A Review of Methionine and Cysteine: Their Roles as Sinks for Reactive Oxygen and Nitrogen Species, Including Bioinformatic Analysis of Their Percent Compositions in Metabolic Proteins

Abstract

This review focuses on the roles of the two primary sulfur-containing amino acids, cysteine and methionine, in regulating reactive oxygen/nitrogen species (RONS). RONS are highly reactive oxygen/nitrogen-containing free radicals and compounds. Endogenous and exogenous antioxidants, including sulfur-containing amino acids, protect cells against the harmful effects of RONS on cellular macromolecules. This study thoroughly reviews the mechanisms by which these two sulfur-containing amino acids neutralize RONS. Additionally, a bioinformatic analysis of the percentage compositions of cysteine and methionine in metabolic proteins of humans and 12 closely related species was conducted using a “Biopython” script to assess their potential role as sinks for RONS, maintaining the structure and function of metabolic proteins. A total of 119 proteins from various metabolic pathways, including glycolysis, pyruvate to acetyl CoA conversion, tricarboxylic acid cycle, oxidative phosphorylation, pentose phosphate pathway, gluconeogenesis, glycogen metabolism, fatty acid metabolism, amino acid catabolism, nucleotide biosynthesis, and ROS scavengers were included in the bioinformatics analysis. This review shows that methionine and cysteine play crucial roles in neutralizing RONS. The bioinformatic analysis revealed that the percentage compositions of methionine and cysteine are higher in key redox enzymes like dehydrogenases, enzymes involved in oxidative phosphorylation, and those participating in the committed steps of metabolic pathways.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12291-024-01284-y.

Introduction

Many biological processes, such as cellular oxidative metabolism, produce free radicals [1]. These free radicals are reactive oxygen/nitrogen species (RONS). They also are formed in response to external stimuli such as environmental toxins and radiation. Although free radicals play important roles in the body's immune response and activating signaling pathways involved in inflammation, an excess of them has damaging effects. RONS damage biomolecules inside cells and are responsible for aging and various chronic diseases such as cancer, cardiovascular diseases, diabetes, and neurodegenerative [2–4]. To counter the harmful effects of free radicals, cells produce antioxidants. The correct balance of oxidants and antioxidants is needed to avoid oxidative stress that reduces lifespan. Methionine (Met) and cysteine (Cys) are the two sulfur-containing amino acids (SAAs) that also exhibit antioxidant properties [5]. These SAAs are present in many metabolic proteins and enzymes, and their potential role as antioxidants through various reversible redox pathways suggests that proteins with a relatively high percentage of Met and Cys may have an additional role in protecting proteins’ structures and functions against oxidative stress. Moreover, these two SAAs are the precursors of endogenous antioxidants like glutathione (GSH). This review focuses on two aspects—(i) a literature review on the role of Met and Cys as RONS sinks and (ii) the percent composition of Met and Cys in human metabolic proteins; the percentage of Cys or Met in proteins could be related to their capacity to scavenge ROS. The percent composition of Cys and Met are 1.39 and 2.21, respectively in globular proteins, and 0.47 and 1.56, respectively in outer membrane proteins [6]. It is expected that the percent composition of these SAAs will be higher in a metabolic protein if they have an additional role i.e., neutralizing RONS beside structural and/or catalytical roles. Herein, we describe RONS and oxidative stress, exogenous and endogenous antioxidants, and oxidation resistance of Met and Cys. Additionally, a bioinformatic analysis for determining the percent composition of Cys and Met in proteins involved in ten metabolic pathways and ROS scavenger proteins was performed to further our understanding of the role played by these two sulfur-containing amino acids in the metabolic adaptation to redox stress. More than 100 human metabolic proteins and their homologs from 12 closely related species were included in the present study.

RONS and Oxidative Stress

Reactive oxygen species (ROS) are highly reactive free radicals or compounds of oxygen such as singlet oxygen (¹O₂), superoxide radical (O₂·⁻), hydrogen peroxide (H₂O₂), and hydroxyl radical (HO·) [7–10]. There also exists reactive nitrogen species (RNS) such as nitric oxide (NO) produced from L-arginine by nitric oxide synthase, and peroxynitrite (ONOO^–) forms when NO reacts with superoxide (O₂·⁻) [3, 7, 11]. ROS and RNS combinedly are known as RONS and are formed through biological oxidation–reduction (redox) reactions. RONS are constantly generated at low amounts within cells via non-enzymatic and enzymatic processes such as neutrophil and macrophage activation during inflammation, metal-catalyzed reactions, and mitochondrial electron transport reactions [12, 13]. Production of RONS also increases due to external factors such as cigarette smoke, heavy metals, xenobiotics, atmospheric pollutants, or irradiation with UV light or X-rays [8, 14, 15].

RONS are utilized in biological processes, including signal transduction (redox signaling), initiation of cell differentiation, and disease progression [16–18]. However, an imbalance between the production of RONS in tissues and cells and the ability of a biological system to detoxify these RONS products can lead to what is referred to as oxidative stress [19, 20]. The overaccumulation of RONS causes toxic effects by damaging lipids, DNA, and proteins [18, 21–23]. During oxidative stress, Cys residues of cellular proteins oxidize [14], which leads to alternation in “off–on” switching of enzyme activity [24]. Oxidative stress is a common factor in many diseases and disorders, including cancer, hypertension, diabetes mellitus, atherosclerosis, emphysema, bronchitis, viral infection, Alzheimer’s, and Parkinson’s disease [25–28]. However, it should be noted that the role of RONS in cancer is somewhat complex. RONS participate in cell signaling and excess of it may induce oxidative stress and cancer cell death. To avoid this, cancer cells increase antioxidants to keep RONS at a level conducive to tumor growth [29].

Antioxidants

Antioxidants are biomolecules or low-molecular-weight molecules that can counter RONS and eliminate their harmful effects [20]. They neutralize RONS in a redox reaction either by deactivating their radical properties and converting them into less harmful molecules or by increasing the effectiveness of enzymes that can destroy free radicals [30]. Antioxidants can be categorized as endogenous (produced by the body) and exogenous (obtained in the diet) [31] and are either enzymatic or non-enzymatic. Examples of common antioxidants and their categorizations are provided in Table 1.

Table 1.

Types of antioxidants

Type	Enzymatic	Non-enzymatic
Endogenous	Catalase Glutathione peroxidase (GPx) Glutathione reductase (GR) Glutathione-s-transferase (GPST) Peroxiredoxin (Prx) Super oxide dismutase (SOD) Thioredoxin reductase (TR)	Alpha-lipoic acid Bilirubin Coenzyme Q Ferritin Glutathione (GSH) Uric acid Thioredoxin (Trx)
Exogenous		Carotenoids Flavonoids N-acetyl-L-cysteine Polyphenols Riboflavin Vitamin C Vitamin E Zinc

The first line of defense antioxidants that act to neutralize RONS are endogenous enzymes. These enzymatic antioxidants convert free radicals in the cell into harmless molecules like molecular oxygen and water. The most common two-step mechanism is that of superoxide dismutase (SOD) and catalase, which are metalloproteins containing redox-active metal centers [32, 33]. Cytosolic and mitochondrial SODs transform RONS molecules into molecular oxygen and hydrogen peroxide by catalyzing the dismutation of superoxide. Then, catalase catalyzes the decomposition of hydrogen peroxide molecules into water and molecular oxygen. SOD is present in all aerobic organisms and subcellular components susceptible to oxidative stress. SODs are classified into three types: Cu/Zn-SOD, Mn-SOD, and Fe-SOD, which are widely expressed in the human lung. Mn-SOD and Fe-SOD are structurally similar whereas Cu/Zn-SOD varies significantly; the formers are homotetrameric, whereas Cu/Zn SOD is homodimeric [32, 33]. Catalase is considered a peroxide oxidoreductase and is an iron-containing tetrameric heme enzyme that removes the peroxide generated during aerobic respiration and as a metabolic by-product of many oxidases in the peroxisome [34]. Another class of endogenous peroxide scavengers in the cytosol and mitochondria is the selenium-containing glutathione peroxidases (GPx). These enzymes catalyze the reduction of hydrogen peroxide to water by glutathione (GSH) (vide infra) following the scheme shown in Fig. 1 [35]. Also, peroxides can be directly reduced by the thioredoxin-dependent peroxidases or peroxiredoxins (Prx). The oxidized Prx is reduced indirectly by NADPH-dependent thioredoxin reductase (TR). Prx is abundant in mammalian cells and protective against oxidative stress [36]; they are found to be induced in response to oxidative stress. Therefore, these antioxidant enzymes play indispensable roles in protecting biological systems against oxidative stress.

Fig. 1.

Scavenging of peroxide by endogenous enzymatic and non-enzymatic antioxidants

The second line of defense antioxidants are non-enzymatic antioxidants, often called free radical scavengers. These scavengers neutralize free radical chain reaction initiators or break chain reaction propagation. They donate electrons or hydrogen to reduce RONS, becoming a less reactive radical species themselves. The lesser reactive species made by scavenging RONS are easily neutralized [33]. These antioxidants can be either exogenous or endogenous (Table 1). The most common exogenous antioxidants are polyphenols, tocopherols, and ascorbic acid. Polyphenols are the most abundant and strong antioxidants and include flavonoids, phenolic acids, polyphenolic amides, and other polyphenols like resveratrol in red wine [37]. Vitamin E is an important exogenous membrane-bound antioxidant and a lipid-soluble peroxyl radical (ROO•) scavenger. Vitamin E refers to a group of structurally related plant-derived natural products comprised of tocopherols and tocotrienols. The chroman ring of vitamin E contains a hydroxyl group, and the reduction of peroxyl radicals occurs via the donation of a hydrogen radical (i.e., hydrogen atom, H·) from the hydroxyl group. The reduced form of vitamin E is regenerated from the oxidized form (tocopheryl radical) with the help of hydrogen donors such as ascorbic acid (vitamin C, the natural water-soluble vitamin) and coenzyme Q, which are also free-radical scavengers [31, 38, 39].

N-acetyl-L-cysteine, a synthetic antioxidant, also acts directly as a scavenger of free radicals. It inhibits ROS via free-radical scavenging with a redox potential of its thiol group or by stimulating GSH biosynthesis. This promotes detoxification and presents a potential treatment option for diseases characterized by the generation of free oxygen radicals [40, 41] Studies have shown that it reduces ROS-mediated oxidative damage of DNA [42].

GSH is a key endogenous non-enzymatic antioxidant crucial for combating oxidative stress. It is a tripeptide and is synthesized endogenously from Cys, glutamic acid, and glycine [43] Within cells, GSH exists in two forms: reduced glutathione (GSH) and oxidized (GSSG). As ROS levels rise, more GSH is converted into GSSG, turning RONS into chemically stable molecules that can safely be eliminated. As mentioned above, this conversion is facilitated by the enzyme GPx, which reduces hydrogen peroxide (H₂O₂) into water. The regeneration of GSH from GSSG is carried out by NADPH-dependent glutathione reductase (GR) (Fig. 1). NAPDH is produced by glucose-6-phosphate dehydrogenase (G6PD) in the pentose phosphate pathway (PPP). In addition to these functions, GSH converts vitamins E and C to their active forms and acts as the cofactor in the detoxification process of several enzymes [39].

After oxidative damage occurs, newly synthesized enzymes formed in response to the damage serve as the third line of antioxidant defense [33]. These enzymes play a crucial role in repairing damage to membranes and nucleic acids, while also helping to eliminate debris that could be toxic to healthy tissues. Key components of the third-line defense antioxidants include polymerases, glycosylases, nucleases, proteinases, proteases, and peptidases, which are present in the cytosol and mitochondria of most mammals. Finally, the fourth line of defense involves an adaptogenic mechanism. Adaptogens act as signals to the cell, indicating the presence of RONS. This signal triggers a cellular response that transports appropriate antioxidants to the site of RONS, allowing for their neutralization [33].

Methionine as a RONS Sink

The sulfur-containing, hydrophobic, flexible amino acid Met has long been considered important for protein synthesis initiation and stabilization of protein structures through hydrophobic interactions with nonpolar aliphatic and aromatic amino acids. Met’s catalytic and regulatory roles have also been reported [11, 44]. Additionally, Met can undergo two-electron oxidation to form methionine sulfoxide (MetO) or one-electron oxidation to produce Met radical cations, depending on the nature of the oxidizing species; the oxidation of Met residues is essentially pH-independent [45, 46]. Recent studies have revealed that Met in proteins also acts as an antioxidant and can neutralize varieties of ROS [47]. Although many RNS do not react with Met residues specifically, it has been reported that they may still affect the redox balance in that the weaker RNS, nitric oxide, reacts with superoxide to produce peroxynitrite (ONOO⁻), which is a known oxidizer of Met [11, 48]. Thus, the sulfur of Met can readily be oxidized to MetO in the presence of RONS, thus scavenging the reactive species. MetO exists in two stereoisomeric forms, MetO-S and MetO-R [49]. These oxidized Met i.e., MetO can be reduced back to Met by methionine sulfoxide reductases (Msr), which catalyze a thioredoxin-dependent reduction of MetO. There are two reductases—methionine sulfoxide reductases A (MsrA) and methionine sulfoxide reductases B (MsrB), one for each stereoisomer-MetO-S and MetO-R, respectively [5, 50] The reduction of MetO occurs via a cascade of redox reactions, with the ultimate reducing power coming from the oxidation of NADPH to NADP⁺ (Fig. 2). This redox cycle allows Met to serve as an effective ROS sink [47, 51, 52], mediates the regulation of various cellular processes [53].

Fig. 2.

The redox reaction of methionine

Met plays a crucial role as an endogenous antioxidant, protecting the structure and function of proteins under oxidative stress. Many proteins display high concentrations of Met residues on the protein surface to protect inner residues from RONS and prevent denaturation and loss of function [45, 54]. In the case of α-2-macroglobulin, a protease inhibitor, exposure to RONS revealed that the surface Met residues protect the functionally important core Met from RONS. Similarly, the structural and biochemical analyses of glutamine synthetase demonstrated that oxidized Met residues were present toward the enzyme surface, while Met residues located more inward were not oxidized. Researchers found that Met residues were lined along the active-site bay of glutamine synthetase, and react with multiple ROS, including hydrogen peroxide. If it were not for the presence of surface Met, ROS would be able to inactivate the enzyme [47, 53]. Thus, Met plays a pivotal role in protecting the catalytic activity of enzymes. Met’s role as a ROS sink is also noted in an important family of enzymes, i.e., aminoacyl-tRNA synthetases. The alanyl-tRNA synthetase of E. coli can maintain its catalytic and editing functions under oxidative stress because of the antioxidant properties of SAAs [55]. These examples further solidify the significance of Met as an effective antioxidant.

To confirm that Met does indeed detoxify ROS, an experiment was conducted with E. coli cultures containing either normal cells or norleucine-substituted cells; norleucine is the carbon analog of Met. The control (normal cells) and norleucine-substituted cultures were exposed to hypochlorous acid, hydrogen peroxide, and ionizing radiation. The results showed that the normal cells maintained cellular function, while the culture containing norleucine-substituted cells died more rapidly than the control cells [56]. Furthermore, MsrA levels were found to be lower in patients with Alzheimer’s disease, which is known to be associated with oxidative damage [47]. Additionally, in knockout msrA mice, oxidative stress was significantly increased and caused enhanced oxidative modification to proteins [45]. The reversible redox process of Met, enzymatically catalyzed by Msr, is a novel molecular mechanism for regulating cell function; the oxidation of Met, in the presence of ROS, affects the functions of signaling proteins such as ion channels [11, 57] The reversible Met oxidation and reduction are also crucial for regulating the cellular immune system. [58]. Taken together, Met in proteins plays variable roles and its role as an antioxidant is as important as its role in protein initiation, structure stabilization, and as a catalytic and regulatory amino acid side chain.

Cysteine as a RONS Sink

Cys has a defensive function against RONS. One of the least abundant amino acids, Cys is the only amino acid that contains a thiol/sulfhydryl (–SH) group. This thiol-containing amino acid is critical for protein structure, function, and regulation [24]. One of the important characteristics of Cys residues in proteins is that they undergo redox reactions and form disulfide bonds. The thiol group of Cys is ionizable, and its deprotonated, negatively charged thiolate form acts as a nucleophile and is readily oxidized, mediated either enzymatically for thiols with pK_a higher than physiological pH or non-enzymatically or Cys thiols with pK_a lower than physiological pH. Additionally, the thiol/thiolate group exhibits a high affinity for metal ions and can be alkylated by electrophiles and oxidized by RONS [24, 59, 60]. Exposure to RONS leads to varying degrees of oxidation of free thiols (− SH) of Cys and results in multiple types of redox post-translational modifications. These modifications include reversible ones such as S-sulfenylation (SOH), S-nitrosylation (SNO), S-glutathionylation (SSG), disulfide bond formation (S–S), and S-polysulfidation (SSnH, n ≥ 1), as well as irreversible modifications like S-sulfinylation (SO₂H) and S-sulfonylation (SO₃H) [61–64].

Cys is a crucial and distinctive RONS sink due to the nucleophilic properties of its thiol/thiolate group and the reversibility of the redox reactions it undergoes. The thiol side chain can form a disulfide bond with another Cys, often one in close proximity, in a reversible redox reaction (24, 65). Disulfide bridges are vital for stabilizing protein structure and, in some cases, Cys protects against oxidative stress by forming intracellular disulfide bonds with other Cys residues and interacting with amide nitrogens in the polypeptide backbone [66]. Cys residues in low-molecular-weight antioxidants like glutathione (GSH) and in the active sites of proteins such as thioredoxin and peroxiredoxin play essential roles in safeguarding cells from oxidative damage. Notably, the free thiols of Cys residues exposed on protein surfaces act as key intracellular antioxidants. Research has shown that the concentration of protein thiols (Cys residues) in the mitochondrial matrix is significantly higher than that of GSH, indicating that Cys thiols in proteins may serve as effective RONS scavengers [67, 68].

The reversible redox process is key to the elimination of excess ROS and oxidative stress. Redox regulation via Cys residues is observed in pyruvate kinase M2, an enzyme involved in the rate-limiting step in glycolysis [69]. Also, Cys residues can coordinate with metal ions in metalloproteins such as zinc-finger proteins and Fe–S clusters and offer structural stability. When Cys is oxidized, it releases the bound zinc (II) and rebinds the zinc ion when it is reduced back to thiol form. This reversible redox process results in an alteration in conformational change and function of the protein [70]. Thus, Cys residues in proteins, either at the catalytic or regulatory site, act as redox switches. Such thiol switches are involved in posttranslational modification signaling pathways including the phosphorylation-dephosphorylation pathway and are ROS-dependent [71, 72]. For example, the protein tyrosine phosphatase catalyzes the dephosphorylation of phosphotyrosine in proteins. It contains a Cys residue at the catalytic site, which acts as a nucleophile in the dephosphorylation reaction. The active thiol moiety is prone to oxidation in the presence of RONS and is first converted into sulfenyl (–SOH), which is further oxidized to sulfinyl (–SO₂H) or sulfonyl (–SO₃H). The irreversible oxidation of Cys residues leads to impaired protein function [73]. Similarly, oxidative modification of a conserved and functionally essential Cys residue to sulfinyl and sulfonyl forms allows DJ-1, known as PARK7 and a member of the large and functionally diverse DJ-1/PfpI superfamily, to act as a sensor of cellular redox homeostasis. [74] These findings indicate that oxidative modification of Cys residues plays a key role in the metabolic adaptation to redox stress.

Additionally, S-glutathionylation and S-nitrosylation of Cys residues occur in the presence of RONS. Post-translational S-glutathionylation is a reversible modification of Cys residues in basic environments, where glutathione (GSH), an endogenous antioxidant, conjugates with the thiolate anions of Cys residues in target proteins. This modification of Cys helps prevent protein degradation caused by sulfhydryl overoxidation during oxidative stress [61, 63, 64] Similarly, S-nitrosylation, another redox post-translational modification of Cys, occurs under nitrosative stress [61, 64, 75]. These reversible modifications of redox-active proteins, such as protein disulfide isomerase, play crucial roles in cell signaling and are modulated by RONS. These redox post-translational modifications in proteins might act as a “redox switch,” altering protein function as a defense mechanism against oxidative and nitrosative stress.

Protein Cys residues readily react with H₂O₂, a signaling molecule. H₂O₂ activates cell regulatory and signaling pathways [76, 77]. Under oxidative stress, H₂O₂ oxidizes the thiol group (–SH) of Cys resulting in the formation of sulfenic acid and H₂O [60]. The antioxidant property of Cys is mainly exhibited through glutathione (GSH), hydrogen sulfide (H₂S), and taurine (Fig. 3), and Cys is the limiting amino acid in the synthesis of these three antioxidants [5]. GSH is synthesized from Cys via the transsulfuration pathway [78]. Transsulfuration is a reaction that involves the transfer of sulfur from homocysteine to serine to form Cys (Fig. 3a). Cystathionine is the intermediate of the reaction and is catalyzed by cystathionine β-synthase (CBS) and cystathionine- γ-lyase (CSE). GSH is synthesized from Cys by two ATP-dependent enzymatic reactions. First, γ-glutamylcysteine synthetase catalyzes the reaction between Cys and glutamate to form -glutamylcysteine. Then, GSH synthase catalyzes the reaction between γ-glutamylcysteine and glycine to form GSH (Fig. 3b) [5]. The reduced GSH is reversibly oxidized to GSSG in the presence of peroxides (Fig. 3b).

Fig. 3.

The antioxidant properties of cysteine via glutathione, H₂S, and taurine

H₂S is a gasotransmitter molecule that is necessary for cellular signaling. It can be used as an antioxidant, anti-inflammatory, and protective agent. H₂S is produced from L-cysteine by the action of two enzymes—CBS and CSE. Also, 3‐mercaptopyruvate sulfurtransferase and Cys amino acid transferase produce H₂S from Cys via the formation of 3-mercaptopyruvate (Fig. 3c) [78]. H₂S neutralizes RONS directly and is also able to enhance the production of GSH [5, 79].

Taurine is a free amino acid that acts as an antioxidant, anti-inflammatory, and detoxifying agent. Taurine is either obtained from diet or synthesized via Cys metabolism. Synthesis occurs in three steps (Fig. 3c). In the first step, Cys is converted to cysteinesulfinate (CSA) by cysteine dioxygenase (CDO). Then the carboxyl group is removed from CSA by cysteinesulfinate decarboxylase and hypotaurine is formed, which then is oxidized to taurine. Taurine acts as an antioxidant as it reduces RONS [5]. One mechanism by which taurine reduces the production of superoxide is by increasing the activity of antioxidant enzymes like SOD. When the efficiency of antioxidant enzymes is increased in the intracellular membrane of mitochondria, the rate of superoxide degradation is greater [80]. In addition, taurine can allow GPx to be reactivated [5]. Therefore, Cys residues in proteins as well as in low-molecular-weight antioxidants are involved in protecting cells against oxidative stress.

Percent Composition of Met and Cys in Metabolic Proteins

The composition of SAAs in metabolic proteins was examined to explore their percentage compositions and possible roles in defending against oxidative stress. UniProt (UniProt Consortium) [81] and the National Center for Biotechnology Information’s Protein database [82] were used to collect amino acid sequences of metabolic proteins of homo sapiens. The 119 proteins included in this analysis were selected for their involvement in various metabolic pathways, with complete amino acid sequences available. These proteins encompass a range of processes: glycolysis (10 proteins), conversion of pyruvate to acetyl CoA (three proteins), tricarboxylic acid (TCA) cycle (eight proteins), oxidative phosphorylation (six proteins), pentose phosphate pathway (seven proteins), gluconeogenesis (five proteins), glycogen metabolism (12 proteins), fatty acid metabolism (24 proteins), amino acid catabolism (17 proteins), nucleotide biosynthesis (19 proteins), and ROS scavengers (eight proteins). If multiple protein isoforms were listed, isoform 1 or A was used. Large proteins were listed based on their subunits. The percentage composition of Met and Cys residues were calculated as follows:

where i stands for the 20 amino acid residues; n_i is the number of residues of each type, and N is the total number of residues. The summation is through all the residues in each protein. Proteins were classified based on type (globular versus transmembrane) and function (redox versus non-redox). The average percentage compositions of Cys and Met were determined for each protein type and function. The software ProtParam [83]. The amino acid sequence of a protein, i.e., the primary structure dictates its three-dimensional conformation, which in turn governs the function of a protein [84].

To assess whether the observed percentage compositions of Cys and Met in human proteins are consistent across other organisms, a Biopython [85] script was used to calculate percentages of Cys and Met in homologous proteins from 12 additional species (taxonomy id in parenthesis)-Ciona intestinalis (7719), Danio rerio (7955), Drosophila melanogaster (7227), Gallus gallus (9031), Homo sapiens (9606), Latimeria chalumnae (7897), Mus musculus (10090), Nematostella vectensis (45351), Ornithorhynchus anatinus (9258), Pongo abelii (9601), Saccharomyces cerevisiae (4932), Strongylocentrotus purpuratus (7668), and Xenopus tropicalis (8364).

The compiled results of the percentage compositions of Cys and Met in human proteins are provided in Tables 2 and 3. The results for individual pathways are provided in Table 2 while the average values for different types of proteins (redox/non-redox, globular/membrane, and cytoplasmic/mitochondrial) are shown in Table 3. Additionally, the percentage compositions of Cys and Met in individual proteins for each metabolic pathway are included in Table S1. Table S2 contains the percentages of Cys and Met in metabolic proteins of Homo sapiens and 12 additional species; the average values are provided in parentheses in Table 2. Analysis of these results suggested a small variation in the average percentages of Cys or Met among proteins involved in different metabolic pathways, with average percentages varying from 1.5 to 2.1 for Cys and 2.3 to 3.5 for Met (Table 2). The average percentage of Met (3.5 ± 1.6) was higher for enzymes involved in oxidative phosphorylation, whereas the percentage of Cys was relatively low (1.6 ± 1.1). The overall higher percentages of SAAs (Cys and Met) in enzymes involved in oxidative phosphorylation may be attributed to the fact that cellular respiration is the major source of ROS; higher contents of SAAs can protect the structure and function of enzymes involved in cellular respiration by acting as ROS sinks. This is consistent with previous findings that the mitochondrial proteins are rich in SAAs and reversible oxidative modification of SAAs within proteins helps regulate and maintain redox homeostasis (86, 87). It is reported that the average Met content of proteins encoded by the mitochondrial genome is 6%, [53] which is closer to what was observed for the cytochrome c oxidase subunit I (mitochondrion) [% Met = 6.2]. However, the percentage of Cys for this enzyme was significantly low, at only 0.2%. Interestingly, the average Cys and Met contents were not exceptionally high for all ROS scavenger proteins (Table 2). The total Cys and Met contents were slightly above 4% for SOD, where the percentage of Cys (3.3%) was significantly higher than the % Met (1.3%) (Table S1). The percentage compositions of Cys and Met of human metabolic proteins were very similar to their homologous proteins (Table 2).

Table 2.

The average and standard deviation of the percent composition of cysteine and methionine in various human proteins involved in metabolic pathways

Metabolic pathway	Average % Cys	Average % Met
Glycolysis	1.6 ± 0.7 (1.8 ± 0.4)	2.7 ± 0.9 (2.7 ± 0.6)
Pyruvate to acetyl CoA	2.1 ± 0.9 (2.4 ± 0.4)	2.6 ± 0.8 (2.8 ± 0.4)
Citric acid cycle	1.5 ± 0.7 (1.4 ± 0.4)	2.8 ± 0.9 (3.1 ± 0.5)
Oxidative phosphorylation	1.6 ± 1.1 (1.6 ± 0.4)	3.5 ± 1.6 (3.3 ± 0.6)
Pentose phosphate pathway	1.5 ± 0.4 (1.6 ± 0.5)	3.1 ± 1.5 (2.3 ± 0.5)
Gluconeogenesis	1.9 ± 0.5 (2.0 ± 0.3)	2.8 ± 0.8 (3.0 ± 0.4)
Glycogen metabolism	1.5 ± 0.7 (1.7 ± 0.3)	2.4 ± 0.4 (2.5 ± 0.5)
Fatty acid metabolism	1.8 ± 0.7 (2.2 ± 0.4)	2.6 ± 0.6 (2.7 ± 0.4)
Amino acid catabolism	1.5 ± 0.8 (1.8 ± 0.4)	2.3 ± 1.0 (2.7 ± 0.5)
Nucleotide biosynthesis	1.8 ± 0.7 (1.8 ± 0.3)	2.3 ± 0.6 (2.5 ± 0.5)
ROS Scavengers	2.1 ± 0.8 (2.1 ± 0.4)	1.8 ± 1.0 (1.9 ± 0.5)

The average values of the % Cys and % Met in metabolic proteins of Homo sapiens, along with data from 12 additional species (Table S2) are provided in parentheses

Table 3.

The average and standard deviation of percent composition of cysteine and methionine in redox and non-redox proteins, globular and membrane proteins, and cytoplasmic and mitochondrial proteins

Type of protein	Average % Cys	Average % Met
Redox	1.6 ± 0.6	2.7 ± 0.5
Non-redox	1.5 ± 0.5	2.6 ± 0.4
Globular	1.7 ± 0.3	2.5 ± 0.4
Membrane	2.2 ± 0.8	2.5 ± 0.7
Cytoplasmic	1.7 ± 0.3	2.5 ± 0.5
Mitochondrial	1.5 ± 0.6	2.7 ± 0.4

Proteomic methionine usage analysis revealed a higher percentage of Met in proteins at key sites of major free radical production, such as the mitochondrial respiratory chain [88, 89]. Recent studies also suggested that the oxidation of protein Met has a role in antioxidant defense and cell regulation. In particular, the oxidation of surface-exposed Met residues protects other functionally essential residues from oxidative damage. The reversible conversion between Met and MetO regulates protein biological activity by changing catalytic efficiency and altering the protein's surface hydrophobicity [5, 45, 57, 90].

In general, the average % Met was higher than the average % Cys for most metabolic proteins. However, there was insignificant variation in the average percentage of Cys or Met residues among different classes of metabolic proteins, such as redox and non-redox proteins (Table 3). However, a slight increase in the average composition of Cys in membrane proteins compared to globular proteins was observed. This difference in % Cys in globular proteins (1.7 ± 0.3) and the membrane proteins (2.2 + 0.8) is statistically significant [df = 13, t-Stat = 1.97, p (one-tail) = 0.036]. The reported compositions of Cys and Met in globular proteins are 1.39% and 2.21%, respectively, while in transmembrane proteins, they are 0.47% and 1.56%, respectively [6]. The study included 377 outer membrane proteins and 674 globular proteins. Compared to these values, the average composition of Met in metabolic proteins is 2.5% for both globular and membrane proteins, whereas Cys is 1.7% for globular proteins and 2.2% for membrane proteins. The observed higher percentages of Cys and Met in metabolic proteins may be necessary to maintain the structure and function of metabolic proteins under oxidative stress; ROS induces the cleavage of peptide bonds in proteins via the diamide pathway and the α-amidation pathway [91]. The oxidative post-translational modifications of Cys and Met and their role as redox switches that modulate the cell's response to oxidative stress are well-established [45, 60, 90, 92].

Important observations were made from individual metabolic pathways. The percentage compositions of Cys and Met in hexokinase were 2.7% and 4.1%, respectively (Table S1; Glycolysis). This enzyme is involved in the committed step of glycolysis, and the higher percentage compositions of these two SAAs indicate their possible role in regulating the interplay between redox homeostasis and glucose metabolism. The total percentage compositions of Cys and Met in pyruvate dehydrogenase (E1 component, subunit alpha), a redox enzyme, were relatively higher (6.7%) compared to other enzymes involved in the oxidative decarboxylation of pyruvate (Table S1: Pyruvate to Acetyl-CoA). Similarly, the total % contents of Cys and Met in the redox enzymes of the citric acid cycle—isocitrate dehydrogenase (4.6%), succinate dehydrogenase (4.5%), and malate dehydrogenase (3.8%) were relatively higher suggesting their possible role as RONS sinks. Moreover, Cys and Met contents were higher for citrate synthase, which catalyzes the condensation of oxalate and acetyl CoA to form citrate (Table S1: Citric Acid Cycle). The formation of citrate is the committed step of the cycle and is regulated by allosteric effectors. As expected, the total percent content of Cys and Met was much higher for the enzymes involved in oxidative phosphorylation, especially for the four redox enzymes—NADH-ubiquinone oxidoreductase, succinate dehydrogenase, ubiquinol-cytochrome c reductase, and cytochrome c oxidase subunit I, which were 5.8%, 5.0%, 6.6%, and 6.4%, respectively (Table S1: Oxidative phosphorylation). These high SAAs contents indicate their role beyond electron transfer from NADH to molecular oxygen. Similarly, the two dehydrogenases in the pentose phosphate pathways, glucose 6-phosphate dehydrogenase and phosphogluconate dehydrogenase, which are important for the regeneration of NADPH also contained a higher content of SAAs (≥ 4.5%; Table S1: Pentose Phosphate Pathway). A significantly high percentage of SAAs, with Cys (1.8%) and Met (6.6%) were found in phosphopentose epimerase/ribulose-phosphate 3-epimerase. This metalloprotein has been shown to protect cells from oxidative stress [93]. For a key rate-limiting gluconeogenic enzyme, phosphoenolpyruvate carboxykinase that catalyzes the conversion of oxaloacetate to phosphoenolpyruvate and water, the percentage compositions of Cys and Met were 2.3% and 3.5%, with total SAAs of 5.7% (Table S1, Gluconeogenesis). In glycogen metabolism, the total SAAs content was relatively higher for α-1,6-glucosidase (4.7%) and adenylate cyclase (5.4%), whereas the total content of SAA was 3.5% for glycogen phosphorylase, the rate-limiting enzyme. For fatty acid metabolism, amino acid metabolism, and nucleic acid metabolism, the percentages of SAAs varied among different enzymes but were uniformly higher for dehydrogenases (Table S1). The results of human enzymes are consistent with that of other species (Table S2).

The endogenous Cys and Met have antioxidant activity, and it is known that these SAAs in many proteins can neutralize RONS through reversible redox reactions. It was observed that dehydrogenases and enzymes involved in committed steps of metabolic pathways have a higher content of SAAs. Although this study did not reveal a correlation between the percentage of Met and Cys in an entire enzyme group (redox versus non-redox) and their activity as RONS sinks, many metabolic enzymes contain higher percentages of SAAs that may help protect cells against oxidative stress. Future research directions should include a detailed analysis of the percentage of Met and Cys residues on the surface and in the core of metabolic proteins, which could provide additional insights. Although the mitochondrial genome encodes only about 1% of the total mitochondrial proteome, a thorough analysis of SAAs in proteins (surface versus core) encoded by the nuclear genome compared to those encoded by the mitochondrial genome could also provide clearer and quantitative information on the role of these SAAs as RONS sinks. Most studies to date have been conducted in vitro, with limited in vivo studies; thus, the functional consequences of oxidative modifications of surface-exposed SAAs in proteins involved in antioxidant defense warrant further in vivo investigation (92). Similarly, the roles of SAAs in redox homeostasis and oxidative damage require thorough in vivo studies. While S-glutathionylation of Cys residues in the presence of glutathione (GSH) has been well studied, the response of these SAAs to other antioxidants in neutralizing RONS remains unexplored, representing a significant area for future research. Moreover, investigating how SAAs function as free amino acids and within proteins to influence inflammatory processes and immune responses through their antioxidant properties could lead to novel therapeutic approaches and advancements in protein therapeutics.

Supplementary Information

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