antioxidants Article Species-Specific, pH-Independent, Standard Redox Potential of Selenocysteine and Selenocysteamine Tamás Pálla 1,2, Arash Mirzahosseini 1,2 and Béla Noszál 1,2,* 1 Department of Pharmaceutical Chemistry, Semmelweis University, H-1092 Budapest, Hungary; [email protected] (T.P.); [email protected] (A.M.) 2 Research Group of Drugs of Abuse and Doping Agents, Hungarian Academy of Sciences, H-1051 Budapest, Hungary * Correspondence: [email protected]; 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 different, 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), compoundsrespectively. yieldsThe selenocystamineconstitutional formulae (CysASeSeCysA) of the studied and selenocystine selenium-containing (CysSeSeCys), compounds respectively. are Thedepicted constitutional in Figure formulae1. of the studied selenium-containing compounds are depicted in Figure1. Figure 1. The structure of the investigated selenols (left) and diselenides (right). Glutathione is known as a “gold standard”standard” in thiol-disulfidethiol-disulfide biochemistry, hence, every thiol-containing antioxidantantioxidant isis comparedcompared toto glutathione.glutathione. Moreover, Moreover, the species-specificspecies-specific physico-chemical acid-base [9] [9] and redox [5,6] [5,6] parameters of glutathione are well-characterized. However, previous works examining selenoproteins or selenium-containing peptides found that the diselenide bridge cannot be reducedreduced by glutathione [[10,11].10,11]. We We fo foundund that even a large excess of glutathione was ineineffectiveffective in reducing the studiedstudied diselenidediselenide compounds, confirmingconfirming the above observation. Thus, a more powerful reducing agentagent was needed to investigate selenolate-diselenide redox equilibria.equilibria. Based Based on on literature literature recommendations recommendations [8,12 ],[8,12], dithiothreitol dithiothreitol (DTT), a(DTT), stronger a reducingstronger agent,reducing was agent, chosen. was Dithiothreitol chosen. Dithiothreitol undergoesa unde one-steprgoes two-electron a one-step redoxtwo-el reaction,ectron redox resulting reaction, in the formation of oxidized dithiothreitol (DTT ) with an intramolecular disulfide in a six-membered ring. resulting in the formation of oxidized dithiothreitolox (DTTox) with an intramolecular disulfide in a Duringsix-membered the redox ring. processes During between the redox dithiothreitol processes and between other agents, dithiothreitol the presence and ofother the intermediateagents, the 2 compound,presence of the where intermediate only one ofcompound, the thiolate wher groupse only of one DTT of− theis oxidized thiolate groups is negligible of DTT [132−]. is The oxidized main physico-chemical properties of DTT are already described (logK = 10.1; logK = 9.2 [14]; E = 0.323 is negligible [13]. The main physico-chemical properties of DTT1 are already2 described (log◦ K1− = 10.1; V at pH = 7.0 [15]). logK2 = 9.2 [14]; E° = −0.323 V at pH = 7.0 [15]). To obtainobtain datadata describingdescribing selenolate-diselenideselenolate-diselenide redox equilibria comparablecomparable to those of 2 thiolate-disulfidethiolate-disulfide systems,systems, firstfirst thethe glutathione-DTTglutathione-DTT2− redoxredox reaction reaction was was investigated investigated (Figure (Figure 22).). 2 The general step-by-step scheme of selenolate-diselenide redox equilibria with DTT − is depicted in Figure3, where RSe − and RSeSeR stand for the reduced and oxidized form of the selenium-containing 2 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 Figure 2. The redox reaction between oxidized glutathione and dithiothreitol (DTT)2− dominant microspecies at the pH of blood. The general step-by-step scheme of selenolate-diselenide redox equilibria with DTT2− 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 thiolate22 − and disulfide, Figure 2. TheThe redox reaction between oxidizedoxidized glutathioneglutathione andand dithiothreitoldithiothreitol (DTT)(DTT) − dominant respectively).microspecies at the pH of blood. The general step-by-step scheme of selenolate-diselenide redox equilibria with DTT2− 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 DTT2− is analogous (where RS− and RSSR stand for the thiolate and disulfide, respectively). 2− 2 Figure 3. TheThe two-step two-step redox redox equilibria equilibria between between a adiselenide diselenide compound compound and and DTT DTT (reaction− (reaction I and I and II) II)followed followed by bytheir their aggregated aggregated one-step one-step net netreaction reaction (III). (III). Identical Identical reaction reaction schemes schemes are areapplicable applicable for fordisulfide disulfide analogues. analogues. The arrow The arrow in reaction in reaction II indica II indicatestes the low the probability low probability of formation of formation of the sulfur– of the sulfur–seleniumselenium bridge. bridge. 2 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 2 arediselenides more stable are more than stable the selenylsulfides than the selenylsulfi [16], thedes reaction [16], the betweenreaction between diselenides diselenides and DTT and− can DTT be2− Figure 3. The two-step redox equilibria between a diselenide compound and DTT2− (reaction I and II) reasonablycan be reasonably discussed discussed in terms ofin the terms net reaction.of the ne Thet reaction. conditional The equilibrium conditiona constantl equilibrium describing constant this followed by their aggregated