CHEMPHYSCHEM ARTICLES DOI: 10.1002/cphc.201402383
Femtosecond Dynamics in the Lactim Tautomer of Phycocyanobilin: A Long-Wavelength Absorbing Model Compound for the Phytochrome Chromophore Patrick Singer,[b] Sonja Fey,[a] Andreas H. Gçller,[c] Gudrun Hermann,*[a] and Rolf Diller*[b] Transient UV/Vis absorption spectroscopy is used to study the primary dynamics of the ring-A methyl imino ether of phycocyanobilin (PCB-AIE), which was shown to mimic the far-red absorbance of the Pfr chromophore in phytochromes (R. Micura, K. Grubmayr, Bioorg. Med. Chem. Lett. 1994, 4, 2517– 2522). After excitation at 615 nm, the excited electronic state is found to decay with t1 = 0.4 ps followed by electronic groundstate relaxation with t2 = 1.2 and t3 = 6.7 ps. Compared with phycocyanobilin (PCB), the initial kinetics of PCB-AIE is much
faster. Thus, the lactim structure of PCB-AIE seems to be a suitable model that could not only explain the bathochromic shift in the ground-state absorption but also the short reaction of the Pfr as compared to the Pr chromophore in phytochrome. In addition, the equivalence of ring-A and ring-D lactim tautomers with respect to a red-shifted absorbance relative to the lactam tautomers is demonstrated by semiempirical calculations.
1. Introduction Light plays a key role for the survival of most living organisms on earth. It is captured and transformed into chemical energy via the process of photosynthesis, one of the most important terrestrial chemical reactions. In addition, photons serve as bits of information about the quality and quantity of light in the surrounding environment. Signal-transducing photoreceptors then use this information to adjust metabolic and developmental changes to the ambient light environment.[1, 2] One of the most prominent signaling light sensors includes the phytochrome family, which regulates a wide variety of physiological responses and cellular processes in plants, bacteria and fungi.[3–6] For light perception phytochromes utilize an openchain tetrapyrrole (bilin) chromophore such as phycocyanobilin (PCB), phytochromobilin (PfB) or biliverdin (BV), which is covalently bonded to the protein moiety by a thioether linkage. In the case of the canonical phytochromes, the photoresponses are mediated by switching reversibly between two forms, the thermally stable red light-absorbing form (Pr) and the thermally instable far-red light-absorbing form (Pfr). Based on the crys[a] Dr. S. Fey, Dr. G. Hermann Institute for Biochemistry and Biophysics Friedrich-Schiller University Jena Philosophenweg 12, 07743 Jena (Germany) E-mail: [email protected] [b] P. Singer, Prof. Dr. R. Diller Department of Physics University of Kaiserslautern Erwin-Schrçdinger-Strasse, Geb. 46 67663 Kaiserslautern (Germany) E-mail: [email protected] [c] Dr. A. H. Gçller Bayer Healthcare AG, 42096 Wuppertal (Germany) Supporting Information for this article is available on the WWW under http://dx.doi.org/10.1002/cphc.201402383.
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tal structure of the Pr form, the chromophore adopts the ZZZ,ssa conformation and is located in the photosensory module in a solvent shielded binding pocket, constituted by three protein sub-domains (PAS, GAF, PHY).[7–9] Upon red-light absorption the Pr chromophore undergoes a rapid Z!E isomerization. As NMR and other spectroscopic studies on chemically locked bilin chromophores reveal, photoisomerization occurs at the C(15)=C(16) methine bridge between rings C and D (Figure 1) resulting in a ZZE,ssa geometry of the Pfr chromophore.[10–16] On the other hand, crystallographic studies on the Pfr form of a cyanobacterial phytochrome, lacking the PAS domain, indicate a 5E-syn conformation of the bilin chromophore, thus suggesting the rotation of ring A about the C(4)=C(5) methine bridge during Pr-to-Pfr photoconversion.[3, 17] However, very recent work disproves an A-ring rotation in this bacteriophytochrome even though chemical shifts characteristic of the D-ring rotation in canonical phytochromes were not unequivocally detected.[18] The reaction pathway for photoconversion of Pr to Pfr is distinct from that for the back reaction of Pfr to Pr, involving different intermediates and time scales. The Pr-to-Pfr photoreaction was shown to occur along two distinct excited-state regions on the S1 potential energy surface resulting in formation of the primary ground state product, lumi-R, which has already adopted the Pfr-like chromophore configuration, within 30 ps (or even longer)[19–27] and 15 % quantum yield.[19, 21–23, 25, 27, 28] On the other hand, the initial photoprocesses of the Pfr-to-Pr back reaction proceed on a much faster time scale.[19, 20, 22, 29–31] Thus, the first ground-state intermediate is observed to arise directly from excited-state Pfr in a barrierless reaction with a time constant of 500 fs. Most likely, this 500 fs process already involves the E!Z isomerization of the ChemPhysChem 2014, 15, 3824 – 3831
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Figure 1. Chemical structures of phycocyanobilin (PCB) and the ring-A methyl imino ether (PCB-AIE): (1) phycocyanobilin, (2) ring-A methyl imino ether in the all-Z, all-syn conformation. In the Pr chromophore of plant phytochromes (PfB) the ethyl group (CH2CH3) at the D-pyrrole ring of PCB is substituted by a vinyl group ( CH=CH2) and the chromophore is in the all-Z,ssa conformation.
Pfr chromophore into the Pr-like geometry. The isomerized intermediate has a lifetime of 3 ps and undergoes further relaxation/conversion in the electronic ground state to form finally the Pr state.[22] The clear difference in the initial reaction kinetics of the Pfr-to-Pr back-reaction with respect to the forward reaction suggests different electronic structures of the two chromophore species. To analyze the structural basis for the distinct kinetics in the Pr-to-Pfr and the reverse Pfr-to-Pr photoreaction, we have investigated the ultrafast reaction dynamics of a model compound that can mimic the far-red absorbance and the dynamic properties of the Pfr state.[32–34] This model compound is the ring-A imino ether of phycocyanobilin (PCB-AIE). As mentioned above, PCB itself occurs as the chromophore in cyanobacterial phytochromes, but in a slightly modified form it also comprises the chromophore in plant phytochromes, from which it differs only by substitution of an ethyl by a vinyl group in the side chain at ring D (Figure 1). Compared with PCB, the crucial change in the structure of PCB-AIE is the tautomerization of the ring-A moiety from the 2,3-dihydrolactam into the lactim form and the stabilization of the lactim tautomer by formation of the methyl imino ether. This chemical transformation results in an elongation of the p-conjugation and a high donor strength of the ring-A lactim oxygen (N=C O). In consequence a significant red shift of 90 nm in the long-wavelength absorption band is observed, which is comparable to the red-shifted absorption of the Pfr relative to the Pr form. Therefore, the lactim derivative of PCB was proposed as appropriate model for the chromophore in the far-red absorbing form (Pfr) of phytochrome.[32–34] Herein, we report on the transient absorption kinetics of PCB-AIE dissolved in methanol with femtosecond time-resolution. These kinetic properties are compared with the excited electronic-state dynamics of PCB, the corresponding lactam structure, which was intensively studied in our previous work.[35–38] Based on a significantly faster time scale found for the initial kinetics in the lactim model, it is discussed whether a lactim-like structure of the Pfr chromophore might explain the differences in the isomerization dynamics !
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between the Pr-to-Pfr forward and back reaction in phytochromes. In this context the red shift upon lactam–lactim tautomerization was also verified by semiempirical calculations for the free, not-stabilized ring-A tautomer and, in particular, the corresponding ring-D lactim tautomer, which would reflect the relevant tautomer in the Pfr form of phytochromes more likely. The free, unmodified tautomers carrying still the kinetically mobile proton at the lactim-OH group are not stable per se in solution and the stable ring-D lactim ether is much more difficult to access by chemical synthesis than the ring-A counterpart. Therefore the work on those tautomer forms remains restricted to the analysis of the red shift in the computed absorbance spectra only.
2. Results and Discussion 2.1. Steady-State Spectra of PCB-AIE versus PCB Figure 2 presents the experimental steady-state absorption spectrum of PCB-AIE in methanol compared to that of PCB. With a peak at 688 nm, the long-wavelength absorption band (Q-band) of PCB-AIE is red-shifted by 86 nm relative to the corresponding band of PCB while the Soret band appears at 368 nm with only a minor bathochromic shift.
Figure 2. Stationary absorption spectra of phycocyanobilin (PCB) and ring-A methyl imino ether (PCB-AIE) in methanol. The spectra are characterized by peaks of the long-wavelength Q band (Soret band) at 688 nm (368 nm) for PCB-AIE (c) and 603 nm (364 nm) for PCB (g). The excitation wavelength (615 nm) is indicated.
2.2. Excited-State Dynamics of PCB-AIE versus PCB Figure 3 shows the absorption difference spectra of PCB-AIE in methanol at selected delay times upon excitation at 615 nm. Negative and positive difference signals are assigned to ground-state bleaching and excited-state absorptions, respectively. Negative signals appear instantaneously after excitation in the spectral region around the ground-state absorption accompanied by positive signals in the adjacent wavelength regions. The observed absorption changes decrease significantly ChemPhysChem 2014, 15, 3824 – 3831
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www.chemphyschem.org The transient absorption data DA(t, l) is globally best fitted with a sum of three exponentials. Applying only two exponentials led to significant residuals. This result is further supported by SVD analysis, which identified three relevant components (not shown). Thus, the kinetics of photoexcited PCB-AIE can be described by a three-component model with time constants of t1 = 0.4 ps, t2 = 1.2 ps and t3 = 6.7 ps. Decay-associated spectra (DAS) of the individual kinetic components are displayed in Figure 5, and characterized as follows: 1) A1 is positive between about 620 and 690 nm and negative below 600 and above 690 nm, 2) in terms of sign and partially in shape, A2 and A3 are mirror images of A1, and 3) A0 is basically zero with very small positive contributions below 550 nm.
Figure 3. Difference spectra of ring-A methyl imino ether of PCB (PCB-AIE) in methanol recorded at selected delay times after excitation at 615 nm. The data around 600–660 nm is subject to higher noise due to contributions from pump pulse scattering. The arrows indicate the temporal signal progression at specific spectral positions.
Figure 4. Transient absorption changes at specific probe wavelengths after excitation of ring-A methyl imino ether of PCB (PCB-AIE) at 615 nm. Solid lines represent the calculated functions obtained from the best-fit parameters of the global analysis shown in Figure 5.
Figure 5. Decay-associated spectra of the kinetic components in photoexcited ring-A methyl imino ether of PCB (PCB-AIE) for excitation at 615 nm. The spectra refer to components of t1 = 0.4 ps, t2 = 1.2 ps, and t3 = 6.7 ps. For A0 data with pump-pulse scattering (shaded area) is omitted. For comparison the static (inverted) absorption spectrum is included.
on the time scale of 10 ps, after 60 ps the photoreaction appears to be largely completed with only very small residual signals remaining. Figure 4 displays the transient absorption changes at different probe wavelengths (450, 670 and 760 nm), together with the result of the global fit (see below). The transient at 670 nm reveals negative absorption changes immediately after excitation, which then undergo a slower decay. This kinetic behavior reflects the ultrafast depletion of the ground state absorption and/or stimulated emission, followed by ground state recovery. The transient at 450 nm shows the inverted behavior, that is, a basically instantaneous rise of the excited-state absorption which decays on the picosecond time scale. At 760 nm, the transient is characterized by a small initial bleach, followed by a delayed signal rise and a subsequent decay on the picosecond time scale.
A1 displays the decay of excited-state absorption between 620 and 690 nm and the loss of stimulated emission at wavelengths > 750 nm. Because of the simultaneity of the two processes, the decay of excited-state absorption and the concurrent loss of stimulated emission, the associated electronic state can be ascribed to the S1-excited state. Furthermore, A2 and A3 associated intermediates are directly formed from the S1-excited state as can be concluded from the mirror images of the amplitudes in A1 and A2 as well as A3. Based on the lack of stimulated emission A2 and A3 can be assigned to electronic ground-state intermediates, which are formed within 0.4 ps and decay with 1.2 ps and 6.7 ps lifetimes back to the initial ground state. Accordingly, the key primary dynamics of PCBAIE can be described by the kinetic model given in Figure 6. Note that an alternative sequential reaction scheme, possibly involving a dark state and an unrelaxed/hot electronic ground state, cannot be excluded. However, just as the parallel model
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in Pr and particularly Pfr being fixed by specific interactions with the local protein environment. Two further aspects remain to be discussed in this context. First in plant and cyanobacterial phytochromes, the primary reaction step is initiated by Z!E photoisomerization of the meFigure 6. Level scheme suggested for the relaxation processes in photoexcited PCB-AIE (A) in comparison with thine bridge between rings C the excited-state processes in PCB (B). The first excited state PCB-AIE* decays with t1 = 0.4 ps into two intermediate states (I1 and I2) that in turn repopulate the ground state with t2 = 1.2 ps and t3 = 6.7 ps under 615 nm excitaand D[3–5, 9, 10, 12, 14] in the bilin tion. In PCB, the excited-state population PCBA* undergoes a photoreaction to form PCBB/PCBC with a much chromophore. Hence, lactam/ slower time constant of t1’ 30 ps.[35, 36] Subsequently, PCBB/PCBC revert into PCBA with t2’ 350 ps until the initial lactim tautomerization should insteady state concentration of PCBA is reached again. volve ring D rather than ring A. Furthermore all four chromophore nitrogens are protonated in both Pr and Pfr discussed above, such a scheme would equally incorporate the forms.[10, 39, 40] Therefore the question arises whether the geofast decay of the S1-excited state (stimulated emission) with 0.4 ps accompanied by the 1.2 ps and 7 ps dynamics of the metrically similar ring-D lactim tautomer does also reveal a sigtwo ground-state intermediates. nificant red shift in the long-wavelength absorbance and a simiWhen compared with the corresponding lactam structure of lar fast reaction dynamics as the ring-A tautomer. Since the PCB, the excited-state dynamics of PCB-AIE is significantly ring-D lactim imino ether (PCB-DIE) is much more difficult to faster. As we have previously shown, PCB exists in three differobtain by chemical synthesis in the required amount than the ent ground-state species (PCBA, PCBB, PCBC), of which only the ring-A counterpart this problem was addressed by calculating most prevalent species PCBA undergoes a photoreaction and the absorption spectrum and analyzing this spectrum for forms species PCBB and PCBC with a time constant of a red-shifted absorbance as compared to the corresponding 30 ps.[35, 36, 38] In a thermally driven back-reaction with a signifilactam tautomer. In addition to the ring-D imino ether, also the cantly longer time constant of 350 ps, PCBB and PCBC then free ring-A and ring-D lactim derivatives were included in the revert into PCBA until the initial steady-state equilibrium is calculations. The reason for this is that the free lactim tautomers are not stable per se in solution and only available after reached again (cf. Figure 6). The difference in the kinetics of fixation of the kinetically mobile proton of the lactim hydroxyl the lactam and lactim tautomers of PCB is reminiscent of the group as in case of the imino ethers. However, in the proteinexcited-state dynamics in phytochromes. As already mentioned bound chromophore as in phytochromes, tautomerization in the introduction, the initial kinetics of the Pfr-to-Pr photoshould more likely involve the free lactim derivative, in which transformation occurs on a much faster time scale than the forthe lactim hydroxyl group is fixed by specific interactions with ward Pr-to-Pfr transformation, suggesting that the two chroprotein sites in its direct surrounding rather than by an ether mophores do not only differ in their configuration but also in bond. their electronic structure, and hence in the shapes of their excited-state potential energy surfaces. PCB itself is found as the chromophore in cyanobacterial 2.3. Computation of PCB Lactam/Lactim Tautomer Spectra phytochromes and in an only slightly modified form also in The structures of all lactim tautomers, which were included the plant phytochromes (Figure 1). It is therefore tempting to into the spectra calculations are depicted in Figure 7. As speculate that the Pfr- as opposed to the Pr-chromophore shown there, in the lowest-energy tautomers of the ring-A adopts a lactim-like structure, which causes the extremely fast imino ether and lactim the acidic hydrogen atoms are found reaction kinetics in Pfr upon conversion into Pr as well as the located on the nitrogen atoms of rings B and D, whereas in Pr to Pfr bathochromic shift. It can be argued that chromothe ring D species those hydrogen atoms are placed on the niphore–protein interactions, also necessary in generating the trogen atoms of rings A and C. The last pattern also applies for unique spectral properties of phytochromes, are not taken into the protonated ring-D lactim, which is examined as an examconsideration with studies on a model chromophore and ple of a protonated lactim species. Figure 8 displays the calcutherefore question this hypothesis. In this respect it is to be lated spectra for the lactam in comparison with the different noted that the primary photochemistry of a protein-bound lactim tautomers. The spectra are based on the results obchromophore is manifested in the chemical constitution of the tained from the Gaussian 09 implementation of ZINDO.[41–43] chromophore itself, whereas the detailed energetics and dynamics of the photochemical processes is affected by the interThe individual spectra together with the relative oscillatory play with the protein sites in the surrounding. That means that strengths of the calculated transitions as well as the absorption the 2,3-dihydrobilindione chromophore in phytochrome maxima as such can be found in the Supporting Information (Figure 1) should retain the capability of lactam/lactim tauto(Figure S1, Table S1). It should be added here that the comparimerization with the lactam/lactim-like chromophore structures son of the calculated spectra is only possible at a qualitative 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 7. Chemical structures of ring-A and ring-D lactim derivatives of phycocyanobilin (PCB) used for calculating the electronic spectra. (I) ring-A methyl imino-ether (2) with R = CH3 and ring-A lactim (3) with R = H), (II) ring-D methyl imino ether (4) with R = CH3 and ring-D lactim (5) with R = H as well as (III) protonated ring-D lactim (6). The structures are shown in the all-Z, all-syn configuration/conformation. The calculated structures have protonated propionic acids at rings B and C and a methyl group (CH3) instead of an ethyl group (CH2CH3) at ring D.
Figure 8. Overlay of the electronic spectra calculated for the ring-A and ringD lactim derivatives (2)–(6) in comparison with the lactam derivative (1) of phycocyanobilin (PCB). The spectra were computed in vacuum using AM1 coordinates with ZINDO implemented in Gaussian09. For a clear representation the spectrum of the protonated ring-D lactim (6) is shown in a separate plot and directly compared with the lactam tautomer of PCB (1) there. (1) PCB lactam tautomer, (2) ring-A methyl imino ether, (3) ring-A lactim tautomer, (4) ring-D methyl imino ether, (5) ring-D lactim tautomer, and (6) protonated ring-D lactim tautomer. For the detailed structures compare Figures 1 and 7. A colored image of the superimposed spectra is given in the Supporting Information (Figure S2).
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level. The reason is that the theoretical methods needed for quantitative predictions like multi-reference configuration interaction approaches (MRCI) are not applicable for systems of the size of PCB. Therefore, the more approximate but well established ZINDO method was employed for the calculations. In addition the experimental spectra are adiabatic in nature, whereas the calculated ones are vertical and therefore blueshifted. Nevertheless, it is evident that the calculated spectra for the lactam tautomer of PCB (1) and the ring-A imino ether, PCB-AIE (2), reproduce both the general shapes of the experimental spectra (Figure 2) with the more intense band in the blue (Soret band) as compared to the red (Q-band) spectral region and the significant red shift in the lactim tautomer. It is therefore to be expected that the calculations predict reliably the spectral trends in the lactam versus lactim spectra of the virtual species. As can be seen from Figure 8, the lactim tautomers (2) to (5) show pronounced red shifts in their long-wavelength absorption bands as compared to the lactam tautomer (1). The most remarkable red shift from 539 to 630 nm occurs for PCB-AIE (2). For PCB-DIE (4) this shift is smaller and only extends to 574 nm. The same tendency is found for the free lactims at the ring-A (3) as opposed to the ring-D site (5), whereas the spectra of the lactims and their respective imino ethers are nearly congruent with just slight red shifts of less than 10 nm. For all structures the long-wavelength absorptions are due to exactly one electronic transition. In accordance with the experimental spectra, the position of the blue band remains almost unchanged between all structures. It is predicted to be between 333 (4) and 338 nm (3). Due the narrow Gaussian shape line fitting applied additional blue bands appear in the calculated spectra, which have their counterparts in the experimental PCB (1) and PCB-AIE (2) spectra in shallow shoulders at around 390 and 430 nm. These bands are red-shifted in the calculated spectra relative to the lactam spectrum (1), too (Figure 8). While only moderate red shifts in the long-wavelength absorption are predicted for ring-D versus ring-A lactims this situation is different in the protonated ring-D lactim (6), (Figure 7). Protonation of the ring-D lactim results in a considerChemPhysChem 2014, 15, 3824 – 3831
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Table 1. Geometrical parameters defining the planarity of the minimum energy structures in the different lactam/lactim tautomers of PCB (1)–(6). The calculations are made for the all-Z, all-syn configuration/conformation. The angles are given in degree and the distances in . Geometrical parameter
Lactam (1)
Ring-A iminoether (2)
Ring-A lactim (3)
Ring-D iminoether (4)
Ring-D lactim (5)
Ring-D lactim (protonated) (6)
dihedral ring (AB)[a] dihedral ring (BC) dihedral ring (CD) dihedral (NANBNCND)[b] r(OAOD)[c] r(CACD)[d]
37.2 24.2 5.1 59.8 6.95 6.51
16.1 8.9 2.4 28.9 3.55 4.31
13.5 8.4 3.7 28.2 3.47 4.12
11.9 14.7 16.1 43.7 4.89 4.89
11.7 11.5 15.5 40.6 4.55 4.70
5.8 2.8 139.4 55.2 4.8 6.5
[a] Dihedrals are defined as the angle between the straight lines given by the nitrogen and bridge head carbon of the first and second ring system. [b] Dihedrals are defined as the angle between the straight lines given by the nitrogen of rings A and B relative to rings C and D. [c] Distances between the oxygen atoms at rings A and D (C=O and OH/OR groups); [d] Distances between the carbon atoms at rings A and D (C=O and COH/COR groups).
able red shift of more than 100 nm (Figure 8), which even exceeds that of the ring-A lactim tautomers in the unprotonated forms (2), (3). The blue band is located near 380 nm (Figure 8). Its position roughly corresponds to the second blue band with lower absorptivity in the spectra of the unprotonated lactim tautomers (2)–(5). The main blue band at 333–338 nm in those spectra loses absorptivity so that in the protonated ring-D lactim (6) the second blue band at 380 nm dominates that at around 330 nm. The gradual red shifts in the lactim tautomers (2)–(6) can be correlated with their three-dimensional structures. Table 1 lists the dihedral angles and distances defining the overall deviations from planarity, which in turn affect the length of the conjugated p-electron path. As already shown earlier, the p-electron path is strictly localized in so far as all bonds of the tetrapyrrolic ring system keep their single and double bond character and conjugation does not lead to bond length equilibration.[38] With respect to torsional strains on the conjugated pelectron path Table 1 reveals that the dihedral angles between the planes defined by the nitrogens of rings A-B-C and rings B-C-D is 608 for the lactam tautomer (1), about 298 for the ring-A lactims (2), (3) as well as about 448 and 418 for the ringD lactim tautomers (4), (5). Electrostatic repulsion between the carbonyl groups is probably the main driving force for the torsional deformation in the lactam tautomer (1). As evident from the dihedral angle between rings A and B, in particular ring-A is strongly bent out of the plane. The largest torsional deformation in this tautomer results in the most blue-shifted longwavelength absorption band. On the other hand, the ring-A and ring-D lactim tautomers (2)–(5) are almost mirror images of each other, apart from the different double bond pattern in rings A and D. While the ring-A lactim tautomers have 11 conjugated double bonds, the corresponding ring-D tautomers have only 10, which leads to a slightly reduced conjugation path. This, together with the higher tilting in the ring-D lactims (4), (5), results in the lower red shifts in these tautomers as compared to the ring-A tautomers (2), (3). Images of the different three-dimensional structures are added in the Supporting Information (Figure S3). The computed spectra clearly indicate a significant red shift of the long-wavelength absorption maximum also in the ring 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
D imino ether and lactim tautomers (4), (5) even if the extent of this shift is smaller than for the ring-A counterpart. However, as further evidenced protonation of the ring-D lactim still increases this red shift and exceeds the value of the unprotonated ring-A tautomer. Furthermore, the value of the calculated red shift becomes comparable to that observed in the Pfr as compared to Pr spectrum of phytochromes.[3–6] Taken the experimental findings, reported here, together it seems conceivable that a lactim-like structure is also retained in the Pfr chromophore. At least such structural feature can definitely explain both the far-red absorbance and in particular the significantly faster photochemistry of the Pfr as compared to the Pr state in phytochromes. It could be argued that the Pr and Pfr structures recently derived by magic-angle spinning NMR studies, which used labeled PCB chromophores, do not provide direct evidence of a lactam/lactim tautomerization upon Pr-to-Pfr photoconversion.[10] However, it is unclear whether ring-D lactam/lactim tautomers could be distinguished by this technique since it is very difficult to discriminate between them by means of 13C and 1H chemical shifts in NMR spectra.[44]
3. Conclusions We have addressed a fundamental question in the phototransformation of phytochromes concerning both, the bathochromically shifted ground-state absorption of the far-red light-absorbing form, Pfr, compared to the red-light-absorbing form, Pr, as well as the substantially different time scales of the respective primary photoreactions. The excited-state dynamics of the lactim tautomer of PCB is significantly faster when compared with the lactam tautomer.[35, 36] Immediately after excitation the ring-A methyl imino ether, PCB-AIE, studied as model of a fixed ring-A tautomer forms two ground-state intermediates within 400 fs, which in turn undergo decay with lifetimes of 1.2 and 6.7 ps. The difference in the early-time dynamics between the lactam and lactim tautomers of PCB is reminiscent of that in the excitedstate chemistry of phytochromes, revealing that the Pfr-to-Pr back reaction occurs in 700 fs and is clearly faster than the Pr-to-Pfr forward reaction.[19, 20, 22, 29] It is therefore suggested ChemPhysChem 2014, 15, 3824 – 3831
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CHEMPHYSCHEM ARTICLES that the chromophore in the Pfr-form of phytochrome could adopt a lactim-like structure, which is responsible for the red shift in the long-wavelength absorption and in particular for the extremely fast primary reaction time. Interactions with the protein moiety in the native chromoprotein are expected to assist the stabilization of the lactim-like structure and to control the specific photochemistry. The strict comparison with the chromophores in phytochrome would require that lactam/lactim tautomerization takes place at pyrrole ring-D. In this context it is shown that the spectra of the ring-D imino ether, PCB-DIE, and the free lactim tautomers, which were computed by semiempirical methods, also exhibit a red shift of the long-wavelength absorption band relative to that of the lactam tautomer. This red shift is smaller than that of the ring-A lactim form but it is significantly increased when the lactim tautomer gets protonated. Thus, also tautomerization of the ring-D lactam into its lactim form provides a qualitative model that can mimic the longwavelength absorption of the Pfr chromophore, whereas the lactim structure itself is well primed for an ultrafast primary reaction step.
Experimental Section Sample Preparation Phycocyanobilin (PCB) was isolated from the cyanobacterium Spirulina geitleri as described earlier.[35] The ring-A methyl imino ether of PCB (PCB-AIE) was prepared with slight modifications according to the procedure given by Micura et al.[32] In brief, starting from 10 mg of PCB, the dimethyl ester of PCB was produced by esterification with methanol and BF3 as catalyst. The dimethyl ester was then dissolved in chloroform (8 mL), a solution of zinc acetate (10 mg) in methanol (1 mL) and acetic anhydride (1 mL). This mixture was heated under refluxing conditions (30 min) and neutralized by addition of hydrogen carbonate (40 mL of a saturated solution). The product formed was subsequently purified by thin layer chromatography on silica gel 60 TLC plates in dichlormethane:ethyl acetate = 7:1. PCB-AIE is characterized by Rf = 0.5 in thin layer chromatography on silica gel 60, a mol peak of MW = 628.33 g mol1 in the MALDI-TOF analysis (Ultraflex III TOF/TOF; Bruker Daltonics) and absorption peaks of l1 = 688 nm and l2 = 368 nm in methanol (Figure 2). For the femtosecond experiments PCB-AIE was dissolved in methanol. To avoid photodegradation, steady state UV/Vis transmission spectra were recorded regularly.
Transient UV/Vis Absorption Spectroscopy Transient femtosecond absorption pump–probe spectroscopy was performed with a regenerative Ti:Sa amplifier laser system (CPA 2001 Clark-MXR Inc.) with pulses of 150 fs duration (FWHM), 0.8 mJ energy at 780 nm and 1 kHz repetition rate. Short excitation pulses were generated by a homebuilt two-stage NOPA (non-collinear parametric amplifier), tunable in the spectral range between 350– 800 nm. Excitation wavelength of PCB-AIE was set to lexc = 615 nm for excitation of the high-energy edge of the Q-band absorption (600–800 nm), (Figure 2). The optical density of the sample was set to 1 (1 mm cuvette) in the absorption maximum of the Q band. The focal width of the actinic laser beam was 250 mm 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemphyschem.org (FWHM) with single pulse energy of 100 nJ at the sample position and pulse duration of 70 fs (FWHM). Broad-band continuum probe pulses (spectral range: 450–850 nm) were generated by means of self-phase modulation in a sapphire window. Pump pulses were time-delayed on a motorized translation stage (Physik Instrumente, PI) with respect to the probe pulse. Wavelength-dependent time zero was determined by recording the cross-phasemodulation signal progression in a H2O sample. The spectrally dispersed probe pulses were detected with an integrated 512 pixel PDA camera device (Stresing, Hamamatsu), electronically synchronized with the laser system. The experiments were performed at room temperature with pump-probe cross-correlation of 200 fs (FWHM). To ensure fresh-sample conditions for each pump–probe event, a flow-cell system (Harrick) was used. During the measurement, every second pump pulse was blocked in order to obtain pump-induced absorption changes DA(t, l) as a function of delay time and probe wavelength. The global analysis of the recorded transient absorption data was carried out between 0.2 and 100 ps in the range 450–850 nm by fitting DA(t, l) with a sum of exponentials via Equation (1): DAðt; lÞ ¼ A0 ðt; lÞ þ
XN i¼1
Ai ðlÞet=ti
ð1Þ
with DA(t, l) corresponding to the decay associated spectra (DAS). In addition, a singular value decomposition analysis (SVD) was applied to determine the number of spectral species.
Quantum Chemical Calculations The absorbance spectra were calculated for the bis-lactam form of PCB, the ring-A and ring-D imino ethers as well as ring-A and ringD lactim forms of PCB (cf. Figure 7). The calculations were carried out for the ZZZ,sss configuration/conformation with protonated propionic acid side chains at rings B and C as well as with the ethyl group at ring-D substituted by a single methyl group. In addition, the protonated ring-D lactim form in the all-Z, all-syn conformation was also included into the calculations. The structures of each PCB form were minimized with the semiempirical NDDO Hamiltonian AM1[45] implemented in the ArgusLab software package. As starting geometry an initial 3D structure in the ZZZ,sss configuration/conformation was derived from extensive minima searches.[37] Vertical excitations on the AM1 optimized coordinates were calculated with (i) the ZINDO method[41, 43] implemented in ArgusLab for 10 singlet states based on the 10 highest occupied and 10 lowest unoccupied orbitals with random phase approximation ansatz and (ii) the ZINDO method implemented in Gaussian 09[42] for 20 excited states. The calculations were performed in the gas phase. The spectra as obtained from ZINDO/Gausian 09 were transformed with SpecDis 1.6[46] using Gaussian line fitting with a sigma/gamma ratio of 0.16 eV in the wavelength region between 250 and 800 nm and plotted with OriginPro 8G. All calculations were carried out at the high performance computation cluster of the University of Jena.
Acknowledgements We thank Prof. em. Dr. H. Falk, Johannes Kepler Universitt Linz, Austria for helpful discussions. R.D. and P.S. thank Research Initiative Rheinland Pfalz in Membrane Biology (RIMB) for financial support and H. Feurich for technical assistance. ChemPhysChem 2014, 15, 3824 – 3831
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CHEMPHYSCHEM ARTICLES Keywords: femtochemistry · lactam/lactim tautomerization · photochemistry · phytochromes · quantum chemistry [1] W. R. Briggs, J. L. Spudich, Handbook of Photosensory Receptors, WileyVCH, Weinheim, 2005. [2] A. Mçglich, X. J. Yang, R. A. Ayers, K. Moffat, Annu. Rev. Plant Biol. 2010, 61, 21 – 47. [3] R. D. Vierstra, J. R. Zhang, Trends Plant Sci. 2011, 16, 417 – 426. [4] N. C. Rockwell, J. C. Lagarias, ChemPhysChem 2010, 11, 1172 – 1180. [5] N. C. Rockwell, Y. S. Su, J. C. Lagarias, Annu. Rev. Plant Biol. 2006, 57, 837 – 858. [6] A. Nagatani, Curr. Opin. Plant Biol. 2010, 13, 565 – 570. [7] J. R. Wagner, J. S. Brunzelle, K. T. Forest, R. D. Vierstra, Nature 2005, 438, 325 – 331. [8] X. Yang, J. Kuk, K. Moffat, Proc. Natl. Acad. Sci. USA 2008, 105, 14715 – 14720. [9] L. O. Essen, J. Mailliet, J. Hughes, Proc. Natl. Acad. Sci. USA 2008, 105, 14709 – 14714. [10] T. Rohmer, C. Lang, J. Hughes, L. O. Essen, W. Grtner, J. Matysik, Proc. Natl. Acad. Sci. USA 2008, 105, 15229 – 15234. [11] C. Song, G. Psakis, C. Lang, J. Mailliet, W. Grtner, J. Hughes, J. Matysik, Proc. Natl. Acad. Sci. USA 2011, 108, 3842 – 3847. [12] W. Rdiger, F. Thmmler, E. Cmiel, S. Schneider, Proc. Natl. Acad. Sci.Biol. 1983, 80, 6244 – 6248. [13] J. C. Lagarias, H. Rapoport, J. Am. Chem. Soc. 1980, 102, 4821 – 4828. [14] K. Inomata, H. Khawn, L. Y. Chen, H. Kinoshita, B. Zienicke, I. Molina, T. Lamparter, Biochemistry 2009, 48, 2817 – 2827. [15] C. Song, G. Psakis, C. Lang, J. Mailliet, J. Zaanen, W. Grtner, J. Hughes, J. Matysik, Biochemistry 2011, 50, 10987 – 10989. [16] R. A. Matute, R. Contreras, L. Gonzlez, J. Phys. Chem. Lett. 2010, 1, 796 – 801. [17] A. T. Ulijasz, G. Cornilescu, C. C. Cornilescu, J. R. Zhang, M. Rivera, J. L. Markley, R. D. Vierstra, Nature 2010, 463, 250 – U143. [18] C. Song, G. Psakis, J. Kopycki, C. Lang, J. Matysik, J. Hughes, J. Biol. Chem. 2014, 289, 2552 – 2562. [19] M. Bischoff, G. Hermann, S. Rentsch, D. Strehlow, Biochemistry 2001, 40, 181 – 186. [20] K. Heyne, J. Herbst, D. Stehlik, B. Esteban, T. Lamparter, J. Hughes, R. Diller, Biophys. J. 2002, 82, 1004 – 1016. [21] C. Schumann, R. Groß, N. Michael, T. Lamparter, R. Diller, ChemPhysChem 2007, 8, 1657 – 1663. [22] M. G. Mller, I. Lindner, I. Martin, W. Grtner, A. R. Holzwarth, Biophys. J. 2008, 94, 4370 – 4382. [23] J. Dasgupta, R. R. Frontiera, K. C. Taylor, J. C. Lagarias, R. A. Mathies, Proc. Natl. Acad. Sci. USA 2009, 106, 1784 – 1789. [24] K. C. Toh, E. A. Stojkovic, A. B. Rupenyan, I. H. M. van Stokkum, M. Salumbides, M. L. Groot, K. Moffat, J. T. M. Kennis, J. Phys. Chem. A 2011, 115, 3778 – 3786. [25] J. J. van Thor, K. L. Ronayne, M. Towrie, J. Am. Chem. Soc. 2007, 129, 126 – 132. [26] Y. Yang, M. Linke, T. von Haimberger, J. Hahn, R. Matute, L. Gonzalez, P. Schmieder, K. Heyne, J. Am. Chem. Soc. 2012, 134, 1408 – 1411.
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www.chemphyschem.org [27] A. R. Holzwarth, E. Venuti, S. E. Braslavsky, K. Schaffner, Biochim. Biophys. Acta Bioenerg. 1992, 1140, 59 – 68. [28] F. Andel, K. C. Hasson, F. Gai, P. A. Anfinrud, R. A. Mathies, Biospectroscopy 1997, 3, 421 – 433. [29] M. Bischoff, G. Hermann, S. Rentsch, D. Strehlow, J. Phys. Chem. A 1998, 102, 4399 – 4404. [30] C. Schumann, R. Groß, M. M. N. Wolf, R. Diller, N. Michael, T. Lamparter, Biophys. J. 2008, 94, 3189 – 3197. [31] K. M. Spillane, J. Dasgupta, R. A. Mathies, Biophys. J. 2012, 102, 709 – 717. [32] R. Micura, K. Grubmayr, Bioorg. Med. Chem. Lett. 1994, 4, 2517 – 2522. [33] R. Micura, K. Grubmayr, Angew. Chem. Int. Ed. Engl. 1995, 34, 1733 – 1735; Angew. Chem. 1995, 107, 1896 – 1898. [34] M. Stanek, K. Grubmayr, Chem. Eur. J. 1998, 4, 1660 – 1666. [35] M. Bischoff, G. Hermann, S. Rentsch, D. Strehlow, S. Winter, H. Chosrowjan, J. Phys. Chem. B 2000, 104, 1810 – 1816. [36] B. Dietzek, R. Maksimenka, G. Hermann, W. Kiefer, J. Popp, M. Schmitt, ChemPhysChem 2004, 5, 1171 – 1177. [37] A. H. Gçller, D. Strehlow, G. Hermann, ChemPhysChem 2001, 2, 665 – 671. [38] A. H. Gçller, D. Strehlow, G. Hermann, ChemPhysChem 2005, 6, 1259 – 1268. [39] H. M. Strauss, J. Hughes, P. Schmieder, Biochemistry 2005, 44, 8244 – 8250. [40] T. Rohmer, H. Strauss, J. Hughes, H. de Groot, W. Grtner, P. Schmieder, J. Matysik, J. Phys. Chem. B 2006, 110, 20580 – 20585. [41] J. Ridley, M. Zerner, Theor. Chim. Acta 1973, 32, 111 – 134. [42] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery Jr., J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, . Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, D. J. Fox, Gaussian Inc., Wallingford, CT. 2009. [43] M. C. Zerner, Rev. Comput. Chem. 1991, 2, 313 – 365. [44] H. Falk, U. Zrunek, Monatsh. Chem. 1983, 114, 983 – 998. [45] M. J. Dewar, E. G. Zoebisch, E. F. Healy, J. J. Stewart, J. Am. Chem. Soc. 1985, 107, 3902 – 3909. [46] T. Bruhn, A. Schaumlçffel, Y. Hemberger, G. Bringmann SpecDis version 1.60, University of Wuerzburg. 2012.
Received: June 2, 2014 Published online on September 4, 2014
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Femtosecond Dynamics in the Lactim Tautomer of Phycocyanobilin: A Long-Wavelength Absorbing Model Compound for the Phytochrome Chromophore Patrick Singer,[b] Sonja Fey,[a] Andreas H. Gçller,[c] Gudrun Hermann,*[a] and Rolf Diller*[b] Transient UV/Vis absorption spectroscopy is used to study the primary dynamics of the ring-A methyl imino ether of phycocyanobilin (PCB-AIE), which was shown to mimic the far-red absorbance of the Pfr chromophore in phytochromes (R. Micura, K. Grubmayr, Bioorg. Med. Chem. Lett. 1994, 4, 2517– 2522). After excitation at 615 nm, the excited electronic state is found to decay with t1 = 0.4 ps followed by electronic groundstate relaxation with t2 = 1.2 and t3 = 6.7 ps. Compared with phycocyanobilin (PCB), the initial kinetics of PCB-AIE is much
faster. Thus, the lactim structure of PCB-AIE seems to be a suitable model that could not only explain the bathochromic shift in the ground-state absorption but also the short reaction of the Pfr as compared to the Pr chromophore in phytochrome. In addition, the equivalence of ring-A and ring-D lactim tautomers with respect to a red-shifted absorbance relative to the lactam tautomers is demonstrated by semiempirical calculations.
1. Introduction Light plays a key role for the survival of most living organisms on earth. It is captured and transformed into chemical energy via the process of photosynthesis, one of the most important terrestrial chemical reactions. In addition, photons serve as bits of information about the quality and quantity of light in the surrounding environment. Signal-transducing photoreceptors then use this information to adjust metabolic and developmental changes to the ambient light environment.[1, 2] One of the most prominent signaling light sensors includes the phytochrome family, which regulates a wide variety of physiological responses and cellular processes in plants, bacteria and fungi.[3–6] For light perception phytochromes utilize an openchain tetrapyrrole (bilin) chromophore such as phycocyanobilin (PCB), phytochromobilin (PfB) or biliverdin (BV), which is covalently bonded to the protein moiety by a thioether linkage. In the case of the canonical phytochromes, the photoresponses are mediated by switching reversibly between two forms, the thermally stable red light-absorbing form (Pr) and the thermally instable far-red light-absorbing form (Pfr). Based on the crys[a] Dr. S. Fey, Dr. G. Hermann Institute for Biochemistry and Biophysics Friedrich-Schiller University Jena Philosophenweg 12, 07743 Jena (Germany) E-mail: [email protected] [b] P. Singer, Prof. Dr. R. Diller Department of Physics University of Kaiserslautern Erwin-Schrçdinger-Strasse, Geb. 46 67663 Kaiserslautern (Germany) E-mail: [email protected] [c] Dr. A. H. Gçller Bayer Healthcare AG, 42096 Wuppertal (Germany) Supporting Information for this article is available on the WWW under http://dx.doi.org/10.1002/cphc.201402383.
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tal structure of the Pr form, the chromophore adopts the ZZZ,ssa conformation and is located in the photosensory module in a solvent shielded binding pocket, constituted by three protein sub-domains (PAS, GAF, PHY).[7–9] Upon red-light absorption the Pr chromophore undergoes a rapid Z!E isomerization. As NMR and other spectroscopic studies on chemically locked bilin chromophores reveal, photoisomerization occurs at the C(15)=C(16) methine bridge between rings C and D (Figure 1) resulting in a ZZE,ssa geometry of the Pfr chromophore.[10–16] On the other hand, crystallographic studies on the Pfr form of a cyanobacterial phytochrome, lacking the PAS domain, indicate a 5E-syn conformation of the bilin chromophore, thus suggesting the rotation of ring A about the C(4)=C(5) methine bridge during Pr-to-Pfr photoconversion.[3, 17] However, very recent work disproves an A-ring rotation in this bacteriophytochrome even though chemical shifts characteristic of the D-ring rotation in canonical phytochromes were not unequivocally detected.[18] The reaction pathway for photoconversion of Pr to Pfr is distinct from that for the back reaction of Pfr to Pr, involving different intermediates and time scales. The Pr-to-Pfr photoreaction was shown to occur along two distinct excited-state regions on the S1 potential energy surface resulting in formation of the primary ground state product, lumi-R, which has already adopted the Pfr-like chromophore configuration, within 30 ps (or even longer)[19–27] and 15 % quantum yield.[19, 21–23, 25, 27, 28] On the other hand, the initial photoprocesses of the Pfr-to-Pr back reaction proceed on a much faster time scale.[19, 20, 22, 29–31] Thus, the first ground-state intermediate is observed to arise directly from excited-state Pfr in a barrierless reaction with a time constant of 500 fs. Most likely, this 500 fs process already involves the E!Z isomerization of the ChemPhysChem 2014, 15, 3824 – 3831
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Figure 1. Chemical structures of phycocyanobilin (PCB) and the ring-A methyl imino ether (PCB-AIE): (1) phycocyanobilin, (2) ring-A methyl imino ether in the all-Z, all-syn conformation. In the Pr chromophore of plant phytochromes (PfB) the ethyl group (CH2CH3) at the D-pyrrole ring of PCB is substituted by a vinyl group ( CH=CH2) and the chromophore is in the all-Z,ssa conformation.
Pfr chromophore into the Pr-like geometry. The isomerized intermediate has a lifetime of 3 ps and undergoes further relaxation/conversion in the electronic ground state to form finally the Pr state.[22] The clear difference in the initial reaction kinetics of the Pfr-to-Pr back-reaction with respect to the forward reaction suggests different electronic structures of the two chromophore species. To analyze the structural basis for the distinct kinetics in the Pr-to-Pfr and the reverse Pfr-to-Pr photoreaction, we have investigated the ultrafast reaction dynamics of a model compound that can mimic the far-red absorbance and the dynamic properties of the Pfr state.[32–34] This model compound is the ring-A imino ether of phycocyanobilin (PCB-AIE). As mentioned above, PCB itself occurs as the chromophore in cyanobacterial phytochromes, but in a slightly modified form it also comprises the chromophore in plant phytochromes, from which it differs only by substitution of an ethyl by a vinyl group in the side chain at ring D (Figure 1). Compared with PCB, the crucial change in the structure of PCB-AIE is the tautomerization of the ring-A moiety from the 2,3-dihydrolactam into the lactim form and the stabilization of the lactim tautomer by formation of the methyl imino ether. This chemical transformation results in an elongation of the p-conjugation and a high donor strength of the ring-A lactim oxygen (N=C O). In consequence a significant red shift of 90 nm in the long-wavelength absorption band is observed, which is comparable to the red-shifted absorption of the Pfr relative to the Pr form. Therefore, the lactim derivative of PCB was proposed as appropriate model for the chromophore in the far-red absorbing form (Pfr) of phytochrome.[32–34] Herein, we report on the transient absorption kinetics of PCB-AIE dissolved in methanol with femtosecond time-resolution. These kinetic properties are compared with the excited electronic-state dynamics of PCB, the corresponding lactam structure, which was intensively studied in our previous work.[35–38] Based on a significantly faster time scale found for the initial kinetics in the lactim model, it is discussed whether a lactim-like structure of the Pfr chromophore might explain the differences in the isomerization dynamics !
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between the Pr-to-Pfr forward and back reaction in phytochromes. In this context the red shift upon lactam–lactim tautomerization was also verified by semiempirical calculations for the free, not-stabilized ring-A tautomer and, in particular, the corresponding ring-D lactim tautomer, which would reflect the relevant tautomer in the Pfr form of phytochromes more likely. The free, unmodified tautomers carrying still the kinetically mobile proton at the lactim-OH group are not stable per se in solution and the stable ring-D lactim ether is much more difficult to access by chemical synthesis than the ring-A counterpart. Therefore the work on those tautomer forms remains restricted to the analysis of the red shift in the computed absorbance spectra only.
2. Results and Discussion 2.1. Steady-State Spectra of PCB-AIE versus PCB Figure 2 presents the experimental steady-state absorption spectrum of PCB-AIE in methanol compared to that of PCB. With a peak at 688 nm, the long-wavelength absorption band (Q-band) of PCB-AIE is red-shifted by 86 nm relative to the corresponding band of PCB while the Soret band appears at 368 nm with only a minor bathochromic shift.
Figure 2. Stationary absorption spectra of phycocyanobilin (PCB) and ring-A methyl imino ether (PCB-AIE) in methanol. The spectra are characterized by peaks of the long-wavelength Q band (Soret band) at 688 nm (368 nm) for PCB-AIE (c) and 603 nm (364 nm) for PCB (g). The excitation wavelength (615 nm) is indicated.
2.2. Excited-State Dynamics of PCB-AIE versus PCB Figure 3 shows the absorption difference spectra of PCB-AIE in methanol at selected delay times upon excitation at 615 nm. Negative and positive difference signals are assigned to ground-state bleaching and excited-state absorptions, respectively. Negative signals appear instantaneously after excitation in the spectral region around the ground-state absorption accompanied by positive signals in the adjacent wavelength regions. The observed absorption changes decrease significantly ChemPhysChem 2014, 15, 3824 – 3831
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www.chemphyschem.org The transient absorption data DA(t, l) is globally best fitted with a sum of three exponentials. Applying only two exponentials led to significant residuals. This result is further supported by SVD analysis, which identified three relevant components (not shown). Thus, the kinetics of photoexcited PCB-AIE can be described by a three-component model with time constants of t1 = 0.4 ps, t2 = 1.2 ps and t3 = 6.7 ps. Decay-associated spectra (DAS) of the individual kinetic components are displayed in Figure 5, and characterized as follows: 1) A1 is positive between about 620 and 690 nm and negative below 600 and above 690 nm, 2) in terms of sign and partially in shape, A2 and A3 are mirror images of A1, and 3) A0 is basically zero with very small positive contributions below 550 nm.
Figure 3. Difference spectra of ring-A methyl imino ether of PCB (PCB-AIE) in methanol recorded at selected delay times after excitation at 615 nm. The data around 600–660 nm is subject to higher noise due to contributions from pump pulse scattering. The arrows indicate the temporal signal progression at specific spectral positions.
Figure 4. Transient absorption changes at specific probe wavelengths after excitation of ring-A methyl imino ether of PCB (PCB-AIE) at 615 nm. Solid lines represent the calculated functions obtained from the best-fit parameters of the global analysis shown in Figure 5.
Figure 5. Decay-associated spectra of the kinetic components in photoexcited ring-A methyl imino ether of PCB (PCB-AIE) for excitation at 615 nm. The spectra refer to components of t1 = 0.4 ps, t2 = 1.2 ps, and t3 = 6.7 ps. For A0 data with pump-pulse scattering (shaded area) is omitted. For comparison the static (inverted) absorption spectrum is included.
on the time scale of 10 ps, after 60 ps the photoreaction appears to be largely completed with only very small residual signals remaining. Figure 4 displays the transient absorption changes at different probe wavelengths (450, 670 and 760 nm), together with the result of the global fit (see below). The transient at 670 nm reveals negative absorption changes immediately after excitation, which then undergo a slower decay. This kinetic behavior reflects the ultrafast depletion of the ground state absorption and/or stimulated emission, followed by ground state recovery. The transient at 450 nm shows the inverted behavior, that is, a basically instantaneous rise of the excited-state absorption which decays on the picosecond time scale. At 760 nm, the transient is characterized by a small initial bleach, followed by a delayed signal rise and a subsequent decay on the picosecond time scale.
A1 displays the decay of excited-state absorption between 620 and 690 nm and the loss of stimulated emission at wavelengths > 750 nm. Because of the simultaneity of the two processes, the decay of excited-state absorption and the concurrent loss of stimulated emission, the associated electronic state can be ascribed to the S1-excited state. Furthermore, A2 and A3 associated intermediates are directly formed from the S1-excited state as can be concluded from the mirror images of the amplitudes in A1 and A2 as well as A3. Based on the lack of stimulated emission A2 and A3 can be assigned to electronic ground-state intermediates, which are formed within 0.4 ps and decay with 1.2 ps and 6.7 ps lifetimes back to the initial ground state. Accordingly, the key primary dynamics of PCBAIE can be described by the kinetic model given in Figure 6. Note that an alternative sequential reaction scheme, possibly involving a dark state and an unrelaxed/hot electronic ground state, cannot be excluded. However, just as the parallel model
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in Pr and particularly Pfr being fixed by specific interactions with the local protein environment. Two further aspects remain to be discussed in this context. First in plant and cyanobacterial phytochromes, the primary reaction step is initiated by Z!E photoisomerization of the meFigure 6. Level scheme suggested for the relaxation processes in photoexcited PCB-AIE (A) in comparison with thine bridge between rings C the excited-state processes in PCB (B). The first excited state PCB-AIE* decays with t1 = 0.4 ps into two intermediate states (I1 and I2) that in turn repopulate the ground state with t2 = 1.2 ps and t3 = 6.7 ps under 615 nm excitaand D[3–5, 9, 10, 12, 14] in the bilin tion. In PCB, the excited-state population PCBA* undergoes a photoreaction to form PCBB/PCBC with a much chromophore. Hence, lactam/ slower time constant of t1’ 30 ps.[35, 36] Subsequently, PCBB/PCBC revert into PCBA with t2’ 350 ps until the initial lactim tautomerization should insteady state concentration of PCBA is reached again. volve ring D rather than ring A. Furthermore all four chromophore nitrogens are protonated in both Pr and Pfr discussed above, such a scheme would equally incorporate the forms.[10, 39, 40] Therefore the question arises whether the geofast decay of the S1-excited state (stimulated emission) with 0.4 ps accompanied by the 1.2 ps and 7 ps dynamics of the metrically similar ring-D lactim tautomer does also reveal a sigtwo ground-state intermediates. nificant red shift in the long-wavelength absorbance and a simiWhen compared with the corresponding lactam structure of lar fast reaction dynamics as the ring-A tautomer. Since the PCB, the excited-state dynamics of PCB-AIE is significantly ring-D lactim imino ether (PCB-DIE) is much more difficult to faster. As we have previously shown, PCB exists in three differobtain by chemical synthesis in the required amount than the ent ground-state species (PCBA, PCBB, PCBC), of which only the ring-A counterpart this problem was addressed by calculating most prevalent species PCBA undergoes a photoreaction and the absorption spectrum and analyzing this spectrum for forms species PCBB and PCBC with a time constant of a red-shifted absorbance as compared to the corresponding 30 ps.[35, 36, 38] In a thermally driven back-reaction with a signifilactam tautomer. In addition to the ring-D imino ether, also the cantly longer time constant of 350 ps, PCBB and PCBC then free ring-A and ring-D lactim derivatives were included in the revert into PCBA until the initial steady-state equilibrium is calculations. The reason for this is that the free lactim tautomers are not stable per se in solution and only available after reached again (cf. Figure 6). The difference in the kinetics of fixation of the kinetically mobile proton of the lactim hydroxyl the lactam and lactim tautomers of PCB is reminiscent of the group as in case of the imino ethers. However, in the proteinexcited-state dynamics in phytochromes. As already mentioned bound chromophore as in phytochromes, tautomerization in the introduction, the initial kinetics of the Pfr-to-Pr photoshould more likely involve the free lactim derivative, in which transformation occurs on a much faster time scale than the forthe lactim hydroxyl group is fixed by specific interactions with ward Pr-to-Pfr transformation, suggesting that the two chroprotein sites in its direct surrounding rather than by an ether mophores do not only differ in their configuration but also in bond. their electronic structure, and hence in the shapes of their excited-state potential energy surfaces. PCB itself is found as the chromophore in cyanobacterial 2.3. Computation of PCB Lactam/Lactim Tautomer Spectra phytochromes and in an only slightly modified form also in The structures of all lactim tautomers, which were included the plant phytochromes (Figure 1). It is therefore tempting to into the spectra calculations are depicted in Figure 7. As speculate that the Pfr- as opposed to the Pr-chromophore shown there, in the lowest-energy tautomers of the ring-A adopts a lactim-like structure, which causes the extremely fast imino ether and lactim the acidic hydrogen atoms are found reaction kinetics in Pfr upon conversion into Pr as well as the located on the nitrogen atoms of rings B and D, whereas in Pr to Pfr bathochromic shift. It can be argued that chromothe ring D species those hydrogen atoms are placed on the niphore–protein interactions, also necessary in generating the trogen atoms of rings A and C. The last pattern also applies for unique spectral properties of phytochromes, are not taken into the protonated ring-D lactim, which is examined as an examconsideration with studies on a model chromophore and ple of a protonated lactim species. Figure 8 displays the calcutherefore question this hypothesis. In this respect it is to be lated spectra for the lactam in comparison with the different noted that the primary photochemistry of a protein-bound lactim tautomers. The spectra are based on the results obchromophore is manifested in the chemical constitution of the tained from the Gaussian 09 implementation of ZINDO.[41–43] chromophore itself, whereas the detailed energetics and dynamics of the photochemical processes is affected by the interThe individual spectra together with the relative oscillatory play with the protein sites in the surrounding. That means that strengths of the calculated transitions as well as the absorption the 2,3-dihydrobilindione chromophore in phytochrome maxima as such can be found in the Supporting Information (Figure 1) should retain the capability of lactam/lactim tauto(Figure S1, Table S1). It should be added here that the comparimerization with the lactam/lactim-like chromophore structures son of the calculated spectra is only possible at a qualitative 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 7. Chemical structures of ring-A and ring-D lactim derivatives of phycocyanobilin (PCB) used for calculating the electronic spectra. (I) ring-A methyl imino-ether (2) with R = CH3 and ring-A lactim (3) with R = H), (II) ring-D methyl imino ether (4) with R = CH3 and ring-D lactim (5) with R = H as well as (III) protonated ring-D lactim (6). The structures are shown in the all-Z, all-syn configuration/conformation. The calculated structures have protonated propionic acids at rings B and C and a methyl group (CH3) instead of an ethyl group (CH2CH3) at ring D.
Figure 8. Overlay of the electronic spectra calculated for the ring-A and ringD lactim derivatives (2)–(6) in comparison with the lactam derivative (1) of phycocyanobilin (PCB). The spectra were computed in vacuum using AM1 coordinates with ZINDO implemented in Gaussian09. For a clear representation the spectrum of the protonated ring-D lactim (6) is shown in a separate plot and directly compared with the lactam tautomer of PCB (1) there. (1) PCB lactam tautomer, (2) ring-A methyl imino ether, (3) ring-A lactim tautomer, (4) ring-D methyl imino ether, (5) ring-D lactim tautomer, and (6) protonated ring-D lactim tautomer. For the detailed structures compare Figures 1 and 7. A colored image of the superimposed spectra is given in the Supporting Information (Figure S2).
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level. The reason is that the theoretical methods needed for quantitative predictions like multi-reference configuration interaction approaches (MRCI) are not applicable for systems of the size of PCB. Therefore, the more approximate but well established ZINDO method was employed for the calculations. In addition the experimental spectra are adiabatic in nature, whereas the calculated ones are vertical and therefore blueshifted. Nevertheless, it is evident that the calculated spectra for the lactam tautomer of PCB (1) and the ring-A imino ether, PCB-AIE (2), reproduce both the general shapes of the experimental spectra (Figure 2) with the more intense band in the blue (Soret band) as compared to the red (Q-band) spectral region and the significant red shift in the lactim tautomer. It is therefore to be expected that the calculations predict reliably the spectral trends in the lactam versus lactim spectra of the virtual species. As can be seen from Figure 8, the lactim tautomers (2) to (5) show pronounced red shifts in their long-wavelength absorption bands as compared to the lactam tautomer (1). The most remarkable red shift from 539 to 630 nm occurs for PCB-AIE (2). For PCB-DIE (4) this shift is smaller and only extends to 574 nm. The same tendency is found for the free lactims at the ring-A (3) as opposed to the ring-D site (5), whereas the spectra of the lactims and their respective imino ethers are nearly congruent with just slight red shifts of less than 10 nm. For all structures the long-wavelength absorptions are due to exactly one electronic transition. In accordance with the experimental spectra, the position of the blue band remains almost unchanged between all structures. It is predicted to be between 333 (4) and 338 nm (3). Due the narrow Gaussian shape line fitting applied additional blue bands appear in the calculated spectra, which have their counterparts in the experimental PCB (1) and PCB-AIE (2) spectra in shallow shoulders at around 390 and 430 nm. These bands are red-shifted in the calculated spectra relative to the lactam spectrum (1), too (Figure 8). While only moderate red shifts in the long-wavelength absorption are predicted for ring-D versus ring-A lactims this situation is different in the protonated ring-D lactim (6), (Figure 7). Protonation of the ring-D lactim results in a considerChemPhysChem 2014, 15, 3824 – 3831
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Table 1. Geometrical parameters defining the planarity of the minimum energy structures in the different lactam/lactim tautomers of PCB (1)–(6). The calculations are made for the all-Z, all-syn configuration/conformation. The angles are given in degree and the distances in . Geometrical parameter
Lactam (1)
Ring-A iminoether (2)
Ring-A lactim (3)
Ring-D iminoether (4)
Ring-D lactim (5)
Ring-D lactim (protonated) (6)
dihedral ring (AB)[a] dihedral ring (BC) dihedral ring (CD) dihedral (NANBNCND)[b] r(OAOD)[c] r(CACD)[d]
37.2 24.2 5.1 59.8 6.95 6.51
16.1 8.9 2.4 28.9 3.55 4.31
13.5 8.4 3.7 28.2 3.47 4.12
11.9 14.7 16.1 43.7 4.89 4.89
11.7 11.5 15.5 40.6 4.55 4.70
5.8 2.8 139.4 55.2 4.8 6.5
[a] Dihedrals are defined as the angle between the straight lines given by the nitrogen and bridge head carbon of the first and second ring system. [b] Dihedrals are defined as the angle between the straight lines given by the nitrogen of rings A and B relative to rings C and D. [c] Distances between the oxygen atoms at rings A and D (C=O and OH/OR groups); [d] Distances between the carbon atoms at rings A and D (C=O and COH/COR groups).
able red shift of more than 100 nm (Figure 8), which even exceeds that of the ring-A lactim tautomers in the unprotonated forms (2), (3). The blue band is located near 380 nm (Figure 8). Its position roughly corresponds to the second blue band with lower absorptivity in the spectra of the unprotonated lactim tautomers (2)–(5). The main blue band at 333–338 nm in those spectra loses absorptivity so that in the protonated ring-D lactim (6) the second blue band at 380 nm dominates that at around 330 nm. The gradual red shifts in the lactim tautomers (2)–(6) can be correlated with their three-dimensional structures. Table 1 lists the dihedral angles and distances defining the overall deviations from planarity, which in turn affect the length of the conjugated p-electron path. As already shown earlier, the p-electron path is strictly localized in so far as all bonds of the tetrapyrrolic ring system keep their single and double bond character and conjugation does not lead to bond length equilibration.[38] With respect to torsional strains on the conjugated pelectron path Table 1 reveals that the dihedral angles between the planes defined by the nitrogens of rings A-B-C and rings B-C-D is 608 for the lactam tautomer (1), about 298 for the ring-A lactims (2), (3) as well as about 448 and 418 for the ringD lactim tautomers (4), (5). Electrostatic repulsion between the carbonyl groups is probably the main driving force for the torsional deformation in the lactam tautomer (1). As evident from the dihedral angle between rings A and B, in particular ring-A is strongly bent out of the plane. The largest torsional deformation in this tautomer results in the most blue-shifted longwavelength absorption band. On the other hand, the ring-A and ring-D lactim tautomers (2)–(5) are almost mirror images of each other, apart from the different double bond pattern in rings A and D. While the ring-A lactim tautomers have 11 conjugated double bonds, the corresponding ring-D tautomers have only 10, which leads to a slightly reduced conjugation path. This, together with the higher tilting in the ring-D lactims (4), (5), results in the lower red shifts in these tautomers as compared to the ring-A tautomers (2), (3). Images of the different three-dimensional structures are added in the Supporting Information (Figure S3). The computed spectra clearly indicate a significant red shift of the long-wavelength absorption maximum also in the ring 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
D imino ether and lactim tautomers (4), (5) even if the extent of this shift is smaller than for the ring-A counterpart. However, as further evidenced protonation of the ring-D lactim still increases this red shift and exceeds the value of the unprotonated ring-A tautomer. Furthermore, the value of the calculated red shift becomes comparable to that observed in the Pfr as compared to Pr spectrum of phytochromes.[3–6] Taken the experimental findings, reported here, together it seems conceivable that a lactim-like structure is also retained in the Pfr chromophore. At least such structural feature can definitely explain both the far-red absorbance and in particular the significantly faster photochemistry of the Pfr as compared to the Pr state in phytochromes. It could be argued that the Pr and Pfr structures recently derived by magic-angle spinning NMR studies, which used labeled PCB chromophores, do not provide direct evidence of a lactam/lactim tautomerization upon Pr-to-Pfr photoconversion.[10] However, it is unclear whether ring-D lactam/lactim tautomers could be distinguished by this technique since it is very difficult to discriminate between them by means of 13C and 1H chemical shifts in NMR spectra.[44]
3. Conclusions We have addressed a fundamental question in the phototransformation of phytochromes concerning both, the bathochromically shifted ground-state absorption of the far-red light-absorbing form, Pfr, compared to the red-light-absorbing form, Pr, as well as the substantially different time scales of the respective primary photoreactions. The excited-state dynamics of the lactim tautomer of PCB is significantly faster when compared with the lactam tautomer.[35, 36] Immediately after excitation the ring-A methyl imino ether, PCB-AIE, studied as model of a fixed ring-A tautomer forms two ground-state intermediates within 400 fs, which in turn undergo decay with lifetimes of 1.2 and 6.7 ps. The difference in the early-time dynamics between the lactam and lactim tautomers of PCB is reminiscent of that in the excitedstate chemistry of phytochromes, revealing that the Pfr-to-Pr back reaction occurs in 700 fs and is clearly faster than the Pr-to-Pfr forward reaction.[19, 20, 22, 29] It is therefore suggested ChemPhysChem 2014, 15, 3824 – 3831
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CHEMPHYSCHEM ARTICLES that the chromophore in the Pfr-form of phytochrome could adopt a lactim-like structure, which is responsible for the red shift in the long-wavelength absorption and in particular for the extremely fast primary reaction time. Interactions with the protein moiety in the native chromoprotein are expected to assist the stabilization of the lactim-like structure and to control the specific photochemistry. The strict comparison with the chromophores in phytochrome would require that lactam/lactim tautomerization takes place at pyrrole ring-D. In this context it is shown that the spectra of the ring-D imino ether, PCB-DIE, and the free lactim tautomers, which were computed by semiempirical methods, also exhibit a red shift of the long-wavelength absorption band relative to that of the lactam tautomer. This red shift is smaller than that of the ring-A lactim form but it is significantly increased when the lactim tautomer gets protonated. Thus, also tautomerization of the ring-D lactam into its lactim form provides a qualitative model that can mimic the longwavelength absorption of the Pfr chromophore, whereas the lactim structure itself is well primed for an ultrafast primary reaction step.
Experimental Section Sample Preparation Phycocyanobilin (PCB) was isolated from the cyanobacterium Spirulina geitleri as described earlier.[35] The ring-A methyl imino ether of PCB (PCB-AIE) was prepared with slight modifications according to the procedure given by Micura et al.[32] In brief, starting from 10 mg of PCB, the dimethyl ester of PCB was produced by esterification with methanol and BF3 as catalyst. The dimethyl ester was then dissolved in chloroform (8 mL), a solution of zinc acetate (10 mg) in methanol (1 mL) and acetic anhydride (1 mL). This mixture was heated under refluxing conditions (30 min) and neutralized by addition of hydrogen carbonate (40 mL of a saturated solution). The product formed was subsequently purified by thin layer chromatography on silica gel 60 TLC plates in dichlormethane:ethyl acetate = 7:1. PCB-AIE is characterized by Rf = 0.5 in thin layer chromatography on silica gel 60, a mol peak of MW = 628.33 g mol1 in the MALDI-TOF analysis (Ultraflex III TOF/TOF; Bruker Daltonics) and absorption peaks of l1 = 688 nm and l2 = 368 nm in methanol (Figure 2). For the femtosecond experiments PCB-AIE was dissolved in methanol. To avoid photodegradation, steady state UV/Vis transmission spectra were recorded regularly.
Transient UV/Vis Absorption Spectroscopy Transient femtosecond absorption pump–probe spectroscopy was performed with a regenerative Ti:Sa amplifier laser system (CPA 2001 Clark-MXR Inc.) with pulses of 150 fs duration (FWHM), 0.8 mJ energy at 780 nm and 1 kHz repetition rate. Short excitation pulses were generated by a homebuilt two-stage NOPA (non-collinear parametric amplifier), tunable in the spectral range between 350– 800 nm. Excitation wavelength of PCB-AIE was set to lexc = 615 nm for excitation of the high-energy edge of the Q-band absorption (600–800 nm), (Figure 2). The optical density of the sample was set to 1 (1 mm cuvette) in the absorption maximum of the Q band. The focal width of the actinic laser beam was 250 mm 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemphyschem.org (FWHM) with single pulse energy of 100 nJ at the sample position and pulse duration of 70 fs (FWHM). Broad-band continuum probe pulses (spectral range: 450–850 nm) were generated by means of self-phase modulation in a sapphire window. Pump pulses were time-delayed on a motorized translation stage (Physik Instrumente, PI) with respect to the probe pulse. Wavelength-dependent time zero was determined by recording the cross-phasemodulation signal progression in a H2O sample. The spectrally dispersed probe pulses were detected with an integrated 512 pixel PDA camera device (Stresing, Hamamatsu), electronically synchronized with the laser system. The experiments were performed at room temperature with pump-probe cross-correlation of 200 fs (FWHM). To ensure fresh-sample conditions for each pump–probe event, a flow-cell system (Harrick) was used. During the measurement, every second pump pulse was blocked in order to obtain pump-induced absorption changes DA(t, l) as a function of delay time and probe wavelength. The global analysis of the recorded transient absorption data was carried out between 0.2 and 100 ps in the range 450–850 nm by fitting DA(t, l) with a sum of exponentials via Equation (1): DAðt; lÞ ¼ A0 ðt; lÞ þ
XN i¼1
Ai ðlÞet=ti
ð1Þ
with DA(t, l) corresponding to the decay associated spectra (DAS). In addition, a singular value decomposition analysis (SVD) was applied to determine the number of spectral species.
Quantum Chemical Calculations The absorbance spectra were calculated for the bis-lactam form of PCB, the ring-A and ring-D imino ethers as well as ring-A and ringD lactim forms of PCB (cf. Figure 7). The calculations were carried out for the ZZZ,sss configuration/conformation with protonated propionic acid side chains at rings B and C as well as with the ethyl group at ring-D substituted by a single methyl group. In addition, the protonated ring-D lactim form in the all-Z, all-syn conformation was also included into the calculations. The structures of each PCB form were minimized with the semiempirical NDDO Hamiltonian AM1[45] implemented in the ArgusLab software package. As starting geometry an initial 3D structure in the ZZZ,sss configuration/conformation was derived from extensive minima searches.[37] Vertical excitations on the AM1 optimized coordinates were calculated with (i) the ZINDO method[41, 43] implemented in ArgusLab for 10 singlet states based on the 10 highest occupied and 10 lowest unoccupied orbitals with random phase approximation ansatz and (ii) the ZINDO method implemented in Gaussian 09[42] for 20 excited states. The calculations were performed in the gas phase. The spectra as obtained from ZINDO/Gausian 09 were transformed with SpecDis 1.6[46] using Gaussian line fitting with a sigma/gamma ratio of 0.16 eV in the wavelength region between 250 and 800 nm and plotted with OriginPro 8G. All calculations were carried out at the high performance computation cluster of the University of Jena.
Acknowledgements We thank Prof. em. Dr. H. Falk, Johannes Kepler Universitt Linz, Austria for helpful discussions. R.D. and P.S. thank Research Initiative Rheinland Pfalz in Membrane Biology (RIMB) for financial support and H. Feurich for technical assistance. ChemPhysChem 2014, 15, 3824 – 3831
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Received: June 2, 2014 Published online on September 4, 2014
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