Molecular Basis of the Antioxidant Capability of Glutathione Unraveled

Article pubs.acs.org/JPCB

Molecular Basis of the Antioxidant Capability of Glutathione Unraveled via Aerosol VUV Photoelectron Spectroscopy Po-Chiao Chang,† Youqing Yu,† Zhong-Hang Wu,† Ping-Cheng Lin,† Wei-Ren Chen,† Chien-Cheng Su,† Meng-Sin Chen,† Yu-Lin Li,† Tzu-Ping Huang,§ Yin-Yu Lee,§ and Chia C. Wang*,†,‡ †

Department of Chemistry and ‡Aerosol Science Research Center, National Sun Yat-sen University, Kaohsiung, Taiwan 80424, ROC National Synchrotron Radiation Research Center, Hsinchu, Taiwan 30076, ROC

§

S Supporting Information *

ABSTRACT: Glutathione (GSH), the most abundant nonenzymatic antioxidant in living systems, actively scavenges various exogenous/endogenous oxidizing species, defending important biomolecules against oxidative damages. Although it is well established that the antioxidant activity of GSH originates from the cysteinyl thiol (−SH) group, the molecular origin that makes the thiol group of GSH a stronger reducing agent than other thiol-containing proteins is unclear. To gain insights into the molecular basis underlying GSH’s superior antioxidant capability, here we report, for the first time, the valence electronic structures of solvated GSH in the aqueous aerosol form via the aerosol vacuum ultraviolet photoelectron spectroscopy technique. The pH-dependent electronic evolution of GSH is obtained, and the possible correlations between GSH and its constituting amino acids are interrogated. The valence band maxima (VBMs) for GSH aqueous aerosols are found at 7.81, 7.61, 7.52, and 5.51 ± 0.10 eV at a pH of 1.00, 2.74, 7.00, and 12.00, respectively, which appear to be lower than the values of their corresponding hybrid counterparts collectively contributed from the three isolated constituting amino acids of GSH. An additional photoelectron feature is observed for GSH aqueous aerosols at pH = 12.00, where the thiol group on its Cys residue becomes deprotonated and the relatively well-separated feature allows its vertical ionization energy (VIE) to be determined as 6.70 ± 0.05 eV. Compared to a VIE of 6.97 ± 0.05 eV obtained for a similar thiolate feature observed previously for isolated Cys aqueous aerosols (Su et al. VUV Photoelectron Spectroscopy of Cysteine Aqueous Aerosols: A Microscopic View of Its Nucleophilicity at Varying pH Conditions. J. Phys. Chem. Lett. 2015, 6, 817−823), a 0.27 eV reduction in the VIE is found for GSH, indicating that the outermost electron corresponding to the nonbonding electron on the thiolate group can be removed more readily from the GSH tripeptide than that from Cys alone. The possible origins underlying the decrease in the VBM of GSH with respect to that of each corresponding hybrid counterpart and the decrease in the VIE of the thiolate feature of GSH with respect to that of the isolated Cys are discussed, providing hints to understand the superior antioxidant capability of GSH from a molecular level.

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INTRODUCTION Nature has built glutathione (γ-L-glutamyl-L-cysteinyl-glycine, GSH) as the master endogenous antioxidant in living systems, including both the animal and plant realms.2−5 It exists in nearly every mammalian cell, with a relatively high concentration, ranging from 1 to 10 mM. As the first line of defense for inhaled xenobiotics as well as for endogenous oxidants released from lung inflammatory cells, the GSH level in the epithelial lining fluid of the lower respiratory tract can even be 2 orders of magnitude higher than that in the blood plasma for the same individual.6 Structurally, GSH is a tripeptide comprising glycine (Gly), cysteine (Cys), and glutamic acid (Glu). With the unique γ-amide linkage between the γ-carboxyl group of Glu and amine group of Cys, GSH is markedly stable as compared to its GluCysGly isomer with the regular α-amide bond.7,8 The biochemical activities of GSH and its multifaceted functionalities are mostly attributed to the highly nucleophilic © 2016 American Chemical Society

thiol (−SH) functional group on the side chain of its Cys residue.9 As the master endogenous antioxidant, GSH readily undergoes redox reactions with various exogenous/endogenous oxidizing species via thiol oxidation and transforms into its oxidized disulfide form, GSSG. Common reactive oxidants that are often scavenged by GSH can be either free radicals or nonfree radicals, and they can be broadly categorized into two major families, namely, reactive oxygen species and reactive nitrogen species.10 Once these oxidizing species enter the human body, they may cause varying extents of oxidative stress, perturb the redox equilibrium status to alter the cell cycle, and eventually lead to the malfunction of biomolecules if not properly reduced/detoxified. Received: May 8, 2016 Revised: August 6, 2016 Published: September 2, 2016 10181

DOI: 10.1021/acs.jpcb.6b04631 J. Phys. Chem. B 2016, 120, 10181−10191

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The Journal of Physical Chemistry B GSH, as the major antioxidant in the human body, effectively detoxifies various harmful oxidants, protecting functional biomolecules against oxidative damages and thus assuring their normal biological function. The metabolism of GSH has been found to be crucial in maintaining brain functions by reducing the oxidative stress in the brain.11−13 The critical role of hepatic GSH in sustaining the overall systemic GSH homeostasis and in maintaining liver function has also been reported.14 Besides its antioxidant capability, GSH also plays several important biological roles, including mediating the signaling pathways of proteins,15−17 maintaining the cellular redox equilibrium, and governing the cell proliferation and apoptosis cycles.18,19 These biochemical activities of GSH are achieved mostly through redox processes, in which GSH serves as a charge donor. For redox-based biological processes, the redox potential is crucial, as the functions of redox-sensitive biomolecules are often delicately regulated by this parameter.20 Although the redox potential of a substance can be obtained from the conventional electrochemistry approach, from a microscopic perspective, it requires further information regarding the molecular orbitals of both the charge donor and acceptor from which the charge is transferred to fully access the thermodynamic and kinetic properties of a particular redox process, as outlined in Marcus’ charge-transfer theory.21,22 In this regard, the valence electronic properties of GSH and the energetics of the orbital from which its electron is removed play decisive roles in governing the various redox-based biochemical activities of GSH. In the meantime, GSH is not the only thiol-containing species to exploit the −SH group to carry out the designated biological functions. Cysteinyl thiols have been established as the strongest nucleophile among all functional groups of proteins and have been identified as the key active site responsible for the redox-based functions of many other thiolcontaining proteins.23−32 Nevertheless, the highly nucleophilic and bioactive nature of thiols on Cys at the same time means that they are also highly vulnerable and subject to attack by reactive and electrophilic oxidizing species and must be well protected so that they can carry out their designated biological functions correctly without disruptions. Considering the crucial importance of thiols for both GSH and other thiol-containing proteins, several issues of fundamental importance arise. First, because the thiol group is the common functional group in GSH as well as in other thiol-containing proteins, how can the thiol of GSH preferentially react with oxidizing species over that of other thiol-containing proteins and protect them from oxidative damage? Second, besides the presence of the redoxactive Cys residue, does the presence of other constituting amino acids play any possible functional role in making GSH one of the most powerful antioxidants in nature? The conformational structures and thermodynamic properties of GSH have been studied previously. For example, the structures of gas-phase neutral and anionic GSH were investigated by Ko et al.33 by combining negative-ion photoelectron spectroscopy and quantum calculations. To clarify the roles of electron delocalization and solvent effects in the antioxidant potential of GSH, Fiser et al. studied the bond dissociation of GSH and its related fragments theoretically.34 The energetics associated with GSH formation and the related thermodynamic properties, including the standard reaction enthalpies, ΔrHo, and reaction free energies, ΔrGo, were also studied by Fiser et al. via quantum chemical calculations, from which the stability of γpeptides over that of α- and β-peptides was addressed.8

Furthermore, the structure−function correlation of GSH and its five structural analogues was studied by combining potentiometry, UV absorption, and NMR spectroscopy,35 whereas the conformations of GSH and its oxidized form, GSSG, in the solution phase were studied theoretically by VilaViçosa et al. using the constant pH molecular mechanics/ molecular dynamics (MD) simulation approach.36 However, despite these previous studies on GSH, the valence electronic structures of GSH in the solution phase, which are expected to play a decisive role in its biochemical activities, remain unavailable. To gain insights into the molecular origin of the antioxidant capacity of GSH, here we report for the first time the valence electronic structures of GSH and its pH-dependent electronic evolutions in the aqueous aerosol phase via the recently developed aerosol vacuum ultraviolet (VUV) photoelectron spectrometer, equipped with a high-resolution hemispherical electron energy analyzer.1 Although photoelectron spectroscopy has been established as a mature technique to probe the electronic properties of gas, liquid, and solid species, as well as of small clusters, in the past decades, the applications of photoelectron spectroscopy, including the angle-resolved velocity-mapped photoelectron imaging technique to study suspended aerosols and nanoscaled particulate matter, have been developed only relatively recently.1,37−43 Wilson et al. applied VUV photoelectron imaging to probe photoelectrons of low kinetic energy ejected from NaCl nanoparticles, with diameters ranging between 50 and 500 nm, and reported sizedependent asymmetry in the electron angular distribution.39 From the photoelectron imaging studies on dehydrated Gly and phenylalanine-glycine-glycine (Phe-Gly-Gly) nanoparticles, the ionization energies and molecular polarizabilities of these dried nanoparticles were determined by Wilson et al.38 On the other hand, Gaie-Levrel et al. combined the aerodynamic focusing technique with photoelectron−photoion coincidence spectroscopy to study VUV state-selected photoionization of thermally desorbed biomolecules.40 In an attempt to better understand the inelastic scattering of electrons with low electron kinetic energy (eKE) in aerosols, Signorell and coworkers applied table-top-based VUV photoelectron imaging to study the inelastic mean free paths of electrons with eKE of 1− 3 eV in water aerosols.42,43 Recently, a breakthrough was achieved by Ellis et al., who integrated the femtosecond extreme ultraviolet generated via the high harmonic generation scheme, aerodynamic focusing, and photoelectron imaging technique to probe the ultrafast relaxation dynamics of solvated electrons in several nanoparticles and nanodroplets,44 bringing aerosol photoelectron spectroscopy into the time domain. Instead of using velocity-mapped photoelectron imaging as the detection scheme, we recently constructed an aerosol VUV photoelectron spectroscopy apparatus utilizing a hemispherical type of energy analyzer for eKE measurements and investigated the electronic structures of Cys in aqueous aerosols, from which a microscopic understanding of the pH-dependent nucleophilicity of Cys was revealed.1 These preliminary studies have collectively demonstrated aerosol photoelectron spectroscopy as a powerful tool to probe the electronic structures, ionization energies, ultrafast electron relaxation dynamics, and other important properties of aerosols in the suspended nanoscaled aerosol phase. Moreover, considering that numerous chemical and biochemical reactions take place in aqueous environments, it is essential and critical to assess the solvated electronic properties 10182


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stagnation pressure ranging between 15 and 35 psig. The GSH aqueous aerosols generated in this manner exhibit an average aerodynamic diameter of the order of ∼100 nm and a number density of the order of ∼107 particles/cm3 (Figure S1), as measured by a scanning mobility particle sizer (model 3936; TSI Inc.) prior to entering the aerosol VUV photoelectron spectroscopy apparatus. The GSH aqueous aerosols were then directed into an AADL system located in the source chamber of the aerosol VUV photoelectron spectroscopy apparatus. The AADL system used in this study is composed of an inlet nozzle of 300 μm; 4 orifices of 5.0, 4.5, 4.0, and 3.5 mm diameter, with each separated by 50 mm; and an accelerating nozzle of 3.0 mm, allowing the aqueous aerosols employed in this study to achieve optimal aerodynamic focusing. In the AADL system, the GSH aqueous aerosols were collimated into a highly focused aerosol beam, which was steered into the main photoelectron investigation chamber after passing through the aerosol source chamber and a differential pumping region. In the present experimental setup, the aqueous aerosols of an initial average diameter of 100 nm are expected to arrive at the photoionization region after a flight time of 2 ms, at a beam speed of 145 m/s, as calculated using the Aerodynamic Lens Calculator by Wang and McMurry.55 The size and temperature of the aqueous aerosols are expected to deviate from their initial statuses as they are subjected to evaporative cooling along their way toward the photoionization point. The evaporation of water molecules from liquid water droplets has been considered previously by Faubel et al.56 and Smith et al.57 in the liquid microjet studies. According to the kinetic theory of evaporation outlined by the Hertz-Knudsen equation and the present experimental conditions, aqueous aerosols of an initial average diameter of ∼100 nm are expected to reduce to ∼94 nm at the point of photoionization (Figure S2). Accompanied by solvent evaporation, the aqueous aerosols are estimated to cool down to ∼193 K at the point of photoionization, implying that the aqueous aerosols most likely reside in a deeply supercooled status (see the Supporting Information for detailed discussions on the nature of the aqueous aerosols).56−58 The liquid nature of the aqueous aerosols can be further ascertained by comparing the observed binding energy (B.E.) of condensed water to the previously reported values of B.E. for liquid54,59−61 and ice62 water and via the explicit pH-dependent electronic structures of solutes in aqueous aerosols. The pH value of the solution was adjusted by adding HCl or NaOH. The possible shift in the pH value as a result of solvent evaporation is less than 0.1. The VUV radiation generated from the undulator (U9) at the National Synchrotron Radiation Research Center (BL21B2; Hsinchu, Taiwan) was used as the photoionization source, which provides 1016−1017 photons/s/mm2/mrad2 in the range of 5−30 eV, with a 0.1% bandwidth at a 360 mA storage ring current under the top-up injection mode. The photoelectrons were detected parallel to the polarization vector of the VUV synchrotron radiation, and their kinetic energies were analyzed with a hemispherical energy analyzer (R3000; VG Scienta Ltd., Uppsala, Sweden). The absolute energy scale of the observed photoelectron spectra was calibrated according to the (0,0,0) and (1,0,0) vibrational fine structures of the 1b1 state of gaseous water molecules.63 All photoelectron spectra presented in the present study were measured at a photon energy of 25 eV, as this photon energy provided the desired spectral range to assess the valence electronic properties of the chosen solutes.

of solutes. Although the electronic properties of several biological molecules, including glycine,45 lysine,46 imidazole47,48 (the functional group of histidine), and nucleic acids,49 have been studied in the solution phase via liquid microjet photoemission spectroscopy,50−52 aerosol photoelectron spectroscopy provides a different approach to probe the electronic properties of solvated species in the form of aqueous aerosols.1 A comparison between aerosol photoelectron spectroscopy and liquid microjet photoelectron spectroscopy has been addressed previously,1 including the sample generation scheme, the electrokinetic charging issue, and the potential implications. In brief, aerosol photoelectron spectroscopy provides a nanoscaled aqueous environment to study the electronic properties of solvated species, in contrast to the liquid droplets of micrometer diameter studied in the microjet experiments. The electrokinetic charging issue commonly encountered in the microjet experiment can be readily eliminated in aerosol photoelectron spectroscopy.53,54 With the size-selection capacity of the adjustable aerodynamic lens (AADL) system, the aerosol VUV photoelectron spectroscopy technique allows one to probe the finite-size effect for aerosols in a sizecontrolled way.42,43 From another perspective related to the fundamental biophysical and biochemical properties of peptides and proteins, there has been a continuing effort by researchers to track how the fundamental properties of the building block amino acids evolve when they grow into larger peptides and proteins. To evaluate the possible correlations/contributions of the constituting amino acids to the resulting peptides, here, we also independently measure the VUV photoelectron spectra of two constituting amino acids of GSH, Gly and Glu, which have remained unavailable up to date. The hybrid photoelectron spectra contributed from the constituting amino acids of GSH are obtained and directly compared to the photoelectron spectra of GSH. By doing so, the possible roles of the constituting amino acids in the ultimate electronic properties of the GSH tripeptide are interrogated.

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EXPERIMENTAL SECTION The experimental details of the aerosol VUV photoelectron spectroscopy apparatus (Figure 1) have been described in detail elsewhere.1 In brief, a 0.2 M GSH (Sigma-Aldrich, >99%) solution was prepared under a specified pH condition and introduced into the aqueous nanoaerosol phase via a constantoutput liquid-feed atomizer (model 9306; TSI Inc., WA), using neon of ultrahigh purity (>99.999%) as the carrier gas, with a

Figure 1. Schematic view of the aerosol VUV photoelectron spectroscopy apparatus. 10183


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RESULTS AND DISCUSSION VUV Photoelectron Spectroscopy of GSH under Varying pH Conditions. Being fabricated from three specific amino acids, Glu, Cys, and Gly, GSH exhibits four possible protonation/deprotonation sites, including (1) the α-carboxyl group of the Glu residue (site I in Scheme 1), (2) the amine

terms of the B.E. between 12.5 and 5.5 eV, as this spectral range provides the proper window to assess the valence electronic structures of GSH without interference by the intense signals of water vapor that are inevitably present in aqueous aerosols. To reveal the spectral characteristics of GSH with satisfactory clarity, the photoelectron features of condensed water are shown only partially in Figure 2 (see the extended views of GSH photoelectron spectra, covering a broader spectral range, in Figure S3A−D). The photoelectron spectra of GSH recorded under the four pH conditions demonstrate an explicit pH-dependent behavior, manifested as the broad-band feature ranging between 11 and 7 eV, whose intensity maximum shifts progressively toward the lower B.E. side with increasing pH (Figure 2A−D). The intensity maximum for the broad GSH feature is situated at 9.57, 9.35, 9.32, and 8.95 ± 0.05 eV, with a full width at halfmaximum ranging between 1.56 and 1.70. Such a spectral shift readily reflects the modifications in the electronic properties of GSH when its local chemical environments are altered upon pH changes. The spectral shift is particularly explicit when the pH is altered from 1.00 to 2.74 and 7.00 to 12.00. The former pH change corresponds to a transformation of the dominating GSH species from the fully protonated GSH+ cation to the neutral GSH zwitterion, whereas the latter case corresponds to a transition from the GSH− anion to the fully deprotonated GSH3− trianion. Between pH values of 2.74 and 7.00, only a minor spectral difference is observed, likely because the chemical environment of GSH experiences only moderate modifications in this pH range. At pH = 2.74, the major GSH species include ∼70.2% of the GSH zwitterion, with one of its two α-carboxyl groups deprotonated (structures B and C in Scheme 1), and ∼15.4% of the GSH− anion, with both the αcarboxyl groups deprotonated (structure D in Scheme 1), whereas at pH = 7.00, the GSH− anion (structure D in Scheme 1) becomes the most abundant species (∼98.1%). At pH = 12 (Figure 2D), a new spectral feature becomes prominent at 6.70 ± 0.05 eV, indicating that an additional molecular orbital becomes subjected to ionization, and the outermost electron of GSH can be ejected more readily from this orbital. Because under this pH condition GSH3−, with the deprotonated thiolate group, becomes the most dominating species (∼99.7%) (structure G in Scheme 1), this newly emerged photoelectron feature at 6.70 ± 0.05 eV appears to be closely correlated to the deprotonated thiolate group of the Cys residue of GSH (Cys ns′). Although the pH-dependent electronic evolutions of GSH are shown clearly from the experimental GSH photoelectron spectra, to gain further insights into the valence electronic properties of GSH and the possible correlations with its constituting amino acids, it is necessary to also have a detailed understanding of the electronic properties of its three constituting amino acids individually. Whereas the valence electronic properties of Cys have been studied by us previously, in the form of aqueous aerosols,1 the valence electronic properties of the other two constituting amino acids of GSH, Gly and Glu, in the aqueous solution phase and their possible electronic evolutions remain unavailable. To address this issue, we also record the VUV photoelectron spectra of Gly and Glu in the aqueous aerosol form and their pH dependence for the first time (see the additional photoelectron spectroscopy results in Figures S4 and S5 and additonal discussions in the Supporting Information). The experimental procedure for obtaining the photoelectron spectra

Scheme 1. Molecular Structures of the Seven Possible Forms of GSH (A−G) Illustrated in the Deprotonation Sequence of GSH with Increasing pH Values (from Top to Bottom)a

a

The charge statuses at the four protonation/deprotonation sites are denoted in terms of I, II, III, and IV.

group of the Glu residue (site II in Scheme 1), (3) the side chain thiol group of the Cys residue (site III in Scheme 1), and (4) the α-carboxyl group of the Gly residue (site IV in Scheme 1). Accordingly, seven possible GSH species of different charged statuses may exist under varying pH ranges (structures A−G in Scheme 1, with their charge statuses at the four protonation/deprotonation sites specified in terms of I, II, III, and IV). To probe the valence electronic properties of GSH and the possible electronic evolutions with varying charged statuses, we measure the VUV photoelectron spectra of GSH in the aqueous aerosol phase under four chosen pH conditions, that is, 1.00, 2.74, 7.00, and 12.00 (Figure 2A−D). Each pH condition represents a dominating charge status of GSH, specified along with the corresponding photoelectron spectra of GSH. The corresponding molecular structures of these GSH species are illustrated in Scheme 1 (see the relative percentages of the seven GSH species under each chosen pH condition in Table S1). The observed photoelectron spectra are expressed in 10184


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Figure 2. (A−D) VUV photoelectron spectra of GSH aqueous aerosols under four pH conditions, including (A) pH = 1.00, (B) pH = 2.74, (C) pH = 7.00, and (D) pH = 12.00. (E−H) Hybrid photoelectron spectra simulated from the experimental spectra of three constituting amino acids of GSH, including Gly, Glu, and Cys aqueous aerosols of the corresponding dominating charged forms at a pH of 1.00, 2.74, 7.00, and 12.00. Symbol: GSH experimental spectra and hybrid spectra, -■-; Cys ns′, green curve; Cys ns, orange curve; Glu nO, violet dashed-dotted curve; Gly nO, violet dotted curve; Cys nO, violet dashed curve; Glu nO′, purple curve; Glu nN, blue dashed-dotted curve; Gly nN, blue dotted curve; Cys nN, blue dashdotted curve; pure condensed water, cyan curve; condensed water in solute-containing aqueous aerosols, green curve; gas-phase water, gray curve; cumulative curve, pink curve.

of solvated Gly and Glu aqueous aerosols is the same as that used for measuring GSH aqueous aerosols, except that the Gly and Glu aqueous aerosols were prepared and their spectra recorded at specifically chosen pH values according to their own specific pKa values, allowing solvated Gly and Glu of specific charge states to be interrogated. The deconvolutions of the photoelectron spectra of Gly (Figure S4) and Glu (Figure S5) aqueous aerosols reveal the electronic evolution of each spectral component when their dominating charge form evolves with pH (see the deconvolution results of Gly and Glu in Tables S2 and S3 and additional discussions in the Supporting Information). In an attempt to assess the possible contributions of the three constituting amino acids of GSH to its electronic structure, we next simulate a hybrid photoelectron spectrum by summing the

experimental VUV photoelectron spectra of the three constituting amino acids of GSH, Gly, Cys, and Glu, of each corresponding charged form under each specified pH condition (Figure 2E−H). Specifically, the hybrid photoelectron spectrum is obtained by adding the VUV photoelectron spectra of Gly (Figure S4), Glu (Figure S5), and Cys (ref 1) aqueous aerosols of the corresponding dominating charged form under each specified pH condition. Prior to summation, the photoelectron spectrum of each individual amino acid is normalized to the nonbonding oxygen orbital (nO) of its αcarboxyl group to ensure that each amino acid has the same weighing factor and contributes equally to the hybrid photoelectron spectrum. Comparing the experimentally observed GSH photoelectron spectrum (Figure 2A−D) with the hybrid photoelectron 10185


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for isolated Cys at a high pH value1 and Cys is the only constituting amino acid of GSH that exhibits such an additional spectral feature at a high pH, one may attribute this new feature at 6.70 eV to the same molecular origin associated with the thiolate group of the Cys residue (denoted as Cys nS′). However, in contrast to the B.E. of Cys nS′ at 6.97 eV for the fully deprotonated Cys2− (Figure 3A), the B.E. of Cys nS′ for

spectrum contributed from its three isolated constituting amino acids (Figure 2E−H) reveals that the photoelectron spectrum of GSH under each pH condition resembles its corresponding hybrid counterpart to a large extent. The pH-dependent shift in the broad feature of GSH toward a lower B.E. is agreeably revealed from the hybrid photoelectron spectra, indicating that the electronic properties of GSH are inherently contributed and governed by the electronic properties of its constituent amino acids. The spectral components of the three constituting amino acids, derived from the spectral deconvolution of each amino acid, including Gly (Table S2), Glu (Table S3), and Cys (Table S4)1 aqueous aerosols, are shown together underneath the hybrid spectra (Figure 2E−H), allowing one to learn the possible contributions of different spectral components to the broad hybrid feature and possibly also to the observed broad feature of GSH. On evaluating the numerous spectral components underlying the broad hybrid band (Figure 2E−H), the outermost electron orbital of GSH appears to be the nonbonding electron associated with either thiol (Cys ns, orange curve in Figure 2E−G), when the thiol remains in the protonated −SH form, or the thiolate group (Cys ns′, green curve in Figure 2H), when the thiol becomes deprotonated at a high pH and the Cys ns′ orbital becomes available for ionization. This likely explains why the thiol or its deprotonated thiolate on Cys serves as the redox-active site for GSH. However, as the various possible spectral components of GSH are energetically close-lying, one cannot differentiate the contribution of each spectral component from the broad GSH feature unambiguously. For the same reason, the vertical ionization energy (VIE) of the outermost electron orbital of GSH cannot be determined precisely, except for the thiolate feature (Cys ns′) observed at pH = 12.00, which is relatively well separated from the broad GSH feature. The VIE of Cys ns′ is found to be 6.70 ± 0.05 eV. Additional insights may be extracted by evaluating the valence band maximum (VBM) from the photoelectron spectra of GSH aqueous aerosols and its evolution with pH. VBM is a term commonly adapted to describe the onset position of the valence band or the minimum energy required to remove the outermost electron from condensed matter. The VBM for GSH aqueous aerosol is found to be 7.81, 7.61, 7.52, and 5.51 ± 0.10 eV at a pH of 1.00, 2.74, 7.00, and 12.00, respectively. This pHdependent VBM evolution trend again implies that GSH can begin to lose its outermost electron more readily with increasing pH. In contrast, the VBM for the hybrid photoelectron feature is found to be 7.91, 7.86, 7.81, and 5.98 ± 0.10 eV under the conditions corresponding to the specific charge status of each amino acid constituent at a pH of 1.00, 2.74, 7.00, and 12.00, respectively. This lowering of the VBM of GSH with respect to that of its hybrid counterpart made from its three isolated constituting amino acids implies that when the three constituting amino acids are linked together via peptide bonds to form the GSH tripeptide, their electronic structural properties are altered, as their local chemical environments change, and the resulting GSH tripeptide may lose its outermost electron from the thiol or thiolate group of its Cys residue more readily than the same spectral feature from the single Cys amino acid. Such an electronic modification of the outermost valence electrons of GSH is most explicitly revealed from the new spectral feature at 6.70 eV observed at pH = 12.00, at which GSH exists mostly (∼99.7%) in the fully deprotonated GSH3− form. Because a similar spectral feature has also been observed

Figure 3. Comparison of the photoelectron feature newly emerged upon formation of the thiolate group for (A) fully deprotonated Cys and (B) fully deprotonated GSH. Symbol notation: experiment, -■-; ns′, green curve; cumulative curve, pink curve.

the fully deprotonated GSH is decreased to 6.70 eV (Figure 3B). This 0.27 eV reduction in the B.E. of the outermost Cys nS′ orbital of GSH is of biological significance, as this indicates that the outermost electron associated with the thiol group (Cys nS′) can be more easily removed from the GSH tripeptide than that from the isolated Cys amino acid. Ab Initio Calculation of Microhydrated GSH Using the NEPCM Approach. To verify the observed pH-dependent ionization energy evolution of GSH as well as the reduced ionization energy of GSH with respect to that of single Cys, we carry out ab initio calculations at the B3LYP/6-311++G (d,p) level via the nonequilibrium polarizable continuum medium (NEPCM) approach.1,48 Diffuse functions (+, +) are included in the basis set so that the hydrogen-bond effect for the solvated molecules can be better captured. Jagoda-Cwiklik et al. have applied the NEPCM model previously to calculate the VIE of imidazole in solution by considering one to five water molecules for the microhydrated imadazole structure and 10186


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Figure 4. Optimized structure of microhydrated GSH in various possible charged forms, hydrogen bonded with 17 explicit water molecules. (A) GSH+, (B) GSH, (C) GSH−, (D) GSH2−, (E) GSH3−, and (F) Cys2−.

suggested that five water molecules can reasonably reproduce the experimentally observed VIE value.48 Nevertheless, considering the relatively larger size of GSH, 17 explicit water molecules are considered for the microhydrated GSH structure of varying charged statuses. The geometries of the energetically most stable GSH structures of varying charged statuses are adapted from the MD simulation study of Lampela et al.64 The optimized microhydrated structures of the energetically lowestlying GSHs of varying charged forms hydrogen bonded with 17 water molecules are then obtained, including the GSH+ cation (Figure 4A), GSH zwitterion (Figure 4B), GSH− anion (Figure 4C), GSH2− dianion (Figure 4D), and the fully deprotonated GSH3− (Figure 4E). The VIEs for the solvated GSH+, GSH zwitterion, GSH−, GSH2−, and GSH3− thus simulated were calculated to be 8.04, 8.06, 7.77, 6.57, and 6.47 eV, respectively. Although the experimental VIE values can be determined explicitly only for pH = 12.00, the progressively reduced computed VIEs of GSH with increasing deprotonation agree qualitatively well with the pH-dependent evolution trend in the VBM. Moreover, to verify the observed 0.27 eV reduction in VIE for the outermost electron on the thiolate group (Cys nS′) of fully deprotonated GSH3− with respect to that of the same orbital for Cys2−, we calculate the VIE for the microhydrated Cys2− that is also hydrogen bonded with 17 water molecules (Figure 4F) and obtain a VIE value of 6.65 eV. Compared to the calculated VIE for GSH3− at 6.47 eV, a VIE reduction of

0.18 eV is derived theoretically for GSH, consistent with the experimentally observed trend. Therefore, here, we provide direct experimental and theoretical evidence to show that GSH is a stronger reducing agent than the isolated Cys amino acid, and this results from the higher tendency of GSH to lose/ donate its outer layer electrons from its thiol or thiolate group. Possible Correlations between GSH and Its Constituting Amino Acids. From the experimental and theoretical results, we show that the valence electronic properties of GSH are correlated to a large extent with its constituting amino acids. Also, the progressively reduced VBM of GSH with respect to that of its corresponding hybrid counterparts simulated from its three constituting amino acids indicates that GSH can lose its electron preferentially than the free amino acids, and the electron is removed preferentially from the thiol/thiolate group of its Cys residue. To gain insights into the possible molecular basis that makes GSH a stronger reducing agent than other thiol-containing peptides and proteins, we tackle the possible correlations between GSH and its constituting amino acids by evaluating how the electronic properties of GSH may be affected by the constituting amino acids in any possible way. Cys has long been established as the key component of GSH responsible for its antioxidant activities. The present study provides direct evidence to explain the redox-active role of Cys in GSH. From the numerous possible spectral components underlying the hybrid spectral features, which are derived from 10187


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The Journal of Physical Chemistry B spectral deconvolution from the individual constituting amino acids (Tables S2−S4), one can readily identify that the nonbonding electron from the thiol group (Cys ns) is the outermost electronic orbital when the thiol group on Cys remains in the protonated −SH form (orange curves in Figure 2E−G). As soon as the thiolate group is formed, the thiolate feature (Cys ns′) becomes the outermost valence layer to lose the electron (green curves in Figure 2H). Moreover, upon comparing GSH with the free Cys amino acid, the progressively decreased VBM of the outermost electrons associated with the thiol group of GSH with respect to the same spectral feature from the isolated Cys suggests that GSH can lose its electron from the thiol group preferentially over that from the isolated Cys. The reduced VIE of the thiolate feature (Cys ns′) of GSH with respect to that of the isolated Cys also implies the same trend. The cysteinyl thiol group has been established as the strongest nucleophile in proteins, which becomes even stronger when thiol is deprotonated to the thiolate form. From the previous VUV photoelectron spectroscopic study on Cys aqueous aerosols, we provided a microscopic understanding of the nucleophilicity of Cys under varying pH conditions and showed that the enhanced nucleophilicity of thiolate is associated with a new molecular orbital of lower B.E. (Cys nS′). Here, we further show that the thiol and thiolate groups of GSH become even more nucleophilic and reducing than those of the single Cys amino acid. To tackle the possible origin contributing to such an electronic modification of GSH, it is constructive to have a perspective view on the charge status evolution with changing pH for all relevant species. The evolution of each possible charged state as a function of the pH value is calculated for the three constituting amino acids of GSH, including Gly (Figure 5A), Cys (Figure 5B), and Glu (Figure 5C), as well as for the GSH tripeptide (Figure 5D), according to the pKa values of each molecule.65−68 Under the physiological pH range, whereas Gly and Cys mainly reside in their neutral zwitterionic forms (Figure 5A,B, orange curves), Glu exists almost exclusively in the negatively charged anionic Glu− form (Figure 5C, green curve) due to the additional γ-carboxyl group in its side chain. The GSH tripeptide also resides mostly in the negatively charged anionic form under the physiological pH condition (Figure 5D, green curve). By evaluating the charge-state evolution of the three constituting amino acids together with that of GSH, it is anticipated that Glu may play a role in making GSH mostly negatively charged across a broad pH range. This in turn makes GSH always more negatively charged than single Cys across a broad pH range, including physiologically relevant pH conditions. Therefore, even though Cys is directly involved the antioxidant activity of GSH, the presence of Glu likely provides additional shielding through its extra carboxylate group such that the outermost electrons on the thiol/thiolate group of GSH may become less tightly bound and can therefore be removed more easily than those from Cys alone.

Figure 5. Fractions of all possible protonation/deprotonation species of (A) Gly, (B) Cys, (C) Glu, and (D) GSH as a function of pH values. The corresponding charge state for each species is denoted above the corresponding fraction curve. Color code for the charge state: +1, red curve; 0, orange curve; −1, green curve; −2, blue curve; −3, purple curve. The minor isomer of Cys−, (Cys−)N, is shown in dark cyan, the minor isomer of neutral GSH, (GSH)Glu_COO−, is shown in magenta, and the minor isomer of the GSH2− dianion, (GSH2−)Glu_NH2, is shown in cyan.

0.05 eV at pH values of 1.00, 2.74, 7.00, and 12.00, respectively, reflecting the progressively modified electronic structures with changing charged statuses of GSH. By comparing the observed GSH photoelectron spectra with the hybrid spectra additively contributed from its three constituting amino acids, we show that the main electronic structure of GSH is inherently contributed and governed by its constituting amino acids. An additional photoelectron spectral feature is observed at 6.70 ± 0.05 eV at a high pH, which is associated with the deprotonated thiolate group on the Cys residue of GSH. Compared with a VIE of 6.97 ± 0.05 eV observed previously for the thiolate group of free Cys amino acid, a 0.27 eV reduction in VIE is found. The experimentally observed pH dependence on the valence electronic structures of GSH is further supported by ab initio calculations of GSH hydrogen bonded with 17 water

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CONCLUSIONS In this work, we report the first valence electronic structural study of the most abundant endogenous antioxidant, GSH, in the aqueous nanoaerosol form via the recently developed aerosol VUV photoelectron spectroscopy technique. The primary photoelectron feature of GSH is observed to shift progressively toward a lower B.E. with an increasing pH value. The VBM of GSH is found to be 7.81, 7.61, 7.52, and 5.51 ± 10188


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molecules using the NEPCM approach. Such electronic modifications of GSH may provide a possible molecular origin to explain its superior antioxidant ability, as they indicate that GSH can begin to lose its outermost electrons to other oxidizing species more readily. The intrinsic origin underlying the reduced VBM of GSH with respect to that of its constituting amino acids is discussed by considering the nature of its constituting amino acids. As the outermost electronic features of GSH originate from the thiol/thiolate functional group of its Cys residue, it is not surprising that Cys is the active residue of GSH responsible for the redox-based chemical activities of GSH, including its antioxidant capability. However, from the charge status evolutions for all relevant species, the negatively charged Glu seems to play a role in modifying the electronic properties of GSH by providing additional screening to make the outermost electron on the thiol/thiolate group of GSH less tightly bound. This work reports the first pHdependent valence electronic structure of GSH in aqueous aerosols and illustrates the crucial importance of probing the valence electronic properties of the redox-active biomolecules under physiologically relevant aqueous conditions. Finally, we demonstrate the aerosol VUV photoelectron spectroscopy apparatus, equipped with a high-resolution hemispherical energy analyzer, to be a powerful investigation tool in unraveling detailed electronic structural information of solvated species in the aqueous nanoaerosol form.

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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.6b04631. Additional discussions and supporting figures and tables (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +886-7-5252000 ext. 3941. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by grants from the Ministry of Science and Technology of ROC under project numbers NSC 1002113-M-110-001-MY3, NSC 102-2113-M-110-005-MY2, and MOST 104-2113-M-110-011. C.C.W. thanks Dr. Cheng-Maw Cheng for helpful discussions.

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REFERENCES

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Molecular Basis of the Antioxidant Capability of Glutathione Unraveled

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