Monomethylarsonous Acid Destroys a Tetrathiolate Zinc Finger Much

The ingested inorganic arsenic is metabolized to methylated derivatives, which are considered to .... The stocks of 0.1 M arsenite and methyloxoarsine...
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Chem. Res. Toxicol. 2008, 21, 600–606

Monomethylarsonous Acid Destroys a Tetrathiolate Zinc Finger Much More Efficiently than Inorganic Arsenite: Mechanistic Considerations and Consequences for DNA Repair Inhibition Katarzyna Pia¸tek,† Tanja Schwerdtle,‡ Andrea Hartwig,‡ and Wojciech Bal*,†,§ Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawin´skiego 5a, 02-106 Warsaw, Poland, Institute of Food Technology and Food Chemistry, Technical UniVersity Berlin, GustaV-Meyer-Allee 25, D-13355 Berlin, Germany, and Central Institute for Labour ProtectionsNational Research Institute, Czerniakowska 16, 00-701 Warsaw, Poland ReceiVed September 1, 2007

Arsenic compounds are human carcinogens. The ingested inorganic arsenic is metabolized to methylated derivatives, which are considered to be more toxic than the inorganic species. Interactions of trivalent arsenicals with thiol groups of proteins are believed to be important for arsenic carcinogenesis, but inorganic arsenite appears to bind to thiol groups more strongly than the methylated AsIII species. Inhibition of the nucleotide excision repair pathway of DNA repair (NER) is likely to be of primary importance in arsenic carcinogenesis. Previously, we demonstrated that methylated AsIII compounds are more efficient than arsenite in releasing zinc from ZnXPAzf, the zinc finger of XPA, a crucial member of the NER complex [Schwerdtle, T., Walter, I., and Hartwig, A. (2003) Arsenite and its biomethylated metabolites interfere with the formation and repair of stable BPDE-induced DNA adducts in human cells and impair XPAzf and Fpg. DNA Repair (Amsterdam) 2, 1449–1463]. In this work, we used ESI-MS to compare aerobic reactivities of arsenite and monomethylarsonous acid (MMAIII) toward ZnXPAzf on the molecular level. We demonstrated that equimolar MMAIII released ZnII from ZnXPAzf easily, forming mono- and diarsenical derivatives of XPAzf. This reaction was accompanied by oxidation of unprotected thiol groups of the monomethylarsinated peptide to intramolecular disulfides. The estimated affinity of MMAIII to XPAzf is 30-fold higher than that established previously for arsenite binding to the thiol groups. No binding of arsenite to the thiol groups of XPAzf was observed under our experimental conditions, and a 10-fold excess of arsenite was required to partially oxidize ZnXPAzf. These results indicate a particular susceptibility of tetrathiolate zinc fingers to MMAIII, thereby providing a novel molecular pathway in arsenic carcinogenesis. Introduction Inorganic arsenic is an established human carcinogen (1). Numerous epidemiological studies have shown that arsenic can cause many types of cancer via exposure to contaminated drinking water and/or ambient air (2–6). Still, the precise mechanism of its action remains unknown, and potential coexposure to factors such as ultraviolet (UV) radiation or organic mutagen-carcinogens, particularly polycyclic aromatic hydrocarbons, contribute further to the complexity. Thus, in various in vitro and in vivo test systems, arsenic has been shown to potentiate the genotoxicity and/or mutagenicity of environmental DNA-damaging agents (7–10). One mechanism by which arsenic is assumed to act is through an indirect pathway, by inhibiting DNA repair mechanisms necessary to remove lesions generated by these environmental carcinogens. The formation of bulky lesions, causing local distortion of the DNA double helix, is believed to be responsible for the carcinogenicity of various environmental agents including UV radiation or benzo[a]pyrene (B[a]P).1 These lesions are removed by nucleotide * To whom correspondence should be addressed. Tel: +48-22-5922353. Fax: +48-22-6584636. E-mail: [email protected]. † Polish Academy of Sciences. ‡ Technical University Berlin. § Central Institute for Labour ProtectionsNational Research Institute. 1 Abbreviations: B[a]P, benzo[a]pyrene; BPDE, benzo[a]pyrene diol epoxide; DMA, dimethylarsinous acid; ERCC1, excision repair cross

excision repair (NER); if unrepaired, they can lead to mutations. NER is a rather complex DNA repair mechanism, consisting of several stages. It starts with damage recognition, followed by incision on both sides of the lesion, excision of the damaged oligonucleotide (24–32 nucleotides), DNA synthesis, and finally ligation to fill the gap (summarized in ref 11). Numerous studies have shown that inorganic arsenic inhibits the repair of bulky DNA adducts in cultured cells and laboratory animals (12–19). In humans, arsenic exposure via drinking water was correlated in a dose-dependent manner to decreased expression of NER genes [excision repair cross complementing protein 1 (ERCC1), Xeroderma pigmentosum group B protein (XPB), and Xeroderma pigmentosum group F protein (XPF)] and diminished repair of lesions in lymphocytes (20, 21). Another mechanism, probably contributing to arsenic carcinogenesis, is biomethylation of inorganic arsenic. In humans and many other mammals, inorganic arsenic is metabolized through successive oxidative methylation and reduction steps to its trivalent and pentavalent mono- and dimethylated metabolites (reviewed in refs 22 and 23). As in various in vitro and in vivo test systems, the trivalent methylated metabolites complementing protein 1; MMAIII, monomethylarsonous acid; MMC, mitomycin C; NER, nucleotide excision repair; RPA, replication protein; TFIIH, transcription factor IIH; XPA, Xeroderma pigmentosum group A protein; XPB, Xeroderma pigmentosum group B protein; XPF, Xeroderma pigmentosum group F protein; ZF, zinc finger.

10.1021/tx7003135 CCC: $40.75  2008 American Chemical Society Published on Web 01/26/2008

MMAIII Reaction with Tetrathiolate Zinc Finger

monomethylarsonous (MMAIII) and dimethylarsinous (DMAIII) acid show stronger toxicity as compared to inorganic arsenite (24–27). Biomethylation of arsenic is no longer believed to be a detoxification process but most probably enhances genotoxicity and/or carcinogenicity induced by inorganic arsenic. Concerning DNA repair inhibition, one of our previous studies demonstrated for the first time that trivalent methylated arsenicals are even more efficient in inhibiting NER of benzo[a]pyrene diol epoxide (BPDE)-induced lesions as compared to inorganic arsenite (18). Searching for molecular mechanisms for the observed NER inhibition by the arsenicals, repair proteins harboring cysteines in functional important domains are potential molecular targets, since arsenic is generally assumed to show high reactivity toward thiols. The current understanding of reactions of arsenicals with thiols has recently been summarized by Kitchin and Wallace (28). Briefly, AsV compounds are reduced by thiols, such as GSH, stoichiometrically to trivalent arsenicals, with an expense of two thiol groups that yield a disulfide (29, 30). GSH appears to be the major reductant of AsV in vivo (31–34). AsIII compounds can exchange their hydroxyl groups for thiols. These reactions are reversible. Collectively, inorganic and methylated AsIII compounds react with thiols according to the following pattern (35):

(CH3)xAs(OH)3-x + (3 - x)RSH T (CH3)xAs(SR)3-x + (3 - x)H2O (1) The impact of methylation on the stability of As-S bonds has been discussed (36). The substitution of a hydroxyl group on AsIII with a methyl group is expected to increase the partial negative charge on the arsenic atom and thus weaken AsIII-S bonds in MMAIII and DMAIII, as compared to inorganic AsIII. There is direct support for such a tendency in AsV species, as the addition of one and two methyl groups increases the pKa value of the As-attached hydroxyl group by 2 and 4 log units, respectively. The corresponding data for AsIII species are not available, however. Reactions of inorganic AsIII with monothiols, such as glutathione and cysteine, were reported to yield complexes with three and one thiolate attached to arsenic, depending on the pH (30, 37). Intramolecular bidentate complexes of trivalent arsenicals and simple dithiols were demonstrated qualitatively to extract As from its GSH complex (38). Arsenite, MMAIII, and DMAIII were found to bind to thiol groups in the major biological polythiol, metallothionein (MT), tri-, bi-, and monodentately, respectively. Final stoichiometries, observed for sufficient amounts of arsenicals, corresponded to saturation of all thiolates: six, 10, and 20 molecules per MT molecule for arsenite, MMAIII, and DMAIII, respectively (39–42). Kitchin and Wallace recently studied reactions of arsenite with peptides containing one, two, three, and four Cys residues. They established that the strongest binding was provided by Cys3 and Cys4 peptides, with micromolar dissociation constants (43). A stepwise formation of AsIII-S3 complexes was observed for Cys1 and Cys3 peptides, studied for this purpose, provided by three Cys1 peptide molecules and one Cys3 peptide molecule, respectively (28). They also reported on the interaction of arsenite with a series of Cys2 peptides having a variable distance between their two Cys residues (44). They found that the binding constant values exhibited no systematic dependence on distances between zero and 14 residues but were uniformly (6-94-fold) higher from that measured for a Cys1 peptide and for a Cys2 peptide with Cys residues separated by 19 residues. Xeroderma pigmentosum group A protein (XPA) is a protein participating in the NER complex (45). Its absence or malfunc-

Chem. Res. Toxicol., Vol. 21, No. 3, 2008 601 Scheme 1. Sequence of XPAzf with Distances between Subsequent Cys Residues Marked with Numbers

tion is associated with xeroderma pigmentosum type A, a severe human disorder characterized by UV hypersensitivity and enhanced cancer risk (46). The XPA activity was inhibited in subcellular assays by several redox-capable metal ions (47, 48). The single zinc finger (ZF) of XPA, of a Cys4 type, is essential for the XPA function and is thus a likely target for oxidative inhibition. Its solution structure was determined by NMR and EXAFS (49, 50). In previous studies, we demonstrated a suitability of the synthetic 37 residue peptide (XPAzf), representing the XPA ZF sequence 101–137 as a model to study molecular mechanisms of XPA damage by metal ions and oxidants, including NiII, CoII, CdII, and reducible selenium compounds (51–53). The sequence of XPAzf is presented in Scheme 1. We also demonstrated that XPAzf released ZnII ions (and thus lost its native structure) in the presence of inorganic and methylated AsIII but not AsV compounds, but relative susceptibilities to these arsenicals varied significantly (18). MMAIII released zinc most vigorously of all compounds tested. To our knowledge, nobody else has studied interactions of methylated arsenicals with zinc-thiolate repair proteins. Recently, we demonstrated the suitability of an electrosprayinduced mass spectrometry (ESI-MS) methodology to study kinetics of the ZnXPAzf reaction with hydrogen peroxide (54) and S-nitrosoglutathione (55). In this study, we applied ESIMS accordingly, to comparatively examine elements of the molecular mechanisms of reactions of arsenite and MMAIII with XPAzf and ZnXPAzf.

Materials and Methods Caution: Inorganic arsenic is classified as a human carcinogen. The following chemicals are hazardous and should be handled carefully: sodium arsenite and methyloxoarsine. Materials. The N-terminally acetylated and C-terminally amidated 37-residue peptide acetyl-DYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHK-amide (XPAzf) was custom-synthesized by Schafer-N Co. (Copenhagen, Denmark) and repurified using HPLC. The identity of this peptide was verified by ESI-MS, and its purity was assessed by HPLC to exceed 98%. AsNaO2 (g99% purity) was obtained from Fluka Chemie (Buchs, Germany). Methyloxoarsine (CH3AsIIIO) was kindly provided by W. R. Cullen (University of British Columbia, Vancouver, Canada); its purity was g99%. Methyloxoarsine underwent hydration in water solution, yielding MMAIII. Ammonium acetate was purchased from SigmaAldrich Chemical Co. Acetonitrile was obtained from LaboratoryScan Analytical Sciences (Dublin, Ireland); trifluoroacetic acid (TFA) and ZnSO4 × 7H2O were purchased from Merck KGaA (Darmstadt, Germany). All solutions were prepared using analysis grade water (Baker). Stock solutions of ZnCl2 were calibrated using atomic absorption spectroscopy by the Department of Analytical Chemistry, Warsaw Technical University. Sample Preparation. Weighed solid samples of XPAzf were dissolved in 10 mM ammonium acetate buffer, pH 7.4, under a low-oxygen atmosphere (Coy glovebox), to prepare 160 µM stock solutions, which were aliquoted and frozen at -20 °C. The samples for studying reactions of ZnXPAzf were reconstituted with equimolar amounts of ZnII ions. The stocks of 0.1 M arsenite and methyloxoarsine were prepared fresh before each experiment. The samples were mixed, and reactions were conducted under ambient air. ESI-MS. Reaction mixtures, containing 10 µM ZnXPAzf (unless specified otherwise) and appropriate concentrations of arsenicals

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Figure 1. Representative ESI-MS spectra, integrated over stationary phases of reactions. (A) 10 µM concentration of XPAzf + 10 µM arsenite, (B) 10 µM ZnXPAzf + 100 µM arsenite, (C) 10 µM XPAzf + 10 µM MMAIII, and (D) 10 µM ZnXPAzf + 10 µM MMAIII. Peak labels are according to Table 1: I, XPAzf + XPAzf(SS) + XPAzf(SS)2; II, ZnXPAzf; III, (AsCH3)XPAzf + (AsCH3)XPAzf(SS); and IV, (AsCH3)2XPAzf. Minor peaks tailing the label peaks are their adducts with H2O molecules and Na+ and K+ ions.

Table 1. Characteristics of Isotopic Clusters of Signals Related to XPAzf, Detected in ESI-MS Spectra group of signals Ia Ib Ic II IIIa IIIb IV

m/z monoisotopic

m/z main

assignment

1111.4 1110.9 1110.4 1126.4 1133.4 1132.9 1155.4

1111.9 1111.4 1110.9 1127.2 1133.9 1133.4 1155.9

XPAzf XPAzf(SS) XPAzf(SS)2 ZnXPAzf (AsCH3)XPAzf (AsCH3)XPAzf(SS) (AsCH3)2XPAzf

double disulfide, XPAzf(SS)2, as evidenced by the isotopic analysis of the corresponding signal (see Figure 2 for examples of analysis). No evidence for arsenic adducts or other products was present. On the other hand, 10 µM ZnXPAzf was completely refractory to an action of 10 µM arsenite and only partially susceptible to a 10-fold excess of arsenite (100 µM). Intramolecular XPAzf disulfides were the only reaction products seen in the latter case, at an approximate 2:1 ratio of XPAzf(SS)2 to XPAzf(SS). The reaction progressed up to 25% of ZnXPAzf decomposition in 30 min. Reactions with MMAIII. When XPAzf was incubated with equimolar MMAIII, the arsenical was completely consumed in the reaction with the peptide, yielding a mixture of single and double monomethylarsinated XPAzf (peaks III and IV in Table 1), as well as the remaining apopeptide, which was fully oxidized to XPAzf(SS)2. Also, the single monomethylarsinated XPAzf was converted to (AsCH3)2XPAzf(SS). The reaction was completed within the dead time of measurement equal to 25 s. Mixtures of single and double monomethylarsinated XPAzf were also found to be major products of reactions of MMAIII with ZnXPAzf, accompanied by residual amounts of the apopeptide. Four MMAIII concentrations, 10, 40, 60, and 100 µM, were used in experiments with 10 µM ZnXPAzf. The increase of excess of MMAIII over the complex resulted primarily in the increase of reaction rate, so that a quantitative kinetic analysis could be performed only for the lowest MMAIII concentration. The kinetic plot of this reaction is presented in Figure 3. Table 2 presents the values of apparent reaction rates obtained from three independent experiments. Similarly to the reaction of XPAzf with MMAIII, the thiol groups in apopeptide and single monomethylarsinated peptide

in 100 mM ammonium acetate buffer, pH 7.4, were continuously injected into Premier ESI-MS spectrometer (Micromass, Manchester, United Kingdom), usually at 4–6 µL/min, using a Hamilton syringe pump. The spectrometer parameters were as follows: cone voltage, 35 V; source block temperature, 80 °C; and analyzer vacuum pressure, 5.8 nB. The time span of one scan was 1.03 s, and typically 1500–1800 scans were collected. Bovine pancreatic trypsin inhibitor was used as an internal mass standard. Data Analysis. The kinetic data were obtained by summing up MS signals over 50, 100, or 200 scan segments, as appropriate. The 4+ molecular ions were found to provide the major signal intensities for all species, with minor signals corresponding to 3+ molecular ions. The proportions of signal intensities were similar within the 4+ and 3+ groups. Therefore, 4+ signals were used for further analysis. The signals belonging to individual species, including their adducts with H2O molecules and Na+ and K+ ions, were integrated for each segment separately and expressed as fractions of the total.

Results The reactions of XPAzf and ZnXPAzf with arsenite and MMAIII were studied separately. Overall, four sets of peaks were detectable in ESI-MS spectra. They were interpreted on the basis of theoretical isotopic distributions of potential reaction products. Each group consisted of the major isotopic cluster, tailed by a set of clusters corresponding to adducts of the major species with H2O molecules and Na+ and K+ ions. The peak shapes and relative intensities of adduct clusters were nearly identical for all sets of peaks, as presented in Figure 1. The major peaks are characterized in Table 1. Individual reactions are described below. Reactions with Arsenite. The addition of equimolar arsenite to 10 µM XPAzf led to a full oxidation of the peptide to its

Figure 2. Examples of isotopic analysis of composite ESI-MS signals. (Top) Black, experimental peak III; red, the best fit, composed of 75% green theoretical (AsCH3)XPAzf(SS) and 25% blue theoretical (AsCH3)XPAzf. (Bottom) Black, experimental peak I; red, the best fit composed of 100% XPAzf(SS)2. Line widths of theoretical components, 0.17 Da, were adjusted to fit the instrumental resolution of the spectra.

MMAIII Reaction with Tetrathiolate Zinc Finger

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Figure 3. Kinetic plot of a reaction of 10 µM ZnXPAzf with 10 µM MMAIII, obtained by integration of ESI-MS signals (symbols), together with fits (lines). Peak labels are according to Table 1, with their disulfide status omitted for clarity: I includes XPAzf(SS) + XPAzf(SS)2, and III includes (AsCH3)XPAzf(SS).

Table 2. Apparent First Order Rates for Parallel Processes Occurring during the Reaction of 10 µM ZnXPAzf with 10 µM MMAIII at 25 °C process

rate (s-1)

decay of ZnXPAzf formation of (CH3As)XPAzf formation of (CH3As)2XPAzf

0.018 ( 0.005 0.031 ( 0.006 0.012 ( 0.004

Table 3. Final Relative Concentrations of Products of the Reaction of XPAzf and ZnXPAzf with MMAIIIa

substrate

initial (MMAIII) (µM)

XPAzf ZnXPAzf ZnXPAzf ZnXPAzf ZnXPAzf

10 10 40 60 100

ratios of final concentrations IV/I IV/II IV/III 3.4 4.0 4.2 4.3 23.3

n.a. 2.7 7.7 11.9 14.0

0.9 1.0b 1.2 1.3 3.2

proportions of disulfide forms Ic:Ib:Ia IIIb:IIIa 100:0:0 60:40:0 100:0:0 100:0:0 100:0:0

a Substrates and products are labeled according to Table 1. average of three independent experiments.

100:0 78:22 86:14 100:0 92:8 b

(20%,

were largely oxidized to intramolecular disulfides, as shown in Table 3. This table shows that the final relative concentrations of reaction products were affected by MMAIII excess to a much smaller degree than reaction rates. The formation of the disulfide bond in the single monomethylarsinated peptide removes it from the equilibrium defined by eq 1 and affects the equilibrium condition of the formation of (CH3As)2XPAzf according to eq 2 (water molecules omitted for simplicity).

2MMA + ZnXPAzf T (CH3As)2XPAzf + ZnII

(2)

Nevertheless, we calculated a tentative equilibrium constant for this process; KaA2L-Zn ) 4.2 ( 2.5 × 104 M–1 or, conversely, KdA2L-Zn ) 33 ( 23 µM.

Discussion The XPAzf apopeptide provides two pairs of CxxC dithiol units as targets for arsenicals. They are separated by a 17-residue long stretch of sequence (Scheme 1). Inorganic arsenite has been considered to form more stable complexes with thiols than its methylated derivatives (36), and thus, one might expect that arsenite should react with XPAzf more vigorously than MMAIII. However, all previous studies were performed under anaerobic conditions. In our case, samples were prepared under nitrogen to prevent adventitious oxidation of thiols and arsenicals, but

dioxygen access was allowed during incubations of reaction mixtures. This approach seems to be more realistic in terms of reproducing actual physiological conditions than strict anaerobicity. We have not detected AsIII adducts in arsenite reactions, which indicates a low stability of putative AsIII complexes of XPAzf. The literature data suggest micromolar values for Kd values of AsIII complexes of peptides possessing pairs of thiols separated by merely two intervening residues (43, 44). Under our experimental conditions, such Kd values should result in nearly complete complexation (80–90%). Instead, we only detected XPAzf oxidation to its intramolecular disulfides. Previous ESI-MS studies on metallothioneins indicate that AsIII thiolate clusters do not decompose in the course of measurements (39–42). Furthermore, we used particularly mild electrospray conditions, as confirmed by the presence of multiple adducts with H2O molecules and Na+ and K+ ions in the spectra (Figure 1). Therefore, it seems likely that the AsIII complex of XPAzf is particularly susceptible to dioxygen oxidation, perhaps via the AsIII/AsV redox pair. All four Cys thiols are brought together in space by the coordination of the ZnII ion in ZnXPAzf, but this coordination inhibits thiol reactivity significantly. For example, oxidation of XPAzf thiols by H2O2 was much slower in ZnXPAzf and CdXPAzf complexes, as compared to XPAzf (51, 52). In accord with this principle, ZnXPAzf was totally resistant to equimolar arsenite and was decomposed slowly and only to a low extent by the 10-fold excess of arsenite. Similarly to the apopeptide case, XPAzf disulfides were the only detectable reaction products. A slow course of this reaction, as compared to the rates of formation of arsenite complexes with peptides and metallothionein, further confirms the oxidative mechanism of ZnXPAzf decomposition by arsenite in the presence of dioxygen (28, 42). The reaction of XPAzf with MMAIII yielded CH3AsIII complexes that were perfectly stable under ESI-MS conditions. The formation of complexes containing one and two CH3AsIII groups results from the bidentate character of MMAIII, established in previous glutathione and metallothionein studies (34, 35, 40). These final products formed very quickly and were stable afterward. The CH3AsXPAzf species underwent full oxidation of its two uncoordinated thiol groups within the dead time of the experiments (Table 3). Therefore, one can state that methylarsinated thiol groups are not susceptible to dioxygen oxidation under conditions deleterious to unprotected thiol groups. As expected, the presence of ZnII ions slowed down the reaction of XPAzf thiols with MMAIII. Still, the process of formation of CH3AsIII complexes was completed within several minutes (Table 2 and Figure 3) with relative abundances similar to those detected for the apopeptide (Table 3). As indicated in the Results section, those thiol groups that did not form bonds with arsenic were mostly oxidized to intramolecular disulfides, but the CH3As-S bonds were perfectly resistant to dioxygen. As a result, the level of arsenic complexes increased along with the increase of MMAIII, but the extent of oxidation of free thiols was independent of that. Notably, the analysis of m/z values demonstrated with absolute certainty that no oxidation of peptide-bound AsIII to AsV occurred. Previous studies demonstrated the reversibility of AsIII interactions with thiols in the absence of dioxygen (reviewed in the Introduction section). The concurrent presence of dioxygen and MMAIII under our conditions resulted in the formation of a mixed species (CH3As)XPAzf(SS) in which one pair of thiols is bonded to CH3As and another forms a disulfide.

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Scheme 2. Flow of the Reaction of ZnXPAzf with MMAIII

ZnXPAzf is perfectly resistant to thiol group oxidation under such conditions, as demonstrated by control runs, as well as our previous studies (51, 54, 55). Therefore, the release of ZnII from ZnXPAzf is caused by an assault of one MMAIII molecule, consuming two thiol groups. This conclusion is also in an excellent agreement with the results of our recent study on zinc release from ZnXPAzf by H2O2 (54). The apparent rate constants presented in Table 2 support the flow of reaction presented in Scheme 2. The disulfide formation in (CH3As)XPAzf(SS) is somewhat faster than the attachment of the second (CH3As) group. This results in a partial inhibition of the formation of (CH3As)2XPAzf. Therefore, the latter complex is in fact even more stable than the Kd value of 33 µM indicates. This is a very striking result. In fact, the formation of (CH3As)2XPAzf by the reaction of MMAIII with ZnXPAzf is very peculiar, because of a very high stability of the Zn(II) complex, expressed by its nanomolar conditional dissociation constant KdZnL at pH 7.4 [the values of 1.2 ( 0.2 and 0.17 ( 0.07 nM were determined in 10 and 50 mM phosphate buffers, respectively (51)]. The formal dissociation constant for the reaction of dissociation of (CH3As)2XPAzf into XPAzf and MMAIII, omitting the presence of ZnII ions, given by KdA2L ) KdA2L-Zn × KdZnL, is equal to 5.6 × 10-15 M–2 (using the 0.17 nM value for KdZnL, as more appropriate in terms of ionic strength). The square root of KdA2L, 75 nM, gives an average dissociation constant for the first and second MMAIII molecule. Taking statistical considerations into account, the monomolecular affinity of MMAIII to XPAzf is at least 30-fold higher from the highest (micromolar) values reported by Kitchin and Wallace for arsenite binding to thiol peptides (28, 43, 44). Moreover, the stoichiometry of (CH3As)2XPAzf, clearly confirmed by the analysis of ESI-MS signals, leaves no space for enhancement of its formation by any associated irreversible processes (such as oxidative ones). Instead, it is likely that the formation of (CH3As)-S2 chelates is facilitated entropically because ZnII brings all four sulfur targets of arsenic close to each other in space, but the charge of the Zn2+ ion is not sufficient to compensate electrostatics of four thiolates. As a result, one of these thiolates may be labile in terms of coordination and activated for nucleophilic substitutions (56, 57). The arsenicals form their chelates in a stepwise manner (28, 42). Therefore, the AsIII species can attach easily to the labile thiol and then take advantage of the clustering of the other thiols by ZnII to form the chelate. The above considerations are equally valid for arsenite and MMAIII, while the effective zinc release from ZnXPAzf occurred only for MMAIII in our experiments. We observed this preference for methylated trivalent arsenicals previously, and even monodentate DMAIII released zinc more effectively than arsenite (18). Pentavalent arsenicals, which should be able to oxidize thiols, were inactive in that assay, somewhat contrary to chemical intuitions. As noted in the Introduction section, inorganic arsenite should form stronger thiolate complexes than its methylated derivatives (36), but the opposite is obviously true for their reactions with XPAzf and its ZnII complex. We do not have an explanation for these puzzling facts at this stage of our research, but they are reproducible and detectable with various methodologies.

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As mentioned above, we performed our experiments with dioxygen access during sample incubations. The purpose of this experimental design was to reproduce physiological conditions more realistically than anaerobically. The next logical step on this path will include glutathione as the major physiological arsenic carrier and a good donor of AsIII to dithiols (29–38). Despite the fact that interaction of XPA with damaged DNA and its exact function in NER is not fully elucidated, XPA is known to be absolutely essential for NER. Of all seven XP genetic complementation groups, XPA is the most common and causes the most severe XP disease. XPA patients are particularly prone to a high risk of skin cancer, due to the deficiency of the XPA protein and consequently unrepaired UV damage. XPA plays a central role in the first steps of NER; the protein contains binding sites for repair proteins including ERCC1, transcription factor IIH (TFIIH), and replication protein (RPA) and is believed to coordinate these factors in the NER preincision complex. Furthermore, XPA binds specifically to damaged DNA, including lesions induced by UVC and B[a]P (summarized in ref 11). Two studies have recently investigated the impact of arsenite on XPA binding to a UVC- (48) or mitomycin C (MMC)damaged (58) oligonucleotide and demonstrated no decrease of XPA binding or XPA binding inhibition only at high micromolar to millimolar concentrations of arsenite, respectively; thereby, arsenic led to a significant decrease in XPA binding only at a 100:1 or greater molar ratio of arsenic to XPA. Similarly, in our present study, ZnXPAzf was completely refractory to an action of an equimolar concentration of arsenite, and only partially susceptible to a 10-fold excess of arsenite. Moreover, the observed oxidation of the cysteines by arsenite and the loss of zinc might be reversible under cellular conditions in the presence of reducing agents and zinc. In contrast, a covalent binding of MMAIII, which was demonstrated in an equimolar concentration in the present study, is most probably quite stable under cellular conditions and might completely inactivate the XPA function. This model could explain the stronger effects of MMAIII on NER of BPDE-induced lesions in cultured human cells (18). Further investigations are necessary to clarify whether XPA is inhibited under cellular conditions. Acknowledgment. This work was sponsored in part by the Polish Ministry of Science and Higher Education Grant 3 T09A 009 26 and by the Deutsche Forschungsgemeinschaft, Grants HA 2372/3-4 and SCHW 903/3-2.

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