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Antioxidant Functions of Ergothioneine

Takeshi Saito1, Kazuhisa Sakakibara2, Yoshihisa Fukuoka2, and Kensei Kobayashi2 

1Institute for Advanced Studies, 22-11 Jyogu-machi, Yanagawa, Fukuoka, 832-0068, Japan; 2Department of Chemistry, Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama, 240-8501, Japan 

Correspondence: (T.S.) 

Saito T et al. Reactive Oxygen Species 10(29):211–220, 2020; ©2020 Cell Med Press

(Received: July 14, 2020; Revised: October 19, 2020; Accepted: October 22, 2020) 

ABSTRACT The function of ergothioneine (EGT) has been in controversy since the discovery of EGT in 1909. The present common understanding is that the functions of EGT remain unclear and that EGT does not seem to provide any advantage over other antioxidants. In this study, the ultraviolet-absorption spectra of EGT were measured at various pH. Thermodynamical parameters such as the chemical structure and bond distances of EGT were derived by BMK/6-311+G**. The molecular orbital quantum parameters such as electron distribution, frontier electron density, highest occupied molecular orbital (HOMO), lowest unoccupied molecular orbital (LUMO), and singly occupied molecular orbital (SOMO) were derived by HF/STO-3G. The results show that EGT exists as a resonance system mixing of thione state and ionic state in solution not only at physiological pH but also in a wide acidic pH range. EGT is very stable because of the resonance system, whereas the ionic state of EGT forms the metal complexes and the EGT-disulfide in the presence of divalent metal ions. The efficient delocalization of π-electrons on the imidazole ring of EGT increases the HOMO energy level and the rate constant of EGT towards hydroxyl (OH) radical. EGT has a high antioxidant activity by donating the highest active electron on the S atom of the imidazole ring of EGT to OH radical. Because OH radical reacts faster with EGT than any other biomolecules such as proteins and DNA, EGT consequently protects the biomolecules against oxidative damage due to the OH radical. 

KEYWORDS Antioxidant function; Ergothioneine; Frontier electron density; Rate constant; Resonance system 

ABBREVIATIONS DFT, Density Functional Theory; EGT, ergothioneine; GSH, reduced glutathione; HF, HartreeFock; HOMO, highest occupied molecular orbital; LUMO, lowest unoccupied molecular orbital; OH, hydroxyl;SOMO, singly occupied molecular orbital; UV, ultraviolet 


  1. Introduction
  2. Materials and Methods

2.1. Chemicals

2.2. Ultraviolet Absorption Spectrometry

2.3. Calculations

  1. Results

3.1. Ultraviolet Absorption Spectra of EGT

3.2. Chemical Structure and Bond Lengths of Imidazole Ring of EGT

3.3. Electron Distributions and Frontier Electron Density Distribution of EGT

3.4. Frontier Electron Density Distribution of Free Simple Aliphatic Amino Acids

3.5. Rate Constants of EGT towards OH Radical

  1. Discussion

4.1. Existence of EGT as Thione Form

4.2. Symmetrical Structure of Imidazole Ring of EGT

4.3. EGT as Resonance System Mixing of Thione State and Ionic State

4.4. Activity and Stability of EGT and Recovery of EGT

4.5. Protection against Oxidative Modifications of Amino Acid Residues and DNA Bases

  1. Conclusion


The function of ergothioneine (EGT) has been in controversy for more than a century. In 1958, Melville summarized the halfcentury work since the discovery of EGT [1] as follows: “The meaning of the activities of EGT is not yet clear. Numerous attempts to define a biological activity for EGT have been recorded. Many of these have given negative results, but a few have yielded interesting positive effects” [2]. Similar reviews were made for the next half century. Akanmuet al. wrote in 1991 that the function of EGT in animal and plant tissues is unknown [3]. Cheah and Halliwell mentioned in 2012that the true physiological role of EGT had yet to be fully elucidated [4]. Servilloet al. wrote in 2015 thatthe redox mechanism of EGC remained unclear and that EGC does not seem to provide any advantage over other antioxidants[5]. What causes the difficulty in knowing the real antioxidant properties of EGT? 

Present common understanding of EGT is as follows: EGT exists as a tautomer between its thiol and thione forms in solution and exists predominantly as the thione in solution at physiological pH [4–7]. Thiol-disulfide of EGT is formed in solution at very low pH in the presence of copper [8]. Because of the thione form, EGT is stable and does not undergo oxidation as rapidly as other thiolcompounds such as reduced glutathione (GSH) [4]. Therefore, EGT does not significantly contribute to the antioxidant thiol defense system in vivo [7]. On the other hand, it is well known that EGT has high activity against free radical species. EGT reacts with hydroxyl (OH) radical within the diffusion time of OH radical [3, 9]. EGT has higher antioxidant activity towards peroxyl radicals and peroxynitrite (ONOOˉ) as compared to GSH, trolox, and uricacid[10]. EGT-thione forms easily the complexes with divalent metal ions such as Co2+, Ni2+, Zn2+ and Cu2+ through sulfur atom in solution at the neutral pH [11, 12]. In order to know the real antioxidant properties of EGT, we have to unravel why EGT lacking antioxidant power against oxygen and superoxide anion (O2˙ˉ) has such a high antioxidant power towards radicals. 

In this study, ultraviolet (UV) absorption spectra of EGT were measured to revalidate the existing form of EGT in solution. Although we already knew the UV absorption spectra of EGT [8], which suggested that EGT existed only as the thione form in solution at neutral pH region as well as in the acidic region, it has been ignored. Precise chemical structure of EGT was obtained by BMK/6-311+G** to study why EGT is so stable, and why the stable EGT forms the disulfide with metal complexes. Quantum orbital parameters of EGT were derived by HF/STO-3G to unravel why a stable EGT has such a high antioxidant power towards OH radical, and how EGT reacts with OH radical.


2.1. Chemicals 

Hydrochloric acid and sodium hydroxide solution were used to adjust pH of the EGT aqueous solution. L-(+)-Ergothioneine was from Funakoshi (Tokyo, Japan). Water used in the present study was purified with Milli-Q® system (Millipore-Sigma, Burlington, MA, USA). 

2.2. Ultraviolet Absorption Spectrometry 

Ultravioletabsorption spectra of a mixed aqueous solution of 0.87 mM L-EGT (0.2 ml) and the pH preparation solution (3.6 ml) were measured with a JASCO V-550 UV-visible spectrophotometer (Easton, MD, USA). 

2.3. Calculations 

We adopted two calculation methods, Density Functional Theory (DFT) method [13]and HartreeFock (HF) method [14, 15]. The DFT method provides the chemical thermodynamic properties such as the chemical structure, the reaction energy, the stability, and activityof EGT. The Hamiltonian of the HF model provides the electronic structure such as a wave function φ and the frontier electron density as φ2as the solution to the Schrödinger equation. Hence, the HF method derives the spread of the delocalized electrons and the most active electron on the orbital of EGT, which are essential for the study of a fast reaction between EGT and OH radical. 

We applied the BMK/6-311+G** in the DFT methods to get the 3D chemical structure and bond lengths of EGT, because BMK/6-311+G**calculations are assumed to be able to evaluate reliable thermodynamic properties and structures of the chemical systems containing EGT and reactive species such as OH radical [16, 17]. 

We applied the HF/STO-3G to getthe shapes and phases of molecular orbitals, frontier electron density, highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) for EGT, GSH, and 20 kinds of proteinogenic amino acids. Singly occupied molecular orbital (SOMO) for OH radical were derived by the unrestricted HF method, UHF/STO-3G//CCSD (T)/cc-PV5Z, because the orbitals of α-spin and β-spin are different from each other.Current general idea is that theclassical HF/STO-3Gmethod is not good as the modern theoretical study because of its insufficient accuracy. However, the HF/STO-3G method provides sufficient results with reasonable accuracy for the present purpose, because the change in correlation energy is not so large and electron correlations are negligible for the low molecular substances under consideration. The HOMO and LUMO/SOMO play a major role in governing the chemical reactions [18–20]. The higher the HOMO is, the more electron-donating the molecule is. The difference between the HOMO value of a substance and the SOMO value of OH radical is related to the rate constant of the substance towards the OH radical. 

All theoretical calculations have been carried out on a Linux-based computer where Gaussian 09 program was installed [21].


3.1. UltravioletAbsorption Spectra of EGT 

Figure 1 shows the ultraviolet absorption spectra of EGT at various pH, in which the peak maxima is normalized to 1in order to see the change of the spectrum shape with the pH values. Figure 2 shows variation inmaximum absorption wavelength (λmax) of EGT by pH. The present relationship between λmax and the pH values is in good agreement with the pastdata [8], in which difference is a systematic error of only 1 nm. We now conclude that the ultraviolet absorption spectra of EGT are almost the same with a sharp maximum at 257nm in solution over a wide pH range of 1 to 9.


FIGURE 1. Ultraviolet absorption spectra of EGT in aqueous solution at different pH.


FIGURE 2. Variations in the absorption maximum of EGT by changing pH. ●: the present study; ■: Heath and Toennies (1958) [8]; ▲: Carsson et al (1974) [22]. 

3.2. Chemical Structure and Bond Lengths of Imidazole Ring of EGT 

Figure 3 shows the chemical structure, and the optimized chemical structure of EGT- thione derived by BMK/6-311+G**. The bond lengths are shown in angstrom.


FIGURE 3. Chemical structure (left) and the optimized chemical structure of EGT-thione derived by BMK/6-311+G** (right). 

3.3.Electron Distributions and Frontier Electron Density Distribution of EGT 

Figure 4 shows the electron distributions in the HOMO of EGT-thionederived by HF/STO-3G (left) and by BMK/6-311+G** (right). The color (red or blue) represents the phase of the orbital electron distribution. The square of the wave function gives a probability of finding an electron at a point in the quantum orbital. The frontier electron density of Fukui [20] is twice the square of the wave function. Figure 5 shows the frontier electron density distribution of EGT-thione derived by HF/STO-3G. The number on the horizontal axis in Figure 5 corresponds to the number on the ring of EGT in Figure 3.


FIGURE 4. Electron distributions in the HOMO of EGT-thione derived by (a) HF/STO-3G (left) or (b) BMK/6-311+G** (right).


FIGURE 5. Frontier electron density distribution of EGT-thione. 

3.4. Frontier Electron Density Distribution of Free Simple Aliphatic Amino Acids 

Figure 6 shows the frontier electron density distribution of HOMO for the CH-NH2-COOH part of free aliphatic amino acids such as glycine (Gly), alanine (Ala), serine (Ser), valine (Val), threonine (Thr), and isoleucine (Ile).


FIGURE 6. Frontier electron density distribution of the CH-NH2-COOH part of free simple aliphatic amino acids. ●: Gly; ○: Ala; ■: Ser; □: Val; ▲: Thr; △: Leu; and ◆: Ile. 

3.5.Rate Constants of EGT towards OH Radical 

Figure shows the relationship between the calculated HOMO-SOMO differences and the observed rate constants in the pH range of 6.0 to 7.5 for EGT-thione, GSH, and 20 proteinogenic amino acids.The red points show the mean values of different experiments when the observed rate constants are distributed within 30% [23–25]. The experimental rate constants for EGT, GSH, tryptophan (Trp), and cysteine(Cys) are widely scattered (the references are given in Table 1). The green vertical lines show the ranges of the maximum and minimum values among the scattered experimental data. The solid red curve shows the least squares fitting of the red point data. The relationship between the rate constants and the HOMO-SOMO differences is given by the following formula: rate constants = 5.39 × 1010 – 6.32 × 1010 log (|HOMO-SOMO|). Table 1 shows the derived rate constants for EGT-thione, GSH, Trp, and Cys.


FIGURE 7. Relationship between the HOMO-SOMO differences and the observed rate constants in the pH range of 6.0 to 7.5 for EGT-thione, GSH, and 20 proteinogenic amino acids indicated by official one letter abbreviations.

TABLE 1. The rate constants (M–1 s–1) towards OH radical in pH 6–7.5 of the present results and the experimental data
Compound Present result Experimental data and reference
EGT-thione 1.90 × 1010 1.60 × 1010 [3], 1.20 × 1010 [9]
GSH 7.85 × 109 3.48 × 10[26], 1.30 × 1010 [6]
Cys 6.81 × 109 2.10 × 10[27], 5.35 × 10[28], 1.90 × 1010 [6], 4.00 × 1010 [29]
Trp 1.22 × 1010 7.80 × 10[24], 1.40 × 1010 [30]


4.1. Existence of EGT as Thione Form 

The observed ultraviolet absorption spectra (Figure 1) and the dependence of the maximum absorption wavelength (λmax) of EGT on the pH values (Figure 2) confirm that EGT exists as the thione form in solution not only at physiological pH but also in a wide acidic pH range, even at pH 1. EGT does not exist as a tautomer between its thiol and thione forms, and EGT never functions as the thiol-disulfide antioxidant system in the same way as thiol compounds such as GSH and Cys. 

4.2. Symmetrical Structure of Imidazole Ring of EGT 

Figure shows the bond lengths of the imidazole ring of EGT-thione. The solid red marks show the present results derived by BMK/6-311+G**. Our results are in good agreement with the DFT values from B3LYP/6-311G(d, p) by Chinh et al. [31], and the experimental values from the X-ray diffraction experiment by Sugihara et al. [32].


FIGURE 8. Symmetrical structure of imidazole of EGT. ●: the present result by BMK6-311+G**; : values by B3LYP/6-311G(d,p) [31]; and : data by diffraction experiment [32]. 

The imidazole ring of EGT is very symmetrical as seen in Figure 8, in which the N(1)-C(2) bond length is almost the same as the C(2)-N(3) bond length, and the N(3)-C(4) bond length is almost the same as the C(5)-N(1) bond length. Because of the symmetrical ring structure, the efficient π-electrons are delocalized and the bond alteration cannot be recognized. Extension of the π-electron conjugation in the imidazole ring increases aromaticity of the ring and the HOMO energy level of EGT. 

4.3. EGT as Resonance System Mixing of Thione State and Ionic State 

Figure 9 shows the bond lengths between C and S for EGT, the thiourea derivatives (TUD, a–j), the normal single (C-S) bond compounds and the normal double (C=S) bond compounds. TUD means compounds forming R1-N-C(=S)-N-R2, and the normal (C-S) bond means the compounds such as H3C(-S)H, [H3C(-S)]4C, R1-C(-S)C-R2, R1-O(C-S)-R2 and so on. As the mean normal (C=S) bond length, Abrahams proposed 1.61 (angstromunits) [33]. We selected the compounds such as O=C(=S),S=(C=S) and R1-C(=S)-R2 where R=H, N, CnH2n+1 and so on. As a typical example of the normal (C=S) compound, the bond lengths of CH3C(=S)H are shown by ▼ (our BMK value) and ▽ (observed value [34]). We select the 32 points of the normal single and double bond lengths with no special criteria from several corresponding compounds [33, 35]. Vertical bars show the mean values and one standard deviation of the bond length distribution. A very narrow distribution of TUD indicates that the (C=S) bond of TUD is insensitive to the kind of the substituent.


FIGURE 9. Bond lengths of EGT, thiourea derivatives (TUD), normal single (C-S) bond, and normal double (C=S) bond compounds. ●: EGT by BMK; ○: EGT from diffraction experiment[32]; ■: thiourea (TU) [36]; ◆: TUD with ring (a–e from the left) [37–39]; ◇: simple TUD (f–j from the left) [40–42]; □: single (C-S) bond compounds; △: double (C=S) bond compounds; ▼: our BMK value; and ▽: observed value for CH3C(=S)H (k) [34]. 

As shown in Figure 9, the mean (C=S) bond length of TUD (a–j), 1.684 ± 0.0083 is an intermediate value between the normal (S-C) bond length of 1.809 ± 0.026 and the normal (S=C) bond length of 1.592 ± 0.032. The intermediate characteristics are the typical phenomena of thiourea resonance system [43]. EGT exists as a resonance system mixing thione state and ionic state as shown in Figure 10.


FIGURE 10. Resonance state of EGT mixing of thione state and ionic state. A drawing of this figure is a matter of convenience. The negative charge means strictly a high electron density state in the resonance. 

Two papers have referred to the resonance of EGT [31, 32]. However, they are still considering EGT in the scheme of thiol-thione tautomeric equilibrium. The ionic state of EGT is completely different from the thiol form. EGT is stabilized because of the resonance system. The ionic state in the resonance system forms a dimer with a disulfide bond (ES-SE) in the presence of divalent metal ions [9] and the complexes (ES-M-SE) with metal ions such as Cu2+, Hg2+, Zn2+, Cd2+, Co2+ and Ni2+ in the same way as thiourea complexes [11, 44], in which 1 mol metal is bonded with 2 mol EGT through the sulfur atom [12].

4.4. Activity and Stability of EGT and Recovery of EGT

OH radical takes easily the highest active electron, 3Py of the S atom on the imidazole ring of EGT in Figure 5. However, the ring of EGT does not break because of the stable resonance system. The OH radical has no H atom to take from the S atom of EGT. As a result, EGT is a strong electron donor without H supply. EGT denotes one electron to OH radical. EGT is converted to a cation radical (・S), and the OH radical is converted eventually to water: E=S +OH˙→ E=・S+ OHˉ; OHˉ + H+→ H2O. Because the cation radical, *S is a strong electrophilic molecule, it transforms automatically into the stable anion, E=S-H by releasing a proton (H+) and by concomitantly taking an electron into *S, as shown in Figure 11. The E=S-H returns to the former EGT by reacting with H+ in the solvent.


FIGURE 11. Conversion of E=*S+ to E=S-H. 

4.5. Protection against Oxidative Modifications of Amino Acid Residues and DNA Bases 

The complete process and the pathology of the protein and DNA modifications, which cause a variety of diseases, are not yet fully known because of very complicated multiple processes [45–49]. However, the first simple reaction between a biomolecule and a radical is essential from the viewpoint of protecting the oxidative modification. As shown in Figure 6, active electrons are distributed on the N and C terminals of amino acids. Because the terminals are bound as peptide in proton, the α-C part is a unique open site for reaction. Moreover, the solid angle of the α-C part is less than half the solid angle of free amino acids, 4π. As a result, the rate constants of amino acid residues in protein are two to three orders of magnitude smaller than that of EGT. The OH radical reacts with DNA by adding to the double bonds of DNA at a rate constant of 3 to 10 ×109 M–1s–1, or by abstracting one H atom from the methyl group of thymine and each C-H bond of 2′-deoxyribose at arate constant of 2×109 M–1s–1 [48]. Because OH radical reacts first with EGT at a rate constant of 1.90×1010 M–1s–1, EGT protects consequently proteins and DNA against oxidative modifications by OH radical.


EGT never functions as thiol-disulfide antioxidant cycle because EGT exits as a resonance system mixing thione state and ionic statein solution not only at physiological pH but also in a wide pH range. Ionic state in the resonance system of EGT forms a dimer with a disulfide bond (ES-SE) and complexes (ES-M-SE) in the presence of divalent metal ions. EGT has high antioxidative power towards OH radical by donating the most active electron on the S atom of EGT to the OH radical without breaking the imidazole ringof EGT. EGT returns quickly to the original EGT after one electron donating. Our results enable a unified understanding of the past experimental results that have been thought to be contradictory and conflicting with each other. 


The authors declare no conflict of interest. 


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