4. Arsenic binding to proteins

The trivalent arsenite has a tendency to bind to sulfhydryl groups. The cysteine residues are a direct target of arsenite in proteins and peptides [28]. The chemical reaction involved in arsenic binding to cysteines has been well recognized. Some of the chemicals like arsine halides used in warfare during the First World War owe their toxicity to their ability of binding to protein dithiols. To defy the toxic effects of such warfare agents, the British government approved the use of β-chlorovinyldichloroarsine (dithioglycerol) which has the ability to form stable complexes with arsenic. The competitive binding of arsenic to dithioglycerol rescues cellular proteins from binding to arsenic [29, 30].

Arsenic affinity for proteins can result in conformational changes in the protein and loss of protein–protein and protein-DNA interactions. Escherichia coli consists of a repressor protein ArsR in which each subunit within its α-helix contains two cysteine residues. The unraveling of this α-helix is required in order to accommodate trivalent arsenite for binding to the protein. The unraveling of the helix causes the conformational change in the protein that dissociates ArsR from DNA resulting in induction of gene expression. Arsenite binds to three cysteine (Cys32, Cys34 and Cys37) residues in ArsR, where Cys32 and Cys37 are present in the α-helix of the DNA-binding sites (Figure 5). The Cys residues in the protein are located in such a way that arsenite is unable to bind unless the protein unwinds for a conformational change [31].

Figure 5. A homology model for arsenical resistance operon repressor protein from E. coli (ARSR\_ECOLI) showing arsenic bound to Cys residues. PDB ID: 1SMT with 40% sequence identity spanning residues 8–90 was used to build the model. The coordinates were downloaded from https://swissmodel.expasy.org/repository/uniprot//P37309 and refined to introduce the arsenic atom.

#### 4.1. Binding sites of arsenic in proteins

vector-transfected JB6 cells, indicating the role of activation of JNKs in arsenic-induced apoptosis. Studies have found that both arsenite and arsenate can cause transactivation of activation protein-1 (AP-1). Since increased activation of AP-1 by arsenite could be inhibited by either treating cells with MAP kinase Erk kinase (MEK)1 inhibitor or overexpression of dominantnegative protein kinase C α (PKCα), this induction appears to occur through activation of mitogen-activated protein (MAP) kinases and PKC. Moreover, in AP-1-luciferase reporter transgenic mice, transactivation of AP-1 was caused by both arsenite and arsenate. Recent data also indicates that PKC, upstream from the MAP kinases, may be involved in mediating arseniteinduced signal transduction. Activation of PKC requires it to be translocated from the cytosol to the membrane, and this phenomenon is observed within 15 minutes when cells are treated with arsenite. Moreover arsenite-induced AP-1 activity, phosphorylation of Erks, JNKs and p38 kinase were blocked once PKC activation was inhibited. These results suggest that PKC plays a critical

Nuclear factor kappa B (NF-κB) is a rapidly induced stress-responsive transcription factor that may play an important role in arsenic-induced signal transduction, cell transformation and apoptosis [27]. Reports suggest that in arsenic-induced oxidative stress, H2O2 and superoxide are the predominant reactive species in endothelia cells and may be the mediators for the activation of the NF-κB pathway. It was also shown that arsenic could induce activation of NF-κB in different cell culture models. Expression of a dominant-negative inhibitory kappa-Β-

The trivalent arsenite has a tendency to bind to sulfhydryl groups. The cysteine residues are a direct target of arsenite in proteins and peptides [28]. The chemical reaction involved in arsenic binding to cysteines has been well recognized. Some of the chemicals like arsine halides used in warfare during the First World War owe their toxicity to their ability of binding to protein dithiols. To defy the toxic effects of such warfare agents, the British government approved the use of β-chlorovinyldichloroarsine (dithioglycerol) which has the ability to form stable complexes with arsenic. The competitive binding of arsenic to dithioglycerol rescues cellular pro-

Arsenic affinity for proteins can result in conformational changes in the protein and loss of protein–protein and protein-DNA interactions. Escherichia coli consists of a repressor protein ArsR in which each subunit within its α-helix contains two cysteine residues. The unraveling of this α-helix is required in order to accommodate trivalent arsenite for binding to the protein. The unraveling of the helix causes the conformational change in the protein that dissociates ArsR from DNA resulting in induction of gene expression. Arsenite binds to three cysteine (Cys32, Cys34 and Cys37) residues in ArsR, where Cys32 and Cys37 are present in the α-helix of the DNA-binding sites (Figure 5). The Cys residues in the protein are located in such a way that arsenite is unable to bind unless the protein unwinds for a conformational change [31].

role in arsenite-induced activation of MAP kinases [25, 26].

α blocked arsenic-induced activation of NF-κB and apoptosis [26].

4. Arsenic binding to proteins

64 Arsenic - Analytical and Toxicological Studies

teins from binding to arsenic [29, 30].

Cysteine and histidine residues are thought to be the most frequent targets of metals like zinc, copper and iron resulting in such metals binding to peptides and proteins [32, 33]. However, the binding of arsenic to histidine is not well understood and is yet to be established. There is no change in nuclear magnetic resonance (NMR) spectra once arsenic was added to a buffered solution of histidine signifying the absence of interaction between histidine and arsenic [34]. Many studies have also used site-directed mutagenesis to replace cysteine residues with serine residues on the reason that interaction between arsenic and serine is very weak. Arsenic is however known to bind to zinc finger protein in C3H1 motif and not in C2H2 motif, releases zinc, and thus decreases the capacity of the protein to bind to DNA. Selenocysteine—a cysteine analogue—also has the ability to bind to arsenic species. This amino acid is present in selenoproteins and has a lower pKa which increases nucleophilicity. The amino acid residues in the vicinity of cysteine (or selenocysteine) act as proton donors [35–37].

Studies with some enzymes reveal that serine residues can be potential targets of arsenic species, thereby inhibiting their function. It was found that in serine hydrolases and the arsenic moieties interacting with hydroxyl containing serine, pentavalent forms of arsenic rather than trivalent forms were prevalent. The complex between the serine residue and the pentavalent arsenic consists of a tripartite oxyanion hole in the proximity of the active site [38].
