X-ray Emission Spectroscopy of Proteinogenic Amino Acids at All

Article pubs.acs.org/JPCB

X-ray Emission Spectroscopy of Proteinogenic Amino Acids at All Relevant Absorption Edges F. Meyer,†,◆ M. Blum,‡ A. Benkert,†,§ D. Hauschild,§,∥ Y. L. Jeyachandran,⊥,¶ R. G. Wilks,# W. Yang,∇ M. Bar̈ ,#,○ C. Heske,‡,§,∥ F. Reinert,† M. Zharnikov,*,⊥ and L. Weinhardt*,‡,§,∥ †

Experimentelle Physik VII, Universität Würzburg, Am Hubland, 97074 Würzburg, Germany Department of Chemistry and Biochemistry, University of Nevada, Las Vegas (UNLV), 4505 Maryland Parkway, Las Vegas, Nevada 89154-4003, United States § Institute for Photon Science and Synchrotron Radiation (IPS), Karlsruhe Institute of Technology (KIT), Hermann-v.-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany ∥ Institute for Chemical Technology and Polymer Chemistry (ITCP), Karlsruhe Institute of Technology (KIT), Engesserstr. 18/20, 76128 Karlsruhe, Germany ⊥ Applied Physical Chemistry, Heidelberg University, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany # Renewable Energy, Helmholtz-Zentrum Berlin für Materialien und Energie GmbH, Hahn-Meitner-Platz 1, 14109 Berlin, Germany ∇ Advanced Light Source, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, California 94720, United States ○ Institut für Physik, Brandenburgische Technische Universität Cottbus-Senftenberg, Platz der Deutschen Einheit 1, 03046 Cottbus, Germany ‡

S Supporting Information *

ABSTRACT: Nonresonant N K, O K, C K, and S L2,3 X-ray emission spectra of the 20 most common proteinogenic amino acids in their solid zwitterionic form are reported. They represent a comprehensive database that can serve as a reliable basis for the X-ray absorption spectroscopy (XES) studies of peptides and proteins. At the most important N and O K edges, clear similarities and differences between the spectra of certain amino acids are observed and associated with the specific chemical structure of these molecules and their functional groups. Analysis of these spectra allows the generation of spectral fingerprints of the protonated amino group, the deprotonated carboxylic group, and, using a building block approach, the specific nitrogen- and oxygen-containing functional groups in the side chains of the amino acids. Some of these fingerprints are compared to the spectra of reference compounds with the respective functional groups; they exhibit reasonable similarity, underlining the validity of the spectral fingerprint approach. The C K and S L2,3 XES spectra are found to be specific for each amino acid, in accordance with the different local environments of the involved C and S atoms, respectively.

1. INTRODUCTION X-ray absorption spectroscopy (XAS) and X-ray emission spectroscopy (XES), investigating the unoccupied and occupied electronic states, respectively, are powerful tools to study the electronic structure of a variety of systems ranging from solidstate materials to molecules and gases. In particular, the investigation of biomolecules and biologically relevant compounds using these techniques is a popular research field, especially when combining XAS and XES with resonant inelastic soft X-ray scattering (RIXS). Using suitable experimental setups, such molecules (e.g., amino acids) can also be probed in a solution.1−7 Along with nucleobases, amino acids represent the most important building blocks of nature, composing peptides and proteins. Studies of these larger biomolecules by XAS, XES, and/or RIXS are, however, generally difficult because of their complexity and a large © 2017 American Chemical Society

number of functional groups involved, which is also reflected in the complexity of the spectra. A rational way to proceed is to reduce the complexity by cataloguing and understanding the spectra of their building blocks, that is, amino acids, as well as by developing basic rules describing the modification of these spectra upon the formation of peptide bonds between individual amino acids. This has been successfully accomplished in the case of XAS. Comprehensive spectral databases for proteinogenic amino acids, first at the C K edge8 and, later, at all of the relevant absorption edges9 (except for the S L2,3 edge of cysteine and methionine) were recorded and analyzed in detail. Further, Received: May 5, 2017 Revised: June 12, 2017 Published: June 14, 2017 6549

DOI: 10.1021/acs.jpcb.7b04291 J. Phys. Chem. B 2017, 121, 6549−6556

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The Journal of Physical Chemistry B

Figure 1. Molecular formulas of the 20 studied amino acids in the zwitterionic form characteristic of the solid state, along with the respective abbreviations given in parentheses. In lysine and arginine, not the α-amino group but a group in the side chain is protonated.

al., who studied not only aqueous glycine but also lysine and showed that the N K XES spectra of these amino acids are strongly affected by ultrafast proton dynamics.5 Further, taking aqueous cysteine as a representative amino acid, we have demonstrated earlier that the XES spectra and the RIXS maps of these compounds can be adequately described within the building block model,7 an approach that is commonly used in XAS16 but not widely established for XES. This approach relies on the element-specific nature and the local probing character of XES and suggests that the spectrum of a complex molecule can be approximated by a superposition of specific contributions of individual functional groups (building blocks), taking these contributions as the spectral fingerprints of individual moieties. It was found that the only limitation of this approach for XES of amino acids is the need to include all of the nearestneighbor atoms of a particular group, as these atoms can affect the group-specific fingerprint spectra to a noticeable extent.7 To form the basis of a systematic building block approach also with XES (i.e., for the occupied electronic states), we present and analyze the XES spectra of the 20 most common

these libraries were used as a basis to interpret and understand the spectra of homopeptides and heteropeptides, and even those of proteins.10−14 At the same time, a software tool, XSpecSim, has been developed that allows the prediction of XAS spectra of peptides and proteins of arbitrary sequence from the libraries of the spectra of the constituent amino acids.11 In addition, the spectral databases for amino acids serve as a basis for dedicated adsorption and solution studies involving these species as well as derived peptides and proteins. In particular, the basic issue of protein conformation and interactions with salts in a solution can be addressed.15 In contrast, a database for proteinogenic amino acids is not yet available for XES, even though some important results for individual amino acids, in different ionic forms and different phases (solid state and solution), have been published.4,5,7 In particular, taking aqueous glycine in different ionic states as a test system, Gråsjö et al. demonstrated that protonation has a strong influence on the local electronic structure of amino acids, and that specific functional groups give distinct spectral fingerprints.4 These conclusions were later verified by Blum et 6550


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Figure 2. Nonresonant N K XES spectra of the 20 studied amino acids and spectral fingerprints of specific functional groups: (a) spectra of amino acids with only one nitrogen atom, except for proline; (b) superposition of the spectra from (a) (thin gray solid lines) as well as the average of these spectra (black solid line) representing a spectral fingerprint of a protonated primary amine group (NH3+); (c) spectra of the amino acids with more than one nitrogen atom and proline; (d) spectral fingerprints of the residual (except for NH3+), nitrogen-containing functional groups in the amino acids with more than one nitrogen atom. The fingerprints, termed “side chain” spectra in the main text and marked by * in (d), were obtained by subtracting the weighted NH3+ contribution shown in (b) from the original spectra presented in (c). The side-chain spectrum of histidine is compared to that of aqueous (pH = 10.3) imidazole (red solid line). The spectra of proline and arginine in (d) are reproductions of the spectra in (c), as the subtraction procedure is not applicable to these amino acids due to the lack of NH3+ group.

amino acids with traces of water cannot be ruled out completely. The XES spectra of the amino acids were acquired with nonresonant excitation at the N, O, and C K edges using the excitation energies of 424, 557, and 322 eV, respectively. For the S L2,3 edge, the excitation energies of 182 and 202 eV were used for methionine and cysteine, respectively. The excitation energy and emission energy scales were calibrated simultaneously using the reference measurements and literature values for N2,18 TiO2,19 and CdS.20 The experiments were performed with the custom-designed Solid and Liquid Spectroscopic Analysis endstation21 at Beamline 8.0.1 of the Advanced Light Source, Lawrence Berkeley National Laboratory. This endstation is equipped with a variable line space soft X-ray spectrometer, which is built in a slit-less design and optimized for maximum transmission at the biologically relevant absorption edges (O, N, and C K, as well as S L2,3).22 To minimize radiation damage to the amino acids, the samples were continuously moved under the beam while recording the spectra. On the basis of previous experience, scanning speeds of >200 μm/s (corresponding to an exposure time of ∼0.15 s and a dose of ∼3.7 MGy for an individual sample spot)23 were chosen to avoid the spectral contributions of decomposed molecules.

proteinogenic amino acids (Figure 1) in the solid state at all of the relevant absorption edges. As a solid, the zwitterionic configuration (i.e., a deprotonated carboxylic group and a protonated amine group) is dominant in these compounds.17 This is also the typical configuration of amino acids in aqueous solutions at the physiologically relevant (neutral) pH values, even though, for some amino acids, differences exist regarding the protonation and deprotonation of certain functional groups as compared to the solid state. In view of the large amount of the experimental data and their spectral database character, our goal is to describe general trends and derive specific fingerprint spectra of the individual functional groups, which are common for all amino acids or specific for some of them. In this context, we do not distinguish the amino acids by the character of their side chain (aliphatic, aromatic, alkaline, etc.), but rather consider them in view of the available nitrogen-, oxygen-, carbon-, and sulfur-containing groups, locally probed at the N K, O K, C K, and S L2,3 edges, respectively.

2. EXPERIMENTAL SECTION All of the amino acid samples, except for cysteine, were prepared by pressing amino acid powders (Alfa-Aesar, stated purity >98%) into pellets without further purification. In the case of cysteine, a thin film was evaporated in high vacuum (∼3 × 10−8 mbar) onto a polished copper plate. For all amino acids, the L-enantiomers were used. Although the sample preparation did not involve any solvents and was performed in a dry environment, a small contamination of the strongly hydrophilic

3. RESULTS AND DISCUSSION 3.1. N K Edge. The nonresonant N K XES spectra of the 20 amino acids presented in Figure 1 are compiled in Figure 2. According to the number and local environments of the 6551


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nitrogen atoms, in the NH3+ group and the side chain, the area of the spectrum was normalized to an area twice as large as that of the NH3+ fingerprint before subtraction. The results of the subtraction procedure are shown in Figure 2d. In view of the local character of XES, we note that this remaining spectral signature is related to the nitrogencontaining functional groups within the side chain of the amino acid (rather than to the entire side chain). Accordingly, the side-chain N K XES spectra of asparagine and glutamine in Figure 2d show large similarities because a primary amide group (H2N−CO−H) is attached to the end of the side chain in both cases. Thus, these spectra can be considered as a spectral fingerprint of the amide group. They consist of a dominant peak at ∼394.9 eV, which is attributed to the lone pair orbital of the neutral amino group, and two broader features at ∼386 and 388 eV. Similar to the above spectra, the spectrum of lysine in Figure 2c is dominated by the lone pair peak at ∼395.5 eV, which is shifted to higher energies compared to the analogous feature for asparagine and glutamine. The low-energy part of this spectrum consists of a single broad emission feature centered at ∼389.9 eV. Subtracting the contribution of the protonated amino group in the side chain (note that, in lysine, it is not the α-amino group but the side chain amino group that is protonated; see Figure 1), one is left with the fingerprint of the neutral amino group (Figure 2d), which differs significantly from that of its protonated counterpart in Figure 2b. Interestingly, the derived fingerprint of the neutral amino group has a certain similarity to the spectrum of aqueous glycine at pH 12.7,5 which is understandable because the amino group of glycine in a basic solution is neutral. Proline, histidine, and tryptophan are amino acids with heterocyclic side chains. In particular, the side chain of histidine consists of an imidazole ring structure linked via a hydrocarbon to the α-carbon. The imidazole ring consists of two nitrogen, three carbon, and three hydrogen atoms. The side-chain spectrum of histidine in Figure 2d is comprised of four main features, namely, a dominant peak at ∼394.3 eV, a high-energy shoulder at ∼395.7 eV, and two well-separated emission features at ∼391.3 and ∼386.8 eV. The side-chain N K XES spectrum of tryptophan in Figure 2d shows several emission features. The most prominent peak is located at ∼397.3 eV, which is the highest emission energy observed in the N K XES spectra of all of the 20 proteinogenic amino acids used in this study. This feature most likely represents a weakly bound aromatic orbital associated with the extended π orbital structure of the aromatic rings. The remaining emission features overlap strongly and create a broad emission band, with the most distinct peaks located at ∼392.7 and ∼388.8 eV. In our previous work, we have shown that the local probing character of XES and RIXS allows us to associate the respective spectra of cysteine with the spectra of suitable reference molecules.7 Referring to this work, the side chain N K XES spectrum of histidine in Figure 2d can be compared to that of imidazole (Imd), representing the same heterocycle as the terminal group of the side chain of histidine. To illustrate this, a N K XES spectrum of a 2 M imidazole solution (pH 10.3) is presented in Figure 2d, drawn in red upon the side-chain spectrum of histidine. The similarities between the two spectra strongly support both the building block principle and the derived NH3+ spectral fingerprint. Note that the spectrum of imidazole had to be measured in the liquid phase because the

nitrogen atoms in these molecules, the spectra can be divided into two groups, namely, the spectra of the amino acids with only one nitrogen atom (in the NH3+ moiety, Figure 2a) and the spectra of the amino acids with more than one nitrogen atom (Figure 2c). The only exception is proline, in which the amine group is part of the heterocycle and in a secondary configuration instead of a primary one (see Figure 1). Its spectrum is shown in Figure 2c. The spectra of the first group in Figure 2a exhibit a very similar N K XES signature, which can be related to the protonated α-amino group (NH3+), a common building block in all of these amino acids. The strong similarities of the shown spectra evidence, in agreement with previous work,4,5,7 the local probing character of XES and also demonstrate that the local electronic structure in the vicinity of the nitrogen atom of the amino group is quite similar for all of the amino acids and is only weakly influenced by the side chain. For most of the amino acids in Figure 2a, the spectrum consists of three emission features, namely, a broad peak at ∼389.8 eV, a weak feature at ∼393.0 eV, and a dominant peak at 395.0 eV. Shape, relative intensity, and exact energy position of these features vary from one amino acid to another. Nevertheless, from the 13 spectra shown in Figure 2a, an averaged N K XES spectrum of the protonated α-amino group (NH3+) can be created, as shown in Figure 2b. The averaged fingerprint shows the same three spectral features as the spectra of the amino acids. These features can be assigned on the basis of a recent XES study of aqueous glycine,5 exploiting the close similarity of the N K XES spectrum for pH 6.5 (zwitterionic state of glycine) and our fingerprint spectrum in Figure 2b. Adapting the conclusions of ref 5, the fingerprint spectrum is highly influenced by ultrafast dissociation processes taking place on the timescale of the corehole lifetime. The low-energy part of the spectrum reflects the electronic structure of the protonated α-amino group (NH3+, with no dissociation-related features), whereas the high-energy part of the spectrum is characteristic of the neutral configuration of the α-amino group (NH2) created by the dissociation. Further, according to ref 5, the low-energy part of the spectrum (∼390.0 eV) is the result of several transitions from lower lying valence orbitals, whereas the dominant peak at ∼394.8 eV can be assigned to the nitrogen lone pair orbital of the neutral amino group (NH2). Compared with the amino acids of the first group (Figure 2a,b), those of the second group, namely, arginine (Arg), asparagine (Asn), glutamine (Gln), histidine (His), lysine (Lys), proline (Pro), and tryptophan (Trp), show more complex and unique N K XES spectra (Figure 2c), underlining the sensitivity of these spectra to the chemical composition of the particular amino acid. Except for proline, all of the amino acids of the second group contain at least two nitrogen atoms, one of which forms the α-amino group. The other nitrogen atoms have a different chemical environment in specific functional groups of the side chain. Because, at nonresonant excitation, each nitrogen atom in the molecule can be excited, the emission spectra in Figure 2c represent the weighted sums of spectral components related to the chemically different nitrogen atoms. In the building block approach,7 the spectral signature of the nitrogen atoms of the side chain can be computed by subtracting the spectral contribution of the protonated α-amino group (Figure 2b) from the original spectrum (Figure 2c), using suitable weight factors. These factors were set according to the number of nitrogen atoms in a particular amino acid. For example, for amino acids hosting two 6552


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Figure 3. Nonresonant O K XES spectra of the 20 studied amino acids and spectral fingerprints of specific functional groups: (a) spectra of the amino acids containing only one carboxylic group with two oxygen atoms; (b) superposition of the spectra from (a) (thin gray solid lines) as well as the average of these spectra (black solid line), representing a spectral fingerprint of a deprotonated carboxyl group (COO−); (c) spectra of the amino acids with more than two oxygen atoms (one carboxylic group); (d) spectral fingerprints of the non-COO− oxygen species in the amino acids with more than two oxygen atoms. The fingerprints, termed side-chain spectra in the main text and marked by * in (d), are obtained by subtracting the weighted COO− contribution in (b) from the original spectra presented in panel (c). All spectra in (a)−(c) are aligned with respect to the energy position of the high-intensity features at an emission energy of 526.7 eV. The spectral fingerprints in the case of serine, threonine, and tyrosine are compared with that of methanol (red solid line).

at first sight, which suggests, analogous to the XAS case,9 that they might be too insensitive to serve as fingerprints of individual amino acids and to be useful to identify them in derived peptides and proteins. Nevertheless, a similar grouping of the O K XES spectra as for the N K edge case can be performed, with the spectra of the first and second groups presented in Figure 3a,c, respectively. To account for small deviations in the energy positions of the spectra (up to 50−100 meV), all of the spectra were aligned with the maximum of the dominant feature set to 526.7 eV. These shifts might be due to a slight uncertainty in energy calibration, but they could also be indicative of a “true” shift caused by the differences in the various molecules. Figure 3a shows the emission spectra of all of the amino acids with only two oxygen atoms in the carboxylic group. The spectral signature is very similar for all these acids, which also indicates that the local electronic structure surrounding the carboxylic group is very similar. Accordingly, like for the N K edge, a spectral fingerprint of the deprotonated carboxylic group can be created by generating the average spectrum, as shown in Figure 3b. The dominant feature at ∼526.65 eV and the shoulder at ∼525.2 eV in this spectrum are most likely related to the HOMO and HOMO − 1 orbitals, respectively, with the electron density distributed over the entire carboxylic group. The broad feature centered at ∼522.2 eV stems from the superposition of several lower lying orbitals.24 The O K XES spectra of all of the amino acids with more than two oxygen atoms (i.e., beyond the carboxylic group), namely, asparagine (Asn), aspartic acid (Asp), glutamine (Gln), glutamic acid (Glu), serine (ser), threonine (Thr), and tyrosine

high vapor pressure of this compound prevented the solid state measurements in UHV. Arginine was excluded from the subtraction procedure because, in this compound, it is not the α-amino group but the guanidinium group at the end of the side chain that is protonated (see Figure 1). Consequently, we just reproduced the original spectrum of this amino acid in Figure 2d. This spectrum represents a superposition of the contributions from the protonated guanidinium group, containing three nitrogen atoms, and the neutral α-amino group. The dominant peak at ∼395.6 eV is accompanied by a low-energy shoulder at ∼393.9 eV, whereas the stronger bound orbitals form the broad emission features at ∼391.9, ∼389.6, and ∼387.5 eV. Another special case, excluded from the subtraction procedure as well, is proline because it has only one nitrogen atom in the α-amine group; however, the aliphatic side chain (hydrocarbons) are back-connected to this group, creating a heterocyclic ring structure. The result is a N K XES spectrum (Figure 2c,d) that differs greatly from the spectra of the protonated α-amino group of the other amino acids (Figure 2a). The distinctly different spectrum indicates that the ring structure highly influences the local electronic structure of the nitrogen atom, and that the entire heterocyclic ring has to be treated as a spectral fingerprint. The N K XES spectrum shows four major emission features, with the most pronounced peak located at ∼395.4 eV. The other three peaks are centered at ∼393.7, ∼392.4, and ∼389.1 eV. 3.2. O K Edge. The O K XES spectra of the amino acids are compiled in Figure 3, along with the derived spectral fingerprints (see below). All these spectra look quite similar 6553


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hosts a hydroxide group. The respective spectrum is shown in red at the bottom of Figure 3d. Although its spectral shape is different from those of serine and threonine, the occurrence and energy positions of the main emission features show a close resemblance. Due to the large intensity of the major spectral feature in the O K XES spectra, the implementation of the building block approach is more complicated than for the N K edge. However, meaningful fingerprint spectra (without the negative intensity) showing features that are in accordance with those of suitable reference molecules could be derived. 3.3. C K Edge. The nonresonant C K XES spectra of the 20 amino acids in this study are compiled in Figure 4. All of these

(Tyr), are compiled in Figure 3c. As mentioned above, they look similar to the spectra of the amino acids with two oxygen atoms only, but some distinct differences exist. To clarify these differences and to generate spectral fingerprints of the oxygencontaining groups in the side chain, the same subtraction procedure as for the N K XES case was applied to the spectra in Figure 3c, using analogous weighting factors reflecting the number of oxygen atoms in a particular amino acid as compared to the COO− fingerprint. However, to obtain a meaningful result (no negative intensities and kinks) upon subtraction of the COO− fingerprint spectrum (Figure 3b) from the O K XES spectra shown in Figure 3c, small energy shifts of this fingerprint with respect to the spectra of the amino acids in Figure 3c were necessary, namely, +0.20 eV for Asn, +0.23 eV for Gln, +0.40 eV for Asp, +0.40 eV for Glu, +0.15 eV for Ser, +0.12 eV for Thr, and +0.00 eV (i.e., no shift) for Tyr. These shifts might be an indicator for the functional group acidity (i.e., its ability to lose a proton), which is lower for the side chain than for the α-carboxylic group. The results of the subtractions are displayed in Figure 3d. Similar to the N K edge case, they can be associated with the oxygen-containing groups in the side chains of the given amino acids, thus representing spectral fingerprints of these groups. In case of aspartic acid and glutamic acid, which have a neutral carboxylic group in their side chain (see Figure 1), the generated side-chain spectra are very similar to one another and represent a spectral fingerprint of this group. This fingerprint shows large similarities to the spectrum of the COO − fingerprint, apart from a red shift of about −0.7 eV, which might be seen as an indicator for the functional group’s acidity, which is lower for the side chain than for the deprotonated αcarboxylic group. Note that the similarity between the derived fingerprints for the protonated and deprotonated carboxylic groups is in contrast to the resonantly excited O K XES spectra, which shows a clear difference between these groups.3 Note also that the derived fingerprint for the protonated carboxylic group differs from the nonresonant spectrum of liquid acetic acid.25 This difference can, however, be related to core holeinduced dynamics, which likely affects the latter spectrum.25 In case of asparagine and glutamine, the resulting spectra are again very similar to one another, in accordance with the similar structure of the side chains and the identity of the oxygencontaining groups in this chain (amide; see Figure 1), but show clear differences to the COO− fingerprint. The side chains of serine, tyrosine, and threonine host an alcohol group (see Figure 1), so that the spectra in Figure 3d should represent a spectral fingerprint of this moiety. However, the subtraction of the COO− fingerprint from the original O K XES spectra results in two different motifs, with similar emission patterns for serine and threonine but a completely different pattern for tyrosine. The most likely reason for this difference is the difference in the nearest-neighbor environment of the alcohol group in the side chains. While the alcohol group in serine and threonine is attached to a methyl group, it is bound to the aromatic ring in tyrosine (see Figure 1). As already shown for proline, an aromatic ring structure can have a quite large effect on the surrounding electronic structure and might also be the reason for the quite specific side-chain spectrum of tyrosine. Referring to the building block model mentioned above, the generated side-chain spectra can be compared to the O K XES spectra of suitable reference molecules. In case of serine and threonine, such a molecule would be methanol, which also

Figure 4. Nonresonant C K XES spectra of the 20 studied amino acids.

spectra have a recognizable “triangular” shape with the center at ∼278 eV. The low-energy flank of the spectra is almost featureless, whereas the high-energy flank shows one to four clearly distinguishable emission lines. These lines are found at different energy positions for different amino acids, which makes their assignment difficult and highly speculative. The origin of individual emission lines can likely only be clarified by calculations and, in some cases, might be supported by the resonant excitation of selected core orbitals, as has been shown previously for cysteine.7 Nevertheless, from the experience gained by the latter example, one can speculate that the small emission feature centered at ∼282.3 eV is (partly) related to the molecular orbitals of the α-carbon in direct vicinity of the protonated amino group. Furthermore, the features located at ∼279.2 eV could, in the case of cysteine,7 be associated with the molecular orbitals localized on (or at least having a high transition probability at) the carbon atom of the carboxylic group. 3.4. S L2,3 Edge. To complete the amino acids database, nonresonant S L2,3 XES spectra of cysteine and methionine were recorded. They are depicted in Figure 5. Although both 6554


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In contrast to the N K and O K XES data, the analogous C K spectra of the amino acids were distinctly different from each other, even though certain general features were identified. The clarification of the emission structure for a particular amino acid will likely need calculations and, in some cases, might be supported by the resonant excitation of selected core orbitals. The latter is also true for the S L2,3 XES spectra of cysteine and methionine, which are additionally complicated by the overlap of the contributions associated with the L2 and L3 emissions. From the methodical viewpoint, the results presented in this study provide further strong evidence for the general applicability of the building block model for the interpretation of XES data of complex molecules, including amino acids in particular. At the same time, in some cases, there is a necessity to take into account all nearest-neighbor atoms, providing a certain limitation of the building block model in its specific application to XES. From the practical viewpoint, the presented comprehensive database provides a reliable basis for further XES and RIXS studies of amino acids, peptides, and proteins in different states and environments. Accordingly, we believe that the given study represents an important step in the implementation of soft Xray spectroscopies to address specific questions of biological significance. In addition, the generated spectral fingerprints of individual functional groups can be useful for other classes of compounds as well.

Figure 5. Nonresonant S L2,3 XES spectra of cysteine and methionine. The excitation energy was set to 182 eV for methionine and to 202 eV for cysteine. Note that the sharp, low-intensity peak at ∼151.5 eV in the spectrum of cysteine is an artifact, stemming from a higher harmonic of the primary excitation energy. It is not present in the spectrum of methionine acquired at the lower excitation energy.

molecules contain only a single sulfur atom, the S L2,3 XES spectra are quite complicated, which is partly related to the spin−orbit splitting of the S 2p core level. At nonresonant excitation, both S 2p3/2 and S 2p1/2 states are excited, creating an emission spectrum by superposition of the L2 and L3 contributions, which are identical, but weighted 2:1 and shifted energetically by the spin−orbit splitting (∼1.2 eV).26 Note that, upon resonant excitation, the L3 emission can be excited separately from the L2 emission, as demonstrated in ref 7.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b04291. Nonresonant N K, O K, C K, and S L2,3 XES spectra of the 20 most common proteinogenic amino acids in their solid zwitterionic form as well as the NH3+ and COO− fingerprints (ZIP)

4. CONCLUSIONS In summary, we have presented and analyzed a comprehensive XES spectral database for all of the 20 proteinogenic amino acids in the zwitterionic form. The spectra, collected from polycrystalline powder samples, were acquired with nonresonant excitation at all of the relevant emission edges, namely, the N K, O K, C K, and S L2,3 edges. At the most important N and O K edges, clear similarities between the spectra of certain amino acids are observed, whereas the spectra of other amino acids exhibit significant differences. These similarities and differences are associated with the specific chemical structure of these molecules, and the presence of nitrogen- and oxygen-containing functional groups in the side chain, in particular. Due to the large sample set, a spectral fingerprint of the protonated amino group could be created by generating the averaged sum of the N K XES spectra of 13 amino acids. A similar spectral fingerprint could be derived for the deprotonated carboxylic group by averaging the O K XES spectra of 13 amino acids. In contrast, the N K and O K XES spectra of the amino acids containing additional nitrogen- and/ or oxygen-containing functional groups in the side chains are more complex. By subtracting the weighted spectral fingerprints of the protonated amino group and the deprotonated carboxylic group from the original XES spectra of these amino acids, the spectral contributions of the side chain could be created, representing a spectral fingerprint of a particular nitrogen- or oxygen-containing functional group. Some of these fingerprints were compared to the spectra of reference compounds with the same functional group and exhibited reasonable similarity, underlining the validity of the spectral fingerprint approach for the present study.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (M.Z.). *E-mail: [email protected] (L.W). ORCID

D. Hauschild: 0000-0001-9088-8944 M. Bar̈ : 0000-0001-8581-0691 M. Zharnikov: 0000-0002-3708-7571 Present Addresses ◆

Fraunhofer-Institut fü r Werkstoffmechanik IWM, Wöhlerstraße 11, 79108 Freiburg, Germany (F.M.). ¶ Department of Physics, Bharathiar University, Coimbatore 641046, India (Y.L.J.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the German Research Society (DFG; projects nos. RE 1469/7-1 and ZH 63/16-1). R.G.W. and M.B. acknowledge the financial support by the Impuls- und Vernetzungsfonds of the Helmholtz-Association (VH-NG423). The ALS is supported by the Department of Energy, Basic Energy Sciences, Contract No. DE-AC02-05CH11231. 6555


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