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Optically Detected Magnetic Resonance of the Chlorophyll Triplet State in the Water-Soluble Chlorophyll Protein from Lepidium virginicum. Evidence for Excitonic Interaction Among the Four Pigments Alessandro Agostini, Daniel M. Palm, Harald Paulsen, and Donatella Carbonera J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b01906 • Publication Date (Web): 21 May 2018 Downloaded from http://pubs.acs.org on May 21, 2018

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The Journal of Physical Chemistry

Optically Detected Magnetic Resonance of the Chlorophyll Triplet State in the Water-Soluble Chlorophyll Protein from Lepidium virginicum. Evidence for Excitonic Interaction Among the Four Pigments

Alessandro Agostini1,2, Daniel M. Palm2, Harald Paulsen2, and Donatella Carbonera1* 1

Department of Chemical Sciences, University of Padova, Via Marzolo 1, 35131 Padova, Italy

2

Institute of Molecular Physiology, Johannes-Gutenberg University Mainz, Johannes-von-Müller-Weg 6, 55128

Mainz, Germany *Author to whom correspondence should be addressed. Telephone: +39 0498275144. Fax: +39 0498275161. Email: [email protected]

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ABSTRACT Optically detected magnetic resonance (ODMR) of triplet states populated by photoexcitation in the Water-Soluble Chlorophyll Protein (WSCP) from Lepidium virginicum has been performed, using both absorption and fluorescence detection. Well resolved Triplet minus Singlet (T-S) spectra have been obtained, which have been interpreted in terms of electronic interactions among the four chlorophylls (Chls) forming two dimers in WSCP tetramer. Localization of the triplet state on a single Chl leads to a redistribution of the oscillator strenght in the remaining three Chls of the complex. Comparing the spectra with those obtained on a substoichiometric WSCP complex containing only two Chls per protein tetramer, we proved that, to interpret the optical spectra of WSCP fully loaded with four Chls, the interactions between the two dimers have to be taken into account and cannot be considered negligible. The results show that WSCP may well be considered an ideal model system to study ChlChl interactions, in view also of the possibility to modify the number and molecular structure of the bound porphyrin chromophores.


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Introduction Water-Soluble Chlorophyll Proteins (WSCPs) from Brassicaceae are non-photosynthetic proteins,1,2 which tetramerize upon binding of four chlorophyll (Chl) molecules.3,4 The crystal strucure of WSCP,3 reported in Figure 1, reveals that the Chls are bound in a cavity in the interior of the tetrameric complex, and are organized in two dimers (center-center distance within the dimer of 10 Å, with the Chl planes inclined at an angle of ~ 27°) separated by a longer inter-dimer distance (ca. 20 Å). The “open sandwich”5 configuration of each Chl dimer is responsible for a distribution of the oscillator strength which differs from that typical of Chl special pairs in photosynthetic reaction centers, since it shows the main excitonic band at higher energies.6 This distribution of the oscillator strength is associated to a lengthening of the lifetime of the excited singlet state,7 with a consequent increase of the ISC probability8 to the triplet state. We recently proved that the Chl triplet state (3Chl) in WSCP is localized on a single Chl. This Chl is accessible to small neutral molecules in solution9 and therefore, effectively photosensitizes singlet oxygen.10 The photobleaching of the bound Chls is efficiently prevented due to a steric shielding by the phytyl chains either of the more reactive portions of the chlorin ring or of the Mg atom.10


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Figure 1: A) Structure of Lepidium virginicum WSCP 3 with the Chls a in spheres and the polypeptide chains in cartoons. The four monomers are shown in yellow, green, blue, and red (A, B, C, and D, respectively3). B) and C) detail of the two open sandwich dimers from two views 45° apart.

The binding of solely Chl11,12 in a low Chl/protein ratio4,13,14 and the remarkable photostability of the complex,10,11 make WSCP a suitable model system to study Chl-Chl interactions in a protein medium, either in its singlet or triplet excited states. Indeed, the properties of the excited states of this complex have been thoroughly investigated by means of advanced optical spectroscopies,7,15–19 however the results have been mostly interpreted in terms of single open sandwich dimers, considering the two dimers on the tetrameric protein too far apart to have strong interactions.18,19


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The recent finding that the tetrameric complex can be reconstituted with only two Chls per tetramer,13 which maintain the open sandwich configuration within the protein scaffold, has opened the possibility for an experimental evaluation of the inter-dimer interaction, since the two/four Chl systems differ from each other just for the absence/presence of the inter-dimer interaction. In this work we make use of Optically Detected Magnetic Resonance (ODMR), both fluorescence detected (FDMR) and absorption detected (ADMR), to characterize the triplet state populated in WSCP. The technique, being a double resonance spectroscopy, is highly selective and allows to monitor at the same time the magnetic and optical properties of a molecule carrying a triplet state. FDMR has been largely used to reveal the spectral heterogeneity of triplet states in protein complexes due to different local environments, since the fluorescence detection allows to detect even very low triplet populations.20–25 ADMR in turn, has been successfully employed in the past to study excitonic redistribution occurring after photoinduced triplet formation in photosynthetic systems.26–28 In fact, the microwave induced triplet minus singlet (T-S) spectrum, which can be detected by ADMR with high resolution, reflects the change in the excitonic interactions upon triplet formation in multi-chromophore systems. Thus, the method is most appropriate to unveil the details of interaction among Chls in WSCP since the localization of the triplet state on one pigment,10 subtracts the pigment itself from the excitonic interaction with the others, perturbing in this way the excitonic landscape.26–30

Experimental and Theoretical Methods Sample preparation. Protein overexpression in E. coli and protein purification have been performed as previously reported.10 The purified WSCP apoprotein was reconstituted at a Chl a to protein stoichiometry of 1:1 (4ChlaWSCP), as previously described.10 To produce the 0.5:1 substoichiometric complexes (2ChlaWSCP), the method previously introduced for Brassica oleracea WSCP,13 has been successfully applied here to L. virginicum WSCP without modifications, obtaining WSCP tetramers with a Chl/protein ratio equal to 0.66. ODMR experiments. Fluorescence detected magnetic resonance (FDMR) and absorption detected magnetic resonance (ADMR) spectra were acquired in a home-built set-up which has been previously described in detail.24,31 The principle of the ODMR technique has been previously reviewed.32,33 To briefly describe the ODMR technique, the sample is excited by light from a halogen lamp, filtered through either a 5 cm CuSO4 solution (FDMR) or a 10 cm water filter (ADMR), focused to the sample ACS Paragon Plus Environment

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cell, immersed in a bath helium cryostat. In FDMR experiments, the fluorescence is detected by a photodiode at 90° geometrywith respect to the excitation light direction, through bandpass filters (FWHM~10 nm), while in ADMR experiments, the absorption is detected with standard straight geometry through a monochromator using the same detector as for FDMR measurements. By sweeping the microwave frequency (MW source HP8559b, sweep oscillator equipped with a HP83522s plug-in and amplified by a TWT Sco-Nucletudes mod 10-46-30 amplifier) while detecting the absorption (ADMR) or the fluorescence (FDMR) changes at specifics wavelengths, the resonance transitions between spin sublevels can be determined and, correspondingly, the Zero Field Splitting (ZFS) parameters of the triplet state. In fact, the change of the optical signal is due to a change of the steady state triplet population induced by the resonance between a couple of levels. This change is accompanied by a corresponding change of the singlet population (for a simplified scheme see Figure S1 in Supplementary Information). The microwaves are on/off amplitude modulated for selective amplification and the signal from the detector is demodulated and amplified using a Lock-In amplifier (EG&G, mod 5210). The analog output is connected to a computer-controlled analog to digital converter. The microwave resonator, where the sample cell is inserted, consists of a slow pitch helix. Once the resonance transitions have been determined, by fixing the microwave field at a resonant frequency and sweeping the absorption detection wavelength, microwave-induced Triplet-minusSinglet (T−S) spectra can be registered.20 Compared to optical time-resolved absorbance spectroscopy on the triplet state, the ODMR technique allows selection (by the resonant microwave field) of specific triplet populations among those present on the same sample. In this way well resolved T-S spectra can be obtained. The samples for the ODMR measurements were diluted with degassed glycerol added to a final concentration of 60% (v/v) immediately before the insertion into the cryostat, in order to obtain homogeneous and transparent matrices upon freezing. After the dilution, the final Chl concentration of the samples was 150 µg/ml for FDMR and 100 µg/ml for ADMR experiments. In all measurements the temperature was 1.8 K. The low temperature is needed to inhibit the relaxation processes between the triplet spin sublevels, thus maintaining the triplet populations far from the Boltzmann equilibrium, which is necessary to reach the sensitivity to detect the ODMR transitions.


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Low temperature absorption spectra. Ground-state absorption spectra were recorded at 1.8 K from 450 to 750 nm (bandwidth of 0.5 nm), concomitantly with the T-S spectra, by measuring the transmittance of the sample and of the blank signal, using the same set-up described above. T-S spectra simulation. T-S spectra of WSCP were simulated in the region corresponding to the Qy transitions following the approach based on the point dipole approximation previously described by Bordignon et al.34 for exciton states of coupled bacteriochlorophylls of the B808-866 complex. The Hamiltonian of the multi-chromophore system has been treated with a first-order perturbation theory, obtaining: = | | + | |

where are site energies, that have been considered the same for the four pigments. is the coupling term, that in the point dipole approximation is: =

! ∙ " #! ∙ " # f ∙ −3 % 4 $

where , are the transition dipole moments at distance " ; is the dielectric constant in the vacuum, and is the relative dielectric constant taken with a value of 2.40 (as derived from a refractive index

value of 1.55,3 typical for protein environments). The local field correction f have been treated with the sphere-cavity approximation: ' =

+ 2 3

The orientation of the transition dipole moments has been retrieved from the structure of WSCP,3 considering the transition dipole moment of the Qy defined by the position of the nitrogen atoms of the pyrrole rings A and C of the Chl a molecular structure (shown in Figure S2 in Supplemantary information), according to Madjet et al.35 A Chl monomer transition dipole moment of 4.67 Debye and site energy ( ) of 15080 cm-1 has been adopted as input parameters for all the Chls, in virtue of the

symmetry of the system.3 These values were adjusted to fit the low temperature absorption spectra.

The exciton spectra are obtained by diagonalization of the Hamiltonian matrix (4x4, 3x3 or 2x2 depending on the number of interacting monomers), whose diagonal elements represent single pigment site energies, and the off-diagonal elements correspond to the transition dipole-dipole interactions between the pigments. ACS Paragon Plus Environment

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Results and Discussion FDMR spectra. Illumination at cryogenic temperatures (1.8 K) of Lepidium virginicum WSCP, reconstituted with either two or four Chl a molecules per tetramer, leads to the formation of a steady state population of 3Chl which can be detected by FDMR, monitoring the emission of the sample while sweeping the microwave field in a microwave frequency range characteristic for 3Chl zero-field transitions.36 The spectra detected at the fluorescence maximum (680 nm), are reported in Figure 2. The positions of the |D|-|E| and |D|+|E| transitions (760 MHz and 981 MHz, respectively) are the same for WSCP reconstituted with either 2 or 4 Chl a molecules, and are comparable to those reported for other Chl a containing systems.36 The values of the ZFS parameters |D| and |E|, (|D| = 0.029 cm-1 and |E| = 0.0037 cm-1), calculated from the position of the detected transitions, are in good agreement with those derived before from the 3Chl a Time Resolved-Electron Paramagnetic Resonance spectrum of L. virginicum WSCP.10 As usually found for ODMR of Chls, the |2E| was very weak (not shown).

Figure 2: 3Chl FDMR spectra of WSCP reconstituted with either 2 (red) or 4 (black) Chls a. Fluorescence collected through a 10 nm band pass filter centered at 680 nm. T= 1.8 K

The spectra show a slight dependence on the detection wavelength (Figure 3), an indication of site heterogeneity of the bound Chls, leading to the observation of 3Chl characterized by different ZFS parameters. The 2ChlaWSCP complex presents a very similar behavior. The linewidth of the FDMR ACS Paragon Plus Environment

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spectra, at each wavelength, is smaller for 4ChlaWSCP complexes (57 vs 66 MHz), showing a smaller variability in the binding site conformations compared to the substoichiometric complex. The latter is likely characterized by a reduced rigidity caused by the empty binding sites. However, it has to be noted that, since the 2ChlaWSCP sample is contaminated by a 32% of 4ChlaWSCP complexes (as derived by a 0.66 Chl/protein measured ratio), a line broadening due to the small difference in the ZFS parameters of the 3Chls in the two complexes cannot be excluded.

Figure 3: 3Chl FDMR spectra detected at different wavelengths of WSCP reconstituted with 2 or 4 Chl a, in red and black line, respectively. Amplitude modulation frequency: 33 Hz, time constant: 100 ms, temperature: 1.8 K. Fluorescence collected through a 10 nm interferential filter centered at 700 nm, 720 nm, or 740 nm. Spectra vertically shifted for better comparison. The vertical dashed line helps to follow the dependence of the resonance maxima on the detection wavelength.

ADMR and T-S spectra. The low-temperature absorption spectra of WSCP proteins, detected at 1.8 K, show a significant dependence of the line shape on the number of Chls bound, the 2ChlaWSCP complex displaying a less intense shoulder in the red region of the Qy band (Figure 4A). This difference was not detected at higher temperature,13 probably due to the broadening of the spectra. To further investigate the interactions among pigments and their effects in the spectroscopic properties of the two samples, the microwave-induced T−S spectra have been collected and analyzed.


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Figure 4: A) Normalized absorption spectra of WSCP reconstituted with either 2 (red) or 4 (black) Chls a. Temperature: 1.8 K. B) 3Chl ADMR spectra detected at 660 nm of WSCP reconstituted with either 2 (red) or 4 (black) Chls a. OD 0.7.

The T−S spectra reported in Figure 5 were measured by selecting the resonance conditions at a microwave frequency corresponding to the maxima of the |D|-|E| transitions in the ADMR spectra (Figure 4B). These latter spectra were detected by sweeping the microwave frequency while monitoring the absorption changes at 660 nm, corresponding to the maximum of the Qy transition as visible in Figure 4A. Both samples show an intense positive signal between 450 and 580 nm due to triplet-triplet (T0→Tn) absorption. Triplet-triplet absorption bands are also the source of the positive signals in the red-most part of the spectra (λ > 680 nm). The intense negative signals in the Qy region correspond to the bleaching of the singlet absorption, as clearly appears from the comparison with the corresponding absorption spectra. However, the peak profile in the T-S spectrum diverges from that of the absorption spectrum, due to the presence of a central narrow signal opposite in sign with respect to the bleaching, appearing as a hole of the whole band. A similar spectral feature has been observed before for excitonically coupled Chl pairs (as the special pair of purple bacteria26 and plants27) and was assigned to the appearing singlet absorption band of half of the Chl dimer, after localization of the triplet state on the other half. Interestingly, localization of the triplet state on a single Chl was suggested to take place also in WSCP on the base of the hyperfine coupling constants determined by


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ENDOR spectroscopy.10 Thus, the T-S spectrum of WSCPs, being suggestive of triplet localization, is in agreement with the previous findings. Remarkably, a clear difference in the T-S spectra of 2ChlaWSCP and 4ChlaWSCP complexes is present, with the hole at 665 nm more pronounced in the 2ChlaWSCP complex. This difference is particularly significant when taking into consideration that the 2ChlaWSCP sample contains a 32% of 4ChlaWSCP complex, as discussed in the next section.

Figure 5: Comparison between the T−S spectra (upper black traces) and absorption spectra (bottom red traces, normalized, inverted and vertically shifted for better comparison) of WSCP reconstituted with either A) 4 or B) 2 Chl a and detected at their |D|-|E| transitions maxima at 760 MHz. Amplitude modulation frequency 33 Hz, time constant: 3 s, temperature 1.8 K, slit 0.8 nm, OD660 0.7.

Additional T-S spectra were collected for both complexes by varying the microwave resonant frequency (Figure 6, only the Qy region is displayed) within the |D|-|E| resonant transition bands observed in the ADMR spectra (Figure 4B). Additional T-S spectra were collected for both complexes by varying the microwave resonant frequency (Figure 6, only the Qy region is displayed) within the |D||E| resonant transition bands observed in the ADMR spectra (Figure 4B). ODMR is a very selective technique. The microwave frequency selects a triplet population with some specific ZFS parameters and absorption characteristics. Since the samples are frozen, different protein conformations are present and may lead to small differences in electronic properties and interactions of the triplet states. In both samples, a comparable microwave frequency dependency of the lineshape of the associated T-S spectrum is observed, indicating a common origin for the spectral shifts due to site selection.


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Figure 6: Comparison between the normalized T−S spectra of WSCP reconstituted with either A) 4 or B) 2 Chl a and detected at various pump frequencies (745 MHz in red, 752 MHz in orange, 760 MHz in black, 780 MHz in blue). Amplitude modulation frequency 33 Hz, time constant: 3 s, temperature 1.8 K.

T-S calculation. In order to reveal the source of the difference in the T-S spectra of 2ChlaWSCP and 4ChlaWSCP complexes, a calculation of the spectra has been performed, in the frame of exciton interaction of the Qy transitions among the Chls bound to the protein. We adopted a point-dipole approximation, that was used in previous studies to satisfactorily describe T-S spectra of photosynthetic reaction centers37 and antenna complexes isolated from bacteria.34,37 This approach, although basic when compared to more advanced methodologies, has been shown to be suitable for describing Chl a Qy-Qy interactions in many systems.38,39 Due to the symmetry of the WSCP tetrameric complex, the four Chl binding sites are equivalent and the triplet state may become localized on whatsoever Chl in the protein with the same probability. Only single excitations are considered in the calculations, according to the low excitation flux used in the experiments. The T-S spectrum consists in the difference between the absorption spectrum of the system after and before triplet formation. The calculation of the singlet-singlet absorption spectrum before and after triplet formation, has been carried out by considering that the excitation to the triplet state subtracts one Chl from the exciton interaction. Thus, we have a 3-Chl exciton system (giving positive bands, as it is accompanying the triplet formation) replacing a 4-Chl exciton system (corresponding to negative bands, bleached after triplet formation). In addition, triplet-triplet absorption bands of the Chl carrying the triplet state are contributing to the positive part of the T-S spectrum. For a qualitative reconstruction


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of the Qy region of T-S spectrum, this contribution can be neglected, because it gives a flat positive offset, as can be inferred from the inspection of the spectrum in the range above 680 nm, where only this contribution is present. The T-S spectra calculated for the two samples in the region of the Qy bands, are reported as stick spectra in Figure 7B and 7C (the calculated values are reported in Table S1 in Supproting Information. Parameters used in the calculations are reported in Materials and Methods. In the case of the 2ChlaWSCP complex, upon triplet formation the positive singlet-singlet absorption contribution to the spectrum is determined by a single Chl, the one which is not carrying the triplet state, while in the case of 4ChlaWSCP the positive singlet-singlet absorption contribution is due to three excitonically coupled Chls. The 2ChlaWSCP sample contains an estimated 32% of 4ChlaWSCP tetrameric complexes, as judged from the measured Chl/protein ratio of 0.66 and considering that only complexes with 2 or 4 Chls can be formed, as demonstrated by Palm et al.13 From this percentage, it can be evaluated that in this sample 52% of the Chls are bound in a 2ChlaWSCP complex, whereas the remaining 48% are bound in a 4ChlaWSCP complex. How can be seen by inspection of the FDMR, ADMR and T-S spectra detected at different microwave frequencies, the overlap of the resonance of the two samples does not allow to discriminate between them at some specific microwave frequency, thus the “pure” 2Chl WSCP T-S spectrum must be obtained by subtraction of the 4Chl WSCP contribution. The pure T-S spectrum of the 2ChlaWSCP complex (Figure 7A, orange line) has been obtained after subtraction of the 4ChlaWSCP spectrum (Figure 5A) from the experimental 2ChlaWSCP spectrum (Figure 5B), both normalized on their area in the 14750-15500 cm-1 interval. The 4ChlaWSCP contribution has been weighted on the distribution of Chls between 2ChlaWSCP and 4ChlaWSCP complexes.


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Figure 7: A) Zoom of the Qy region of the T−S spectra reported in Figure 5. A wavenumber scale is adopted. The pure contribution of the 2ChlaWSCP (orange) to the T-S spectrum of WSCP reconstituted with 2 Chls has been obtained after subtraction of a 4ChlaWSCP contribution (black, weighted on the distribution of Chls between 2ChlaWSCP (52%) and 4ChlaWSCP (48%)) from the 2ChlaWSCP experimental spectrum (red). B) Calculated stick spectra of the 4ChlaWSCP (4Chl interaction in dark green and white diamond, 3-Chl interaction in light green and black diamond). C) Calculated stick spectra of the 2ChlaWSCP (2-Chl interaction in red and white triangle, 1-Chl in orange and black triangle).

Although the oscillatory strength and the relative position of the bands of the T-S spectra are not quantitatively reproduced, probably due to the simple point dipole approximation adopted38,39 and to the fact that contributions of vibrational and triplet-triplet absorption bands have been neglected, the main differences between the two samples are reproduced. In the 2ChlaWSCP sample the positive contribution has an oscillatory strength concentrated in a single band with half the value of the bleaching bands, while in the 4ChlaWSCP spectrum, the positive contribution is shared by three bands, making the appearing hole less pronounced and broader. This can be appreciated analyzing the contribution of the transition dipole moments of individual pigments to the transition dipole moments of the excitonic interactions of the 3-Chl system (Figure 8A). Thus, our calculations support the conclusion that the spectral difference observed between the two complexes is due to a distribution of the oscillator strength that, in the case of the 4ChlaWSCP complex, involves also the three pigments not involved in the triplet formation. This allows us to state that the two open sandwich dimers, coordinated by the WSCP tetramer, are not energetically isolated from each other.


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Figure 8: A) Contribution of the transition dipole moment of the interacting Chls to the transition dipole moments of the excitonic transitions (E1, E2, and E3) upon the localization of the triplet state (3-Chl interaction in Figure 7B). Color code according to B) showing the detail of the two open sandwich dimers (Chl A, B, C, and D in white, green, blue and red, respectively) in L. virginicum WSCP,3 in which phytol chains have been omitted for clarity.

Conclusions The experimental results obtained by ODMR spectroscopy are consistent with the localization of the triplet state on a single Chl in the WSCP protein fully loaded with four Chl molecules, in agreement with our previous ENDOR data.10 This proves that triplet-triplet interactions between Chls in the WSCP are weak, and the triplet state is localized on a single pigment. The spectroscopic comparison of WSCP complexes with different pigment/protein stoichiometries highlighted a difference in the exciton distribution that can be assigned to a non-negligible electronic interaction between the two dimers in the tetrameric complex, confirming that WSCP may be considered an ideal model system to study Chl-Chl interactions, due to the possibility to modify the number of bound Chls. Although the quantitative agreement between the calculated and the experimental T-S spectra is not reached, however a difference between the 2ChlaWSCP and 4ChlaWSCP complexes is predicted and clearly present in the experimental spectra, meaning that the interactions between the two open


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sandwich dimers in 4ChlaWSCP cannot be neglected. Due to the strong spectral effect derived from the localization of the triplet state in a single Chl molecule, the T-S spectra allowed us to highlight the different pigment interactions in the two complexes. Albeit more advanced calculation methods may provide better agreement with experiments and give fine details of pigment interactions, our study opens the way to further work to characterize and compare exciton states of systems consisting of Chls of different structure, such as Chl b, c, d or bacteriochlorophylls, metal free or central metal substituted porphyrins, in a protein medium. Of particular interest is the possibility to change, by site-directed mutagenesis, those residues, which are putatively involved in determining site energies of the pigments and influencing the cluster geometry, with the aim to rationalize the effects of the protein matrix on the spectroscopic properties of Chls in multi-chromophore systems.

Supporting Information Supporting Information contains: details of the principle of the ODMR technique (Figure S1); scheme of the orientation of dipole transition moment of Chl a relatively to the molecular structure (Figure S2); Table with the results of calculation of the dipole-dipole interaction among Chls in WSCP (Table S1).

Acknowledgment The authors acknowledge Chiara De Santi for her contribution to implementing the calculation program. This work has been funded by a grant from the Deutsche Forschungsgemeinschaft to H.P. (Pa 324/10-1). A.A. gratefully acknowledges the Ing. Aldo Gini foundation for supporting his stay in Germany with a travel grant.

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