Magnesium K-Edge NEXAFS Spectroscopy of Chlorophyll a in

Article pubs.acs.org/JPCB

Magnesium K‑Edge NEXAFS Spectroscopy of Chlorophyll a in Solution Katharina Witte,*,† Cornelia Streeck,‡ Ioanna Mantouvalou,† Svetlana A. Suchkova,§,∥,# Heiko Lokstein,⊥ Daniel Grötzsch,† Wjatscheslav Martyanov,† Jan Weser,‡ Birgit Kanngießer,† Burkhard Beckhoff,‡ and Holger Stiel*,⊗ †

Technische Universität Berlin, Institut für Optik und Atomare Physik, 10623 Berlin, Germany Physikalisch-Technische Bundesanstalt, 10587 Berlin, Germany § Humboldt-Universität zu Berlin, School of Analytical Sciences Adlershof (SALSA), 10099 Berlin, Germany ∥ Southern Federal University, International Research Center “Smart materials”, Rostov-na-Donu 344090, Russia ⊥ Charles University, Faculty of Mathematics and Physics, Department of Chemical Physics and Optics, 121 16 Prague, Czech Republic ⊗ Max-Born-Institut für Nichtlineare Optik und Kurzzeitspektroskopie Berlin, 12489 Berlin, Germany ‡

ABSTRACT: The interaction of the central magnesium atom of chlorophyll a (Chl a) with the carbon and nitrogen backbone was investigated by magnesium K near-edge X-ray absorption fine structure (NEXAFS) spectroscopy in fluorescence detection mode. A crude extract of Chl a was measured as a 1 × 10−2 mol/L ethanol solution (which represents an upper limit of concentration without aggregation) and as dried droplets. For the first time, the investigation of Mg bound to Chl a in a liquid environment by means of X-ray absorption spectroscopy is demonstrated. A pre-edge feature in the dissolved as well as in dried Chl a NEXFAS spectra has been identified as a characteristic transition originating from Mg in the Chl a molecule. This result is confirmed by theoretical DFT calculations leading to molecular orbitals (MO) which are mainly situated on the magnesium atom and nitrogen and carbon atoms from the pyrrole rings. The description is the first referring to the MO distribution with respect to the central Mg ion of Chl a and the surrounding atoms. On this basis, new approaches for the investigations of dynamic processes of molecules in solution and structure−function relationships of photosynthetic pigments and pigment− protein complexes in their native environment can be developed.

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INTRODUCTION Porphyrins are an important class of natural pigments responsible for light harvesting, excitation energy transfer, and charge separation in photosynthesis1 or as active sites in oxygen transport and metabolism.2 Certain porphyrins are used in photodynamic tumor therapy3 as well. The functions of porphyrins are mainly determined by interaction of the central metal atom with nitrogen atoms as nearest neighbors, whereas the porphyrin macrocycle as a highly conjugated system is responsible for the intense light absorption bands in the visible range. Among porphyrins, chlorophylls (Chls) play an outstanding role. Chls as a group of cyclic tetrapyrrole pigments can be found in several chemical configurations (Chl a, b, c, d, etc.), which differ in the substituents of the tetrapyrrole rings, the number of double bonds of the conjugated system, and the esterified alcohol chain.4 The Chl macrocycle is the site of redox chemistry in the primary reactions of photosynthesis. In photosynthetic pigment− protein complexes, Chl molecules are embedded in a protein matrix rendering them very effective for light harvesting and charge separation. © 2016 American Chemical Society

The optical absorption properties of porphyrins are well understood. According to Gouterman’s four-orbital theory,5 the optically allowed transitions in porphyrins arise from the two highest occupied (HOMO, HOMO−1) and two lowest unoccupied (LUMO, LUMO+1) molecular orbitals. The interaction of the central metal atom with the macrocycle determines the resultant transition energies. By orbital mixing, an energy splitting into one state with higher and one state with lower energy occurs. The first one is called the Soret or B-band and the latter the Q-band. This simple, 50-year-old model explains the optical absorption behavior of Chls qualitatively. More recently, quantum chemical calculations based on ab initio and density functional methods6 were applied to understand excited-state properties of Chls and Bacterio (B)Chls on a more quantitative level. Besides optical spectroscopy, X-ray based methods can give access to structural, electronic, and chemical information on the Received: June 8, 2016 Revised: October 25, 2016 Published: October 26, 2016 11619

DOI: 10.1021/acs.jpcb.6b05791 J. Phys. Chem. B 2016, 120, 11619−11627

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The Journal of Physical Chemistry B investigated molecular system. The structural information on Chls and Chl containing pigment−protein complexes can be traced back to the analysis of crystallized samples investigated by X-ray diffraction (XRD) analysis.7,8 However, in terms of dynamic processes, a more native environment is required. With X-ray absorption spectroscopy (XAS), molecules can be investigated in solutions that resemble their native environment. XAS can probe transitions from the ground state of the absorbing atom. By tuning the excitation energy across the absorption edge of a selected core level electron, probing of the density of unoccupied states in the vicinity of the absorbing central metal atom and the specific bonds to intramolecular neighbors is possible. The analysis of the spectral region of up to about 50 eV above the absorption edge of a selected atomic core level electron is called near edge X-ray absorption fine structure (NEXAFS) spectroscopy. The analysis of the higher energy region, extending over several 100 eV, is called extended X-ray absorption fine structure (EXAFS) spectroscopy. While NEXAFS yields information about the chemical bonding to the surrounding neighbors and the chemical state of the investigated element, the EXAFS region can reveal specific bond lengths and angles between atoms in a larger radius. There are numerous previous studies on properties of (B)Chl containing pigment−protein complexes in solution, employing optical spectroscopic methods, to elucidate, e.g., transition dipole enhancements due to strong excitonic coupling.9 X-ray spectroscopic investigations on the photosystem II (PS II)8 can be found, too, essentially to study structure and function of the oxygen-evolving manganese cluster. However, NEXAFS investigations on Chl a in solution were performed for the first time in this study. At moderate concentrations (10−3 − 10−2 mol/L), XAS in fluorescence detection mode allows investigations of the signal dynamics as compared to conventional transmittance schemes, calling for both high molecular concentrations and rather small sample dimensions. At very low concentrations, XAS in fluorescence detection mode competes with excitation radiation scattered elastically at the liquid matrix, thus causing a final detection limit. Here, the concentration of Chl a is in a range where aggregation does not occur, and thus, the isolated molecules can be examined. Due to the small amount of Mg per Chl molecule, the detected Mg signal derives from a minor component of the investigated solution. As a first approximation, porphyrins, as well as (metal-) phthalocyanines can be used as model systems for biological redox-active compounds such as Chls in PS II due to their similar molecular structure.10,11 Comparison to other porphyrins can be justified because all transitions, which are probed in the near-edge region, are mainly located on the central tetrapyrrole ring (see Figure 1) and are, therefore, comparable to the general porphyrin structure. NEXAFS spectra of magnesium- and zinc-containing porphyrins have been calculated using a time-dependent density functional theory (TDDFT) as well as a time- dependent Hartree−Fock (TDHF) approach.12 However, the presented results are not readily applicable to the behavior of the Mg atom in Chl because they are dealing with an ideal porphyrin structure where all MOs are highly symmetric. In contrast, the Chl molecule is characterized by its rich peripheral structure that modifies the absorption properties. Because of this, analysis of XAS data using modern quantum chemical approaches like

Figure 1. Structure of chlorophyll a with the central magnesium ion surrounded by four nitrogen atoms as nearest neighbors. The absorption characteristics of Chl a are mainly determined by the interaction of the central Mg ion and the tetrapyrrole ring. Without Mg, the derivative is called pheophytin a.

FEFF,13−15 FDMnes16 and DFT17,18 can provide both structural data and information about electronic/excitonic properties of Chls in solution. In this work, NEXAFS investigations on the Mg K-edge of Chl a in solution are performed and compared to spectra of a Mg-containing salt (MgCl2). To the best of our knowledge, the presented data are the first NEXAFS spectra of dissolved Chl. The main challenges involved are the study of intact liquid biological and radiation-sensitive samples by an X-ray spectroscopic technique. Furthermore, the detection of Mg as a minor component in the Chl a solution can be seen as a proof-of-principle investigation for sample systems where only a small amount of sample is available. To reveal a more comprehensive view on the MO distribution within the Chl a molecule, the experimental results are compared to theoretical predictions using DFT calculations based on molecular orbital data. The combination of the investigation of biological samples in solution and the interpretation of NEXAFS characteristics by theoretical considerations opens up the possibility to realize time-resolved XAS experiments, where ultrafast exciton dynamics in Chl complexes in solution on a molecular length scale can be monitored and interpreted.

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EXPERIMENTAL AND COMPUTATIONAL METHODS Sample Preparation. For the following experiments, large amounts of Chl were required. However, the samples do not require high purity. Thus, crude Chl a was extracted with methanol from cells of the cyanobacterium Thermosynechococcus elongatus. Petroleum ether was added to the extract as well as salt-saturated water. After vigorous shaking, the Chlcontaining phase was removed. This step was repeated several times to remove the majority of carotenoids. Finally, the enriched Chl a was dried under a stream of nitrogen in the dark. For the XAS measurements, the dried Chl a was dissolved in ethanol (EtOH). Prior to XAS measurements, the integrity and concentration of Chl a in EtOH were analyzed using a PerkinElmer Lambda900 spectrometer (see Figure 2). The concentration of Chl a in solution was identified by the absorption at 664 nm (Qy band5). With the extinction coefficient ε of Chl a in 80% ethanol (derived from the coefficient for 80% acetone19) the concentration c of the prepared solution can be calculated: 11620


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The Journal of Physical Chemistry B Table 1. Overview of the Investigated Samplesa sample

state

NEXAFS measurements

Mg (ppm)

Chl a−EtOH Chl a−EtOH

solution dried droplets

fluorescence fluorescence

65

MgCl2(H2O)6− H2O MgCl2(H2O)6− H2O metal Mg

solution

fluorescence

60

dried droplets solid (foil)

fluorescence

Mg (ng/mm2) 2.9 ± 0.6 10 ± 2 117 ± 14

transmission

The concentration of the solutions was in each case 1 × 10−2 mol/L. NEXAFS measurement mode and atomic fraction of Mg are listed. For the dried droplet samples, the amount of Mg quantified by referencefree XRF at the spots of the NEXAFS measurements is given. Results for 1 × 10 μL or 3 × 10 μL (Chl a) and 20 μL (MgCl2) droplets are displayed (beam size: 40 μm × 105 μm for Chl a and 22 μm × 105 μm for MgCl2). a

Figure 2. Vis spectra of dissolved Chl a in ethanol after preparation and after 1 and 2 days, respectively. The shift toward lower wavelengths of the absorption band at 664 nm indicates a partial degradation of Chl a to its derivative pheophytin a. The total measuring time was around 10 min.

⎛ ⎞ A 664nm cChla − 80%EtOH = ⎜ ⎟ ⎝ εChla − 80%EtOH × d ⎠

of MgCl2(H2O)6 solution that corresponds to 4.8 μg of Mg was dried on a substrate for comparison to evaporated Chl a droplets. Computational Methods. In order to analyze the experimental results, density functional theory (DFT) simulations including geometry optimization were performed. The optimization of the structure of the molecule was carried out by the Amsterdam Density Functional (ADF) 201421−23 package using the triple zeta basis set with one polarization function. A popular generalized gradient approximation functional with the latest dispersion correction was used with a more moderate Becke and Johnson damping function.17,18 The approach to calculate the NEXAFS spectrum was based on molecular orbital data, computed by ADF as a result of all-electron single-point calculations. Molecular orbitals of interest were projected onto the three-dimensional cubic grid centered on the absorbing atom. For the Mg K-edge NEXAFS these were two 1s orbitals and several hundred unoccupied orbitals above the lowest unoccupied molecular orbital (LUMO). The spectrum was formed as a result of the calculation of dipole matrix elements for transitions between the occupied and unoccupied molecular orbitals

(1)

The extinction coefficient ε of Chl a in 80% EtOH is εChla−80%EtOH = 78.8 mol−1 L cm−1. Because of the high optical density of the stock solution, 10 μL was diluted 100-fold with an additional 1000 μL of 80% EtOH for obtaining UV−vis spectra, resulting in a stock solution concentration of 1 × 10−2 mol/L. With one magnesium (Mg) atom per Chl a molecule (C55H72O5N4Mg, see Figure 1), this results in an atomic fraction of 65 ppm for Mg in the 1 × 10−2 mol/L solution which was expected to be well above the absolute detection limit for XAS in fluorescence detection mode. Between sample preparation and NEXAFS measurements was a time gap of 48 h, during which a partial degradation from Chl a to its derivative pheophytin a occurred. This was monitored by UV− vis measurements. The further absorption decrease on the second day after preparation (dashed line in Figure 2) indicates an ongoing degradation process.20 Nevertheless, a ratio of at least 60% of intact Chl a in comparison to pheophytin a in this primary solution can be estimated by the deconvolution of the absorption spectra. Hence, the following NEXAFS measurements were performed with a 1 × 10−2 mol/L ethanol solution of a mixture of Chl a and its derivative pheophytin a. The primary solution was also used to prepare dried Chl a samples. These were made by drying 1 × 10 μL and 3 × 10 μL droplets of Chl a solution on a substrate in a dark room for several hours at room temperature, resulting in inhomogeneous films of microcrystalline aggregates. In these samples, a total amount of 2.7 μg and 8.1 μg of Mg, respectively, was present (see Table 1). Since the same primary solution was used, Chl a and pheophytin a coexisted in the dried samples. A metallic Mg foil (Lebow Co.) of 2 μm thickness was chosen as the solid reference sample. MgCl2(H2O)6 was purchased from Merck and was used without any further purification. MgCl2(H2O)6 was chosen as a reference sample and can be investigated as a dried sample or a liquid sample dissolved in water. Crystalline MgCl2(H2O)6 (20.3 mg) was dissolved in 10 mL of doubledistilled water to achieve a 1 × 10−2 mol/L solution yielding a Mg2+ concentration of 60 ppm, comparable to the Mg concentration in the Chl a solution. Likewise, a 20 μL droplet

2

IKNEXAFS ∼

LUMO + N

∑ ∑ k = 1 j = LUMO

|⟨f j |d|̂ ik⟩|2

(2)

where d̂ is the dipole operator, i and f are core (initial) and unoccupied (final) orbitals, respectively, k specifies two Mg 1s orbitals, and j runs over N unoccupied orbitals above the LUMO. N is typically of the order of several hundred, depending on the chosen basis set and the energy range to be calculated. Numerical integration was performed by a separate in-house program.24 An arctangent energy-dependent broadening was applied to the initially obtained discrete spectrum in order to match the resolution of the experimental data. NEXAFS Measurements. Magnesium K-edge NEXAFS measurements were carried out at the plane grating monochromator25,26 beamline for undulator radiation in the Physikalisch-Technische Bundesanstalt (PTB)26 laboratory at the electron storage ring BESSY II in Berlin. This beamline provides soft X-ray radiation of high photon flux and spectral purity in the photon energy range of 78 to 1860 eV. For the measurements, two ultrahigh vacuum chambers consecutively 11621


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The Journal of Physical Chemistry B arranged at the beamline were used.27,28 Both chambers enable measurements in transmission mode by photodiodes and in emission mode by either an absolutely calibrated energydispersive silicon drift detector (SDD), as used in this work, or a wavelength-dispersive spectrometer. The liquid samples were measured in one chamber, and the dried samples were analyzed in the second chamber. A detailed description of the instrumentation can be found elsewhere.28−30 The NEXAFS measurements of the solutions and the dried Chl a and MgCl2(H2O)6 samples were performed in fluorescence detection mode using an SDD (BrukerNano) with a 17 mm2 active chip area. For the sake of highest detection efficiencies in the soft X-ray range, the detector is windowless and requires vacuum conditions of at least 10−6 mbar for the cooled detector chip. The liquid samples were measured by using a newly selfdeveloped ultrahigh vacuum-compatible liquid cell that enables soft X-ray analytical measurements while ensuring the required vacuum conditions. The liquid of interest is located behind an ultrathin window and is probed through this window. Here, 150 nm thick Si3N4 windows (Norcada, Inc.) were used with a transmission of at least 94% in the energy range of interest. The liquid cell features in-cell pressure monitoring to control the distortion of the thin window and to prevent possible window rupture.31 The NEXAFS measurements in fluorescence detection mode were carried out in conventional 45°/45° Xray fluorescence (XRF) beam geometry of the incident beam and detection direction with respect to the sample surface. An additional filter (12 μm beryllium foil) was applied to suppress the high count rate at the SDD induced by mainly C Kα and O Kα fluorescence radiation of the liquid matrix elements. Mg K-edge XAS measurements were performed between 1280 up to 1550 eV with the different liquid, dried, and solid samples, respectively. In the NEXAFS region, from 1300 to 1320 eV, step sizes of 0.25 and 0.5 eV were chosen. For higher energies in the EXAFS region, different step sizes between 0.5 and 5 eV were applied to optimize the total measurement time. Within this energy region, the monochromator provides an energy resolution of at least 0.7 eV depending on the exit slit and the chosen cff value32 of the grating monochromator of the beamline. In fluorescence detection mode, XAS typical data collection times for one X-ray fluorescence spectrum amounted to 30−90 s. Every spectrum was deconvoluted using experimentally determined detector response functions to extract the Mg Kα count rate.33 In particular, for the liquid Chl a samples the Mg fluorescence signal had to be distinguished from the elastic scatter peak in the spectrum, which was in the same order of magnitude due to the substantial scattering cross section of the ethanol solution. The NEXAFS measurement of the Mg foil was performed in transmission mode. The transmitted intensity and the incoming beam intensity were measured by calibrated silicon photon diodes. The incoming beam intensity was monitored during both, transmission and fluorescence mode measurements by a photo diode located at the beamline exit slit behind the monochromator. The quantification of the mass deposition of Mg in the dried samples was performed by reference-free XRF analysis33 using the Mg K(α + β) count rate of the last XRF spectrum in the NEXAFS scan at an excitation energy of 1550 eV. Analysis of the NEXAFS Spectra. The energetic positions of characteristic features in the NEXAFS spectra were determined by fitting with Gaussian distributions and an arc

tangent function for the edge jump using the program ATHENA Version 0.8.061.34 Prior to the fit procedure, the data were normalized with regard to the pre- and postedge regions. In the spectrum obtained by the transmission measurement of the Mg foil, the y-axis is labeled with “norm. μd”. μ is the element-specific absorption coefficient, and d is the sample thickness. This product displays the exponential decrease of the incoming beam intensity due to the absorption in the sample (Lambert−Beers law).

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RESULTS AND DISCUSSION Solid Reference: Mg Foil. Figure 3 shows the Mg K-edge XAS spectrum of a thin Mg foil. The Mg K-edge of metallic Mg

Figure 3. Normalized Mg K-edge absorption spectrum of a 2 μm Mg metal film measured in transmission mode with the total measuring time of 150 min. The energetic position of the Mg K absorption edge at (1303.6 ± 0.7) eV is indicated by the dotted line. The detailed near edge region is displayed in the inset. Feature A corresponds to a Mg 1s → 3p transition and is indicated by a dashed line.

can be identified at (1303.6 ± 0.7) eV (indicated by a dotted black line). The first near-edge structure, A, which is visible as a smooth shoulder shortly above the absorption edge, is positioned at (1305.9 ± 0.7) eV and can be assigned to a Mg 1s→ 3p transition35 (dashed line, inset Figure 3). The oscillations at higher energies (EXAFS region) are due to multiple scattering of ejected photoelectrons at the Mg atoms. The interpretation of the EXAFS region is beyond the scope of this work. Liquid Reference System: 1 × 10−2 mol/L MgCl2(H2O)6 Solution. In Figure 4a, the NEXAFS spectrum at the Mg Kedge of a MgCl2(H2O)6 solution (dotted line) as well as the spectrum of a dried MgCl2(H2O)6 droplet (solid line) are displayed. Due to the full dissociation of MgCl2(H2O)6 in water, the Mg2+ cation is surrounded by water molecules forming a hydration structure, and hence, a long-range order connected with the Mg atoms is absent. This is expressed in a less pronounced structure of the NEXAFS spectrum of the liquid sample in comparison to the dried MgCl2(H2O)6. Compared to the spectrum of the Mg foil, significantly fewer oscillations of the EXAFS region are visible, indicating a stronger scattering by the neighboring atoms of the Mg ions. 11622


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Figure 5. Normalized Mg K-edge NEXAFS spectra of dried Chl a droplets (sample 1: green, 1 × 10 μL of Chl a solution; sample 2: red, 3 × 10 μL of Chl a solution). The decrease of the main peak as well as the intensity of the prepeak indicate progressive radiation damage of the Chl a molecules with each additional measurement. The total measuring time for each spectrum was 28 min.

Figure 4. Normalized Mg K-edge NEXAFS spectra of the MgCl2(H2O)6 solution (dotted line) and dried MgCl2(H2O)6 droplet (solid line, a). Both spectra were measured in fluorescence mode with the total measuring time of 150 min. (b) Pre-edge region is displayed in detail. In the NEXAFS spectrum of the dried MgCl2(H2O)6 a small pre-edge feature at 1306 eV can be resolved (solid line, b).

The main peak in the NEXAFS spectrum is located around (1311.0 ± 0.7) eV, and a pronounced pre-edge feature at (1305.5 ± 0.7) eV can be resolved (arrows) in all spectra. With an ongoing number of measurements, the intensity of the main peak, as well as that of the pre-edge peak, decrease whereas the energetic positions of both features do not shift. This suggests that upon irradiation the environment of the Mg central atom is changing and Chl a molecules are increasingly being damaged. Finally, Mg NEXAFS spectra of Chl a in solution were measured. Two scans were executed; an overview scan over a wide energy range (1280 to 1500 eV) and a second, detailed scan with smaller step size around the absorption edge region. The NEXAFS spectrum of the Chl a solution shows a similar shape in comparison to the spectrum of dried Chl a (Figure 6, left). The stronger noise in the solution NEXAFS spectra can be attributed to the higher scattering background due to the high carbon content of the EtOH solution. However, a noticeable shift of ∼1 eV of the main peak toward higher energies is visible, which was detected also by comparison between liquid and dried MgCl2 solution. This change can be explained by the influence of the surrounding solvent. The released Mg2+ cations that are present due to the decay of Chl a to pheophytin a interact with anions in the EtOH solution. Due to the lower amount of oxygen atoms in EtOH compared to pure water and less free Mg ions; the shift is not as distinctive as in MgCl2 solution. A closer look at the pre-edge region indicates the presence of a small feature in the spectra of dried and dissolved Chl a, which can be analyzed more precisely in the detailed NEXAFS spectra around the absorption edge (Figure 6, right). In the solution spectrum, a pre-edge peak at (1306.0 ± 0.7) eV is discernible. Again a shift toward higher energies of the NEXAFS spectrum of dissolved Chl a is clearly visible, like in the previous measurements (Table 3). In contrast to the results of the measurements with MgCl2 for the NEXAFS spectra of both dissolved and dried Chl a, a characteristic pre-edge peak can be resolved. Due to the correlation of this feature with the central Mg ion, which is still attached to the chlorin ring, this indicates the presence of intact Chl a molecules in the solution, which are neither damaged by radiation nor during sample preparation. These spectra show

A defined first sharp peak (main peak at 1309.0 eV, usually denoted as “white line”) and two more features at higher energies (1311 and 1320 eV) can be resolved in the NEXAFS spectrum of the dried solution. In addition, the main peak of the MgCl2(H2O)6 solution and the energy position of the Mg K absorption edge are shifted to higher energies as compared to the dried MgCl2 (see Table 2). This result is in good agreement Table 2. Energetic Positions of Mg K Absorption Edge, Preedge Features, and Main Peak in the NEXAFS Spectra of the Mg Foil (Figure 3) and the Liquid and Dried MgCl2(H2O)6 Solution (Figure 4)a sample Mg foil MgCl2(H2O)6 (dried) MgCl2(H2O)6 (solution)

K-edge (eV) 1303.6 1307.9 1311.8

pre-edge (eV)

first feature/main peak (eV)

ref (eV)

1306.4

1305.9 1309.0

1304.535 1309.537

1314.5

All positions are determined with an energy resolution of ΔE = 0.7 eV. The given reference values correspond to the results determined for the main peak.

a

with previous work on effects of concentration on the solvation structure of Ca2+ in aqueous solution.36 Due to the relatively low concentration of 10−2 mol/L, the Mg2+ ions are fully dissociated and strongly hydrated. With respect to the results published in ref 36, the intense peak at (1314.5 ± 0.7) eV of the MgCl2(H2O)6 solution can be explained by Mg−O scattering in the first shell around the Mg atoms. A further indication of the absence of Mg2+−Cl− binding is a lack of the pre-edge feature at (1306 ± 0.7) eV, which can be detected in the measurement of the dried MgCl2 (Figure 4b, arrow). Thus, this peak can be used as a fingerprint for an intact crystal or molecular structure meaning that the Mg ion is binding. NEXAFS Spectroscopy of Chl a Solution and Dried Chl a Droplets. For each dried Chl a droplet with different amounts of solution three NEXAFS spectra were recorded at the same position. The spectra of sample 1 (least amount) and 2 (highest amount) are depicted in Figure 5. 11623


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Figure 6. Mg K-edge NEXAFS spectra of a Chl a solution (solid line) and dried Chl a droplets (dashed line). A significant pre-edge peak can be resolved in all spectra and assigned to intact Chl a both in solution and in dried droplets. The total measuring time for each spectrum of the overview scan was 85 min (left) and 28 min for the detailed scans around the absorption edge (right).

of the ligands (C, N atoms) and 3px, y orbitals of the Mg atom. The energetic positions are in good agreement with the TDDFT calculations in ref 12 where the transition Mg 1s → 3pz is calculated to be at 1306.26 eV and the 1s → 3px, 1s → 3py transitions are 1309.37 and 1309.40 eV, respectively. The observed pre-edge feature is typical for the square-planar structure of a metalloporphyrin. Likewise, these results were confirmed by our DFT calculations using the Chl a molecular structure in which the pre-edge feature originates in the MOs mainly located at the nearest nitrogen and carbon atoms in the porphyrin ring-structure. The experimental results, the energetic positions of our DFT calculations, and the assigments of the transitions based on the results of ref 12 are summarized in Table 4.

Table 3. Energetic Positions of Mg K-Edge, Pre-edge Feature, and Main Peak of NEXAFS Measurements of Dissolved (Sol) and Dried Chl a Droplet Samplesa sample

K-edge (eV)

pre-edge (eV)

main peak (eV)

dried Chl a, detailed scan dried Chl a, long scan Chl a sol, detailed scan Chl a sol, long scan

1308.4 1308.7 1308.8 1309.5

1305.5 1306.5 1306.0 1307.1

1310.8 1311.7 1312.1 1312.8

a

The given energies are referring to the spectra shown in Figure 6 and are determined with an energetic resolution of ΔE = 0.7 eV.

the successful examination of the central Mg ion of intact Chl a with NEXAFS spectroscopy. Computational Results and Interpretation of the Spectra. Electronic transitions contributing to the spectrum of Chl a together with the most important molecular orbitals (MO) calculated are presented in Figure 7. Transitions forming the pre-edge around 1306 eV (MOs: 245, 248, 249, 251) originate mostly from the molecular orbitals situated on the magnesium ion as well as nitrogen and carbon atoms from the pyrrole rings. The transitions on the pre-edge are suppressed after the convolution because of the low density of states compared to the higher energy part above the K absorption edge. The first three most intense LUMOs result from spd and sp hybridization of the Mg atom. Peripheral ligands and the phytol chain of Chl a show almost no contribution, while they are the main source of intensity for the main peak at 1312 eV (main peak, MOs: 328, 333 and 334). Spatial distributions of the computed molecular orbitals are presented, too (Figure 7, side columns). The theoretical spectrum is scaled and shifted along the energy axis with respect to the experimental spectra. First, the experimental results were compared to previous work on calculated absorption near-edge spectra of Mg porphyrins.12 Second, DFT calculations of Mg NEXAFS of the correct Chl a molecule structure were computed to verify the results. Following the notation given in ref 12, the pre-edge peak can be assigned to the Mg 1s → 3pz transition belonging predominantly to MO 245, 248, and 249 with A2u symmetry. The shoulder at 1310 eV visible in both experimental and theoretical spectra belongs to the Mg transitions 1s → 3py and 1s → 3px. The related MO 287 is a mixture of 2s, 2px, y orbitals

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CONCLUSION

In this work, Mg K-edge NEXAFS spectra of Chl a in solution were investigated. The results were compared to Mg K-edge NEXAFS measurements of a Mg foil and MgCl2 in solution. In order to elucidate the influence of the solvent on the spectra, dried samples of MgCl2 and Chl a were included in the investigations. Using a novel liquid cell for measurements under vacuum conditions, a Mg K-edge NEXAFS spectrum of intact Chl a molecules has been recorded for the first time (Figure 6). The experimental results are supported by DFT calculations, which confirm that characteristic structures in the NEXAFS spectrum can be assigned to transitions and atoms which are located in the intact Chl molecular structure (Figure 7). Furthermore, the DFT calculations and the experimental data confirm and expand previous simulations of X-ray absorption near-edge spectra of organometallic compounds as reported in ref 12. The reference system MgCl2[H20]6 was measured as solution and as dried droplets on a substrate comparable to the Chl a solution with a similar concentration of 10−2 mol/L. In the NEXAFS spectra of the dried droplets a pronounced pre-edge feature can be resolved which can be assigned to binding between the Mg ion and the nearest neighboring atoms of the respective sample, Cl in MgCl2, and N in Chl a. In contrast, no pre-edge peak in the NEXAFS spectrum of dissolved MgCl2(H2O)6 is resolvable (Figure 4, Table 2). The NEXAFS spectra of Chl a in solution show a stronger noise due to the higher scattering background of the carbon-containing ethanol solution but feature the same characteristic structures that are 11624


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Figure 7. Calculated and experimental NEXAFS spectra of Chl a. The convoluted NEXAFS spectrum calculated with DFT (black dotted line) displays the same characteristic features as in the measured Chl a NEXAFS spectra (top). The prominent pre-edge peak can be assigned to MOs, which are mainly located at the N and C atoms of the porphyrin ring around the central Mg atom, whereas the main peak originates from MOs from the periphery of the Chl molecule (bottom, side columns).

Table 4. Summary of Calculated MOs and Transition Assignment structure pre-edge

shoulder main peak

MO

MO energy (eV)

exptl results (eV)

assignment/symmetry according to ref 12

calcd energy (eV)

245 248 249 251 287

1306.1 1306.7 1306.8 1307.2 1309.9

1306 ± 0.7

Mg 1s → 3pz/A2u

1306.26

1310 ± 0.7

Mg 1s → 3px,y N, C 2s, 2px,y/Eu

1309.37 1309.40

328 334

1311.7 1312.0

1312 ± 0.7

displayed in the NEXAFS spectra of Chl droplet (Figure 6). The prominent pre-edge feature at (1306.5 ± 0.7) eV can be assigned to intact Chl a by means of DFT calculations. These calculations provide proof that this structure can be attributed to the LUMOs of the central Mg ion and the surrounding N and C atoms from the pyrrole rings. Hence, this can be taken as an evidence for the integrity of the respective Chl a molecules. This work demonstrates the feasibility of analyzing the electronic structure of photosynthetic pigments like Chl a in solution. Additionally, the successful measurement of a minor component with a concentration of less than 100 ppm in a dilute sample, providing a small detectable fluorescence signal, gives rise to several fields of application, where only minute amounts of sample are available, as is often the case for biological specimens. A further prospect for future studies concerning the coordination of the central Mg ion in the Chl molecule could be EXAFS investigations of Chl molecules embedded in their native protein matrices. Additionally, the

influence of different solvents and solvent−water mixtures on the coordination of the Mg ion38,39 can be analyzed by using EXAFS spectroscopy. With this method, access to bond lengths and coordination to ligands of the central metal ion can be gained. The successful investigation of a rather intact radiationsensitive specimen without any further sample preparation as well as measurements with soft X-ray radiation on a solution under (ultra)high vacuum conditions leads to new application possibilities in studies of biological samples, like the monitoring of dynamic processes. Both experimental and theoretical results of the present work can be expected to raise confidence in successful optical-pump X-ray probe measurements, for example, with native photosynthetic pigment−protein complexes.40 Monitoring of structural changes assessed by NEXAFS spectra, depending on the delay time between probe and pump pulses in combination with theoretical considerations involving molecular orbitals, may help to elucidate the functions of these 11625


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pigments in their native environments. The current work opens up new prospects to study (components of) the photosynthetic apparatus by monitoring in real time structural changes which are connected with, e.g., energy and/or charge-transfer processes.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +49 30 314 79851. *E-mail: [email protected]. Tel: +49 30 6392 1351. Present Address #

(S.A.S.) Leibnitz-Institut für Analytische Wissenschaften Berlin, 12489 Berlin, Germany.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge Katrin Herrmann (Max-Born-Institute) for help with the UV−vis measurements. Calculations were performed at the high-performance computing cluster “Blokhin” in the International Research Center “Smart Materials” of the Southern Federal University. H.L. acknowledges financial support from the Grant Agency of the Czech Republic, GAČ R, No. P501/12/G055. S.A.S. acknowledges financial support from the DFG project GSC1013-SALSA.

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Magnesium K-Edge NEXAFS Spectroscopy of Chlorophyll a in

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