond favors ultrafast ESPT in GFP-like proteins, such as the GFP S65T/H148D mutant, but the detailed mechanism and its quantum nature remain to be resolved. Here we study in vacuo, light-induced proton transfer from the GFP chromophore in hydrogen-bonded complexes with two anionic proton acceptors, I⁻ and deprotonated trichloroacetic acid (TCA⁻). We address the role of the strong H-bond and the quantum mechanical proton-density distribution in the excited state, which determines the proton-transfer probability. Our study shows that chemical modifications to the molecular network drastically change the proton-transfer probability and it can become strongly wavelength dependent. The proton-transfer branching ratio is found to be 60 % for the TCA complex and 10 % for the iodide complex, being highly dependent on the photon energy in the latter case. Using high-level ab initio calculations, we show that light-induced proton transfer takes place in S₁, revealing intrinsic photoacid properties of the isolated GFP chromophore in strongly bound H-bonded complexes. ESPT is found to be very sensitive to the topography of the highly anharmonic potential in S₁, depending on the quantum-density distribution upon vibrational excitation. We also show that the S₁ potential-energy surface, and hence excited-state proton transfer, can be controlled by altering the chromophore microenvironment.     

trans-cis Photoisomerization of a photoactive yellow protein model chromophore in crystalline phase

We have studied the photoinduced trans/cis isomerization of the protonated form of p-hydroxycinnamic thiophenyl ester, a model chromophore of the photoactive yellow protein (PYP), in crystalline phase, by both fluorescence and infrared spectroscopies. The conversion from trans to cis configuration is revealed by a shift of the fluorescence peak and by inspection of the infrared maker bands. The crystal packing apparently stabilizes the cis photoproduct, suggesting different environmental effects from the solvent molecules for this model chromophore in liquid solutions or from the amino acid residues for the PYP chromophore.     

Solvation of p-coumaric acid in water

The photoactive yellow protein (PYP) is an important model protein for many (photoactive) signaling proteins. Key steps in the PYP photocycle are the isomerization and protonation of its chromophore, p-coumaric acid (pCA). In the ground state of the protein, this chromophore is in the trans configuration with its phenolic oxygen deprotonated. For this paper, we studied four different configurations of pCA solvated in water with ab initio molecular dynamics simulations as implemented in CP2K/Quickstep. We researched the influence of the protonation and isomerization state of pCA on its hydrogen-bonding properties and on the Mulliken charges of the atoms in the simulation. The chromophore isomerization state influenced the hydrogen-bonding less than its protonation state. In general, more charge yielded a higher hydrogen-bond coordination number. Where deprotonation increases both the coordination number and the residence time of the water molecules around the chromophore, protonation showed a somewhat lower coordination number on two of the three pCA oxygens but much higher residence times on all of them. This could be explained by the increased polarization of the OH groups of the molecule. The presence of the chromophore also influenced the charge and polarization of the water molecules around it. This effect was different in the four systems studied and mainly localized in the first solvation shell. We also performed a proton-transfer reaction from hydronium through various other water molecules to the chromophore. In this small charge-separated system, the protonation occurred within 6.5 ps. We identified the transition state for the final step in this protonation series.     

Acid-Base Properties of the Ground and Excited States of Ruthenium(II) Tris(4′-methyl-2,2′-bipyridine-4-carboxylic Acid)

The acid dissociation constant, pK_a, for the ground and excited states of ruthenium tris(4′-methyl-2,2′-bipyridine-4-carboxylic acid) complex have been measured. The ground state pK_a obtained from the pH titration curve of the complex absorption at 454 nm was 2.5. The lifetimes of the excited-state for deprotonated and protonated ruthenium complexes are 595 and 150 ns, respectively. The excited-state pK_a* is obtained from the emission titration curve at 630 nm and corrected for the excited-state lifetime to be 4.2. The increase of 1.7 pH units in the acid dissociation constant in the excited-state indicates that the ligand is much more basic in the excited-state. This result confirms the MLCT assignment for the lowest electronic transition of [Ru(mbpyCOOH)₃]²⁺.