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Species-Specific, pH-Independent, Standard Redox Potential of Selenocysteine and Selenocysteamine

Species-Specific, pH-Independent, Standard Redox Potential of Selenocysteine and Selenocysteamine antioxidants Article Species-Specific, pH-Independent, Standard Redox Potential of Selenocysteine and Selenocysteamine 1 , 2 1 , 2 1 , 2 , Tamás Pálla , Arash Mirzahosseini and Béla Noszál * Department of Pharmaceutical Chemistry, Semmelweis University, H-1092 Budapest, Hungary; palla.tamas@pharma.semmelweis-univ.hu (T.P.); mirzahosseini.arash@pharma.semmelweis-univ.hu (A.M.) Research Group of Drugs of Abuse and Doping Agents, Hungarian Academy of Sciences, H-1051 Budapest, Hungary * Correspondence: noszal.bela@pharma.semmelweis-univ.hu; Tel.: +36-1217-0891 Received: 10 April 2020; Accepted: 21 May 2020; Published: 1 June 2020 Abstract: Microscopic redox equilibrium constants and standard redox potential values were determined to quantify selenolate-diselenide equilibria of biological significance. The highly composite, codependent acid-base and redox equilibria of selenolates could so far be converted into pH-dependent, apparent parameters (equilibrium constants, redox potentials) only. In this work, the selenolate-diselenide redox equilibria of selenocysteamine and selenocysteine against dithiothreitol were analyzed by quantitative nuclear magnetic resonance (NMR) methods to characterize the interfering acid-base and redox equilibria. The directly obtained, pH-dependent, conditional redox equilibrium constants were then decomposed by our method into pH-independent, microscopic constants, which characterize the two-electron redox transitions of selenocysteamine and selenocysteine. The 12 di erent, species-specific parameter values show close correlation with the respective selenolate basicities, providing a tool to estimate otherwise inaccessible site-specific selenolate-diselenide redox potentials of related moieties in large peptides and proteins. Keywords: selenocysteine; redox; disulfide 1. Introduction The role of selenium, the biological trace element and the related selenium-containing proteins (selenoproteins) has been described in numerous antioxidant processes [1]. Selenocysteine, known as the twenty-first amino acid, is the only selenium-containing building block of proteins that contains selenium. The majority of selenoproteins are enzymes (such as glutathione peroxidase [2], iodothyronine deiodinase [3], thioredoxin reductase [4]), most of which are involved in redox reactions. Their selenocysteine residue is the essential unit for their catalytic activity. A new, specific way to quantify redox properties at the submolecular level has recently been introduced to characterize thiols of biological importance (cysteine, cystamine, homocysteine and glutathione [5,6]; ovothiol A and penicillamine [5,7]). A major conclusion of these studies is that there is a close correlation between the thiolate basicities and the thiolate-disulfide redox properties [5–9]. The hypothesis of this work is that such a correlation between acid-base and redox characteristics exists for the selenium-analogues of the above compounds as well, namely for selenocysteine and selenocysteamine. We wish to demonstrate said correlation by determining the species-specific physico-chemical parameters of the acid-base and redox equilibria of the above compounds and extend the correlation to nuclear magnetic resonance (NMR) chemical shift values as well. In this work, the species-specific redox equilibria of two biogenic selenols: selenocysteamine (CysASeH) and selenocysteine (CysSeH) with dithiothreitol (DTT) were studied. The oxidation of these Antioxidants 2020, 9, 465; doi:10.3390/antiox9060465 www.mdpi.com/journal/antioxidants Antioxidants 2020, 9, x FOR PEER REVIEW 2 of 13 In this work, the species-specific redox equilibria of two biogenic selenols: selenocysteamine Antioxidants 2020, 9, 465 2 of 13 (CysASeH) and selenocysteine (CysSeH) with dithiothreitol (DTT) were studied. The oxidation of these compounds yields selenocystamine (CysASeSeCysA) and selenocystine (CysSeSeCys), compounds yields selenocystamine (CysASeSeCysA) and selenocystine (CysSeSeCys), respectively. respectively. The constitutional formulae of the studied selenium-containing compounds are The depicted in constitutional Figure 1. formulae of the studied selenium-containing compounds are depicted in Figure 1. Figure 1. The structure of the investigated selenols (left) and diselenides (right). Figure 1. The structure of the investigated selenols (left) and diselenides (right). Glutathione is known as a “gold standard” in thiol-disulfide biochemistry, hence, every Glutathione is known as a “gold standard” in thiol-disulfide biochemistry, hence, every thiol-containing antioxidant is compared to glutathione. Moreover, the species-specific thiol-containing antioxidant is compared to glutathione. Moreover, the species-specific physico-chemical acid-base [9] and redox [5,6] parameters of glutathione are well-characterized. physico-chemical acid-base [9] and redox [5,6] parameters of glutathione are well-characterized. However, previous works examining selenoproteins or selenium-containing peptides found that the However, previous works examining selenoproteins or selenium-containing peptides found that the diselenide bridge cannot be reduced by glutathione [10,11]. We found that even a large excess of diselenide bridge cannot be reduced by glutathione [10,11]. We found that even a large excess of glutathione was ine ective in reducing the studied diselenide compounds, confirming the above glutathione was ineffective in reducing the studied diselenide compounds, confirming the above observation. Thus, a more powerful reducing agent was needed to investigate selenolate-diselenide observation. Thus, a more powerful reducing agent was needed to investigate selenolate-diselenide redox equilibria. Based on literature recommendations [8,12], dithiothreitol (DTT), a stronger reducing redox equilibria. Based on literature recommendations [8,12], dithiothreitol (DTT), a stronger agent, was chosen. Dithiothreitol undergoes a one-step two-electron redox reaction, resulting in the reducing agent, was chosen. Dithiothreitol undergoes a one-step two-electron redox reaction, formation of oxidized dithiothreitol (DTT ) with an intramolecular disulfide in a six-membered ring. ox resulting in the formation of oxidized dithiothreitol (DTTox) with an intramolecular disulfide in a During the redox processes between dithiothreitol and other agents, the presence of the intermediate six-membered ring. During the redox processes between dithiothreitol and other agents, the compound, where only one of the thiolate groups of DTT is oxidized is negligible [13]. The main 2− presence of the intermediate compound, where only one of the thiolate groups of DTT is oxidized physico-chemical properties of DTT are already described (logK = 10.1; logK = 9.2 [14]; E =0.323 1 2 is negligible [13]. The main physico-chemical properties of DTT are already described (logK1 = 10.1; V at pH = 7.0 [15]). logK2 = 9.2 [14]; E° = −0.323 V at pH = 7.0 [15]). To obtain data describing selenolate-diselenide redox equilibria comparable to those of To obtain data describing selenolate-diselenide redox equilibria comparable to those of thiolate-disulfide systems, first the glutathione-DTT redox reaction was investigated (Figure 2). 2− thiolate-disulfide systems, first the glutathione-DTT redox reaction was investigated (Figure 2). The general step-by-step scheme of selenolate-diselenide redox equilibria with DTT is depicted in Figure 3, where RSe and RSeSeR stand for the reduced and oxidized form of the selenium-containing compounds, respectively. The reaction between disulfides (e.g., oxidized glutathione) and DTT is analogous (where RS and RSSR stand for the thiolate and disulfide, respectively). Antioxidants 2020, 9, x FOR PEER REVIEW 3 of 13 Antioxidants 2020, 9, 465 3 of 13 Antioxidants 2020, 9, x FOR PEER REVIEW 3 of 13 2− Figure 2. The redox reaction between oxidized glutathione and dithiothreitol (DTT) dominant microspecies at the pH of blood. 2− The general step-by-step scheme of selenolate-diselenide redox equilibria with DTT is depicted in Figure 3, where RSe and RSeSeR stand for the reduced and oxidized form of the selenium-containing compounds, respectively. The reaction between disulfides (e.g., oxidized 2− − glutathione) and DTT is analogous (where RS and RSSR stand for the thiolate and disulfide, 2− Figure 2. Figure 2. The The redox redox react reaction ion between between ox oxidized idized g glutathione lutathione and and dith dithiothr iothreitol (DTT eitol (DTT)) dominant dominant respectively). microspecies at the pH of blood. microspecies at the pH of blood. 2− The general step-by-step scheme of selenolate-diselenide redox equilibria with DTT is depicted in Figure 3, where RSe and RSeSeR stand for the reduced and oxidized form of the selenium-containing compounds, respectively. The reaction between disulfides (e.g., oxidized 2− − glutathione) and DTT is analogous (where RS and RSSR stand for the thiolate and disulfide, respectively). 2− 2 Figure Figure 3. 3. The t The two-step wo-step re redox dox equ equilibria ilibria betw between een a adis diselenide elenide com compound pound an and d DTT DTT (reac (reaction tion I and II I and ) II) followed followed by their aggregated one-step net re by their aggregated one-step netaction reaction (III) (III). . IdeIdentical ntical reac rtion eaction schem schemes es are ap areplicable applicable for for disu disulfide lfide analo analogue gues. The arrow in reaction I s. The arrow in reaction I indica II indicates tes the low probability the low probability of formof ation of the su formation of lfu the r– sulfur seleniu –selenium m bridge.bridge. Since DTT 2− is highly driven towards the ring-closing second oxidation step [13], and diselenides Since DTT is highly driven towards the ring-closing second oxidation step [13], and are more stable than the selenylsulfides [16], the reaction between diselenides and DTT can be 2− diselenides are more stable than the selenylsulfides [16], the reaction between diselenides and DTT 2− Figure 3. The two-step redox equilibria between a diselenide compound and DTT (reaction I and II) reasonably discussed in terms of the net reaction. The conditional equilibrium constant describing this can be reasonably discussed in terms of the net reaction. The conditional equilibrium constant followed by their aggregated one-step net reaction (III). Identical reaction schemes are applicable for net reaction is as follows: describing this net reaction is as follows: disulfide analogues. The arrow in reaction II indicate2 s the low probability of formation of the sulfur– [RSe ] [DTT ] ox. h i selenium bridge. K = (1) 3C [RSe ] [DTT ] (1) 𝐾 = DTT [RSeSeR] [DTT ][RSeSeR] 2− Since DTT is highly driven towards the ring-closing second oxidation step [13], and Since redox and acid-base reactions coexist, the observed apparent redox equilibrium constants are 2− diselenides are more stable than the selenylsulfides [16], the reaction between diselenides and DTT pH-dependent. In order to get a clear insight into the redox equilibria, purified from the protonation can be reasonably discussed in terms of the net reaction. The conditional equilibrium constant e ects, an improved evaluation method has been introduced [5,6]. This new method makes it describing this net reaction is as follows: x possible to determine species-specific redox equilibrium constants (k ) and the species-specific, i.e., [RSe ] [DTT ] (1) 𝐾 = [ ][ ] DTT RSeSeR Antioxidants 2020, 9, x FOR PEER REVIEW 4 of 13 Antioxidants 2020, 9, x FOR PEER REVIEW 4 of 13 Since redox and acid-base reactions coexist, the observed apparent redox equilibrium constants are pH-dependent. In order to get a clear insight into the redox equilibria, purified from the Since redox and acid-base reactions coexist, the observed apparent redox equilibrium constants protonation effects, an improved evaluation method has been introduced [5,6]. This new method are pH-dependent. In order to get a clear insight into the redox equilibria, purified from the makes it possible to determine species-specific redox equilibrium constants (k ) and the protonation effects, an improved evaluation method has been introduced [5,6]. This new method species-specific, i.e., the standard redox potential (E°x, where x is the general sign of the appropriate makes it possible to determine species-specific redox equilibrium constants (k ) and the RSe microspecies in Figure 4; Figure 5). This determination method requires all the species-specific species-specific, i.e., the standard redox potential (E°x, where x is the general sign of the appropriate protonation c − onstants, including those of the minor microspecies; otherwise, redox processes could Antioxidants 2020, 9, 465 4 of 13 RSe microspecies in Figure 4; Figure 5). This determination method requires all the species-specific only be characterized at the level of phenomenon. The complete set of the microscopic protonation protonation constants, including those of the minor microspecies; otherwise, redox processes could constants of the studied selenium-containing compounds has recently been established [17]. The only be characterized at the level of phenomenon. The complete set of the microscopic protonation the standard redox potential (E , where x is the general sign of the appropriate RSe microspecies in protonation constants of selenocysteine, selenocysteamine, and glutathione, relevant to this work, constants of the studied selenium-containing compounds has recently been established [17]. The Figures 4 and 5). This determination method requires all the species-specific protonation constants, are co protonation c llected in Table onstants o 1 anfd selenocyst used for feine, selenocysteamine, urther calculations. and glutathione, relevant to this work, including those of the minor microspecies; otherwise, redox processes could only be characterized at are co Here, we llected i re n Table port the co 1 and usemprehensive d for further ca redox lculat char ions. acterization of the selenolate-diselenide the level of phenomenon. The complete set of the microscopic protonation constants of the studied Here, we report the comprehensive redox characterization of the selenolate-diselenide equilibria of selenocysteine and selenocysteamine in terms of pH-independent standard redox selenium-containing compounds has recently been established [17]. The protonation constants of equilibria of selenocysteine and selenocysteamine in terms of pH-independent standard redox potentials (298 K, 95/5 v/v% H2O/D2O and 0.15 mol/L ionic strength). The correlation between the selenocysteine, selenocysteamine, and glutathione, relevant to this work, are collected in Table 1 and potentials (298 K, 95/5 v/v% H2O/D2O and 0.15 mol/L ionic strength). The correlation between the acid-base and redox parameters with NMR chemical shifts is also determined. used for further calculations. acid-base and redox parameters with NMR chemical shifts is also determined. Figure 4. The coexisting protonation and redox microequilibria of selenocysteamine and Figure 4. The coexisting protonation and redox microequilibria of selenocysteamine and Figure 4. The coexisting protonation and redox microequilibria of selenocysteamine and selenocystamine; N, and Se labels denote the amino, and selenolate groups, respectively. selenocystamine; selenocystamine; N, N, and S and Se e la labels bels denote denote th the e amino, and amino, and se selenolate lenolate grou groups, ps, respe respectively ctively. . Figure 5. The protonation microequilibrium schemes of selenocysteine (left) and selenocystine (right). For simplicity, not every protonation microspecies is shown in a structural formula, rather a Figure 5. The protonation microequilibrium schemes of selenocysteine (left) and selenocystine Figure 5. The protonation microequilibrium schemes of selenocysteine (left) and selenocystine (right). (right). For simplicity, not every protonation microspecies is shown in a structural formula, rather a For simplicity, not every protonation microspecies is shown in a structural formula, rather a schematic structure depicts the microspecies with its basic sites where N, Se, and O labels denote the amino, selenolate, and carboxylate groups, respectively. The corresponding species participating in redox equilibria are bordered in color. Antioxidants 2020, 9, 465 5 of 13 Table 1. Protonation constant and standard redox potential values of the studied compounds from previous works. See Figures 4 and 5 for the description of the equilibrium constants. Oxidized Selenocysteine [17] Selenocystine [17] Selenocysteamine [17] Glutathione [9] Glutathione [18] logK 10.18 logK 9.19 logK 10.87 logK 9.65 logK 9.53 1 1 1 1 1 logK 5.52 logK 8.22 logK 6.26 logK 8.78 logK 8.83 2 2 2 2 2 logK 2.01 logK 2.25 logk 10.87 logK 3.52 logK 3.85 3 3 3 3 N Se logk 10.18 logK 1.58 logk 7.55 logK 2.22 logK 3.15 4 4 4 O N Se N 5.02 8.89 6.26 9.49 logK 2.32 logk logk logk logk N 5 O N’ logk 3.13 logk 8.52 E 0.374 mV logK 1.60 B 6 N N Se O N logk 6.81 logk 4.40 Selenocystamine [17] Dithiothreitol [14] logk 9.41 Se O’ N’ 5.52 4.33 logK 9.62 logK 10.1 9.25 logk logk logk N O 1 1 N Se logk 5.69 logK 8.48 logK 9.2 2 2 Se logk 4.40 NO Here, we report the comprehensive redox characterization of the selenolate-diselenide equilibria of selenocysteine and selenocysteamine in terms of pH-independent standard redox potentials (298 K, 95/5 v/v% H O/D O and 0.15 mol/L ionic strength). The correlation between the acid-base and redox 2 2 parameters with NMR chemical shifts is also determined. 2. Methods 2.1. Materials Seleno-l-cystine, oxidized glutathione, sarcosine and tert-butylamine were obtained from Sigma Aldrich, dithiothreitol was obtained from Tokyo Chemical Industry, selenocystamine dihydrochloride was purchased from AKos GmbH. Deuterium oxide (D O) and methanol were purchased from Merck. All reagents were of analytical grade and used without further purification. The deionized water was prepared with a Milli-Q Direct 8 Millipore system. 2.2. Preparation of Solutions for Equilibrium Constant Determination Stock solutions containing one of the reactants (seleno-l-cystine, selenocystamine dihydrochloride, oxidized glutathione or dithiothreitol) were prepared by dissolving in borax bu er. To a stock solution of DTT a known amount of tert-butylamine or sarcosine was added for use as an internal concentration standard and in situ pH indicator (see NMR spectroscopy chapter). A series of solutions was made by mixing the two types of stock solutions followed by pH adjustment using hydrochloric acid or sodium hydroxide [19]. The NMR measurement was carried out immediately after preparing the sample solution to diminish the risk of oxidation by air. To ensure that equilibrium was reached, NMR spectra were recorded instantly, 15 min, 1 h and 24 h after preparation of the sample. As no di erence was detected between the spectra, the onset of the equilibrium is considered immediate, which is in good agreement with the kinetics of thiolate-disulfide transitions [20]. A blank series (with no selenol compound) was also prepared and measured to determine the perturbing e ect of oxidation by air. 2.3. NMR Spectroscopy NMR spectra were recorded on a Varian 600 MHz spectrometer at 298 K. The solvent in every case was H O:D O, 95:5, v/v (0.15 mol/L ionic strength), using internal DSS 2 2 (3-(trimethylsilyl)propane-1-sulfonate sodium) as chemical shift reference. The sample volume was 600 L, the analyte concentration was 1–5 mmol/L. The pH values were determined by internal indicator molecules optimized for NMR [21,22]. The water resonance was diminished by presaturation pulse sequence (nt = 16, np = 64,000, acquisition time = 301 ms, relaxation delay = 15 s). Antioxidants 2020, 9, 465 6 of 13 2.4. Data Analysis For the analysis of quantitative NMR measurements the Lorentzian peak fitting algorithm of the ACD/NMR Processor Academic Edition v12.01 software package (Advanced Chemistry Development, Toronto, ON, Canada) was used with automatic baseline and phase correction, and no apodization. The integrals of the fitted peaks were compared to the integral of the concentration standard peak for concentration determination. For the regression analyses, the software Origin Pro 8 (OriginLab Corp., Northampton, MA, USA) was used. 3. Results Figure 4 shows the species-specific protonation equilibria of selenocysteamine and selenocystamine, along with the selenolate! diselenide oxidation processes. Microspecies that take part in the redox processes are framed. Figure 5 represents the species-specific acid-base equilibria of selenocysteine and selenocystine. The corresponding microspecies in the redox pairs have identical status of the amino and carboxylate sites. Acid-base equilibria are often described with macroscopic protonation constants, which only reveal the stoichiometry of the successively protonated ligands. Microscopic protonation schemes used in this work, however, characterize the site of protonation as well. The protonation microspecies are discerned with their one-letter symbols (a, b, c, etc.) and the microscopic protonation constants are N Se O depicted using k , k , k , etc. The superscript of microscopic protonation constants k indicates the protonating group, while the subscript (if any) shows the site(s) already protonated. Se, N, O denote the selenolate, amino, and carboxylate sites, respectively. The acid-base microequilibria are indispensable constituents in the evaluation of the species-specific, pH-independent redox equilibrium constants, as shown in Equations (7) and (8). Some protonation constant examples for selenocysteine are shown below: [HL ] h i K = (2) 2 + [L ] H [H L ] = K K K = (3) 3 1 2 3 h i 2 + [L ] H [b] k = h i (4) [a] H where K , K , K are successive macroconstants, is one of the cumulative macroconstants, and k is the 1 2 3 3 microconstant representing the amino protonation in CysSeH, when its carboxylate and selenolate sites are unprotonated. The concentrations of the various macrospecies comprise the sum of the concentration of those microspecies that contain the same number of protons, for example, in CysSeSeCys: 0 0 0 0 0 0 [H L] = [ f ] + [ g ] + [ h ] + [ i ] + [ j ] + [ k ] (5) For the selenolate-diselenide (and dithiolate-disulfide for dithiothreitol) redox equilibria, only the apparent (pH-dependent) or conditional equilibrium constants (K ) are directly available, by 3C determining the equilibrium concentration of the compounds (RSeH, DTT, RSeSeR, and DTT ) which ox. correspond to the total concentration of variously protonated reactant species. The pH-dependent, apparent redox equilibrium constants can be decomposed into pH-independent, species-specific equilibrium constants, the number of which is large, but definite. For example, the selenolate moiety in CysSeH is adjacent to two other basic sites: the amino and the carboxylate. Therefore, the number of selenolate-bearing microspecies is four, corresponding to the number of protonation states of Antioxidants 2020, 9, 465 7 of 13 the amino and carboxylate moieties (amino-carboxylate, ammonium-carboxylate, amino-carboxyl. ammonium-carboxyl). Accordingly, the selenolate exists in four di erent electrostatic environments, having thus four di erent oxidizabilities. Considering the opposite direction of the redox equilibrium, dithiothreitol has only one microspecies, bearing both thiols in their deprotonated, oxidizable form. Therefore, the redox equilibria between selenocysteine and dithiothreitol can be characterized with four di erent microscopic, species-specific equilibrium constants. The method for determining the microscopic redox equilibrium constants is analogous for every case. The determination of one of b b the microscopic redox equilibrium constants, k will be demonstrated. The notation k refers to the reaction involving the ”b” microspecies of CysSeH and oxidized dithiothreitol on the products side, and the corresponding ”f “ microspecies of CysSeSeCys and the deprotonated dithiothreitol on the reactants side. Note that ”b” defines that the diselenide microspecies is mandatorily ”f ”, since ”f “ is the only diselenide microspecies with protonation states in the side-chain identical with ”b”. Superscript ”b” therefore unambiguously identifies all four microspecies in the microequilibrium in question. The example of this pH-independent, microscopic selenolate-diselenide equilibrium constant is in Equation (6): [ ] [ ] b DTT ox. k = h i (6) 2 0 [ ] DTT f For the next step of the calculation, the relative abundance of the protonation microspecies is needed. The mole fraction of microspecies ”b” relative to the total CysSeH concentration can be written with the following equation, as can be carried out for every microspecies provided the complete set of protonation microconstants is known. h i k H [b] = [CysSeH] = [CysSeH] (7) b h i h i h i 2 3 + + + 1 + K H + K K H + K K K H 1 1 2 1 2 3 By writing analogous equations as Equation (7) for the remaining microspecies in the formula of k , one can write the equation of the microscopic equilibrium constant expressed with the conditional equilibrium constant as follows: 2 2 2 [CysSeH]  [DTT ] [b] [DTT ] ox. ox. b b k = h i = = K (8) 3C 2 0 0 [ ] [ ] 0 DTT  2 Se Cys  2 DTT [f ] f f DTT DTT Thus, if K and the  values are known, the species-specific redox microconstants can be 3C calculated. The conditional redox equilibrium constant values determined directly from the NMR spectra are listed in Table 2 and Supplementary Materials. These conditional redox equilibrium constants were determined in basic media only (above pH 9) due to the fact that the redox reaction did not commence at lower pH values. Only the negatively charged thiolate in DTT participates in redox reactions, and below pH 9 the thiolate groups are overwhelmingly in protonated form. However, as the conditional redox equilibrium constants provide pH-independent redox microconstants, it is apparent from Equation (8) that the pH range of determination does not impede the calculation. It should be noted however, that the pH-independent redox microconstants pertaining to microspecies that are predominantly present at pH values outside the determined range (i.e., k ) will have a greater inherent uncertainty based on the present calculations. Antioxidants 2020, 9, 465 8 of 13 Table 2. The conditional redox equilibrium constants in logarithmic units at the pH indicated in reaction mixtures of dithiothreitol with selenocysteine, selenocysteamine and glutathione. Selenocysteine Selenocysteamine Glutathione pH logK pH logK pH logK C C C 9.79 2.88 9.66 2.31 9.00 0.00 9.81 2.20 9.67 2.43 8.30 0.41 9.86 2.87 9.67 2.23 8.35 0.03 9.90 2.07 9.68 2.48 9.07 0.20 9.96 1.98 9.69 2.40 9.63 0.41 9.98 2.04 9.80 2.37 10.23 0.05 10.02 2.05 9.89 2.09 11.01 0.83 9.94 2.25 9.98 2.21 10.01 2.16 The microscopic redox equilibrium constants were calculated and derived from the conditional equilibrium constants using analogous equations to Equation (8) based on at least three repeated measurements. The mean values of the calculated microscopic redox equilibrium constants and their standard deviation of determination are listed in Table 3. Note that near physiological pH (i.e., around pH 7) the most abundant CysSe and CysASe selenolate microspecies are ”b” and ”b”, respectively. In fact, these microspecies have maximal relative abundance between pH 6.5 and 10, therefore the pH range of determination in our study is well-conditioned for the extraction of the physiologically relevant redox microconstants. In order to obtain the standard redox potential values of the studied selenol compounds, the standard redox potential of dithiothreitol is needed. Although dithiothreitol has been extensively studied, only its apparent redox potential has been previously determined, therefore we determined the pH-independent, species-specific standard redox potential of DTT /DTT as well using glutathione ox. as the reaction partner. In the state of a chemical equilibrium, the electrode potential of every existing redox system is equal. For example, for the reaction of dithiothreitol and glutathione one can write: 0 0 E = E = E =  (9) A /A E /B DTT /DTT ox. where the A’/A, E’/B symbols denote the microspecies of glutathione disulfide/glutathione redox pairs. We chose to perform further calculations with equations pertaining to glutathione/glutathione disulfide microspecies B/E’, since this redox pair has the highest relative abundance in the pH of interest, and therefore its calculation is least exposed to uncertainty. The above equation adapted for glutathione microspecies ”B” Figure 6 and DTT becomes: RT [DTT ] RT [ E ] ox. E + ln = E + ln (10) E /B DTT /DTT ox. 2 2 zF zF [DTT ] [ ] 0 2 RT [ E ][DTT ] RT 0 0 E = E + ln = E ln k (11) E /B E /B DTT /DTT ox. 2 zF zF [ ] [ ] B DTT ox. where E is the standard redox potential of the redox system denoted in its subscript, R is the universal gas constant, T is the absolute temperature, z is the number of electrons transferred in the redox half-reaction, and F is the Faraday constant. Using the previously determined microscopic protonation constant [9] and standard redox potential [5] of glutathione microspecies ”B”, the species-specific standard redox potential was determined for the DTT /DTT redox system as0.403 0.007 V. ox. Antioxidants 2020, 9, x FOR PEER REVIEW 8 of 13 The microscopic redox equilibrium constants were calculated and derived from the conditional equilibrium constants using analogous equations to Equation (8) based on at least three repeated measurements. The mean values of the calculated microscopic redox equilibrium constants and their standard deviation of determination are listed in Table 3. Note that near physiological pH (i.e., around pH 7) the most abundant CysSe and CysASe selenolate microspecies are ”b” and ”b”, respectively. In fact, these microspecies have maximal relative abundance between pH 6.5 and 10, Antioxidants 2020, 9, 465 9 of 13 therefore the pH range of determination in our study is well-conditioned for the extraction of the physiologically relevant redox microconstants. Table 3. Microspecies-specific, pH-independent equilibrium constants of selenocysteine, Table 3. Microspecies-specific, pH-independent equilibrium constants of selenocysteine, selenocysteamine and glutathione with dithiothreitol in logarithmic units, with standard deviations. selenocysteamine and glutathione with dithiothreitol in logarithmic units, with standard deviations. The parameter of glutathione was determined for the B microspecies only, for further calculation (see The parameter of glutathione was determined for the B microspecies only, for further calculation (see Figure 6 for glutathione microspecies). Figure 6 for glutathione microspecies). Selenocysteine Selenocysteamine Glutathione Selenocysteine Selenocysteamine Glutathione Microspecies logk sd Microspecies logk sd Microspecies logk sd Microspecies logk sd Microspecies logk sd Microspecies logk sd a a 2.68−2.68 0.42 0.42 a a − 3.72 3.72 0.19 0.19 B B 0.99 0.990.24 0.24 b 0.27 0.42 b 0.08 0.19 b 0.27 0.42 b −0.08 0.19 d 1.37 0.42 d −1.37 0.42 f 2.70 0.42 f 2.70 0.42 Figure 6. The structure and protonation states of the referenced glutathione (A, B, top) and glutathione Figure 6. The structure and protonation states of the referenced glutathione (A, B, top) and disulfide (A’, E’, bottom) microspecies in this work. glutathione disulfide (A’, E’, bottom) microspecies in this work. With the knowledge of the selenolate-diselenide species-specific redox equilibrium constants In order to obtain the standard redox potential values of the studied selenol compounds, the and the standard redox potential of the DTT /DTT redox couple, the species-specific standard ox. standard redox potential of dithiothreitol is needed. Although dithiothreitol has been extensively redox potentials of the di erent selenolate-containing microspecies could be calculated. Equation (12) studied, only its apparent redox potential has been previously determined, therefore we determined shows the continued example of CysSeH microspecies ”b”. The comprehensive set of determined 2− the pH-independent, species-specific standard redox potential of DTTox./DTT as well using species-specific standard redox potentials is listed in Table 4. glutathione as the reaction partner. In the state of a chemical equilibrium, the electrode potential of every existing redox system is equal. For example, for the reaction of dithiothreitol and glutathione RT [b] [DTT ] RT ox. one can write: E = E 2 + ln = E 2 + ln k (12) DTT /DTT 0 2 DTT /DTT ox. ox. zF zF [ ] f [DTT ] 𝐸 =𝐸 =𝐸 =⋯ (9) / / / Table 4. The species-specific standard redox potential values for every microspecies of selenocysteine where the A’/A, E’/B symbols denote the microspecies of glutathione disulfide/glutathione redox and selenocysteamine given as mean standard deviation. pairs. We chose to perform further calculations with equations pertaining to Selenocysteine Selenocysteamine Microspecies E (V) Microspecies E (V) a 0.482 0.01 a 0.513 0.006 b 0.395 0.01 b 0.405 0.006 d 0.444 0.01 f 0.323 0.01 Antioxidants 2020, 9, 465 10 of 13 By graphing these standard redox potentials against the concomitant selenolate-specific protonation constants compiled in Table 1 we find a similar correlation to that of thiolates presented in Antioxidants 2020, 9, x FOR PEER REVIEW 10 of 13 Figure 7. Figure 7. Correlation between standard redox potentials and species-specific thiolate protonation Figure 7. Correlation between standard redox potentials and species-specific thiolate protonation constants for various thiol-containing compounds (glutathione, cysteine, cysteamine, homocysteine, constants for various thiol-containing compounds (glutathione, cysteine, cysteamine, homocysteine, penicillamine, ovothiol A) reproduced from [4] with the addition of the data of dithiothreitol (upper penicillamine, ovothiol A) reproduced from [4] with the addition of the data of dithiothreitol (upper line, line, fu fulllcir l circl cles);es); the the correl correlationation between between standar stan d redox dard re potentials dox potentials and the species-specific and the speciselenolate es-specific pr selenolate protonation constants otonation constants for the various for the var selenol-containing ious selenol-containing compounds (lower compou line, nds (lower l empty circine, em les). pty circles). 4. Discussion 4. Discussion In this work, the highly interwoven acid-base and redox pathways of selenolate-diselenide systems were decomposed into species-specific, component equilibria. The standard redox potentials In this work, the highly interwoven acid-base and redox pathways of selenolate-diselenide of biologically relevant selenolate-diselenide couples are determined for the first time; these values systems were decomposed into species-specific, component equilibria. The standard redox characterize the redox processes at the protonation microspecies level. The elucidation of these potentials of biologically relevant selenolate-diselenide couples are determined for the first time; redox microequilibria reveals considerable di erences between the various protonation species. these values characterize the redox processes at the protonation microspecies level. The elucidation The knowledge of species-specific standard redox potentials of selenocysteine and selenocystine, of these redox microequilibria reveals considerable differences between the various protonation in particular, improves our knowledge on redox homeostasis and can lead to better interpretation species. The knowledge of species-specific standard redox potentials of selenocysteine and of several biochemical phenomena. For example the cytoprotective potential of selenoproteins selenocystine, in particular, improves our knowledge on redox homeostasis and can lead to better is thought to entail several mechanisms involving thio/seleno chemistry [23]. One proposed interpretation of several biochemical phenomena. For example the cytoprotective potential of mechanism of action is formation of stable diselenide bonds in thioredoxin reductase acting as selenoproteins is thought to entail several mechanisms involving thio/seleno chemistry [23]. One a ”diselenide trap”, which can be further supported by exact standard redox potentials of the proposed mechanism of action is formation of stable diselenide bonds in thioredoxin reductase reaction partners. New selenoproteins are investigated for their function, likely related to catalyzing acting as a ”diselenide trap”, which can be further supported by exact standard redox potentials of thiol/disulfide exchange in proteins, as the kinetics of selenolate-diselenide transitions is seven orders the reaction partners. New selenoproteins are investigated for their function, likely related to of magnitude greater than that of thiolate-disulfide transitions [24]. In order to understand these catalyzing thiol/disulfide exchange in proteins, as the kinetics of selenolate-diselenide transitions is catalytic e ects purified from the protonation fraction of the thiolate and selenolate moieties at seven orders of magnitude greater than that of thiolate-disulfide transitions [24]. In order to understand these catalytic effects purified from the protonation fraction of the thiolate and selenolate moieties at physiological pH, a comprehensive species-specific characterization is needed. The knowledge of detailed physico-chemical properties of selenocompounds presented in this work Antioxidants 2020, 9, 465 11 of 13 Antioxidants 2020, 9, x FOR PEER REVIEW 11 of 13 can also serve as the basis to develop artificial selenoenzymes [25] of highly selective redox physiological pH, a comprehensive species-specific characterization is needed. The knowledge of capacities. detailed physico-chemical properties of selenocompounds presented in this work can also serve as the The example of selenocystine microspecies ”a′” and ”f′” (Table 4) shows that even side chain basis to develop artificial selenoenzymes [25] of highly selective redox capacities. protonation changes can lead to significantly different redox characteristics, with nearly 160 mV 0 0 The example of selenocystine microspecies ”a ” and ”f ” (Table 4) shows that even side chain difference in standard redox potential values. Therefore, small changes in pH can not only affect the protonation changes can lead to significantly di erent redox characteristics, with nearly 160 mV redox processes of selenolate-diselenide transitions by changing the protonation fraction of the di erence in standard redox potential values. Therefore, small changes in pH can not only a ect selenolate, but also by altering the protonation state of neighboring moieties. the redox processes of selenolate-diselenide transitions by changing the protonation fraction of the The correlation between selenolate basicity and standard redox potentials verifies the previous selenolate, but also by altering the protonation state of neighboring moieties. observations regarding thiolate basicity and its proportionality with thiolate oxidizability. It is The correlation between selenolate basicity and standard redox potentials verifies the previous interesting that the correlation line between selenolate basicity and the concomitant standard redox observations regarding thiolate basicity and its proportionality with thiolate oxidizability. It is potential is parallel to the correlation line of thiolates, however shifted by ca. −246 mV units. This interesting that the correlation line between selenolate basicity and the concomitant standard redox accentuates the fact that selenolates, apart from being less basic, are vastly stronger reducing agents potential is parallel to the correlation line of thiolates, however shifted by ca. 246 mV units. This than thiolates in general. It is noteworthy that the species-specific standard redox potentials of accentuates the fact that selenolates, apart from being less basic, are vastly stronger reducing agents selenolate-containing microspecies also show linear correlation with species-specific NMR chemical than thiolates in general. It is noteworthy that the species-specific standard redox potentials of shift. The chemical shift values were previously determined [16], and the details of the correlation selenolate-containing microspecies also show linear correlation with species-specific NMR chemical 1 13 are shown in Figure 8 and Table 5. The use of chemical shifts in protein NMR (from H, C, and for shift. The chemical shift values were previously determined [16], and the details of the correlation selenoproteins the relatively undisrupted Se spectra) can now serve as sound means to predict 1 13 are shown in Figure 8 and Table 5. The use of chemical shifts in protein NMR (from H, C, and selenolate oxidizability or diselenide reducibilty/stability in proteins: a key parameter to understand for selenoproteins the relatively undisrupted Se spectra) can now serve as sound means to predict and influence oxidative stress. selenolate oxidizability or diselenide reducibilty/stability in proteins: a key parameter to understand The critical issue in designing preventive or therapeutic antioxidants is the narrow path of and influence oxidative stress. redox potentials, effective enough to reduce harmful oxidative agents, but keeps disulfides and The critical issue in designing preventive or therapeutic antioxidants is the narrow path of other reducible units in useful biomolecules intact. Naturally, small reducing agents can hardly be as redox potentials, e ective enough to reduce harmful oxidative agents, but keeps disulfides and other selective as substrate-specific enzymes of the biological antioxidant system; however, a finely tuned reducible units in useful biomolecules intact. Naturally, small reducing agents can hardly be as and designed selenolate-containing compound with an appropriate basicity, redox potential and selective as substrate-specific enzymes of the biological antioxidant system; however, a finely tuned concomitant selectivity can be confined to a narrower range. The correlation between the redox and and designed selenolate-containing compound with an appropriate basicity, redox potential and NMR parameters serves now as a sound basis to better quantify the characteristics of diselenide concomitant selectivity can be confined to a narrower range. The correlation between the redox and moieties in selenoenzymes, allowing thus the development of potent, selective antioxidant NMR parameters serves now as a sound basis to better quantify the characteristics of diselenide compounds for serious ailments related to selenoenzyme deficiencies, such as autism [26]. moieties in selenoenzymes, allowing thus the development of potent, selective antioxidant compounds for serious ailments related to selenoenzyme deficiencies, such as autism [26]. Figure 8. Correlation of selenocysteine standard redox potentials with the corresponding selenocysteine Figure 8. Correlation of selenocysteine standard redox potentials with the corresponding microspecies chemical shifts (left) and selenocystine microspecies chemical shifts (right). selenocysteine microspecies chemical shifts (left) and selenocystine microspecies chemical shifts (right). Antioxidants 2020, 9, 465 12 of 13 Table 5. Correlation data of selenocysteine standard redox potentials with the corresponding selenocysteine microspecies chemical shifts and the corresponding selenocystine microspecies chemical shifts. Selenocysteine 77 1 1 1 13 13 Se H ( CH) H ( CH ) H ( CH ) C ( CH) C ( CH ) 2a 2 2b slope 70.524 5.251 2.208 3.163 24.743 52.487 intercept 196.660 5.755 3.970 4.013 50.137 3.319 r 0.4979 0.9622 0.4838 0.9095 0.8571 0.9180 Selenocystine slope 59.106 5.806 2.446 2.437 16.557 18.030 intercept 310.960 6.338 4.102 4.036 49.893 23.692 r 0.8908 0.9660 0.9720 0.9259 0.5578 0.7718 5. Conclusions The standard redox potentials of diselenide/selenolate-containing microspecies of selenocysteine and selenocysteamine were determined using an indirect approach of measuring redox equilibrium constants with the help of dithiothreitol. These standard redox potentials are pH-independent and show correlation with selenolate acid-base characteristics; an important observation previously demonstrated for thiolate-analogues as well. The acid-base and redox parameters both show correlation with the NMR chemical shifts of the diselenide or selenolate-containing species as well. This can be used as a tool for predicting diselenide behavior solely based on Se NMR data. Supplementary Materials: The following are available online at http://www.mdpi.com/2076-3921/9/6/465/s1. Author Contributions: Conceptualization, T.P., A.M. and B.N.; Methodology, T.P., A.M. and B.N.; Investigation, T.P., A.M. and B.N.; Resources, B.N.; Data Curation, T.P. and A.M.; Writing—Original Draft Preparation, T.P. and A.M; Writing—Review & Editing, T.P., A.M. and B.N.; Visualization, T.P. and A.M.; Supervision, B.N.; Project Administration, B.N.; Funding Acquisition, A.M. and B.N. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: This research was supported by the FIKP-2019 New National Excellence Program of the Ministry of Human Capacities of Hungary. Conflicts of Interest: The authors report no conflict of interest. References 1. Tapiero, H.; Townsend, D.M.; Tew, K.D. The antioxidant role of selenium and seleno-compounds. Biomed. Pharmacother. 2003, 57, 134–144. [CrossRef] 2. Flohe, L.; Günzler, W.; Schock, H. Glutathione peroxidase: A selenoenzyme. FEBS Lett. 1973, 32, 132–134. [CrossRef] 3. Köhrle, J. The deiodinase family: Selenoenzymes regulating thyroid hormone availability and action. Cell. Mol. Life Sci. CMLS 2000, 57, 1853–1863. [CrossRef] [PubMed] 4. Zhong, L.; Arnér, E.S.; Holmgren, A. 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A comparative study of the kinetics of selenol/diselenide and thiol/disulfide exchange reactions. J. Am. Chem. Soc. 1989, 111, 6553–6558. [CrossRef] 25. Huang, X.; Liu, X.; Luo, Q.; Liu, J.; Shen, J. Artificial selenoenzymes: Designed and redesigned. Chem. Soc. Rev. 2011, 40, 1171–1184. [CrossRef] 26. Raymond, L.J.; Deth, R.C.; Ralston, N.V. Potential role of selenoenzymes and antioxidant metabolism in relation to autism etiology and pathology. Autism Res. Treat. 2014, 2014. [CrossRef] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Antioxidants Multidisciplinary Digital Publishing Institute

Species-Specific, pH-Independent, Standard Redox Potential of Selenocysteine and Selenocysteamine

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antioxidants Article Species-Specific, pH-Independent, Standard Redox Potential of Selenocysteine and Selenocysteamine 1 , 2 1 , 2 1 , 2 , Tamás Pálla , Arash Mirzahosseini and Béla Noszál * Department of Pharmaceutical Chemistry, Semmelweis University, H-1092 Budapest, Hungary; palla.tamas@pharma.semmelweis-univ.hu (T.P.); mirzahosseini.arash@pharma.semmelweis-univ.hu (A.M.) Research Group of Drugs of Abuse and Doping Agents, Hungarian Academy of Sciences, H-1051 Budapest, Hungary * Correspondence: noszal.bela@pharma.semmelweis-univ.hu; Tel.: +36-1217-0891 Received: 10 April 2020; Accepted: 21 May 2020; Published: 1 June 2020 Abstract: Microscopic redox equilibrium constants and standard redox potential values were determined to quantify selenolate-diselenide equilibria of biological significance. The highly composite, codependent acid-base and redox equilibria of selenolates could so far be converted into pH-dependent, apparent parameters (equilibrium constants, redox potentials) only. In this work, the selenolate-diselenide redox equilibria of selenocysteamine and selenocysteine against dithiothreitol were analyzed by quantitative nuclear magnetic resonance (NMR) methods to characterize the interfering acid-base and redox equilibria. The directly obtained, pH-dependent, conditional redox equilibrium constants were then decomposed by our method into pH-independent, microscopic constants, which characterize the two-electron redox transitions of selenocysteamine and selenocysteine. The 12 di erent, species-specific parameter values show close correlation with the respective selenolate basicities, providing a tool to estimate otherwise inaccessible site-specific selenolate-diselenide redox potentials of related moieties in large peptides and proteins. Keywords: selenocysteine; redox; disulfide 1. Introduction The role of selenium, the biological trace element and the related selenium-containing proteins (selenoproteins) has been described in numerous antioxidant processes [1]. Selenocysteine, known as the twenty-first amino acid, is the only selenium-containing building block of proteins that contains selenium. The majority of selenoproteins are enzymes (such as glutathione peroxidase [2], iodothyronine deiodinase [3], thioredoxin reductase [4]), most of which are involved in redox reactions. Their selenocysteine residue is the essential unit for their catalytic activity. A new, specific way to quantify redox properties at the submolecular level has recently been introduced to characterize thiols of biological importance (cysteine, cystamine, homocysteine and glutathione [5,6]; ovothiol A and penicillamine [5,7]). A major conclusion of these studies is that there is a close correlation between the thiolate basicities and the thiolate-disulfide redox properties [5–9]. The hypothesis of this work is that such a correlation between acid-base and redox characteristics exists for the selenium-analogues of the above compounds as well, namely for selenocysteine and selenocysteamine. We wish to demonstrate said correlation by determining the species-specific physico-chemical parameters of the acid-base and redox equilibria of the above compounds and extend the correlation to nuclear magnetic resonance (NMR) chemical shift values as well. In this work, the species-specific redox equilibria of two biogenic selenols: selenocysteamine (CysASeH) and selenocysteine (CysSeH) with dithiothreitol (DTT) were studied. The oxidation of these Antioxidants 2020, 9, 465; doi:10.3390/antiox9060465 www.mdpi.com/journal/antioxidants Antioxidants 2020, 9, x FOR PEER REVIEW 2 of 13 In this work, the species-specific redox equilibria of two biogenic selenols: selenocysteamine Antioxidants 2020, 9, 465 2 of 13 (CysASeH) and selenocysteine (CysSeH) with dithiothreitol (DTT) were studied. The oxidation of these compounds yields selenocystamine (CysASeSeCysA) and selenocystine (CysSeSeCys), compounds yields selenocystamine (CysASeSeCysA) and selenocystine (CysSeSeCys), respectively. respectively. The constitutional formulae of the studied selenium-containing compounds are The depicted in constitutional Figure 1. formulae of the studied selenium-containing compounds are depicted in Figure 1. Figure 1. The structure of the investigated selenols (left) and diselenides (right). Figure 1. The structure of the investigated selenols (left) and diselenides (right). Glutathione is known as a “gold standard” in thiol-disulfide biochemistry, hence, every Glutathione is known as a “gold standard” in thiol-disulfide biochemistry, hence, every thiol-containing antioxidant is compared to glutathione. Moreover, the species-specific thiol-containing antioxidant is compared to glutathione. Moreover, the species-specific physico-chemical acid-base [9] and redox [5,6] parameters of glutathione are well-characterized. physico-chemical acid-base [9] and redox [5,6] parameters of glutathione are well-characterized. However, previous works examining selenoproteins or selenium-containing peptides found that the However, previous works examining selenoproteins or selenium-containing peptides found that the diselenide bridge cannot be reduced by glutathione [10,11]. We found that even a large excess of diselenide bridge cannot be reduced by glutathione [10,11]. We found that even a large excess of glutathione was ine ective in reducing the studied diselenide compounds, confirming the above glutathione was ineffective in reducing the studied diselenide compounds, confirming the above observation. Thus, a more powerful reducing agent was needed to investigate selenolate-diselenide observation. Thus, a more powerful reducing agent was needed to investigate selenolate-diselenide redox equilibria. Based on literature recommendations [8,12], dithiothreitol (DTT), a stronger reducing redox equilibria. Based on literature recommendations [8,12], dithiothreitol (DTT), a stronger agent, was chosen. Dithiothreitol undergoes a one-step two-electron redox reaction, resulting in the reducing agent, was chosen. Dithiothreitol undergoes a one-step two-electron redox reaction, formation of oxidized dithiothreitol (DTT ) with an intramolecular disulfide in a six-membered ring. ox resulting in the formation of oxidized dithiothreitol (DTTox) with an intramolecular disulfide in a During the redox processes between dithiothreitol and other agents, the presence of the intermediate six-membered ring. During the redox processes between dithiothreitol and other agents, the compound, where only one of the thiolate groups of DTT is oxidized is negligible [13]. The main 2− presence of the intermediate compound, where only one of the thiolate groups of DTT is oxidized physico-chemical properties of DTT are already described (logK = 10.1; logK = 9.2 [14]; E =0.323 1 2 is negligible [13]. The main physico-chemical properties of DTT are already described (logK1 = 10.1; V at pH = 7.0 [15]). logK2 = 9.2 [14]; E° = −0.323 V at pH = 7.0 [15]). To obtain data describing selenolate-diselenide redox equilibria comparable to those of To obtain data describing selenolate-diselenide redox equilibria comparable to those of thiolate-disulfide systems, first the glutathione-DTT redox reaction was investigated (Figure 2). 2− thiolate-disulfide systems, first the glutathione-DTT redox reaction was investigated (Figure 2). The general step-by-step scheme of selenolate-diselenide redox equilibria with DTT is depicted in Figure 3, where RSe and RSeSeR stand for the reduced and oxidized form of the selenium-containing compounds, respectively. The reaction between disulfides (e.g., oxidized glutathione) and DTT is analogous (where RS and RSSR stand for the thiolate and disulfide, respectively). Antioxidants 2020, 9, x FOR PEER REVIEW 3 of 13 Antioxidants 2020, 9, 465 3 of 13 Antioxidants 2020, 9, x FOR PEER REVIEW 3 of 13 2− Figure 2. The redox reaction between oxidized glutathione and dithiothreitol (DTT) dominant microspecies at the pH of blood. 2− The general step-by-step scheme of selenolate-diselenide redox equilibria with DTT is depicted in Figure 3, where RSe and RSeSeR stand for the reduced and oxidized form of the selenium-containing compounds, respectively. The reaction between disulfides (e.g., oxidized 2− − glutathione) and DTT is analogous (where RS and RSSR stand for the thiolate and disulfide, 2− Figure 2. Figure 2. The The redox redox react reaction ion between between ox oxidized idized g glutathione lutathione and and dith dithiothr iothreitol (DTT eitol (DTT)) dominant dominant respectively). microspecies at the pH of blood. microspecies at the pH of blood. 2− The general step-by-step scheme of selenolate-diselenide redox equilibria with DTT is depicted in Figure 3, where RSe and RSeSeR stand for the reduced and oxidized form of the selenium-containing compounds, respectively. The reaction between disulfides (e.g., oxidized 2− − glutathione) and DTT is analogous (where RS and RSSR stand for the thiolate and disulfide, respectively). 2− 2 Figure Figure 3. 3. The t The two-step wo-step re redox dox equ equilibria ilibria betw between een a adis diselenide elenide com compound pound an and d DTT DTT (reac (reaction tion I and II I and ) II) followed followed by their aggregated one-step net re by their aggregated one-step netaction reaction (III) (III). . IdeIdentical ntical reac rtion eaction schem schemes es are ap areplicable applicable for for disu disulfide lfide analo analogue gues. The arrow in reaction I s. The arrow in reaction I indica II indicates tes the low probability the low probability of formof ation of the su formation of lfu the r– sulfur seleniu –selenium m bridge.bridge. Since DTT 2− is highly driven towards the ring-closing second oxidation step [13], and diselenides Since DTT is highly driven towards the ring-closing second oxidation step [13], and are more stable than the selenylsulfides [16], the reaction between diselenides and DTT can be 2− diselenides are more stable than the selenylsulfides [16], the reaction between diselenides and DTT 2− Figure 3. The two-step redox equilibria between a diselenide compound and DTT (reaction I and II) reasonably discussed in terms of the net reaction. The conditional equilibrium constant describing this can be reasonably discussed in terms of the net reaction. The conditional equilibrium constant followed by their aggregated one-step net reaction (III). Identical reaction schemes are applicable for net reaction is as follows: describing this net reaction is as follows: disulfide analogues. The arrow in reaction II indicate2 s the low probability of formation of the sulfur– [RSe ] [DTT ] ox. h i selenium bridge. K = (1) 3C [RSe ] [DTT ] (1) 𝐾 = DTT [RSeSeR] [DTT ][RSeSeR] 2− Since DTT is highly driven towards the ring-closing second oxidation step [13], and Since redox and acid-base reactions coexist, the observed apparent redox equilibrium constants are 2− diselenides are more stable than the selenylsulfides [16], the reaction between diselenides and DTT pH-dependent. In order to get a clear insight into the redox equilibria, purified from the protonation can be reasonably discussed in terms of the net reaction. The conditional equilibrium constant e ects, an improved evaluation method has been introduced [5,6]. This new method makes it describing this net reaction is as follows: x possible to determine species-specific redox equilibrium constants (k ) and the species-specific, i.e., [RSe ] [DTT ] (1) 𝐾 = [ ][ ] DTT RSeSeR Antioxidants 2020, 9, x FOR PEER REVIEW 4 of 13 Antioxidants 2020, 9, x FOR PEER REVIEW 4 of 13 Since redox and acid-base reactions coexist, the observed apparent redox equilibrium constants are pH-dependent. In order to get a clear insight into the redox equilibria, purified from the Since redox and acid-base reactions coexist, the observed apparent redox equilibrium constants protonation effects, an improved evaluation method has been introduced [5,6]. This new method are pH-dependent. In order to get a clear insight into the redox equilibria, purified from the makes it possible to determine species-specific redox equilibrium constants (k ) and the protonation effects, an improved evaluation method has been introduced [5,6]. This new method species-specific, i.e., the standard redox potential (E°x, where x is the general sign of the appropriate makes it possible to determine species-specific redox equilibrium constants (k ) and the RSe microspecies in Figure 4; Figure 5). This determination method requires all the species-specific species-specific, i.e., the standard redox potential (E°x, where x is the general sign of the appropriate protonation c − onstants, including those of the minor microspecies; otherwise, redox processes could Antioxidants 2020, 9, 465 4 of 13 RSe microspecies in Figure 4; Figure 5). This determination method requires all the species-specific only be characterized at the level of phenomenon. The complete set of the microscopic protonation protonation constants, including those of the minor microspecies; otherwise, redox processes could constants of the studied selenium-containing compounds has recently been established [17]. The only be characterized at the level of phenomenon. The complete set of the microscopic protonation the standard redox potential (E , where x is the general sign of the appropriate RSe microspecies in protonation constants of selenocysteine, selenocysteamine, and glutathione, relevant to this work, constants of the studied selenium-containing compounds has recently been established [17]. The Figures 4 and 5). This determination method requires all the species-specific protonation constants, are co protonation c llected in Table onstants o 1 anfd selenocyst used for feine, selenocysteamine, urther calculations. and glutathione, relevant to this work, including those of the minor microspecies; otherwise, redox processes could only be characterized at are co Here, we llected i re n Table port the co 1 and usemprehensive d for further ca redox lculat char ions. acterization of the selenolate-diselenide the level of phenomenon. The complete set of the microscopic protonation constants of the studied Here, we report the comprehensive redox characterization of the selenolate-diselenide equilibria of selenocysteine and selenocysteamine in terms of pH-independent standard redox selenium-containing compounds has recently been established [17]. The protonation constants of equilibria of selenocysteine and selenocysteamine in terms of pH-independent standard redox potentials (298 K, 95/5 v/v% H2O/D2O and 0.15 mol/L ionic strength). The correlation between the selenocysteine, selenocysteamine, and glutathione, relevant to this work, are collected in Table 1 and potentials (298 K, 95/5 v/v% H2O/D2O and 0.15 mol/L ionic strength). The correlation between the acid-base and redox parameters with NMR chemical shifts is also determined. used for further calculations. acid-base and redox parameters with NMR chemical shifts is also determined. Figure 4. The coexisting protonation and redox microequilibria of selenocysteamine and Figure 4. The coexisting protonation and redox microequilibria of selenocysteamine and Figure 4. The coexisting protonation and redox microequilibria of selenocysteamine and selenocystamine; N, and Se labels denote the amino, and selenolate groups, respectively. selenocystamine; selenocystamine; N, N, and S and Se e la labels bels denote denote th the e amino, and amino, and se selenolate lenolate grou groups, ps, respe respectively ctively. . Figure 5. The protonation microequilibrium schemes of selenocysteine (left) and selenocystine (right). For simplicity, not every protonation microspecies is shown in a structural formula, rather a Figure 5. The protonation microequilibrium schemes of selenocysteine (left) and selenocystine Figure 5. The protonation microequilibrium schemes of selenocysteine (left) and selenocystine (right). (right). For simplicity, not every protonation microspecies is shown in a structural formula, rather a For simplicity, not every protonation microspecies is shown in a structural formula, rather a schematic structure depicts the microspecies with its basic sites where N, Se, and O labels denote the amino, selenolate, and carboxylate groups, respectively. The corresponding species participating in redox equilibria are bordered in color. Antioxidants 2020, 9, 465 5 of 13 Table 1. Protonation constant and standard redox potential values of the studied compounds from previous works. See Figures 4 and 5 for the description of the equilibrium constants. Oxidized Selenocysteine [17] Selenocystine [17] Selenocysteamine [17] Glutathione [9] Glutathione [18] logK 10.18 logK 9.19 logK 10.87 logK 9.65 logK 9.53 1 1 1 1 1 logK 5.52 logK 8.22 logK 6.26 logK 8.78 logK 8.83 2 2 2 2 2 logK 2.01 logK 2.25 logk 10.87 logK 3.52 logK 3.85 3 3 3 3 N Se logk 10.18 logK 1.58 logk 7.55 logK 2.22 logK 3.15 4 4 4 O N Se N 5.02 8.89 6.26 9.49 logK 2.32 logk logk logk logk N 5 O N’ logk 3.13 logk 8.52 E 0.374 mV logK 1.60 B 6 N N Se O N logk 6.81 logk 4.40 Selenocystamine [17] Dithiothreitol [14] logk 9.41 Se O’ N’ 5.52 4.33 logK 9.62 logK 10.1 9.25 logk logk logk N O 1 1 N Se logk 5.69 logK 8.48 logK 9.2 2 2 Se logk 4.40 NO Here, we report the comprehensive redox characterization of the selenolate-diselenide equilibria of selenocysteine and selenocysteamine in terms of pH-independent standard redox potentials (298 K, 95/5 v/v% H O/D O and 0.15 mol/L ionic strength). The correlation between the acid-base and redox 2 2 parameters with NMR chemical shifts is also determined. 2. Methods 2.1. Materials Seleno-l-cystine, oxidized glutathione, sarcosine and tert-butylamine were obtained from Sigma Aldrich, dithiothreitol was obtained from Tokyo Chemical Industry, selenocystamine dihydrochloride was purchased from AKos GmbH. Deuterium oxide (D O) and methanol were purchased from Merck. All reagents were of analytical grade and used without further purification. The deionized water was prepared with a Milli-Q Direct 8 Millipore system. 2.2. Preparation of Solutions for Equilibrium Constant Determination Stock solutions containing one of the reactants (seleno-l-cystine, selenocystamine dihydrochloride, oxidized glutathione or dithiothreitol) were prepared by dissolving in borax bu er. To a stock solution of DTT a known amount of tert-butylamine or sarcosine was added for use as an internal concentration standard and in situ pH indicator (see NMR spectroscopy chapter). A series of solutions was made by mixing the two types of stock solutions followed by pH adjustment using hydrochloric acid or sodium hydroxide [19]. The NMR measurement was carried out immediately after preparing the sample solution to diminish the risk of oxidation by air. To ensure that equilibrium was reached, NMR spectra were recorded instantly, 15 min, 1 h and 24 h after preparation of the sample. As no di erence was detected between the spectra, the onset of the equilibrium is considered immediate, which is in good agreement with the kinetics of thiolate-disulfide transitions [20]. A blank series (with no selenol compound) was also prepared and measured to determine the perturbing e ect of oxidation by air. 2.3. NMR Spectroscopy NMR spectra were recorded on a Varian 600 MHz spectrometer at 298 K. The solvent in every case was H O:D O, 95:5, v/v (0.15 mol/L ionic strength), using internal DSS 2 2 (3-(trimethylsilyl)propane-1-sulfonate sodium) as chemical shift reference. The sample volume was 600 L, the analyte concentration was 1–5 mmol/L. The pH values were determined by internal indicator molecules optimized for NMR [21,22]. The water resonance was diminished by presaturation pulse sequence (nt = 16, np = 64,000, acquisition time = 301 ms, relaxation delay = 15 s). Antioxidants 2020, 9, 465 6 of 13 2.4. Data Analysis For the analysis of quantitative NMR measurements the Lorentzian peak fitting algorithm of the ACD/NMR Processor Academic Edition v12.01 software package (Advanced Chemistry Development, Toronto, ON, Canada) was used with automatic baseline and phase correction, and no apodization. The integrals of the fitted peaks were compared to the integral of the concentration standard peak for concentration determination. For the regression analyses, the software Origin Pro 8 (OriginLab Corp., Northampton, MA, USA) was used. 3. Results Figure 4 shows the species-specific protonation equilibria of selenocysteamine and selenocystamine, along with the selenolate! diselenide oxidation processes. Microspecies that take part in the redox processes are framed. Figure 5 represents the species-specific acid-base equilibria of selenocysteine and selenocystine. The corresponding microspecies in the redox pairs have identical status of the amino and carboxylate sites. Acid-base equilibria are often described with macroscopic protonation constants, which only reveal the stoichiometry of the successively protonated ligands. Microscopic protonation schemes used in this work, however, characterize the site of protonation as well. The protonation microspecies are discerned with their one-letter symbols (a, b, c, etc.) and the microscopic protonation constants are N Se O depicted using k , k , k , etc. The superscript of microscopic protonation constants k indicates the protonating group, while the subscript (if any) shows the site(s) already protonated. Se, N, O denote the selenolate, amino, and carboxylate sites, respectively. The acid-base microequilibria are indispensable constituents in the evaluation of the species-specific, pH-independent redox equilibrium constants, as shown in Equations (7) and (8). Some protonation constant examples for selenocysteine are shown below: [HL ] h i K = (2) 2 + [L ] H [H L ] = K K K = (3) 3 1 2 3 h i 2 + [L ] H [b] k = h i (4) [a] H where K , K , K are successive macroconstants, is one of the cumulative macroconstants, and k is the 1 2 3 3 microconstant representing the amino protonation in CysSeH, when its carboxylate and selenolate sites are unprotonated. The concentrations of the various macrospecies comprise the sum of the concentration of those microspecies that contain the same number of protons, for example, in CysSeSeCys: 0 0 0 0 0 0 [H L] = [ f ] + [ g ] + [ h ] + [ i ] + [ j ] + [ k ] (5) For the selenolate-diselenide (and dithiolate-disulfide for dithiothreitol) redox equilibria, only the apparent (pH-dependent) or conditional equilibrium constants (K ) are directly available, by 3C determining the equilibrium concentration of the compounds (RSeH, DTT, RSeSeR, and DTT ) which ox. correspond to the total concentration of variously protonated reactant species. The pH-dependent, apparent redox equilibrium constants can be decomposed into pH-independent, species-specific equilibrium constants, the number of which is large, but definite. For example, the selenolate moiety in CysSeH is adjacent to two other basic sites: the amino and the carboxylate. Therefore, the number of selenolate-bearing microspecies is four, corresponding to the number of protonation states of Antioxidants 2020, 9, 465 7 of 13 the amino and carboxylate moieties (amino-carboxylate, ammonium-carboxylate, amino-carboxyl. ammonium-carboxyl). Accordingly, the selenolate exists in four di erent electrostatic environments, having thus four di erent oxidizabilities. Considering the opposite direction of the redox equilibrium, dithiothreitol has only one microspecies, bearing both thiols in their deprotonated, oxidizable form. Therefore, the redox equilibria between selenocysteine and dithiothreitol can be characterized with four di erent microscopic, species-specific equilibrium constants. The method for determining the microscopic redox equilibrium constants is analogous for every case. The determination of one of b b the microscopic redox equilibrium constants, k will be demonstrated. The notation k refers to the reaction involving the ”b” microspecies of CysSeH and oxidized dithiothreitol on the products side, and the corresponding ”f “ microspecies of CysSeSeCys and the deprotonated dithiothreitol on the reactants side. Note that ”b” defines that the diselenide microspecies is mandatorily ”f ”, since ”f “ is the only diselenide microspecies with protonation states in the side-chain identical with ”b”. Superscript ”b” therefore unambiguously identifies all four microspecies in the microequilibrium in question. The example of this pH-independent, microscopic selenolate-diselenide equilibrium constant is in Equation (6): [ ] [ ] b DTT ox. k = h i (6) 2 0 [ ] DTT f For the next step of the calculation, the relative abundance of the protonation microspecies is needed. The mole fraction of microspecies ”b” relative to the total CysSeH concentration can be written with the following equation, as can be carried out for every microspecies provided the complete set of protonation microconstants is known. h i k H [b] = [CysSeH] = [CysSeH] (7) b h i h i h i 2 3 + + + 1 + K H + K K H + K K K H 1 1 2 1 2 3 By writing analogous equations as Equation (7) for the remaining microspecies in the formula of k , one can write the equation of the microscopic equilibrium constant expressed with the conditional equilibrium constant as follows: 2 2 2 [CysSeH]  [DTT ] [b] [DTT ] ox. ox. b b k = h i = = K (8) 3C 2 0 0 [ ] [ ] 0 DTT  2 Se Cys  2 DTT [f ] f f DTT DTT Thus, if K and the  values are known, the species-specific redox microconstants can be 3C calculated. The conditional redox equilibrium constant values determined directly from the NMR spectra are listed in Table 2 and Supplementary Materials. These conditional redox equilibrium constants were determined in basic media only (above pH 9) due to the fact that the redox reaction did not commence at lower pH values. Only the negatively charged thiolate in DTT participates in redox reactions, and below pH 9 the thiolate groups are overwhelmingly in protonated form. However, as the conditional redox equilibrium constants provide pH-independent redox microconstants, it is apparent from Equation (8) that the pH range of determination does not impede the calculation. It should be noted however, that the pH-independent redox microconstants pertaining to microspecies that are predominantly present at pH values outside the determined range (i.e., k ) will have a greater inherent uncertainty based on the present calculations. Antioxidants 2020, 9, 465 8 of 13 Table 2. The conditional redox equilibrium constants in logarithmic units at the pH indicated in reaction mixtures of dithiothreitol with selenocysteine, selenocysteamine and glutathione. Selenocysteine Selenocysteamine Glutathione pH logK pH logK pH logK C C C 9.79 2.88 9.66 2.31 9.00 0.00 9.81 2.20 9.67 2.43 8.30 0.41 9.86 2.87 9.67 2.23 8.35 0.03 9.90 2.07 9.68 2.48 9.07 0.20 9.96 1.98 9.69 2.40 9.63 0.41 9.98 2.04 9.80 2.37 10.23 0.05 10.02 2.05 9.89 2.09 11.01 0.83 9.94 2.25 9.98 2.21 10.01 2.16 The microscopic redox equilibrium constants were calculated and derived from the conditional equilibrium constants using analogous equations to Equation (8) based on at least three repeated measurements. The mean values of the calculated microscopic redox equilibrium constants and their standard deviation of determination are listed in Table 3. Note that near physiological pH (i.e., around pH 7) the most abundant CysSe and CysASe selenolate microspecies are ”b” and ”b”, respectively. In fact, these microspecies have maximal relative abundance between pH 6.5 and 10, therefore the pH range of determination in our study is well-conditioned for the extraction of the physiologically relevant redox microconstants. In order to obtain the standard redox potential values of the studied selenol compounds, the standard redox potential of dithiothreitol is needed. Although dithiothreitol has been extensively studied, only its apparent redox potential has been previously determined, therefore we determined the pH-independent, species-specific standard redox potential of DTT /DTT as well using glutathione ox. as the reaction partner. In the state of a chemical equilibrium, the electrode potential of every existing redox system is equal. For example, for the reaction of dithiothreitol and glutathione one can write: 0 0 E = E = E =  (9) A /A E /B DTT /DTT ox. where the A’/A, E’/B symbols denote the microspecies of glutathione disulfide/glutathione redox pairs. We chose to perform further calculations with equations pertaining to glutathione/glutathione disulfide microspecies B/E’, since this redox pair has the highest relative abundance in the pH of interest, and therefore its calculation is least exposed to uncertainty. The above equation adapted for glutathione microspecies ”B” Figure 6 and DTT becomes: RT [DTT ] RT [ E ] ox. E + ln = E + ln (10) E /B DTT /DTT ox. 2 2 zF zF [DTT ] [ ] 0 2 RT [ E ][DTT ] RT 0 0 E = E + ln = E ln k (11) E /B E /B DTT /DTT ox. 2 zF zF [ ] [ ] B DTT ox. where E is the standard redox potential of the redox system denoted in its subscript, R is the universal gas constant, T is the absolute temperature, z is the number of electrons transferred in the redox half-reaction, and F is the Faraday constant. Using the previously determined microscopic protonation constant [9] and standard redox potential [5] of glutathione microspecies ”B”, the species-specific standard redox potential was determined for the DTT /DTT redox system as0.403 0.007 V. ox. Antioxidants 2020, 9, x FOR PEER REVIEW 8 of 13 The microscopic redox equilibrium constants were calculated and derived from the conditional equilibrium constants using analogous equations to Equation (8) based on at least three repeated measurements. The mean values of the calculated microscopic redox equilibrium constants and their standard deviation of determination are listed in Table 3. Note that near physiological pH (i.e., around pH 7) the most abundant CysSe and CysASe selenolate microspecies are ”b” and ”b”, respectively. In fact, these microspecies have maximal relative abundance between pH 6.5 and 10, Antioxidants 2020, 9, 465 9 of 13 therefore the pH range of determination in our study is well-conditioned for the extraction of the physiologically relevant redox microconstants. Table 3. Microspecies-specific, pH-independent equilibrium constants of selenocysteine, Table 3. Microspecies-specific, pH-independent equilibrium constants of selenocysteine, selenocysteamine and glutathione with dithiothreitol in logarithmic units, with standard deviations. selenocysteamine and glutathione with dithiothreitol in logarithmic units, with standard deviations. The parameter of glutathione was determined for the B microspecies only, for further calculation (see The parameter of glutathione was determined for the B microspecies only, for further calculation (see Figure 6 for glutathione microspecies). Figure 6 for glutathione microspecies). Selenocysteine Selenocysteamine Glutathione Selenocysteine Selenocysteamine Glutathione Microspecies logk sd Microspecies logk sd Microspecies logk sd Microspecies logk sd Microspecies logk sd Microspecies logk sd a a 2.68−2.68 0.42 0.42 a a − 3.72 3.72 0.19 0.19 B B 0.99 0.990.24 0.24 b 0.27 0.42 b 0.08 0.19 b 0.27 0.42 b −0.08 0.19 d 1.37 0.42 d −1.37 0.42 f 2.70 0.42 f 2.70 0.42 Figure 6. The structure and protonation states of the referenced glutathione (A, B, top) and glutathione Figure 6. The structure and protonation states of the referenced glutathione (A, B, top) and disulfide (A’, E’, bottom) microspecies in this work. glutathione disulfide (A’, E’, bottom) microspecies in this work. With the knowledge of the selenolate-diselenide species-specific redox equilibrium constants In order to obtain the standard redox potential values of the studied selenol compounds, the and the standard redox potential of the DTT /DTT redox couple, the species-specific standard ox. standard redox potential of dithiothreitol is needed. Although dithiothreitol has been extensively redox potentials of the di erent selenolate-containing microspecies could be calculated. Equation (12) studied, only its apparent redox potential has been previously determined, therefore we determined shows the continued example of CysSeH microspecies ”b”. The comprehensive set of determined 2− the pH-independent, species-specific standard redox potential of DTTox./DTT as well using species-specific standard redox potentials is listed in Table 4. glutathione as the reaction partner. In the state of a chemical equilibrium, the electrode potential of every existing redox system is equal. For example, for the reaction of dithiothreitol and glutathione RT [b] [DTT ] RT ox. one can write: E = E 2 + ln = E 2 + ln k (12) DTT /DTT 0 2 DTT /DTT ox. ox. zF zF [ ] f [DTT ] 𝐸 =𝐸 =𝐸 =⋯ (9) / / / Table 4. The species-specific standard redox potential values for every microspecies of selenocysteine where the A’/A, E’/B symbols denote the microspecies of glutathione disulfide/glutathione redox and selenocysteamine given as mean standard deviation. pairs. We chose to perform further calculations with equations pertaining to Selenocysteine Selenocysteamine Microspecies E (V) Microspecies E (V) a 0.482 0.01 a 0.513 0.006 b 0.395 0.01 b 0.405 0.006 d 0.444 0.01 f 0.323 0.01 Antioxidants 2020, 9, 465 10 of 13 By graphing these standard redox potentials against the concomitant selenolate-specific protonation constants compiled in Table 1 we find a similar correlation to that of thiolates presented in Antioxidants 2020, 9, x FOR PEER REVIEW 10 of 13 Figure 7. Figure 7. Correlation between standard redox potentials and species-specific thiolate protonation Figure 7. Correlation between standard redox potentials and species-specific thiolate protonation constants for various thiol-containing compounds (glutathione, cysteine, cysteamine, homocysteine, constants for various thiol-containing compounds (glutathione, cysteine, cysteamine, homocysteine, penicillamine, ovothiol A) reproduced from [4] with the addition of the data of dithiothreitol (upper penicillamine, ovothiol A) reproduced from [4] with the addition of the data of dithiothreitol (upper line, line, fu fulllcir l circl cles);es); the the correl correlationation between between standar stan d redox dard re potentials dox potentials and the species-specific and the speciselenolate es-specific pr selenolate protonation constants otonation constants for the various for the var selenol-containing ious selenol-containing compounds (lower compou line, nds (lower l empty circine, em les). pty circles). 4. Discussion 4. Discussion In this work, the highly interwoven acid-base and redox pathways of selenolate-diselenide systems were decomposed into species-specific, component equilibria. The standard redox potentials In this work, the highly interwoven acid-base and redox pathways of selenolate-diselenide of biologically relevant selenolate-diselenide couples are determined for the first time; these values systems were decomposed into species-specific, component equilibria. The standard redox characterize the redox processes at the protonation microspecies level. The elucidation of these potentials of biologically relevant selenolate-diselenide couples are determined for the first time; redox microequilibria reveals considerable di erences between the various protonation species. these values characterize the redox processes at the protonation microspecies level. The elucidation The knowledge of species-specific standard redox potentials of selenocysteine and selenocystine, of these redox microequilibria reveals considerable differences between the various protonation in particular, improves our knowledge on redox homeostasis and can lead to better interpretation species. The knowledge of species-specific standard redox potentials of selenocysteine and of several biochemical phenomena. For example the cytoprotective potential of selenoproteins selenocystine, in particular, improves our knowledge on redox homeostasis and can lead to better is thought to entail several mechanisms involving thio/seleno chemistry [23]. One proposed interpretation of several biochemical phenomena. For example the cytoprotective potential of mechanism of action is formation of stable diselenide bonds in thioredoxin reductase acting as selenoproteins is thought to entail several mechanisms involving thio/seleno chemistry [23]. One a ”diselenide trap”, which can be further supported by exact standard redox potentials of the proposed mechanism of action is formation of stable diselenide bonds in thioredoxin reductase reaction partners. New selenoproteins are investigated for their function, likely related to catalyzing acting as a ”diselenide trap”, which can be further supported by exact standard redox potentials of thiol/disulfide exchange in proteins, as the kinetics of selenolate-diselenide transitions is seven orders the reaction partners. New selenoproteins are investigated for their function, likely related to of magnitude greater than that of thiolate-disulfide transitions [24]. In order to understand these catalyzing thiol/disulfide exchange in proteins, as the kinetics of selenolate-diselenide transitions is catalytic e ects purified from the protonation fraction of the thiolate and selenolate moieties at seven orders of magnitude greater than that of thiolate-disulfide transitions [24]. In order to understand these catalytic effects purified from the protonation fraction of the thiolate and selenolate moieties at physiological pH, a comprehensive species-specific characterization is needed. The knowledge of detailed physico-chemical properties of selenocompounds presented in this work Antioxidants 2020, 9, 465 11 of 13 Antioxidants 2020, 9, x FOR PEER REVIEW 11 of 13 can also serve as the basis to develop artificial selenoenzymes [25] of highly selective redox physiological pH, a comprehensive species-specific characterization is needed. The knowledge of capacities. detailed physico-chemical properties of selenocompounds presented in this work can also serve as the The example of selenocystine microspecies ”a′” and ”f′” (Table 4) shows that even side chain basis to develop artificial selenoenzymes [25] of highly selective redox capacities. protonation changes can lead to significantly different redox characteristics, with nearly 160 mV 0 0 The example of selenocystine microspecies ”a ” and ”f ” (Table 4) shows that even side chain difference in standard redox potential values. Therefore, small changes in pH can not only affect the protonation changes can lead to significantly di erent redox characteristics, with nearly 160 mV redox processes of selenolate-diselenide transitions by changing the protonation fraction of the di erence in standard redox potential values. Therefore, small changes in pH can not only a ect selenolate, but also by altering the protonation state of neighboring moieties. the redox processes of selenolate-diselenide transitions by changing the protonation fraction of the The correlation between selenolate basicity and standard redox potentials verifies the previous selenolate, but also by altering the protonation state of neighboring moieties. observations regarding thiolate basicity and its proportionality with thiolate oxidizability. It is The correlation between selenolate basicity and standard redox potentials verifies the previous interesting that the correlation line between selenolate basicity and the concomitant standard redox observations regarding thiolate basicity and its proportionality with thiolate oxidizability. It is potential is parallel to the correlation line of thiolates, however shifted by ca. −246 mV units. This interesting that the correlation line between selenolate basicity and the concomitant standard redox accentuates the fact that selenolates, apart from being less basic, are vastly stronger reducing agents potential is parallel to the correlation line of thiolates, however shifted by ca. 246 mV units. This than thiolates in general. It is noteworthy that the species-specific standard redox potentials of accentuates the fact that selenolates, apart from being less basic, are vastly stronger reducing agents selenolate-containing microspecies also show linear correlation with species-specific NMR chemical than thiolates in general. It is noteworthy that the species-specific standard redox potentials of shift. The chemical shift values were previously determined [16], and the details of the correlation selenolate-containing microspecies also show linear correlation with species-specific NMR chemical 1 13 are shown in Figure 8 and Table 5. The use of chemical shifts in protein NMR (from H, C, and for shift. The chemical shift values were previously determined [16], and the details of the correlation selenoproteins the relatively undisrupted Se spectra) can now serve as sound means to predict 1 13 are shown in Figure 8 and Table 5. The use of chemical shifts in protein NMR (from H, C, and selenolate oxidizability or diselenide reducibilty/stability in proteins: a key parameter to understand for selenoproteins the relatively undisrupted Se spectra) can now serve as sound means to predict and influence oxidative stress. selenolate oxidizability or diselenide reducibilty/stability in proteins: a key parameter to understand The critical issue in designing preventive or therapeutic antioxidants is the narrow path of and influence oxidative stress. redox potentials, effective enough to reduce harmful oxidative agents, but keeps disulfides and The critical issue in designing preventive or therapeutic antioxidants is the narrow path of other reducible units in useful biomolecules intact. Naturally, small reducing agents can hardly be as redox potentials, e ective enough to reduce harmful oxidative agents, but keeps disulfides and other selective as substrate-specific enzymes of the biological antioxidant system; however, a finely tuned reducible units in useful biomolecules intact. Naturally, small reducing agents can hardly be as and designed selenolate-containing compound with an appropriate basicity, redox potential and selective as substrate-specific enzymes of the biological antioxidant system; however, a finely tuned concomitant selectivity can be confined to a narrower range. The correlation between the redox and and designed selenolate-containing compound with an appropriate basicity, redox potential and NMR parameters serves now as a sound basis to better quantify the characteristics of diselenide concomitant selectivity can be confined to a narrower range. The correlation between the redox and moieties in selenoenzymes, allowing thus the development of potent, selective antioxidant NMR parameters serves now as a sound basis to better quantify the characteristics of diselenide compounds for serious ailments related to selenoenzyme deficiencies, such as autism [26]. moieties in selenoenzymes, allowing thus the development of potent, selective antioxidant compounds for serious ailments related to selenoenzyme deficiencies, such as autism [26]. Figure 8. Correlation of selenocysteine standard redox potentials with the corresponding selenocysteine Figure 8. Correlation of selenocysteine standard redox potentials with the corresponding microspecies chemical shifts (left) and selenocystine microspecies chemical shifts (right). selenocysteine microspecies chemical shifts (left) and selenocystine microspecies chemical shifts (right). Antioxidants 2020, 9, 465 12 of 13 Table 5. Correlation data of selenocysteine standard redox potentials with the corresponding selenocysteine microspecies chemical shifts and the corresponding selenocystine microspecies chemical shifts. Selenocysteine 77 1 1 1 13 13 Se H ( CH) H ( CH ) H ( CH ) C ( CH) C ( CH ) 2a 2 2b slope 70.524 5.251 2.208 3.163 24.743 52.487 intercept 196.660 5.755 3.970 4.013 50.137 3.319 r 0.4979 0.9622 0.4838 0.9095 0.8571 0.9180 Selenocystine slope 59.106 5.806 2.446 2.437 16.557 18.030 intercept 310.960 6.338 4.102 4.036 49.893 23.692 r 0.8908 0.9660 0.9720 0.9259 0.5578 0.7718 5. Conclusions The standard redox potentials of diselenide/selenolate-containing microspecies of selenocysteine and selenocysteamine were determined using an indirect approach of measuring redox equilibrium constants with the help of dithiothreitol. These standard redox potentials are pH-independent and show correlation with selenolate acid-base characteristics; an important observation previously demonstrated for thiolate-analogues as well. The acid-base and redox parameters both show correlation with the NMR chemical shifts of the diselenide or selenolate-containing species as well. This can be used as a tool for predicting diselenide behavior solely based on Se NMR data. Supplementary Materials: The following are available online at http://www.mdpi.com/2076-3921/9/6/465/s1. Author Contributions: Conceptualization, T.P., A.M. and B.N.; Methodology, T.P., A.M. and B.N.; Investigation, T.P., A.M. and B.N.; Resources, B.N.; Data Curation, T.P. and A.M.; Writing—Original Draft Preparation, T.P. and A.M; Writing—Review & Editing, T.P., A.M. and B.N.; Visualization, T.P. and A.M.; Supervision, B.N.; Project Administration, B.N.; Funding Acquisition, A.M. and B.N. 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Antioxidants – Multidisciplinary Digital Publishing Institute

Published: Jun 1, 2020

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