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“Building Block Picture” of the Electronic Structure of Aqueous Cysteine Derived from Resonant Inelastic Soft X‑ray Scattering F. Meyer,† M. Blum,‡ A. Benkert,†,§ D. Hauschild,† S. Nagarajan,#,△ R. G. Wilks,∇ J. Andersson,○,▼ W. Yang,◆ M. Zharnikov,# M. Bar̈ ,‡,∇,¶ C. Heske,‡,§,∥,□ F. Reinert,†,⊥ and L. Weinhardt*,‡,§,∥,□ †

Experimentelle Physik VII, Universität Würzburg, Am Hubland, 97074 Würzburg, Germany Department of Chemistry, University of Nevada, Las Vegas, 4505 Maryland Parkway, Las Vegas, Nevada 89154-4003, United States § Institute for Photon Science and Synchrotron Radiation, ∥ANKA Synchrotron Radiation Facility, and ⊥Gemeinschaftslabor für Nanoanalytik, Karlsruhe Institute of Technology (KIT), Hermann-v.-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany # Angewandte Physikalische Chemie, Universität Heidelberg, INF 253,69120 Heidelberg, Germany ∇ Solar Energy Research, Helmholtz-Zentrum Berlin für Materialien und Energie GmbH, Hahn-Meitner-Platz 1, 14109 Berlin, Germany ○ Department of Physics and Astronomy, Uppsala University, Box 516, S-751 20 Uppsala, Sweden ◆ Advanced Light Source, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, California 94720, United States ¶ Institut für Physik und Chemie, Brandenburgische Technische Universität Cottbus-Senftenberg, Platz der Deutschen Einheit 1, 03046 Cottbus, Germany □ Institute for Chemical Technology and Polymer Chemistry, Karlsruhe Institute of Technology, Engesserstrasse 18/20, 76128 Karlsruhe, Germany ‡

ABSTRACT: The electronic structure of the amino acid L-cysteine in an aqueous environment was studied using resonant inelastic soft X-ray scattering (RIXS) in a 2D map representation and analyzed in the framework of a “building block” approach. The element selectivity of RIXS allows a local investigation of the electronic structure of the three functional groups of cysteine, namely, the carboxyl, amino, and thiol groups, by measuring at the O K, N K, and S L2,3 edges, respectively. Variation of the pH value allows an investigation of molecules with protonated and deprotonated functional groups, which can then be compared with simple reference molecules that represent the isolated functional groups. We find that such building blocks can provide an excellent description of X-ray emission spectroscopy (XES) and RIXS spectra, but only if all nearest-neighbor atoms are included. This finding is analogous to the building block principle commonly used in X-ray absorption spectroscopy. The building blocks show a distinct spectral character (fingerprint) and allow a comprehensive interpretation of the cysteine spectra. This simple approach opens the path to investigate the electronic structure of more complex biological molecules in aqueous solutions using XES and RIXS.

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py.3,4,16−27 In particular, two resonant inelastic soft X-ray scattering (RIXS) studies on aqueous glycine8,28 and earlier pioneering X-ray absorption spectroscopy (XAS) work on all proteinogenic amino acids in solution29 were performed. In the present work, we employ a comprehensive spectroscopic approach, namely, the collection of RIXS maps8,30−32 at all accessible absorption edges of the functional groups to investigate the electronic structure of cysteine in aqueous solution. Furthermore, we analyze results from nonresonant soft X-ray emission (XES) and XAS. Conducting

INTRODUCTION Due to their biological relevance and the importance of the electronic structure for their functionality, amino acids have been extensively studied by the spectroscopic community (see, e.g., refs 1−12). Numerous studies of solid amino acids using a wide range of spectroscopies, in particular photoelectron spectroscopy (PES), can be found in the literature.9,10,13−15 These studies were performed in vacuum, either on a thick layer or on self-assembled monolayers, i.e., not in the biologically relevant aqueous environment. Investigating the electronic structure of molecules in aqueous solution represents a technical challenge that has recently been overcome with sophisticated setups to study liquids and solutions with soft Xray absorption, emission, and/or photoelectron spectrosco© 2014 American Chemical Society

Received: September 4, 2014 Revised: October 22, 2014 Published: October 23, 2014 13142

dx.doi.org/10.1021/jp5089417 | J. Phys. Chem. B 2014, 118, 13142−13150

The Journal of Physical Chemistry B

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such experiments in the aqueous environment of a flowthrough liquid cell allows the direct manipulation of the functional groups by changing the pH value of the solution. Making use of the element-specific nature and the local probing character of RIXS and considering the molecule under study to be composed of molecular “building blocks” (e.g., functional groups), we surmise that also the electronic structure of the molecule can be decomposed into specific contributions of these building blocks. This approach is widely used to interpret XAS data,29,33−35 while, in contrast, a similar approach is not established for XES or RIXS (it was briefly mentioned in a study of poly(phenylenevinylene)s by Guo et al.36). RIXS maps (like the ones presented in this paper) probe both unoccupied and occupied valence states and thus offer significantly more spectral information than XAS alone. Consequently, a successful XES/RIXS building block model provides deeper insights into the electronic structure, but is also expected to require a more careful choice of the individual building blocks than for XAS. Cysteine contains a carboxyl, an amino, and a thiol group, which makes it a well-suited candidate to test the building block approach for the interpretation of RIXS and XES spectra. In this paper, small reference molecules (ammonia, hydrogen sulfide, methylamine, and acetic acid) will be used as building blocks (Figure 1), and we will investigate their suitability to

a two-dimensional map, where the X-ray emission intensity is color-coded as a function of the excitation (ordinate) and emission (abscissa) energies.8,31,39 In all maps presented here, the excitation and emission axes were calibrated using reference measurements of N2,40 TiO2,41 and CdS,42 together with the elastically scattered Rayleigh line. To investigate molecules in aqueous solutions, SALSA is equipped with a flow-through liquid cell,37 in which the solution and vacuum are separated by a thin membrane. For the present experiments, we used Si3N4 (for the O K edge, Silson), SiC (for the N K edge, NTT), and pure carbon (for the S L2,3 edge, JAVU AB) as membrane materials. In the flow-through cell, the sample volume is replaced ∼770 times per second, which allows spectra to be recorded without any evidence of beam-damage and beam-heating artifacts.43 Commercial Lcysteine (Alfa Aesar, purity ≥98%) was used at a concentration of 1.16 M without further purification. pH values of 1, 5, 9.5, 12, and 13 (±0.2) were established by adding hydrochloric acid or sodium hydroxide to the solution. The validity of the building block approach is supported by density functional theory (DFT), which was performed using the StoBe-deMon code.44 We used single-point calculations with the modified generalized gradient approximation (GGA) exchange functional of Hammer et al.,45 the GGA correlation functional of Perdew, Burke, and Ernzerhof,46 and the TZVP basis set.

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RESULTS AND DISCUSSION Cysteine (C2SNH6COOH) has three functional groups, each with a different pKa value (1.91 for the carboxylic group, 8.14 for the thiol group, and 10.28 for the amino group47), as shown in Figure 2. For pH values lower than 1.91, the carboxyl group is neutral and the molecule is positively charged (cation). For pH values between 1.91 and 8.14, the molecule is zwitterionic with a positively charged (protonated) amino group and a negatively charged (deprotonated) carboxyl group. Above a pH value of 8.14, the thiol group becomes deprotonated, resulting in a molecular net charge of −1 (anion 1). For pH values above 10.28, the amino group deprotonates and becomes neutral, leaving the molecule with a net charge of −2 (anion 2). In the following, the results for the three individual functional groups (carboxyl, amino, and thiol) will be discussed by focusing on the O K, N K, and S L2,3 edges, respectively, and by employing suitably chosen simple reference molecules (hydrogen sulfide, methylamine, and acetic acid, depicted in Figure 1) as building blocks. Carboxyl Group: O K Edge. Figure 3 shows the O K RIXS maps of cysteine solutions at pH 1 (Figure 3a) and pH 5 (Figure 3b). At pH 5, the cysteine molecule is in a zwitterionic conformation; at pH 1, about 80% of the carboxyl groups are neutralized. Above an excitation energy of about 534 eV, the spectra are dominated by the emission of liquid water, which has been investigated and (controversially) discussed in earlier studies.19,31,48−51 Still, by tuning the excitation energy below the water absorption edge (532.5 eV for pH 1 and 533 eV for

Figure 1. Schematic drawing of the cysteine molecule and the three reference molecules (building blocks) hydrogen sulfide, acetic acid, and methylamine: red, oxygen; blue, nitrogen; yellow, sulfur; dark gray, carbon; white, hydrogen.

describe the RIXS spectra at the O K, N K, and S L2,3 edges. Using cysteine as a case study, we provide a first test of the applicability and limits of such an approach, in particular for biomolecules in an aqueous environment.

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EXPERIMENTAL AND THEORETICAL METHODS RIXS experiments were carried out with the SALSA (solid and liquid spectroscopic analysis) endstation37 at beamline 8.0.1 at the Advanced Light Source, Lawrence Berkeley National Laboratory. The high transmission of SALSA’s variable line space spectrometer38 allows a single XES spectrum of cysteine with a very good signal-to-noise ratio to be recorded within 30 s, and a full XES spectrum can thus be recorded at each excitation energy of a regular XAS spectrum (in this paper, a step size of 0.1 eV was chosen). The data are then presented in

Figure 2. Lewis structures of cysteine in aqueous solution as a function of the pH value, together with the corresponding pKa values.47 13143



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Figure 3. O K RIXS maps of cysteine in aqueous solution at (a) pH 1 and (b) pH 5. The X-ray emission intensity is color-coded and shown as a function of the excitation (ordinate) and emission (abscissa) energies. The dashed white lines pertain to spectra depicted in Figure 4.

pH 5), we are able to selectively excite only the oxygen atoms in the carboxyl group of cysteine. By comparison with published solid-state XAS data,24,52 we can attribute this energy region to the π* resonance of the double bond oxygen. The observed blue shift (0.5 eV) in the absorption onset for the deprotonated carboxyl group (pH 5) is consistent with the shifts observed for acetic acid and glycine upon deprotonation.24,25 At pH 1, the O K RIXS map (Figure 3a) shows a single strong emission peak (526.4 eV) and several weaker peaks between 520.0 and 525.5 eV at the π* resonance (532.5 eV excitation). For pH 5 (Figure 3b), the RIXS map changes substantially, and three distinct peaks with emission energies of 522.4, 525.6, and 526.5 eV become visible. For a better demonstration of these changes, Figure 4 shows four spectra excited at the π* resonance of cysteine at different pH values (b−d), together with two reference spectra of aqueous acetic acid (25%) (a, f) at pH values of 0.2 and 12.8 (established by adding hydrochloric acid or sodium hydroxide to the solution, respectively). The red (b) and green (c) spectra are directly extracted from the RIXS maps (dashed white lines in Figure 3). To enhance the signal-to-noise ratio, the spectra in the excitation energy ranges of 531.9−532.1 eV (pH 1) and 532.8−533.0 eV (pH 5) are summed up. Spectra d and e show cysteine at pH 9.5 and 13 (sum over excitation energies of 532.7−532.9 eV, RIXS maps not shown). The excitation energies of spectra a and f are identical to those of spectra b and e. For spectrum b at pH 1, these energies were chosen such that the zwitterionic contributions are suppressed. We find that the two acetic acid spectra agree very well with the spectra of cysteine in the corresponding configurations. While the overall spectral shape is reproduced, we observe small shifts in the energetic positions of approximately 0.3 eV or less, which can have several origins. First, we presume that differences in hybridization in the two molecules can lead to additional shifts in the (ground-state) energy levels. Second, electronic relaxation in the final state may play a role. Third, cysteine and acetic acid can have different solvent−solute interactions. At pH 5, 9.5, and 13, the carboxyl group of cysteine is deprotonated, which leads to very similar spectra (c−e). Nevertheless, the increasing pH value results in a small increase in the relative intensity of feature Z2. This implies that the orbital related to feature Z2 is also sensitive toward deprotonation at the thiol and amine groups. To further understand the spectral characteristics, Figure 5 shows the calculated wave functions of isolated cysteine and acetic acid, depicting isodensity surfaces of the highest occupied molecular orbitals (HOMOs) and the lowest unoccupied molecular orbitals (LUMOs). At the carboxyl group of both

Figure 4. X-ray emission spectra excited at the π* resonance of the double bond oxygen of cysteine at four different pH values (b, red, pH 1; c, green, pH 5; d, blue, pH 9.5; e, black, pH 13) compared to spectra of acetic acid at pH 0.2 (a, bottom) and pH 12.8 (f, top). Spectra are the result of the sum of excitation energies from 531.9 to 532.1 eV (cysteine at pH 1, acetic acid at pH 0.2), from 532.8 to 533.0 eV (cysteine at pH 5), and from 532.7 to 532.9 eV (cysteine at pH 9.5 and 13, acetic acid at pH 12.8). The different emission features (C1− C4 and Z1−Z3) and their relative shifts are labeled.

molecules, we find them to be similar and group them in Figure 5 accordingly, explaining the strong similarities in the spectral fingerprints. On the basis of the theoretical spectra of acetic acid and acetate25 as well as Figure 5, we can assign the features in our spectra in Figure 4 as given in the right columns in Figure 5. For the HOMO of the cysteine cation, no corresponding orbital at the carboxyl group of acetic acid exists. However, due to its small local contribution at the carboxyl group, we only expect a very weak signal possibly manifested in the small foot at the high-energy side of feature C1 in the spectrum of the cysteine cation. Amino Group: N K Edge. Figure 6 displays the N K edge RIXS maps of cysteine with an ionized (protonated) amino group (Figure 6a, pH 5) and a neutral amino group (Figure 6b, pH 13). We find that the deprotonation of the amino group has 13144



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Figure 5. Molecular structure and frontier orbital isodensity surfaces of cysteine and acetic acid (a) in the protonated state and (b) in the deprotonated state. Red and blue colors of the orbitals denote the sign of the wave function at the carboxyl group, while the remaining part of the wave function is shown in gray. The assignment of the different orbitals to the emission features in Figure 4 is given on the right of each figure.

Figure 6. N K RIXS maps of aqueous cysteine at (a) pH 5 and (b) pH 13. The black solid bars at the upper abscissa indicate the integration regions for the XAS spectra shown in Figure 8.

dramatic effect on the emission spectrum. Most prominently, the sharp emission feature at ∼395 eV is much more intense at pH 13 than at pH 5. These changes are best seen in the nonresonant N K XES and the partial-fluorescence-yield (PFY) XAS spectra of cysteine when compared with those of methylamine and

a very strong impact on the N K RIXS map, as found previously for glycine.8 The main absorption edge of the neutral amino group is significantly lower than for the protonated one, which is consistent with the change in electron binding energy as discussed in ref 4. In addition, we find a pre-edge feature at an excitation energy of 401.9 eV. The deprotonation also has a 13145



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nearest neighbors as in cysteine. As expected, its gas-phase spectrum in Figure 7c (taken at 0.9 bar and room temperature, hνexc = 423.4 eV) thus gives a closer match to that of cysteine. The orbital giving rise to the highest emission energy feature is again assigned to the N lone pair and shows similar relative intensities and energies for all three molecules (methylamine, cysteine, and ammonia), as expected for an orbital that is not directly involved in the chemical bonding. At lower emission energies (i.e., below 393 eV), we now find emission from several hybrid orbitals involving the methyl group and a peak at ∼382.5 eV (N4) that we attribute to the 4a′−1 final state in methylamine.53 Nevertheless, significant differences between the spectra of methylamine and cysteine can also be found in this energy region, e.g., the overall line shape and the relative intensities of N2 and N3. These differences suggest a stronger influence of the remaining molecule on the orbital (and/or vibrational) structure at the amine group (as compared to the carboxyl group), but an impact of the different environments (liquid, gas) of the compared samples can also not be ruled out. As mentioned above, the cysteine molecule with a protonated amine group shows a spectrum significantly different from that of the cysteine molecule with a deprotonated amine group (Figure 7a,b). Most prominently, the intensity of the N1 feature is significantly reduced (but still present) at pH 5. We attribute this change to two effects: First, upon protonation, the lone pair orbital giving rise to the highenergy feature is expected to hybridize with the 1s orbitals from the additional proton, forming a covalent bond. The newly formed hybrid orbital has a significant fraction of s symmetry, which, due to the dipole selection rule, should lead to a strongly reduced emission. Second, as found earlier for glycine,8 dissociation processes on the time scale of the X-ray emission can give rise to a spectral feature similar to that of the lone pair if the proton is removed during the X-ray emission process. On the basis of the building block model, we thus expect such dynamic processes to also play a role in the spectra of cysteine. This can be tested by comparing the PFY N K XAS spectra of cysteine at the two pH values with their decay-channelspecific (DCS) XAS8 counterparts shown in Figure 8. While the PFY spectra are extracted from the RIXS maps in Figure 6 by integrating the emission intensity over the energy range from 380 to 396.5 eV (giving rise to the black spectra in Figure 8), the DCS XAS spectra were generated by only integrating over a 1.2 eV wide window around the N1 emission peak (marked by the black bars in Figure 6, giving rise to the red spectra in

ammonia (Figure 7). In the emission spectrum of cysteine at pH 5 (Figure 7a, hνexc = 421.0 eV), three main features can be

Figure 7. N K XES and XAS spectra of aqueous cysteine (a, pH 5; b, pH 13; hνexc = 421.0 eV), methylamine (c, hνexc = 423.4 eV), and ammonia (d, from ref 32, hνexc = 409.5 eV).

observed (N1−N3), while the corresponding absorption spectrum is dominated by a broad single feature (N7). Upon deprotonation of the amino group (Figure 7b, pH 13, hνexc = 421.0 eV), a blue shift of the emission feature at ∼394.6 eV of about 0.8 eV, accompanied by a substantial increase in relative intensity, is observed. In contrast, the features at lower emission energies exhibit a red shift (of about 1.1 eV). Furthermore, the absorption maximum shifts to lower excitation energies (by about 1 eV), and two additional low-energy features appear (N5 and N6). The simplest building block representation for the amino group is aqueous ammonia, the N K XES (excitation energy 409.5 eV) and XAS spectra of which (taken from ref 32) are shown in Figure 7d. At a pH of 12.3, as used in this experiment, ammonia is primarily present as ammonia. In this configuration, the nitrogen atom has a local environment very similar to that of the nitrogen atom of the amino group of cysteine at pH 13, only differing in one bonding partner (H in ammonia vs C in cysteine), which leads to apparent similarities in both XAS and XES spectra. Most prominently, the feature at ∼395.4 eV in the spectrum of cysteine corresponds to emission from the 3a1 lone pair orbital in the spectrum of aqueous ammonia. The good match can be explained by the fact that, close to the N atom, the corresponding orbital in cysteine is also dominated by the N lone pair. The largest difference between the spectra can be observed in the region between 386 and 393 eV, where cysteine exhibits several emission lines resulting in a broad structure, while ammonia only exhibits one vibrationally broadened emission feature (1e−1).32 The orbitals giving rise to the emission in this region are involved in the molecular bonding and, while degenerate in ammonia due to its three equivalent bonding partners, split into several lines for cysteine. In contrast to that in ammonia, the N atom in methylamine has the same

Figure 8. N K edge partial-fluorescence-yield XAS spectra of aqueous cysteine (black) compared to decay-channel-specific XAS spectra (red, integrated over the region of the black solid bar in Figure 6). Spectra were taken at pH 5 (bottom) and pH 13 (top) and are normalized at an excitation energy of 410 eV. 13146



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Figure 9. S L2,3 RIXS maps of aqueous cysteine at (a) pH 5 and (b) pH 12. The dashed white lines pertain to spectra depicted in Figure 10.

Figure 8). We find that the DCS XAS spectrum of the pH 13 solution closely follows the PFY XAS spectrum. In contrast, we find a significant blue shift of the DCS XAS spectrum of the pH 5 solution as compared to the PFY spectrum. This corroborates the suggested dynamic origin of the N1 feature: Since the duration time of the RIXS process decreases for detuned excitation below the absorption edge,54 spectral features originating from dynamical processes are suppressed below the edge. The process can then be qualitatively summarized as follows:8 Upon excitation, one proton dissociates from the −NH3+ group, which causes a rearrangement of molecular orbitals before the de-excitation takes place and results in the appearance of spectral features similar to those of the −NH2 group. Thiol Group: S L2,3 Edge. The third functional group of cysteine is a thiol group. Figure 9 shows the RIXS maps of cysteine at the sulfur L2,3 edge, measured at pH 5 (Figure 9a) and 12 (Figure 9b). With a pKa value of 8.12,47 the thiol group is neutral at pH 5, while it is ionized (deprotonated) at pH 12. Both maps show the double-edge structure of the S L2,3 absorption edge (with a spin−orbit splitting of about 1.2 eV55). Furthermore, we also observe differences between the two maps: In the case of pH 5, we observe multiple absorption features, while in the case of pH 12, a featureless, steadily rising absorption edge is found. Figure 10 shows resonantly excited S L3 and nonresonantly excited S L2,3 XES spectra of cysteine (at pH 5 and 12). The resonantly excited spectra were extracted from the RIXS maps in Figure 9, as marked by the white dashed lines (excitation energy 163.0 eV). Furthermore, Figure 10 shows the nonresonant and resonant XES (excitation energy 162.8 eV, 1 bar, room temperature) and PES57 spectra of hydrogen sulfide gas as a reference for cysteine at pH 5. The excitation energies for the nonresonant spectra were chosen as 181 eV (hydrogen sulfide gas and cysteine at pH 5) and 186 eV (cysteine at pH 12), and the PES spectrum was aligned with the XES L3 component such that the 5a1−1 peak in PES coincides with the highest energy peak in the XES spectrum (see discussion below). All spectra can be divided into two main regions: a broad and intense low-energy feature (region A, below 153 eV) and several weaker high-energy features (region B, above 153 eV). Note that the regions around the elastically scattered line (∼163 eV) were multiplied by the displayed factors. The L2 and L3 components of the S XES spectra have been separated using a fit with two identical components comprised of a sum of Gaussians, shifted by a fixed spin−orbit splitting (ΔE = 1.2 eV) and with a fixed intensity ratio (L3:L2 = 2:1),

Figure 10. Nonresonant (black, hνexc = 181 eV) and resonant (gray, hνexc = 162.8 eV) S L2,3 emission spectra of hydrogen sulfide (top) and cysteine at pH 5 (center, hνexc = 181 eV, hνexc = 163.0 eV) and 12 (bottom, hνexc = 186 eV, hνexc = 163.0 eV). Fits of the L2 (red) and L3 (blue) contributions to the nonresonant spectra, as well as the residuals (green, 10×). For hydrogen sulfide, the corresponding PES spectrum is depicted (purple), aligned with the resonant X-ray emission spectrum using the 5a1−1 peaks (from ref ).

and are depicted in Figure 10 in blue (L3) and red (L2). The magnified (10×) residuum is shown in green. We find that the L3 contribution derived from the fit of the nonresonant spectra (blue) and the resonantly excited experimental L3 emission (gray) show similar features. However, they differ in peak position and relative intensity, which we assign to spectator shifts and changes in the matrix elements due to the symmetry of the intermediate state upon resonant excitation. By comparing the resonantly excited spectrum of hydrogen sulfide with the corresponding PES spectrum,56 we can assign the different spectral features. Note that the L3 emission does not show any contribution of the 2b1 orbital due to its p symmetry and the dipole selection rule disallowing the decay 13147



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into a p symmetry core hole. Also, the XES spectrum appears to have additional spectral features as compared to the photoemission spectrum, which we attribute to a rapid dissociation of the hydrogen sulfide molecule on the time scale of the X-ray emission process observed earlier in photoemission/Auger measurements57 and leading to a spectral superposition of contributions from intact and dissociated hydrogen sulfide. The comparison of the cysteine pH 5 spectra with those of hydrogen sulfide shows a similar spectral fingerprint in region A and a significantly modified fine structure in region B, reflecting the difference in nearest neighbors of the S atom in the two molecules. In contrast to the pH 5 spectrum, the pH 12 spectrum shows a distinct fine structure in region A and no intensity between 158 and 162 eV. We speculate, using the hydrogen sulfide molecule as a model, that the 5a1-type orbital slightly changes its symmetry after deprotonation, acquiring a more pronounced p-type symmetry, and thus, the corresponding feature vanishes from the L2,3 emission spectrum (indicated by the arrow).

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S.N.: Department of Chemistry, National Institute of Technology, Tiruchirappalli 620015, India. ▼ J.A.: Centre for Quantum Technologies, National University of Singapore, 3 Science Drive 2, 117543 Singapore. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschaft (Project Nos. RE 1469/7-1 and ZH 63/16-1). R.G.W. and M.Bä. acknowledge financial support by the Impuls- und Vernetzungsfonds of the Helmholtz-Association (Grant VHNG-423). The Advanced Light Source (ALS) is supported by the Department of Energy, Basic Energy Sciences, Contract No. DE-AC02-05CH11231.

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CONCLUSION We have investigated the electronic structure of cysteine in aqueous solution using soft X-ray spectroscopy, presenting RIXS maps of all of its functional groups. We compare the results to spectra of simple reference molecules (hydrogen sulfide, ammonia, methylamine, and acetic acid) representing the building blocks of the molecule. Upon protonation/ deprotonation, strong changes in the RIXS maps of each functional group can be observed. On the basis of the similarity of the O K XES spectra of cysteine and acetic acid, we conclude that the spectral fingerprint of the carboxyl group is not sensitive to changes to the rest of the molecule. In contrast, a stronger sensitivity to the rest of the molecule was found in the N K spectra representing the amino group. Nevertheless, the most prominent features can still be used as a fingerprint, e.g., distinguishing between the protonated and deprotonated configurations. The thiol group of cysteine is unique among the proteinogenic amino acids, and we report the first S L2,3 spectra (and RIXS maps) of this functional group. We were able to show the impact of deprotonation on the spectrum and could separate L2 and L3 components in the nonresonantly excited XES spectra. In conclusion, we find the building block approach to be a powerful tool for the qualitative interpretation of XES spectra and RIXS maps of organic molecules. Using small reference molecules with the same bonding symmetry but partly differing nearest neighbors around the probed atom (i.e., ammonia and hydrogen sulfide for the amino and thiol groups, respectively), a first (rough) description of the functional groups of larger organic molecules (here cysteine) can be given. This description is significantly improved when the next nearest neighbors are chosen to be identical (i.e., methylamine and acidic acid as reference molecules for the amino and carboxyl groups, respectively). We expect that this building block approach can directly be applied to large organic molecules: by locally measuring only the relevant functional groups, it will be possible to study otherwise very complex molecules in biologically relevant processes in a natural (i.e., aqueous) environment.

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