Redox Chemistry and Biology of Thiols

Redox Chemistry and Biology of Thiols offers an applied, comprehensive overview of redox chemistry and biology of thiol-dependent processes. Running from basic biology and chemistry of redox phenomena to research methods and highlighting recently identified roles of thiols across cellular and bodily systems, this book draws upon a range of disciplines to illuminate new research directions, new applications of thiol studies, and clinical translation. Sections cover thiol oxidizing species, thiol reactivity and modifications, thioredoxin, glutaredoxin, glutathione, peroxidases, thiol repair enzymes, thiol oxidative signaling, disulfide bond formation, thiol-based redox biosensors, cysteine and hydrogen sulfide metabolism, iron-sulfur cluster biogenesis, thiols in chloroplasts, blood thiols, sugar and polyamine thiols in pathogenic organisms, redox medicine (therapeutic applications of thiols and drug development), as well as methods and bioinformatics tools.

• Runs from basic thiol biology and chemistry to applications and clinical translation
• Provides methods and protocols that will power new research across biomedicine, cell biology, plant biology, drug development, and protein folding and modulation
• Includes chapter contributions from international leaders in the field

• Language: English
• Publisher: Academic Press
• Release date: May 25, 2022
• ISBN: 9780323915663

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Chapter 1: Basic concepts of thiol chemistry and biology

Beatriz Alvareza,b; Gustavo Salinasc,d    a Laboratorio de Enzimología, Facultad de Ciencias, Instituto de Química Biológica, Universidad de la República, Montevideo, Uruguay

b Centro de Investigaciones Biomédicas (CEINBIO), Universidad de la República, Montevideo, Uruguay

c Laboratorio de Biología de Gusanos, Institut Pasteur de Montevideo, Universidad de la República, Montevideo, Uruguay

d Departamento de Biociencias, Facultad de Química, Universidad de la República, Montevideo, Uruguay

Abstract

Thiols (RSH) are present in low molecular weight compounds such as glutathione and in protein cysteine residues. Their high chemical versatility allows thiols and their derivatives to play prominent roles in catalysis, regulation, protein folding, and signaling. Thiol-dependent redox systems are widespread in biological organisms and involved in diverse life processes. In this chapter, the concepts needed to understand the redox chemistry and biology of thiols are introduced.

Keywords

Thiol; Thiolate; Disulfide; Cysteine; Redox; Oxidoreductase; Thioredoxin; Glutathione; Sulfenic acid; Thiyl radical

Acknowledgments

This work was supported by Comisión Sectorial de Investigación Científica (CSIC), Universidad de la República, Uruguay. The authors thank Gerardo Ferrer-Sueta (Universidad de la República) and Marcelo Comini (Institut Pasteur de Montevideo) for valuable insights, Franco Vairoletti (Universidad de la República) for helpful discussions, and Matías Machado (Institut Pasteur de Montevideo) for help with Fig. 8.

1: Thiols in biology

Thiols (RSH) are organosulfur compounds that contain carbon-bonded sulfhydryl (also called sulfanyl) groups. They are the sulfur analogs of alcohols (ROH), and are also known as mercaptans because of their ability to bind to mercury compounds. Thiols are widespread in all biological systems. Low molecular weight (LMW) thiols are important in numerous processes, where they participate in different types of essential redox reactions (Glossary box), alkylation reactions, and metal binding. Some common biologically relevant LMW thiols include the amino acids cysteine (Cys) and homocysteine, the tripeptide glutathione (GSH), coenzyme A, and dihydrolipoic acid (Fig. 1). Other LMW thiols, such as trypanothione, mycothiol, bacillithiol, coenzyme M, and coenzyme B, are also important in some specific organisms. Most LMW thiols derive from Cys. In addition, Cys is the source of sulfur for the formation of Fe/S clusters in proteins. The thiol group is also essential for protein function. Cys protein residues are genetically encoded and present in all organisms. Since Cys is almost always important for protein function or regulation, it is highly conserved in proteins. The unique physicochemical properties of the thiol group, which include acidity, nucleophilicity, and the capacity to bind metal ions and to form disulfides (RSSR) and other oxidation products, make the Cys residue in proteins the most versatile of all amino acids.

Fig. 1

Fig. 1 Structures of ubiquitous LMW thiols.

An enormous body of knowledge in the field of thiol chemistry and biology, both conceptual and methodological, has emerged and been accrued. Thiols are chemically multifaceted and, in addition to their classical functions in protein structure and oxidant scavenging, novel roles in catalysis, folding, and signaling have been acknowledged. The posttranslational modifications of Cys protein residues have been revealed as key regulators of life processes. Research into biological thiols has gained renewed interest based on their roles as health determinants and as potential targets for intervention. The basics for understanding the rich biological chemistry of thiols are introduced in this chapter.

2: The thiol group confers unique properties to the amino acid cysteine

Sulfur belongs to the chalcogen group of the periodic table (group 16), below oxygen and above selenium, and has six electrons in the outermost shell. Sulfur, oxygen, and selenium are present in the genetically encoded amino acids cysteine (Cys), serine (Ser), and selenocysteine (Sec), respectively. These amino acids differ exclusively in the heteroatom of the side chain: Cys contains a –CH2SH thiol, Ser a –CH2OH alcohol, and Sec a –CH2SeH selenol. From oxygen to sulfur to selenium, the atomic radius increases, the electronegativity decreases, and the polarizability increases, impacting on the acidity and reactivity of these amino acids (Table 1).

Table 1

a Carbon: 2.55, hydrogen 2.20.

b pKa of the side chain of Ser, Cys and Sec.

Thiols and selenols, and therefore Cys and Sec, are relatively similar. The particularities of Sec are covered in Chapter 18. In contrast, thiols and alcohols, and thus Cys and Ser, have profound differences. The main differences between thiols and alcohols are covered in the following sections. Besides, due to the small electronegativity difference between sulfur and hydrogen, thiols are less polar and have a lower dipole moment than the corresponding alcohols. Thus, thiols establish very weak hydrogen bonds.

3: Thiols ionize to thiolates

Thiols are much more acidic than alcohols. Thus, thiols ionize to a greater extent than alcohols. This difference can be explained mainly by the larger size of the sulfur atom, which can bear a negative charge better than an oxygen atom. In other words, the conjugate base of thiol (thiolate, RS−) is more stable than the conjugate base of alcohol (alkoxide, RO−).

The ionization (deprotonation or dissociation) of a thiol can be represented by Eq. (1).

si1_e    (1)

Since thiols are weak acids, they do not ionize completely; in equilibrium, a fraction is in its neutral form (RSH) and a fraction in its anionic form or conjugate base (RS−). The ratio between the two forms depends on two factors: the equilibrium constant for the reaction and the pH. The equilibrium constant is known as acid dissociation constant or Ka (Eq. 2), which is a quantitative measure of the strength of an acid in solution.

si2_e    (2)

Usually, − logKa, denoted pKa, is used for convenience. The pKa is a useful parameter since it coincides with the pH at which the concentration of the acid and the conjugate base (RSH and RS− in Eq. 2, respectively) are equal. The more acidic or ionizable the thiol, the higher the acid dissociation constant, the lower the pKa, and the higher the fraction of thiolate at a certain pH. For those thiols that possess two or more groups able to ionize at similar pHs, like Cys, which has both thiol and protonated amine, the microscopic constants corresponding to each separate species can be defined, as discussed in Chapter 2.

The pKa of the thiol group of free Cys has been determined to be 8.3 [1], and thus at this pH, the fractions of [RS−] and [RSH] with respect to total concentration are equal to 0.5. Fig. 2 shows the fraction of each species as a function of pH. At the physiological pH of 7.4, only 11% is ionized to thiolate, while 89% is protonated.

Fig. 2

Fig. 2 Fraction of deprotonated (RS − ) and protonated species (RSH) versus pH for Cys. The values of RS − ( continuous blue line ) and RSH ( dashed red line ) fractions over total concentration were calculated using the acid dissociation equilibrium equation (Eq. 2) and a pKa of 8.3 for Cys. The dotted line denotes a pH of 7.4; the corresponding fractions at that pH are indicated with arrows.

When a Cys is part of a peptide or protein, the pKa of the thiol group tends to increase; for a Cys residue in a typical peptide, the pKa is 9.1 [2]. Nevertheless, it should be noted that the pKa of Cys residues in proteins varies over a broad range, from ~ 3 to 11, because the protein environment can substantially affect the thiol pKa. For instance, in glutaredoxins, the N-terminal solvent exposed Cys residue has a pKa of ∼ 3.8, while the buried C-terminal residue has a pKa of > 10.5; whereas in thioredoxins, the N-terminal Cys residue pKa varies between 6.3 and 7.1 [3]. The protein electrostatic microenvironment can alter the pKa; for example, the proximity of a positively charged residue tends to decrease the thiol pKa, as in the case of the peroxiredoxins [4]. These aspects are discussed in detail in Chapter 2.

It is important to note that the ionization of thiols is a very fast and reversible acid-base reaction, and as such, thermodynamically driven, in contrast to redox reactions, which usually require catalysis and are kinetically driven.

Thiolates are hydrogen bond acceptors and can interact with polar and charged residues. For such types of Cys residues, Ser can be an adequate amino acid replacement in protein engineering experiments. In contrast, a buried, nonsolvent exposed Cys may be better substituted by alanine (Ala) rather than by Ser, given the low polarity of the thiol group. Both amino acid replacements will abrogate the potential redox activity of the Cys residue.

Finally, in most reactions, thiolates are the reacting species.

4: Thiolates are excellent nucleophiles

A nucleophile is a species that donates a pair of electrons to an electrophile to form a new covalent bond. Nucleophilicity is measured by comparing reaction rates: the faster the reaction with a certain electrophile, the stronger the nucleophile. Thiolates (RS−) are excellent nucleophiles, and much better nucleophiles than thiols. This high nucleophilicity can be explained by their negative charge, and also by the relatively low electronegativity and large size of sulfur, which determine that the electron pair to be donated is held less tightly than in oxygen. Furthermore, in comparison to alkoxides, thiolates have a lower degree of water solvation and hence a lower energetic barrier to their reactions. Due to the relatively high acidity of thiols in comparison to alcohols, thiolates are more available at neutral pHs than alkoxides. Finally, the high polarizability of sulfur favors the formation of stable products with relatively soft electrophiles (e.g., mercury compounds) in the context of Pearson's hard and soft acids and bases (HSAB) theory, which proposes that bases of intermediate softness such as thiolate react preferentially with Lewis acids of comparable softness (e.g., transition metals from group 8 onwards).

Since thiolates are much better nucleophiles than thiols, the observed rate constant for a given reaction increases with pH because more thiolate is then available (Fig. 2). If we compare two thiols, the more acidic thiol (the one with the lower pKa) presents a higher fraction of thiolate at a fixed pH. This has given rise to the often-quoted misconception that reactive Cys have low pKa. Indeed, thiols with low pKa present the advantage of higher thiolate availability at a certain pH. However, the intrinsic reactivity of a thiolate whose conjugate thiol has a low pKa is diminished with respect to one with a high pKa. Nucleophilicity actually depends on the electron density on the sulfur atom, hence it correlates with basicity and increases with pKa. In general terms, nucleophilicity and basicity are closely related properties: nucleophilicity involves the formation of a covalent bond with an electron-deficient center other than the proton, and basicity, the formation of a covalent bond with the proton, which is a particular electron-deficient center. These considerations regarding the association between nucleophilicity and basicity apply exclusively to LMW compounds; the reactivity of thiolates in proteins is more difficulty to predict and nucleophilicity is dissociated from basicity, as explained in Chapter 2.

Thiolates participate in concerted SN2-type substitution reactions (substitution, nucleophilic, bimolecular reactions). They also participate in addition reactions, in which there is a single product that contains all the atoms of the reactants. These are discussed in depth in Chapter 2. Some examples are shown in Fig. 3. The substitution reaction with iodoacetamide as an electrophile forming a thioether and iodide (Reaction A) is commonly used to block thiols in analytical procedures. The reaction between a thiolate and hydrogen peroxide to form a sulfenic acid and hydroxide (Reaction B) constitutes the first step in the catalytic mechanism of peroxiredoxins. GSH reacts with 1-chloro-2,4-dinitrobenzene (Reaction C) in the canonical reaction catalyzed by glutathione transferases. The reversible Michael addition-elimination reaction with N-ethylmaleimide (Reaction D) is another reaction commonly used to block thiols. The addition to a carbonyl group to form a thiohemiacetal (Reaction E) occurs in the mechanism of glyceraldehyde-3-phosphate dehydrogenase or in LMW thiols with methylglyoxal. Thiols can also add to α,β-unsaturated carbonyls, as exemplified by the reaction with lipid-derived 4-hydroxy-nonenal (Reaction F); the reactions with these and other Michael acceptors are described in Chapter 9.

Fig. 3

Fig. 3 Examples of substitution and addition reactions of thiolates. S N 2-type substitution reactions: (A) reaction of a generic thiolate with iodoacetamide to form a thioether and iodide; (B) reaction of the peroxidatic cysteine of a peroxiredoxin (Prx) with hydrogen peroxide forming sulfenic acid and hydroxide; (C) glutathione transferase-catalyzed reaction between GS − and 1-chloro-2,4-dinitrobenzene. Addition reactions: (D) Michael addition-elimination reaction of a generic thiol with N -ethylmaleimide to form a thioether; (E) reaction of an active site thiolate in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with the aldehyde of the substrate to form a thiohemiacethal; (F) Michael addition-elimination reaction with the electrophilic lipid derivative 4-hydroxy-nonenal.

5: Thiols can be oxidized

Another remarkable difference between thiol and alcohol (and between Cys and Ser) is that the thiol group can easily participate in redox reactions. Whereas oxidation of an alcohol usually leads to a product in which the oxidation state of the carbon but not of the oxygen atom is increased, oxidation of a thiol affects the sulfur atom. Sulfur, as an element, has the electron configuration [Ne]3s²3p⁴, and has six valence electrons (i.e., in the outermost shell, which are those that can participate in reactions). Thus, sulfur can gain two electrons to complete the octet (oxidation state − 2 when fully reduced), or lose up to six electrons (oxidation state + 6 when fully oxidized). In the thiol group, the sulfur atom is − 2, and thus it is maximally reduced. Thiols can undergo a range of reactions under physiological conditions that render the different oxidation products introduced in Fig. 4, indicating the commonly used names and the oxidation state of sulfur.

Fig. 4

Fig. 4 Selected thiol oxidation products showing the usual names and the oxidation state of the sulfur atoms.

For those unfamiliar with the concept of oxidation state of an atom, it should be explained that it is calculated with a set of arbitrary rules [5], which are more intuitive for ionic bonds, where electrons are entirely gained or lost between different atoms, than for covalent bonds, in which electrons are shared between different atoms. Nevertheless, although not completely rational for organic compounds, the rules are useful. For instance, in thiols, an oxidation state of − 2 is assigned to the sulfur atom, as if it had gained the two electrons at the expense of carbon and hydrogen atoms to which the sulfur is bound. This oxidation state is assigned because sulfur is more electronegative than carbon and hydrogen, but the differences between carbon, hydrogen, and sulfur atoms are marginal, and the polarity of these bonds is low.

Considering the scope of this book, we feel that it is important to introduce the basic concept of a redox reaction, which consists of two coupled half-reactions that cannot occur without each other. Reduction is a half-reaction in which an atom, ion, or molecule gains electrons provided by an electron donor (reductant or reducing agent); oxidation is a half-reaction in which an atom, ion, or molecule loses electrons, donating them to an electron acceptor (oxidant or oxidizing agent). The two half-reactions must occur simultaneously. Since the reductant becomes oxidized and the oxidant becomes reduced, these two half-reactions are defined by two redox couples or pairs. An example of a redox couple is Fe³ +/Fe² +; other examples are H2O2, 2H+/2H2O and NADP+, H+/NADPH. As a convention, a redox couple is denoted with the oxidized species first, followed by the reduced species. In reactions involving organic molecules and sulfur compounds, an oxidation will usually result in a decrease in C glyph_sbnd H or S glyph_sbnd H bonds, or in an increase in C glyph_sbnd O and S glyph_sbnd O bonds. In contrast, a reduction will result in an increase in C glyph_sbnd H or S glyph_sbnd H bonds, and a decrease in C glyph_sbnd O or S glyph_sbnd O bonds.

In a redox reaction, the direction in which the electrons are transferred depends, as in any reaction, on the ΔG. This is determined by the relative tendency of the oxidized species in each redox couple to gain electrons, which is called reduction potential (E). ΔG depends on the difference in reduction potential (ΔE) according to Eq. (3), in which n is the number of electrons transferred in a redox reaction, and F is the Faraday constant.

si3_e    (3)

For a reaction to occur, ΔG has to be negative and ΔE, usually measured in volts, should be positive.

The standard reduction potential (E°) of a given redox couple is a measure of the tendency to gain electrons of the oxidized species in the couple, under standard conditions (1 M in practical terms). The E° is defined with respect to the hydrogen electrode (2H+/H2), to which an arbitrary value of 0 is assigned. E°′ is used instead of E° if the biochemical standard states at pH 7 are used. The higher the E° of a redox couple, the greater the likelihood of the oxidized species to become reduced. When the E° of two redox couples are compared, the reduced species of the couple with lower E° is capable of reducing, thermodynamically, not necessarily kinetically, the oxidized species of the redox couple with higher E°. In other words, under standard conditions, the electrons will tend to move from the reduced species of a redox couple with lower E° to the oxidized species of the redox couple with higher E°.

For a general reaction in which n electrons are transferred from reductant (Bred) to oxidant (Aox) (Eq. 4), the redox potential of the reaction (ΔE) is represented by the Nernst equation (Eq. 5). ΔE depends not only on ΔE° (the difference in the standard reduction potentials of the couples in the reduction minus the oxidation half-reaction, ΔE° = E°Aox/Ared − E°Box/Bred), but also on the concentration of the species of each redox couple. In Eq. (4), F is the Faraday constant, R is the gas constant, and T is the temperature. A positive ΔE is indicative of a thermodynamically favorable reaction.

si4_e    (4)

si5_e

   (5)

These concepts can be exemplified by a redox series connecting the redox couple H2O2, 2H+/2H2O, which has an E°′ of + 1.35 V [6] with the redox couple NADP+, H+/NADPH, which has an E°′ of − 0.32 V. Under standard conditions, the reaction proceeds in the direction shown in Eq. (6), with the electrons flowing from NADPH to H2O2, since ΔE°′ = 1.35 −(− 0.32) = 1.67 V. Under the steady-state conditions of the cell, the ΔE differs from ΔE°′ (Eq. 5). In fact, in the cytosol, NADPH is even more reducing than under standard conditions, given the high NADPH/NADP+ steady-state ratio [7].

si6_e

   (6)

Despite the favorable thermodynamics, the direct reaction between H2O2 and NADPH does not occur at significant rates; actually, it needs enzymatic catalysis. One of the systems that catalyzes the process is the redox series shown in Fig. 5. Hydrogen peroxide is reduced by a selenol residue in glutathione peroxidase, which becomes selenenic acid. Oxidized glutathione peroxidase is reduced by GSH, which becomes GSSG. The flavoprotein glutathione reductase reduces GSSG to GSH, and is reduced by NADPH.

Fig. 5

Fig. 5 Redox series. The standard reduction potential ( E°′ ) is a measure of the tendency to gain electrons. In a redox series, the oxidized species of the redox couple with the highest E°′ , the oxidant (in this example H 2 O 2 ), can thermodynamically oxidize the reduced species of all other redox couples of the series. Conversely, the reduced species of the redox pair with the lowest E°′ , the reductant (in this example NADPH), can thermodynamically reduce the oxidized species of all other redox pairs of the series. The direction of the electrons from NADPH to H 2 O 2 is represented by the grey arrow . The standard redox potential of the two-electron reaction between H 2 O 2 and NADPH is Δ E°′  = 1.35 −(− 0.32) = 1.67 V. Although thermodynamically favored, the direct reaction does not occur at significant rates. Rather, the reduction of H 2 O 2 to water is catalyzed by the selenoenzyme glutathione peroxidase, which is in turn reduced by GSH, whose disulfide is reduced by the flavoenzyme glutathione reductase, which is reduced by NADPH.

For a typical LMW thiol, the standard one-electron reduction potential of a thiyl radical/thiol couple, E°’(RS glyph_rad , H+/RSH), is 0.96 V (pH 7) [8]. This means that only strong one-electron oxidants (e.g., hydroxyl radical, E°’(HO glyph_rad , H+/H2O) = 2.31 V [6]; nitrogen dioxide, E°’(NO2 glyph_rad /NO2−) = 1.04 V [9]) are in principle able to oxidize thiols to thiyl radicals. This also means that thiyl radicals are relatively strong oxidizing species, potentially able to oxidize several biomolecules.

The two-electron reduction potential of a typical LMW disulfide, E°’(RSSR, 2H+/2RSH), is − 0.21 V (pH 7) [8]. This means that thiols are good reductants and that, in principle, two thiols can be reversibly oxidized to form a disulfide (e.g., protein disulfides, glutathione disulfide, or protein-glutathione mixed disulfides). Importantly, the formation of disulfides from thiols is not a direct process but needs to be carried out in at least two steps, as exemplified by Eqs. (7)–(8), in which oxidation of thiols to disulfides by hydrogen peroxide has sulfenic acid (RSOH) as intermediate. Mechanistically, these reactions are SN2 bimolecular substitutions with thiolate as the reacting species, and are performed at very high rates in the peroxiredoxins.

si7_e    (7)

si8_e    (8)

It is very important to stress that reduction potential considerations only denote whether a certain process can, in principle, happen from a thermodynamic point of view, but they cannot imply whether the process may take place from a kinetic point of view. The process may be too slow to occur in the absence of catalysis, and its rate may only become significant with the appropriate enzyme.

6: Redox versatility of thiols

Thiols can be oxidized to a wide range of products. The thiol oxidation pathways are explained in depth in Chapters 5–8 and summarized here.

First, thiols can react with disulfides (R’SSR’) through thiol-disulfide exchange reactions (pathway a in Fig. 6). These reactions are reversible and can occur spontaneously, albeit slowly, or they can be catalyzed by enzymes of the thioredoxin superfamily (see below).

Fig. 6

Fig. 6 Thiol oxidation pathways. Thiols (precisely, thiolates), can undergo reversible thiol-disulfide exchange reactions with disulfides (R’SSR’) (pathway a ). They can react with two-electron oxidants (e.g., H 2 O 2 ) forming sulfenic acids (RSOH) (pathway b ). Sulfenic acids can decay by reacting with another thiol, forming disulfides, with an amine or amide (represented here by R’NH 2 ), forming sulfenamides (RSNHR’), or with another oxidant (e.g., H 2 O 2 ) forming sulfinic (RSO 2 H) and sulfonic (RSO 2 H) acids. Thiols can be oxidized by one-electron oxidants (represented by X glyph_rad ) forming thiyl radical (RS glyph_rad ) (pathway c ), which can react with another thiyl radical, forming disulfides, or with another thiolate, forming the disulfide anion radical (RSSR glyph_rad − ), a reducing radical that can react with O 2 yielding superoxide radical (O 2 glyph_rad − ). Thiyl radicals can also react with O 2 or with nitric oxide (NO glyph_rad ), and can oxidize different biomolecules (represented by A − ). Dashed lines represent processes that may occur in more than one step. Protons are sometimes omitted for clarity.

Second, thiols can react with two-electron oxidants such as H2O2, lipid hydroperoxides (ROOH), and peroxynitrous acid (ONOOH), forming sulfenic acids (RSOH) (pathway b in Fig. 6). In addition to these hydroperoxides, other biologically relevant oxidants are hypochlorous acid (HOCl) and chloramines (RNHCl), which initially form RSCl species that hydrolyze quickly to RSOH. The reaction between a thiol and a hydroperoxide to form a sulfenic acid constitutes the first step in the catalytic mechanism of the peroxiredoxins. Sulfenic acids are typically unstable and can react with another thiol, in the same or in another molecule, yielding disulfides; this is the predominant pathway for sulfenic acid decay and also part of the catalytic mechanism of peroxiredoxins. In particular cases (e.g., protein tyrosine phosphatase 1B [10]), sulfenic acids can react with an amine or amide forming a sulfenamide. Sulfenic acids can also undergo further oxidation to sulfinic (RSO2H) and sulfonic (RSO3H) acids; this latter is usually the result of extensive oxidative damage.

Thiols can also react with one-electron oxidants including hydroxyl radical (HO glyph_rad ), nitrogen dioxide (NO2 glyph_rad ), carbonate radical (CO3 glyph_rad −), and oxoferryl compounds, forming highly reactive thiyl radicals (RS glyph_rad ) (pathway c in Fig. 6). Thiyl radicals are oxidizing and can react with biomolecules, with another thiol, or with ascorbate. When reacting with nitric oxide (NO glyph_rad ), nitrosothiols (RSNO) are formed. Importantly, they can also react with oxygen and participate in oxygen-dependent chain reactions that can amplify the initial oxidation events. The reactivity of thiyl radicals is exploited in the catalytic mechanism of ribonucleotide reductase: a thiyl radical formed in an active site Cys participates in the conversion of ribonucleotides to deoxyribonucleotides.

Some of the products formed from thiol oxidation processes, particularly disulfides and sulfenic acids, can revert to thiols with suitable reductants. Other oxidation products such as sulfinic (RSO2H) and sulfonic (RSO3H) acids constitute final products [11]. Their formation in biological systems is considered irreversible because there is no known reduction mechanism for them, except for sulfiredoxins that can reduce sulfinic acid in peroxiredoxins [12].

Thiol-based redox signaling and catalysis in numerous proteins is based on controlled thiol oxidation, which is revertible with specific reductants. Of the 20 canonical amino acids, sulfur-containing amino acids (methionine or Met, and Cys) are the only ones capable of undergoing revertible two-electron redox reactions. Regulation by oxidation/reduction is very rare for Met residues, with the exception of the sulfoxidation of actin [13]. In contrast, a large number of proteins and enzymes are under redox control by critical Cys residues, or use thiol oxidation as part of their catalytic mechanism (Chapters 12–14). Finally, in some cases, the oxidation of a thiol is spurious, as a consequence of a kinetically-significant reaction between a solvent-exposed bystander thiol and an oxidant; these spurious oxidations may, depending on the extent of oxidation, be repaired by reductants. Bystander solvent-exposed Cys residues tend to be eliminated by evolution [14].

7: The thiol-disulfide exchange reaction can be catalyzed by proteins of the thioredoxin superfamily

The reversible thiol-disulfide exchange reaction is particularly important in biological systems. In this SN2, one-step, bimolecular reaction there is a nucleophilic Cys (a thiolate) that attacks a sulfur atom of a disulfide bond. This S atom of the disulfide is the electrophilic center (i.e., accepts an electron pair from a nucleophile to form a chemical bond). Simultaneously, the bond to the other S atom of the original disulfide is broken and thiolate is released as the leaving group (i.e., the molecular fragment that departs with a pair of electrons in the heterolytic bond cleavage). The reaction has a negatively-charged and linear transition state. As a result of the thiol-disulfide exchange, a new disulfide and a new thiol are formed (Fig. 7).

Fig. 7

Fig. 7 The thiol-disulfide exchange reaction. Generic reaction in which a deprotonated Cys residue (a nucleophilic thiolate, R 1 -S − ) attacks an electrophilic disulfide (R 2 -S-S-R 3 ). The transition state is negatively charged and linear, and is indicated with the double dagger ‡ symbol. A new thiolate (the leaving group, R 3 -S − ) departs and a new disulfide (R 1 -S-S-R 2 ) is formed.

Numerous oxidoreductases catalyze this reaction or use this reaction as part of their catalytic cycle. Many of these oxidoreductases belong to the thioredoxin superfamily that contains the thioredoxin folding unit [15]. This superfamily is found in all living organisms and includes, among other proteins, thioredoxins (Trx) themselves, glutaredoxins (Chapters 11 and 12), and protein disulfide isomerases (Chapter 15) that catalyze thiol-disulfide exchanges, as well as glutathione peroxidases and peroxiredoxins (Chapter 13) that reduce hydrogen peroxide using thiols as reductants. The minimal thioredoxin domain has approximately 80 amino acids and consists of a four-stranded β-sheet core, surrounded by α-helices; additional secondary structural elements can be found at the N- or C-terminal ends, and to a minor extent at the loops between β2-α2, α2-β3, and β4-α3 [16]. The structure of a human Trx is shown in Fig. 8. Note that Trx is the actual protein that gives the superfamily its name.

Fig. 8

Fig. 8 Structure of thioredoxin. Secondary structure of a typical Trx (a β-strand surrounded by α-helices). The two redox-active cysteine residues (Cys N, the nucleophilic Cys, and Cys R, the resolving Cys) are shown in grey with the sulfurs in yellow . The structure shown corresponds to the reduced form of human mitochondrial Trx 2, deposited in the Protein Data Bank (code 1W89), and was generated using the molecular visualization program VMD.

The active sites of Trx contain a pair of redox-active Cys residues usually in a CysGlyProCys motif, located in the N-terminal portion of an α-helix. The Cys closer to the N-terminus is polarized by the helix dipole, which promotes its nucleophilicity. This nucleophilic Cys attacks a disulfide in a target protein forming a mixed Trx-target disulfide and releases a new free thiol in the target protein as a result of the first thiol-disulfide exchange reaction. Then, the second Cys residue of the Trx active site, called the resolving Cys, attacks the nucleophilic Cys of Trx, which is engaged in the mixed disulfide with the target. As a consequence of this second thiol-disulfide exchange reaction, the two Cys of the target protein are reduced and the two Cys of Trx are oxidized to an intramolecular disulfide (Fig. 9).

Fig. 9

Fig. 9 Reduction of a protein disulfide by Trx. The nucleophilic Cys of Trx (depicted in purple ) attacks the target disulfide (in green ), forms an intermolecular mixed disulfide with the target, and a first thiol is released. Then the resolving Cys of Trx forms a disulfide with the initially nucleophilic Cys, and a second thiol is formed in the target protein. Note that the nucleophilic Cys is a nucleophile in the first step, but an electrophile when attacked by the resolving Cys.

8: The thioredoxin and the glutahione-glutaredoxin systems: Pathways that use the thiol-disulfide exchange reaction as a leitmotiv

Although the thioredoxin and glutathione-glutaredoxin systems are covered in depth in Chapters 10–12, it is important to introduce these two major pathways that couple the reducing power of the cell (NADPH) to numerous oxidized targets using a cascade of thiol-disulfide exchange reactions.

As already mentioned, Trx is able to reduce disulfides in specific proteins. Targets of Trx include ribonucleotide reductase (that reduces ribonucleotides to deoxyribonucleotides), peroxiredoxin (that reduces hydrogen peroxide to water), and methionine sulfoxide reductase (that reduces Met sulfoxide to Met). In addition, thioredoxin is a powerful disulfide reductase capable of reducing various other protein disulfides formed under oxidative stress conditions. Trx becomes oxidized to a disulfide after reducing a target disulfide (Fig. 9). Thus, it needs to be re-reduced to be able to participate in a new cycle. This is achieved by thioredoxin reductase, a pyridine-nucleotide thiol-disulfide oxidoreductase, that can reduce the Trx disulfide at the expense of NADPH. Thioredoxin reductase is a complex flavoenzyme that possesses several domains and redox centers, including a redox-active FAD, an N-terminal CX4C redox motif, and in the case of animal thioredoxin reductase, an additional C-terminal Cys-Sec redox-active motif [17]. Fig. 10A presents a scheme of the thioredoxin system.

Fig. 10

Fig. 10 Thioredoxin and glutathione-glutaredoxin systems. These pathways shuttle electrons from NADPH to diverse targets through thiol-disulfide exchange reactions. (A) The thioredoxin system comprises the flavoenzyme thioredoxin reductase and thioredoxin (Trx). Thioredoxin reductase transfers electrons from NADPH to FAD, to a CX 4 C redox center, to the C-terminal GCUG (where U is Sec), and finally, to Trx. Reduced Trx reduces protein disulfides; some of the targets are peroxiredoxin (Prx) and methionine sulfoxide reductase (MSR), that reduce hydroperoxides and Met sulfoxide, respectively. (B) The glutathione-glutaredoxin system comprises the flavoenzyme glutathione reductase, glutathione, and glutaredoxin (Grx). Glutathione reductase transfers electrons from NADPH to FAD, to a CX 4 C redox center, and finally, to oxidized glutathione. GSH can reduce Grx, which in turn reduces some protein disulfides and glutathionylated proteins (PrS-SG). A common target of both systems is the essential enzyme ribonucleotide reductase (RR).

In the glutathione-glutaredoxin system, the protein glutaredoxin (Grx) is a disulfide oxidoreductase capable of reducing mixed protein-glutathione disulfides as well as intra- or interprotein disulfides (e.g., ribonucleotide reductase). Similar to Trx, after reducing its target disulfide, Grx has to be re-reduced. The reductant is glutathione (GSH), which becomes oxidized to glutathione disulfide (GSSG). GSSG is then reduced by glutathione reductase, another pyridine-nucleotide thiol-disulfide oxidoreductase, also a flavoprotein that contains a redox-active FAD and a CX4C redox motif. In addition to Grx, GSH has other direct targets, including glutathione peroxidase. Fig. 10B presents a scheme of the glutathione-glutaredoxin system.

Most organisms possess thioredoxin and/or glutathione-glutaredoxin systems, although there are some lineage-specific redox systems, such as bis-glutathionylspermidine (trypanothione) in trypanosomatids (Chapter 24), as well as bacillithiol and mycothiol in certain bacteria (Chapter 23). Mixed arrays such as linked thioredoxin-glutathione systems are present in other lineages (Chapter 10).

In vertebrates, the thioredoxin and glutathione-glutaredoxin systems are present in the cytosol and in the mitochondrial matrix, maintaining a disulfide-reducing environment in these compartments. In contrast, in the endoplasmic reticulum and in the intermembrane mitochondrial space, specific pathways promote disulfide formation (Chapter 15). This compartmentalization of Cys-reducing and Cys-oxidizing pathways is also present in bacteria: while bacterial cytoplasm promotes disulfide reduction, the periplasm promotes disulfide oxidation (Chapter 16).

9: The concentrations of thiols and oxidized derivatives are under kinetic control

Inside cells, the total concentration of thiols is very high. In vertebrate cells, glutathione is 2–17 mM and 91% reduced [18,19]; particularly in the cytosol, glutathione is ~ 99.97% reduced [20,21]. Protein thiols are even more abundant (10–50 mM) and ~ 90% reduced [18,19]. In contrast, extracellular compartments have higher proportions of disulfides and lower thiol concentrations. For example, in plasma (see Chapter 25), reduced thiols add up to ~ 0.4–0.6 mM and mostly belong to albumin. Total glutathione is ~ 6 μM, of which ~ 3 μM is reduced and ~ 1 μM LMW disulfide. Cys is ~ 240 μM total, of which ~ 10 μM is reduced and ~ 50 μM LMW disulfide. The rest are mixed disulfides formed with protein Cys, mainly albumin [22].

The different thiol-disulfide pairs are typically not in equilibrium with each other with regards to the thiol-disulfide exchange reactions. If they were, the values of the concentration quotients would be the same as the equilibrium constants. For example, the exchange between cysteine and glutathione, and the corresponding equilibrium constant, are represented by Eqs. (9) and (10).

si9_e

   (9)

si10_e    (10)

The equilibrium constant has been measured as 0.265 at neutral pH [23]. However, in plasma, the concentration quotient is ~ 5 [22]. Clearly, these two LMW thiol-disulfide pairs are not in equilibrium. This is due to the existence of kinetic barriers. Indeed, the rate constants of LMW thiol-disulfide exchange reactions are quite low [23]; enzymes that catalyze these reactions are scarce in plasma, and if present, they would catalyze exchange between only some and not all thiol-disulfide pairs [24]. This far-from-equilibrium situation can be extrapolated to other redox pairs in plasma and to other compartments, with exceptions in the case of fast processes involving enzymes, which can sometimes achieve equilibrium.

What determines the biological concentrations of thiols and their oxidized derivatives? The concentrations are the result of several simultaneous processes that affect the proportions as well as the total concentrations. In addition to the rates of the enzymatic and nonenzymatic redox reactions, other processes that impact on the concentrations are the rates of metabolic synthesis and degradation pathways, and the rates of transport between compartments.

In general, redox systems are under kinetic instead of thermodynamic control. Thus, readers are encouraged to question statements that include the terms redox buffer and redox equilibrium. Rather than reduction potentials, a good predictor of the biological significance of a certain process is the comparison of its kinetic rate with the kinetic rates of competing processes.

That said, for beginners, it is relevant to introduce a few very basic kinetic concepts. Given the general reaction represented by Eq. (11), the rate v is defined as the change in concentration of the reactants or products per unit time (Eq. 12). The rate law relates the rate of the reaction to a rate constant and to the concentrations of reactants (Eq. 13). The reaction order is the sum of the exponents of the concentration factors in the rate law (α + β) and needs to be determined experimentally.

si11_e    (11)

si12_e

   (12)

si13_e    (13)

For an elementary reaction representing a molecular event, the order is the number of species entering the reaction itself. For example, the isomerization of a molecule A into a product P is likely to be first-order in A; the rate increases linearly with A concentration, and the time courses of A decay are exponential. A bimolecular reaction between two reagents A and B, such as the SN2 thiol-disulfide exchange reaction shown in Fig. 7, is overall second-order: first-order in A and first-order in B. Importantly, second-order reactions can effectively be considered pseudo-first order if the concentration of one of the reactants is constant and/or much higher than the other reactant. Furthermore, the rate of a reaction can be affected by temperature, pH, ionic strength, and catalysts.

In the case of enzymatic reactions, the rate of the catalyzed reaction is first-order in enzyme, and thus has a linear dependency with the concentration of enzyme; as the concentration of enzyme increases, the rate also increases. Thus, many systems control the rates of processes by modulating the concentrations of active enzymes. Regarding the substrates, the order in a typical enzyme catalyzed reaction is mixed, and the dependence with substrates concentration is hyperbolic: as the concentration of substrates increases, the enzyme becomes saturated as all the active sites become occupied, so that further increases in concentration do not affect the rate. In other words, for the enzyme-catalyzed transformation of a substrate S in the product P, the initial rate of the reaction can be represented by Eq. (14), where [E] and [S] are the total enzyme and substrate concentrations, and kcat and KM are kinetic parameters—kcat represents the maximum number of substrate molecules that can be transformed into product per unit time and per active site, and KM is the substrate concentration at which an enzyme functions at half-maximal rate. A high KM indicates that the enzyme binds the substrate with low apparent affinity.

si14_e    (14)

10: Cys diversity in proteins

The functional diversity of Cys derives from the versatile chemistry of thiols. The reactivity of Cys protein residues, which can deprotonate to thiolates and undergo a variety of reactions, explains that Cys constitutes in most cases a critical residue, usually conserved. This is why mutations involving Cys residues result in genetic diseases more often than expected based on abundance [25]. Since reactivity is a double edged sword, nonfunctional Cys residues in proteins tend to be eliminated by evolution, in particular if they are isolated and exposed to the solvent, while Cys residues clustered in pairs tend to be conserved [14].

The diversity of Cys in proteins is covered in Chapter 3, and is briefly introduced here. Functional Cys residues can be arbitrarily classified in the following categories:

1.Catalytic Cys, which are present in the active sites of enzymes involved in catalysis of redox (e.g., thiol-disulfide oxidoreductases) or nonredox (e.g., Cys peptidases) reactions.

2.Structural Cys, which form permanent protein disulfides that stabilize protein folding and structure, and are usually present extracellularly (secreted or in membrane ectodomains), in the endoplasmic reticulum, and in the intermembrane mitochondrial space.

3.Metal-coordinating Cys, which bind metal ions (e.g., Fe, Zn, Cu, Mo, and Co ions) as in Fe/S-containing proteins or in Zn-fingers transcription factors. They contribute to protein function and stability, and some can undergo redox reactions and modulate protein activity.

4.Posttranslational modification site Cys, which serve regulatory roles by transient changes in their redox state, as in kinases and transcription factors, or are permanently modified by nonredox mechanisms (e.g., protein S-palmitoylation and prenylation). A description of the different post-translational modifications can be found in Chapter 19.

11: Concluding remarks

Thiols have a very rich chemistry. This is due to the very high nucleophilicity of thiolates and to the acidity of thiols, which determines the availability of thiolates at physiological pH. Oxidation can occur by one- and two-electron mechanisms and can lead to several products, some of which can revert to thiols with suitable reductants. The thiol-disulfide exchange reaction plays a prominent role in biology and can be catalyzed by dedicated systems: the thioredoxin and the glutathione-glutaredoxin systems. These systems couple the reduction of disulfides to the oxidation of NADPH via a sequence of thiol-disulfide exchange reactions. The high potential reactivity of protein Cys residues explains their frequent presence in conserved catalytic, regulatory, and metal-binding sites, while nonfunctional Cys tend to be eliminated by evolution. The concentrations of thiols and their oxidized derivatives are under kinetic control in biological systems. The formation of oxidized thiol derivatives is associated not only with oxidative damage and detoxification processes, but also with enzymatic catalytic mechanisms, regulation of protein activity, and redox signaling. Thus, thiols are in the spotlight for potential applications in the fields of medicine and drug discovery.

Glossary box

Addition: A reaction between two or more molecular entities that results in a single product containing all atoms of all components, without the loss of any atoms. The reverse is called elimination.

Electronegativity: The tendency of an atom to attract shared electrons (or electron density) to itself. The higher the electronegativity, the more the electrons are attracted to that nucleus.

Electrophile: A chemical species that accepts an electron pair from a nucleophile to form a chemical bond.

K a : The equilibrium dissociation constant for an acid (a substance that can donate protons).

Leaving group: A molecular fragment that departs with a pair of electrons in heterolytic bond cleavage.

Nucleophile: A chemical species that donates an electron pair to an electrophile to form a chemical bond.

Oxidation: Process in which an atom or molecule loses one or more electrons. Oxidation is a half-reaction that occurs simultaneously with a reduction half-reaction.

Oxidation state: Describes the degree of oxidation (loss of electrons) of an atom in a chemical species. The oxidation state is the hypothetical charge that an atom would have if all bonds to other atoms were completely ionic, with no covalent component. This is far from true for covalent bonds, but the convention is useful.

pKa: −log Ka. It is the pH at which the concentration of an acid and its conjugate base are equal. It is a convenient measure of the strength of an acid: the lower the pKa the stronger its acidic character.

Polarizability: It is a measure of how easily an electron cloud belonging to an atom or molecule is distorted by an electric field and acquires a dipole moment.

Radical: Also known as free radical this is an atom, molecule, or ion that has one or more unpaired electrons. Their formulas include a dot, symbolizing the unpaired electron.

Reaction rate: For a chemical or enzymatic reaction, the rate v is defined as the change in concentration of the reactants or products per unit time.

Redox couple: The oxidized and reduced species that participate in a redox half-reaction.

Reduction: Process in which an atom or molecule gains one or more electrons. A reduction is a half-reaction that occurs simultaneously with an oxidation half-reaction.

Reduction potential: Represents the tendency for a species in a redox couple to be reduced. It is measured in volts. The more positive, the greater the tendency of the couple's oxidized form to accept electrons and become reduced.

Substitution: A reaction in which one atom or group in a molecule is replaced by another atom or group. In an SN2 substitution, bond breaking and making are synchronous, and the reaction is one-step, concerted, and bimolecular.

References

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Chapter 2: Chemical basis of cysteine reactivity and specificity: Acidity and nucleophilicity

Gerardo Ferrer-Suetaa,b    a Laboratorio de Fisicoquímica Biológica, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay

b Centro de Investigaciones Biomédicas (CEINBIO), Universidad de la República, Montevideo, Uruguay

Abstract

The acidity of small alkylthiols depends mainly on the inductive effect of substituents, and the ionization state of neighbor functional groups. Protein thiols have a wide range of pKas, which depend, qualitatively, on the electrostatic environment, due to charged neighboring groups and α-helix macrodipoles and hydrogen bonds. The desolvation of the thiol/thiolate group also causes major alterations of the pKa. Nucleophilicity is the most important kinetic property of the thiolate catalytic cysteines in proteins, it is not related to pKa and depends on the regulation of the solvation as well as hydrogen bonding of protein thiolates. In general, aqueous environments greatly diminish the nucleophilicity of thiolates but the protein environment can delicately modulate it by tweaking its nonbonding and solvation interactions, and importantly enhancing its reactivity when the right substrate is bound and ready to react. This explains why protein thiolate reactivity is highly selective.

Keywords

Acid-base; Nucleophilicity; Thiolate; pKa; Acidity; Selectivity; Catalysis; Solvation; Hydrogen bond; Enzyme catalysis

Acknowledgments

I would like to thank the reviewers of this chapter and the organizers of the course that originated this book, Beatriz Alvarez, Marcelo Comini, Gustavo Salinas, and Madia Trujillo, for the invitation to participate. This text contains bits and pieces of insightful discussions held along the years with excellent colleagues, such as Bruno Manta, Horacio Botti, and Stephanie Portillo.

The uniqueness of cysteine as a protein residue has a large number of contributing factors. We will visit some of them in this chapter under the guise of studying two major aspects of the chemistry of the thiol group, namely its acid-base behavior and the nucleophilic character of its conjugated base, the thiolate.

Acid-base equilibria, as discussed here, will refer to the Brønsted definition by which an acid is a substance able to lose a hydrogen ion (H+) in a reaction and a base is able to gain a hydrogen ion in a reaction. Such definition leads to the acid dissociation equilibrium (or ionization) between an acid and its conjugate base, as illustrated in Eq. (1) for a generic thiol and thiolate.

si1_e    (1)

This equilibrium has an acid dissociation constant, also known as ionization constant, or Ka, defined in Eq. (2).

si2_e    (2)

Taking logarithms and rearranging, Eq. (2) becomes the well-known Henderson-Hasselbalch relationship (Eq. 3), which will be useful later on.

si3_e    (3)

The thiol group of cysteine, as a free amino acid in aqueous solution, is a weak acid. That is also the case of most alkylthiols, which have pKas in a range from above 11 down to approximately 6.5. The effect governing this variation of nearly five units can be understood qualitatively from the inductive effect of the substituents in the alkyl chain. As a general consideration, groups donating electronic density close to the thiol stabilize the protonated form, whereas groups withdrawing electronic density stabilize the thiolate. In Table 1 we have selected a number of thiols with ethanethiol (bold type) as a common skeleton. Without additional substituents, the pKa of ethanethiol is 10.6, but adding two electron-donating methyl groups into the alpha carbon atom increases the pKa by 0.62 units in 2-methyl-2-propanethiol. On the other hand, adding increasingly stronger electron-withdrawing groups, such as acylamino, alcohol, ammonium ion,and ester to the alkyl chain, decreases the pKa. The last entry of Table 1, cysteine methyl ester, is so acidic that the predominant form at neutral pH is the thiolate (pKa = 6.45).

Table 1

Before going on, I would like to make a brief remark about language. In this chapter I will do my best to use the term thiol only to refer to the protonated form, and thiolate when referring to the ionized form. When dealing with equilibrium mixtures dependent on pH, the specific name of the compound, such as cysteine, 2-hydroxyethanethiol, or thioredoxin will be used. Alternative names such as sulfhydryl or mercaptan will not be used in this chapter.

1: Measuring pKa

The problem of measuring the pKa of a thiol-thiolate pair can be addressed in various ways depending on the compound and experimental circumstances, such as availability of material, solubility, etc. The standard methodology for measuring pKa is a potentiometric titration using a pH electrode and acid or base as titrant, but it is only useful with relatively concentrated solutions of low molecular weight compounds. Furthermore, the determination becomes complex when several acid base systems coexist with a narrow range of pKas.

The history of the pKa of cysteine illustrates this point. The potentiometric titration of cysteine yields three pKa values. The lowest, with a value below 2, can be assigned to the carboxylic acid by analogy with other carboxylic acids. The other two need to pertain to the dissociation of the ammonium and thiol groups, but their assignation is ambiguous—they are very close, in the region of 8–10.5, where both primary ammonium ions and thiols are known to dissociate. Deciding which is which is impossible with only data on H+ concentration and volume of titrant consumed; another piece of information needs to be used to decide. Fortunately, many such spectroscopic and reactivity indicators are available and habitually used in the experimental determination of dissociation constants. The ambiguity in the pKas of cysteine was solved by associating the pH measurement with the UV spectra of the species involved. It had been reported that thiols and thiolates have important differences in their UV spectra in the 230–240 nm region [6]. Benesch and Benesch [4] used the UV absorption to determine the pKa of cysteine and other aminothiols and found that the pKas of the ammonium and the thiol can be distinguished but are inextricably related.

As we saw before, the thiol pKa depends on the inductive effect of nearby substituents on the alkyl chain. In that case, the inductive effect will also depend on the protonation state of such substituents, and thus, the amino group (− NH2) will exert a different influence to the ammonium group (–NH3+). Fig. 1A depicts the ionization possibilities of cysteine, starting on the upper left corner with both the thiol and the ammonium groups. Going to the right implies the conversion of thiol into thiolate, whereas going down implies the conversion of ammonium to amino. It can be seen that the two horizontal equilibria should be different owing to the difference in the inductive effect of ammonium and amine. In fact, those equilibrium constants have been measured: pKaA is 8.3, as in Table 1, but pKaD is 9.67, very close to the value of N-acetyl cysteine [3]. Fig. 1B shows the experimental results: first, the spectra at different pH, then the titration curve at 240 nm, and the best fit to a function derived from the four equilibria system proposed by Benesch. The equilibrium constants of Fig. 1A are known as microscopic ionization constants and can be extended to consider also the ionization of the carboxylic acid in the case of cysteine, and even all neighboring functional groups with acid base properties. For instance, Rabenstein et al. [7] studied glutathione by nuclear magnetic resonance and determined a set of eight microscopic ionization constants, two of which involve the ionization of the thiol.

Fig. 1

Fig. 1 Microscopic equilibria of cysteine. (A) The protonation states of the amino and thiol groups are intertwined in such a way that the protonation state of one affects the dissociation equilibrium of the other, yielding a four equilibria scheme. (B) In a spectrophotometric titration, the curve following the thiolate absorbance shows two inflection points coincident with the two dissociation processes of the thiol group, namely K a A and K a D , as marked in the curve. From data published in Portillo-Ledesma S, Sardi F, Manta B, Tourn MV, Clippe A, Knoops B, et al. Deconstructing the catalytic efficiency of peroxiredoxin-5 peroxidatic cysteine. Biochemistry. 2014;53(38):6113–25.

Given the complexity of the problem of assigning a pKa value to a thiol group in a molecule containing other acid-base groups, the aid of spectroscopic techniques is extremely valuable. UV absorption at 240 nm is a workhorse for researchers studying thiol chemistry. It has been used also to measure the pKa of thiols in proteins, such as DsbA [8,9], glutathione transferase [10], arsenate reductase [11], tryparedoxin [12], NrdH-redoxin [13], AhpC [14], Pseudomonas aeruginosa CcmG [15], and human serum albumin [16].

UV absorption as a technique for measuring pKa is rather limited. The molar absorptivity of the thiolate is relatively small (4000–6000 M− 1 cm− 1) implying that a substantial concentration of sample is needed in order to see a good difference in absorbance. Furthermore, 240 nm is a region where other chromophores from the protein may interfere and the concentrated protein solution needs to be stable throughout the pH interval.

Other spectroscopic techniques can have advantages over the UV absorption of the thiolate; for instance, if the protein in question has more than one cysteine residue and they have overlapping or similar pKa values, it would be impossible to distinguish them at 240 nm; you would only know that more than one residue was being titrated because of the larger than expected change in absorbance. That is not a problem for NMR since each magnetic nucleus can, in principle, be identified and also, isotopes can be used to label specific amino acids and make the reading unambiguous. Thus, monitoring the chemical shift of the cysteine β-carbon, either by its hydrogen substituents or by labelling it with ¹³C, as a function of pH, one can detect the protonation state, as done in the three cysteine residues coordinating Zn² + in the HIV-1 nucleocapsid protein [17], in the two active-site cysteines in E. coli Trx1 [18–21], in acyl coenzyme A binding protein [22], and in cDsbD [23]. The subject has been reviewed for cysteine and other ionizable amino acids in proteins [24].

Other spectroscopic techniques have been used less frequently; for instance, Raman dispersion has served to characterize the ionization of E. coli Trx active site cysteines [25].

There are some indirect spectroscopic measurements of protein cysteine pKa, for instance following the intrinsic fluorescence of a protein, due mostly to tryptophan and tyrosine residues. This technique can be used when the cysteine ionization affects the emission wavelength and/or intensity of one or more neighboring fluorescent residues. By themselves, the results are ambiguous, because many different modifications can induce the change in fluorescence, but with a set of adequate control experiments the technique is very sensitive. Intrinsic fluorescence has been used to study the ionization of the active site cysteines of E. coli Trx [26,27] and Trypanosoma brucei Grx1 [28].

A very common method for measuring protein cysteine pKa is by the differential reactivity between the thiol and the thiolate. As we will see in the second part of this chapter, the thiolate is a good nucleophile in aqueous solution, whereas the thiol nucleophilicity is rather poor. There is a diversity of reactants used to measure pKa in protein cysteines, but the most common are alkyl halides such as iodoacetamide (IAM) or iodoacetic acid (IAA). Being relatively small, these alkylating agents can access the active site of enzymes inactivating the catalytic function related to the Cys under scrutiny, for instance, in hexokinase [29], glutaredoxins [30–36], protein tyrosine phosphatases [37], or tryparedoxin [12]. Other alkyl halides having fluorescent substituents, generating a difference in mass [14], or yielding a fluorescent product