*Article* **Interactions of an Artificial Zinc Finger Protein with Cd(II) and Hg(II): Competition and Metal and DNA Binding**

**Bálint Hajdu <sup>1</sup> , Éva Hunyadi-Gulyás <sup>2</sup> and Béla Gyurcsik 1,\***


**\*** Correspondence: gyurcsik@chem.u-szeged.hu; Tel.: +36-62-54-4335

**Abstract:** Cys2His2 zinc finger proteins are important for living organisms, as they—among other functions—specifically recognise DNA when Zn(II) is coordinated to the proteins, stabilising their ββα secondary structure. Therefore, competition with other metal ions may alter their original function. Toxic metal ions such as Cd(II) or Hg(II) might be especially dangerous because of their similar chemical properties to Zn(II). Most competition studies carried out so far have involved small zinc finger peptides. Therefore, we have investigated the interactions of toxic metal ions with a zinc finger proteins consisting of three finger units and the consequences on the DNA binding properties of the protein. Binding of one Cd(II) per finger subunit of the protein was shown by circular dichroism spectroscopy, fluorimetry and electrospray ionisation mass spectrometry. Cd(II) stabilised a similar secondary structure to that of the Zn(II)-bound protein but with a slightly lower affinity. In contrast, Hg(II) could displace Zn(II) quantitatively (log*β* <sup>0</sup> ≥ 16.7), demolishing the secondary structure, and further Hg(II) binding was also observed. Based on electrophoretic gel mobility shift assays, the Cd(II)-bound zinc finger protein could recognise the specific DNA target sequence similarly to the Zn(II)-loaded form but with a ~0.6 log units lower stability constant, while Hg(II) could destroy DNA binding completely.

**Keywords:** zinc finger proteins; cadmium; mercury; metal binding affinity; DNA binding; electrospray ionisation mass spectrometry; spectroscopy; EMSA; FluoZin-3

### **1. Introduction**

Zinc finger proteins (ZFPs) are present in various living organisms, such as amphibians, reptiles and mammals [1–4]. ZFPs are involved in DNA transcription, translation, error correction, metabolism, stimulus generation, cell division and cell death by interacting with other proteins, small molecules, RNA and DNA [5,6]. Zinc finger (ZF) motifs of a ZFP are involved in molecular recognition, while the rest of the protein is most commonly responsible for its function [7–13]. The structure of a ZF motif is stabilised by the tetrahedral coordination of Zn(II) and by the formation of a hydrophobic core [14]. Similar tetrahedral coordination was found in self-assembling peptides offering a Cys2His2 binding site [15]. Cys2His2-type proteins form the most populous family of ZFPs [16]. Their biotechnological significance is highlighted by the fact that a ZF unit recognises three subsequent nucleobases in a double strand (ds) DNA, and several ZF units can be linked together to increase the specificity of the interaction. ZF arrays were first applied as the DNA recognition domains of artificial nucleases linked to the FokI nuclease domain [17]. Since then, numerous gene modification experiments have been performed with nucleases of this type [18–24]. The recognition sequence can further be extended by the chimera of ZF and other DNA binding motifs [25].

Cys2His2 ZFPs can only bind to DNA specifically in their Zn(II)-bound form. Other metal ions inside the living organism may substitute Zn(II), form mixed complexes and/or

**Citation:** Hajdu, B.; Hunyadi-Gulyás, É.; Gyurcsik, B. Interactions of an Artificial Zinc Finger Protein with Cd(II) and Hg(II): Competition and Metal and DNA Binding. *Inorganics* **2023**, *11*, 64. https://doi.org/ 10.3390/inorganics11020064

Academic Editors: Peter Segl'a and Ján Pavlik

Received: 29 December 2022 Revised: 25 January 2023 Accepted: 27 January 2023 Published: 29 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

promote oxidation of the cysteines. In addition, toxic metal ions may also react with ZFPs, rendering the investigation of these interactions crucial. The coordination chemistry and biophysical properties of ZFPs [11,26–28] have been extensively studied, but still there are open questions regarding the stabilities of their complexes and the competition between Zn(II) and non-native metal ions [29].

Cd(II) has a stronger "soft" character than Zn(II). Therefore, it forms the most stable complexes with thiolate ligands, while it can also interact with nitrogen and oxygen donor atoms in biological systems. ZF peptides modelling a Cys2His2 type ZF unit bind Zn(II) about two–three orders of magnitude more strongly than Cd(II). The affinities of the two metal ions are comparable towards the Cys3His binding site, while the Cys4 ZF motifs bind Cd(II) two–three orders of magnitude stronger than Zn(II) [30–33]. Heinz et al. investigated the metal ion coordination of the consensus peptide 1 (CP1) Cys2His2 ZF model. Starting from the apo-peptide, they reported that a biscomplex forms with Cd(II) when the ligand is in excess through the cysteine thiolates, while Cys2His2 coordination is favoured in the monocomplex formed at a 1:1 initial metal to ligand ratio [34]. Cd(II) may disturb DNA recognition of a ZFP and thus the related biological processes, which is one of the possible mechanisms of its toxic effect [35]. Petering et al. and Hanas et al. found that Cd(II) could inhibit DNA binding of the Zn(II)-bound TFIIIA ZFP [35–37]. On the other hand, it was shown that both the high- and low-stability binding sites of TFIIIA bind Cd(II) weaker than Zn(II) by ~2.5 and one order of magnitude, respectively [38]. Investigations with the 3rd ZF subunit of TFIIIA ZFP revealed that once formed, the Cd(II) complex had a similar secondary structure and just a slightly lower DNA binding affinity (not specific) than the Zn(II) complex [39]. The situation is even more complicated in the Sp1 transcription factor consisting of three ZF subunits. In a few publications, Cd(II) has been shown to inhibit DNA binding of Zn(II) saturated Sp1 [40–42], while others did not observe such effect [43]. Kuwahara et al. reported that the Cd(II) complex of Sp1 was also capable of recognizing the specific target DNA, but with slightly lower affinity than the Zn(II) complex [44]. Malgieri et al. reported comparable affinity of Ros87, an eukaryotic Cys2His2-type ZFP, towards Cd(II) and Zn(II). The two complexes shared a similar secondary structure based on UV– Vis, CD and NMR measurements. Furthermore, the Cd(II) complex could recognise the same DNA target as the Zn(II)-loaded Ros87. However, it must be emphasised that this protein had only a single ZF unit linked to other protein elements, which also played a role in DNA binding [45]. A similar phenomenon was observed with the Tramtrack ZFP (consisting of two ZF subunits), although the α-helix content and DNA binding affinity of the Cd(II) complex was lower than that of Zn(II) bound protein [46] based on CD and EMSA measurements. The MTF-1 (metal response element-binding transcription factor-1) consists of six Cys2His2 ZF subunits. Cd(II) could inhibit the DNA-binding of this ZFP both when added to the apo-protein or to the Zn(II)-loaded MTF-1 [43,47]. The three unusual C-terminal fingers (4th–6th) of MTF-1 were investigated by Giedroc et al., where NMR and UV–Vis data revealed that although Cd(II) could bind these subunits, the obtained secondary structure differed significantly from the native ββα-type, most probably due to the unusual amino acid composition of the 5th subunit between the two cysteines [48].

The high affinity of Hg(II) towards sulphur donor groups is well known. However, the fact that Hg(II) can form a very stable complex with Cl− ions under physiological conditions (lg*β*ML<sup>2</sup> = 13.23) makes it difficult to compare the results of equilibrium studies [49]. Depending on the chloride content of the medium, some studies consider the affinity of Hg(II) and Zn(II) for Cys2His2 ZF motifs to be comparable, while in the absence of Cl− ions, Hg(II) forms more stable complexes [50]. Depending on the applied medium and measurement conditions, it has been shown that Hg(II) could not inhibit the DNA binding of TFIIIA during a DNase I footprinting assay [37], while more studies revealed that the secondary structure of the Zn(II)-loaded ZFPs collapsed in the presence of both organic and inorganic Hg(II), and the Hg(II)-bound ZFPs were unable to recognise DNA target sequences [40,44,51].

Solution equilibria of Cys2His2 zinc finger motifs have been widely investigated using the CP1 model peptide and the metal binding properties of naturally occurring ZFP subunits are usually compared to this model. However, CP1 could only give information regarding the metal binding properties of a single ZF subunit but not the protein–DNA interactions, since one ZF subunit cannot provide significant selectivity and affinity towards DNA. Therefore, the trends predicted on the basis of CP-1 and the behaviour experienced with natural ZFPs are contradictory [35–37,40–43,47].

Recently, we quantitatively characterised the Zn(II) and DNA binding properties of 1MEY#, an artificial ZFP consisting of three CP1-like subunits [52]. The amino acid sequence of 1MEY# can be found in Figure S1. With the knowledge of Zn(II) and DNA affinities, here we investigated the interaction between the 1MEY# artificial ZFP and toxic metal ions by spectrometric and electrophoretic methods. The competition of Cd(II) and Hg(II) with Zn(II) for the ZFP is described quantitatively to better understand the possible mechanisms of toxicity.

### **2. Results and Discussion**

### *2.1. Interaction of 1MEY# Zinc Finger Protein with Cd(II)*

The investigated 1MEY# ZFP consists of three CP1-like ZF subunits. The ZF units in 1MEY# differ only in a few amino acids responsible for DNA recognition. Therefore, it is assumed that they have similar metal binding properties. For this reason, the metal binding affinities of a single "average" 1MEY# binding site, referred to as 1MEY# bs, are presented throughout the text, unless otherwise stated. A Zn(II)-loaded Cys2His2 ZFP displays ββα-type secondary structure, while the apo-protein turns into an unordered structure, both resulting in characteristic circular dichroism spectra [53]. This provides a great opportunity to apply circular dichroism (CD) spectroscopy to investigate the effect of Cd(II) on 1MEY# ZFP. First, Zn(II) was removed from 1MEY# by treatment with ~25× excess of EDTA (Section 3.1). In a subsequent ultrafiltration, the apo-protein was transferred into an EDTA-free buffer. The initial Zn(II)-loaded holo-1MEY# protein displayed an ordered ββα structure as revealed by its CD spectrum with two negative peaks around 220 and 205 nm and a positive one at 190 nm (Figure 1a). The CD spectrum of apo-1MEY# represented an unfolded protein with a single negative peak around 200 nm. By adding three equivalents of Cd(II) (i.e., one equivalent per 1MEY# bs), the CD spectrum of the protein adopted a similar pattern to the Zn(II)-loaded protein. This indicated that Cd(II) could induce folding of the Cys2His2 ZF units into an ordered structure. Furthermore, no additional change in the CD spectra was observed upon an increase in the Cd(II) to protein ratio of up to ~120 fold (40 fold compared to the binding site) (Figure 1b). Cd(II) binding to the thiol groups of the ZFP was observed via ligand-to-metal charge transfer bands [54,55] in the 230–250 nm region of the UV–Vis absorption spectra as well (Figure S2).

It has to be mentioned that there are slight differences in the CD spectra of the Cd(II) and Zn(II)-bound 1MEY#. The negative peak around 220 nm disappeared and the intensity of the 205 nm negative peak increased for the Cd(II)-loaded 1MEY#. For a better understanding, we have evaluated the CD spectra using the BeStSel software [56], by means of which the secondary structure compositions of the Zn(II)- and Cd(II)-loaded proteins were obtained (Figure 1c,d). According to the best fit of the data, the percentage of antiparallel β-sheet increased by ~6%, while the percentage of α-helices decreased by ~8.5% in the Cd(II)-bound protein. The cysteines favoured by Cd(II) are located in the antiparallel β-sheet region, and tight binding to these ligands might have caused extension of these structural elements, while the percentage of the helices decreased. Nevertheless, the most significant changes in the spectra occurred in the 220–240 nm region, which may also be attributed to the chiral contribution of the charge transfer transitions [54,55,57]. The programs used for evaluation of the protein CD spectra do not consider these contributions separately; thus, these are finally detected as the change in the secondary structural elements. In our case, this may result in overestimation of the β-sheet content of the protein. Based on the above discussion, we can conclude the 1MEY# ZFP could fold into an ordered

secondary structure in the presence of Cd(II), which is most probably similar to that of the Zn(II)-loaded protein. This finding is consistent with the observations of Malgieri et al. for Ros87 ZFP [45], and Krepkiy et al. for the 3rd finger of TFIIIA [39]. In contrast, in case of the Tramtrack ZFP, Roesijadi et al. could only observe the 220–240 nm changes without the intense peak around 190 nm dedicated mostly to the α-helices [46]. The CD spectra of Cd(II)-1MEY# also suggested that under the measurement conditions, binding to the Cys2His2 site was favoured over the Cys3 or Cys4 coordination mode, which could have also been a possibility for 1MEY# containing altogether six cysteines in the three ZF units. The exclusive coordination to the cysteines would, however, result in the collapse of the finger structures, which did not occur. *Inorganics* **2023**, *11*, x FOR PEER REVIEW 4 of 19

**Figure 1.** (**a**) Circular dichroism spectra of Zn(II)-loaded (black), Cd(II)-loaded (orange) and metalfree (dashed grey) 1MEY# ZFP. The Cd(II)-loaded form in the presence of six equivalents (eqs) Zn(II) per 1MEY# (two eqs per binding site) (light blue) and the Zn(II)-depleted form using five eqs of EDTA per 1MEY# (1.7 eqs per binding site) (dashed black) are also presented. (**b**) CD spectra of Cd(II)-loaded 1MEY# in the presence of excess Cd(II). All CD spectra were normalised to the intensity of the starting Zn(II)-loaded 1MEY# spectrum recorded at 18.8 µM protein concentration. Measured and fitted CD spectra of (**c**) Cd(II)-1MEY# and (**d**) Zn(II)-1MEY# in the 185–250 nm wavelength range. Residual curves showing the differences between the fitted and measured spectra are marked with a red colour. Insets represent the estimated secondary structure composition of the complexes. The fitting was performed by BeStSel program suite [56]. It has to be mentioned that there are slight differences in the CD spectra of the Cd(II)- **Figure 1.** (**a**) Circular dichroism spectra of Zn(II)-loaded (black), Cd(II)-loaded (orange) and metalfree (dashed grey) 1MEY# ZFP. The Cd(II)-loaded form in the presence of six equivalents (eqs) Zn(II) per 1MEY# (two eqs per binding site) (light blue) and the Zn(II)-depleted form using five eqs of EDTA per 1MEY# (1.7 eqs per binding site) (dashed black) are also presented. (**b**) CD spectra of Cd(II)-loaded 1MEY# in the presence of excess Cd(II). All CD spectra were normalised to the intensity of the starting Zn(II)-loaded 1MEY# spectrum recorded at 18.8 µM protein concentration. Measured and fitted CD spectra of (**c**) Cd(II)-1MEY# and (**d**) Zn(II)-1MEY# in the 185–250 nm wavelength range. Residual curves showing the differences between the fitted and measured spectra are marked with a red colour. Insets represent the estimated secondary structure composition of the complexes. The fitting was performed by BeStSel program suite [56].

and Zn(II)-bound 1MEY#. The negative peak around 220 nm disappeared and the intensity of the 205 nm negative peak increased for the Cd(II)-loaded 1MEY#. For a better understanding, we have evaluated the CD spectra using the BeStSel software [56], by means of which the secondary structure compositions of the Zn(II)- and Cd(II)-loaded proteins were obtained (Figure 1c,d). According to the best fit of the data, the percentage of antiparallel β-sheet increased by ~6%, while the percentage of α-helices decreased by ~8.5% By adding six eqs of Zn(II) to the Cd(II)-saturated 1MEY# protein (two eqs per 1MEY# bs), the CD spectrum of the initial Zn(II)-loaded ZFP was recovered, indicating that Zn(II) has significantly higher affinity towards the Cys2His2 binding site than Cd(II). The similarity of the resulting spectrum with that of the initial holoprotein also demonstrated that in the series of the above described experiments, no oxidation of the cysteines of 1MEY# occurred.

in the Cd(II)-bound protein. The cysteines favoured by Cd(II) are located in the antiparallel β-sheet region, and tight binding to these ligands might have caused extension of these structural elements, while the percentage of the helices decreased. Nevertheless, the most significant changes in the spectra occurred in the 220–240 nm region, which may also be FluoZin-3 is considered to be a Zn(II) selective fluorescent probe, which can be applied to detect free Zn(II) replaced by Cd(II) in a competition assay with holo-1MEY#. However, Cd(II) also binds to FluoZin-3. Therefore, the stability of this complex was determined first by titrating four samples containing Zn(II) and FluoZin-3 at various molar ratio

attributed to the chiral contribution of the charge transfer transitions [54,55,57]. The programs used for evaluation of the protein CD spectra do not consider these contributions

ments. In our case, this may result in overestimation of the β-sheet content of the protein. Based on the above discussion, we can conclude the 1MEY# ZFP could fold into an ordered secondary structure in the presence of Cd(II), which is most probably similar to that of the Zn(II)-loaded protein. This finding is consistent with the observations of Malgieri et al. for Ros87 ZFP [45], and Krepkiy et al. for the 3rd finger of TFIIIA [39]. In contrast, in case of the Tramtrack ZFP, Roesijadi et al. could only observe the 220–240 nm changes without the intense peak around 190 nm dedicated mostly to the α-helices [46]. The CD with Cd(II). Assuming only the formation of a monocomplex and excluding formation of the ternary complex formation, a pH-independent stability constant of log*β* = 7.44 ± 0.01 was determined by evaluating the titration curves with the PSEQUAD program [58]. Recalculating this value at pH = 7.0, the obtained value of 7.18 was close to the available literature data for the Cd(II)-FluoZin-3 monocomplex (log*β* 0 = 6.9 [59]) (Figure S3a).

While starting from the apo-1MEY# ZFP, it was demonstrated that a protein coordinated to three Cd(II) could be established. Competition experiments were also performed to monitor the Cd(II) vs. Zn(II) exchange within 1MEY#. Based on the results of the CD spectroscopic, fluorometric and ESI-MS measurements, it was not possible to completely substitute Zn(II) with Cd(II) in the applied concentration range (Figure 2). A gradual metal–ion exchange was observed in the mass spectra with the subsequent formation of Zn2Cd<sup>1</sup> and Zn1Cd<sup>2</sup> mixed complexes and the Cd<sup>3</sup> species upon an increase in the Cd(II) excess. Since previously we could not distinguish the Zn(II) binding ability of the three subunits of 1MEY# ZFP [52], the ZF units (1MEY# bs) were considered to be identical here as well. The evaluation of the fluorometric titrations revealed that the apparent stability constant of the Cd(II)-1MEY# bs complex is ~2 orders of magnitude lower than that of the Zn(II)-1MEY# bs, characterised by a log*β* 0 Zn(II)-1MEY#bs, pH 7.4 of 12.2 [52]. This is a similar effect to that observed for the CP-1 model ZF peptide, where the stability decreased by ~2.5 orders of magnitude (Table 1) [33]. From the CD titration data, a one order of magnitude higher stability value was calculated, but here, only very small changes were observed in the spectra during the metal–ion exchange, decreasing the sensitivity of the method. By fitting the mass spectrometric data, an average log*β* 0 Cd(II)-1MEY# bs pH 7.4 of 10.75 was obtained. Taking into account that the results of the ESI-MS measurements may not always correlate with the solution equilibria due to the different measurement conditions and the potentially different ionisation rates of different species, this value is in a very good agreement with those determined by fluorometric and CD experiments in Table 1. It is also worth mentioning that average log*β* 0 values and single binding site models could not be directly used in the calculation of the Cd(II) binding affinity of the protein from the ESI-MS results, since here, the whole protein is observed. Therefore, statistical considerations were applied (Figure 2d) (Supplementary Section S1). A good agreement between the fitted and experimental ESI-MS data supported the hypothesis of the identity of the ZF units within 1MEY#.

These results indicated reversible Cd(II)/Zn(II) exchange within the CP-1-based 1MEY# ZFP. No cooperativity was observed during the titrations, suggesting that the ZF subunits behaved independently. Furthermore, the secondary structures of the formed complexes were almost identical (Figure 2) (Scheme 1a). The Cd(II) binding affinity of 1MEY# was found to be the highest among the available literature data with Cys2His2 ZFPs (Table 1), although it was still ~1–2 log*β* 0 units lower than the Zn(II) binding under similar conditions (log*β* 0 Zn(II) = 12.2) [52]. The difference between the Zn(II) and Cd(II) binding affinity of CP1 and TFIIIA was reported to be 2.5 [33,38], while in case of the Ros87, it was only 1.2 log*β* 0 units [60].

In good correlation with the results of CD measurements, no signals related to Zn1Cd<sup>1</sup> or Cd<sup>2</sup> species were observed during the analysis of the ESI-MS spectra of 1MEY#. Such species could have been characterised by Cys4 or Cys3 coordination where Cd(II) would bind to the cysteine sidechains of more than one ZF subunit, resulting the collapse of secondary structure and presumably the loss of the DNA-binding function (Scheme 1b). This effect might be responsible for the observed function loss in natural ZFPs with low Zn(II) and Cd(II) affinity towards the Cys2His2 coordination site. For example, in the case of TFIIIA ZFP, out of the nine subunits, only between two and three had higher Zn(II) affinity, and while it was possible to purify a protein with ~9 Zn(II) per ZFP, the purifications in the presence of Cd(II) yielded up to ~4 Cd(II) per ZFP products [35–38] (Table 1). This might be due to the formation of Cd(II)-Cys3 and Cd(II)-Cys4 coordination where Cd(II) bound the cysteines of multiple ZF subunits [32,36].

**Figure 2.** (**a**) Measured (full lines) and calculated (dashed lines) CD spectra of 1MEY# in the course of titration with Cd(ClO₄)₂; *c*1MEY# = 16.4 µM. The endpoint of the titration (fully Cd(II)-loaded 1MEY#, orange line) was established separately starting from apo-1MEY#. (**b**) Measured (symbols) and simulated (full lines) relative fluorescence curves obtained by titrating holo-1MEY# (blue) with Cd(II). Reference titrations were simultaneously conducted to obtain data for the relative fluorescence of the system in the absence of 1MEY# (red) or in the presence of an amount of Zn(ClO₄)₂ equal to the Zn(II) content of 1MEY# (yellow). *c*FluoZin-3 = 6 µM and *c*1MEY# = 1 µM. (**c**) ESI-MS spectra of Zn(II)-loaded 1MEY# in the presence of increasing amounts of Cd(II). Sample amount: 20 µL *c*1MEY# = 2 µM. (**d**) The species distribution diagram calculated from the ESI-MS data (separate points). **Figure 2.** (**a**) Measured (full lines) and calculated (dashed lines) CD spectra of 1MEY# in the course of titration with Cd(ClO<sup>4</sup> )2 ; *c*1MEY# = 16.4 µM. The endpoint of the titration (fully Cd(II)-loaded 1MEY#, orange line) was established separately starting from apo-1MEY#. (**b**) Measured (symbols) and simulated (full lines) relative fluorescence curves obtained by titrating holo-1MEY# (blue) with Cd(II). Reference titrations were simultaneously conducted to obtain data for the relative fluorescence of the system in the absence of 1MEY# (red) or in the presence of an amount of Zn(ClO<sup>4</sup> )<sup>2</sup> equal to the Zn(II) content of 1MEY# (yellow). *c*FluoZin-3 = 6 µM and *c*1MEY# = 1 µM. (**c**) ESI-MS spectra of Zn(II)-loaded 1MEY# in the presence of increasing amounts of Cd(II). Sample amount: 20 µL *c*1MEY# = 2 µM. (**d**) The species distribution diagram calculated from the ESI-MS data (separate points).

These results indicated reversible Cd(II)/Zn(II) exchange within the CP-1-based 1MEY# ZFP. No cooperativity was observed during the titrations, suggesting that the ZF subunits behaved independently. Furthermore, the secondary structures of the formed complexes were almost identical (Figure 2) (Scheme 1a). The Cd(II) binding affinity of 1MEY# was found to be the highest among the available literature data with Cys2His2 **Table 1.** Apparent average Cd(II) and Zn(II) affinities of some Cys2His2 ZFs and ZFPs in log*β* 0 units. cITC: competition with complexones monitored by ITC; FTc: competition with fluorescent complexones monitored by fluorescence spectroscopy; rCD: circular dichroism spectroscopy followed reverse titration; ESI-MS: reverse titration followed by ESI-MS; RT: spectroscopic reverse titration; DT: spectroscopic direct titration; ED: equilibrium dialysis.


<sup>1</sup> Difference between Zn(II) and Cd(II) affinity, if the measurement conditions were identical. <sup>a</sup> High-affinity binding sites. <sup>b</sup> Low-affinity binding sites.

bound the cysteines of multiple ZF subunits [32,36].

**Scheme 1.** (**a**) Schematic representation of the Zn(II)/Cd(II) exchange in the 1MEY# ZFP. The extended scheme including the microspecies that may form during the replacement of the first and second Zn(II) is included in Supplementary Scheme S1. (**b**) A hypothetical reaction scheme where Cd(II) coordinates to the cysteine sidechains of multiple ZF subunits inside the ZFP, resulting the collapse of the secondary structure. Such a reaction has been proven not to take place in the case of the highly stable 1MEY#, but might occur in the case of lower stability natural ZFPs such as TFIIIA. **Scheme 1.** (**a**) Schematic representation of the Zn(II)/Cd(II) exchange in the 1MEY# ZFP. The extended scheme including the microspecies that may form during the replacement of the first and second Zn(II) is included in Supplementary Scheme S1. (**b**) A hypothetical reaction scheme where Cd(II) coordinates to the cysteine sidechains of multiple ZF subunits inside the ZFP, resulting the collapse of the secondary structure. Such a reaction has been proven not to take place in the case of the highly stable 1MEY#, but might occur in the case of lower stability natural ZFPs such as TFIIIA. tive peak assigned to the α-helix around 190 nm completely disappeared (Figure 3a,b). Thus, the formed species, assumed to be the Hg₃1MEY# complex, displayed an unordered structure. Since Hg(II) shows extreme affinity towards the cysteine thiolates, this was the expected outcome, independent of whether Zn(II) was completely displaced from the complex or a ternary species was formed. By the addition of further Hg(II), up to ~5 eq per 1MEY# bs, only small, but continu-

**Table 1.** Apparent average Cd(II) and Zn(II) affinities of some Cys2His2 ZFs and ZFPs in log*β'*units. cITC: competition with complexones monitored by ITC; FTc: competition with fluorescent complexones monitored by fluorescence spectroscopy; rCD: circular dichroism spectroscopy followed reverse titration; ESI-MS: reverse titration followed by ESI-MS; RT: spectroscopic reverse titration; DT:

CP1 8.7 11.2 2.5 RT [33] TFIIIA 5.6 <sup>a</sup> 8.0 <sup>a</sup> 2.4 ED [38]

**<sup>1</sup>** Difference between Zn(II) and Cd(II) affinity, if the measurement conditions were identical. <sup>a</sup> High-

In good correlation with the results of CD measurements, no signals related to Zn1Cd<sup>1</sup> or Cd<sup>2</sup> species were observed during the analysis of the ESI-MS spectra of 1MEY#. Such species could have been characterised by Cys4 or Cys3 coordination where Cd(II) would bind to the cysteine sidechains of more than one ZF subunit, resulting the collapse of secondary structure and presumably the loss of the DNA-binding function (Scheme 1b). This effect might be responsible for the observed function loss in natural ZFPs with low Zn(II) and Cd(II) affinity towards the Cys2His2 coordination site. For example, in the case of TFIIIA ZFP, out of the nine subunits, only between two and three had higher Zn(II) affinity, and while it was possible to purify a protein with ~9 Zn(II) per ZFP, the purifications in the presence of Cd(II) yielded up to ~4 Cd(II) per ZFP products [35−38] (Table 1). This might be due to the formation of Cd(II)-Cys3 and Cd(II)-Cys4 coordination where Cd(II)

<sup>b</sup> Low-affinity binding sites.

**log***β'***Cd(II) log***β'***Zn(II) Δlog***β'***<sup>1</sup> Method Reference**

10.11 ± 0.03 2.11 FTc Present work 11.14 ± 0.03 1.06 rCD Present work

3.8 <sup>b</sup> 4.6 <sup>b</sup> 0.8 ED [38]

8.0 9.2 1.2 RT [51,61] 7.7 DT [45]

12.2 cITC [52]

spectroscopic direct titration; ED: equilibrium dialysis.

1MEY#

Ros87

affinity binding sites.

### *2.2. Hg(II) Binding of the 1MEY# Zinc Finger Protein* ous, changes were observed in the CD spectra, indicating that further thermodynamic events took place. Furthermore, the shape of the CD spectrum changed completely and

A perchlorate salt of Hg(II) was used in experiments with 1MEY# ZFP, aiming to avoid the interference with Cl− ions, i.e., to observe the direct interaction of the toxic metal ion with the ZFP. A significant change in the CD spectra of 1MEY# was observed upon addition of three equivalents of Hg(II) (one eq. per 1MEY# bs), referring to the collapse of the ββα secondary structure of the protein, similar to other investigations [51]. The positive peak assigned to the α-helix around 190 nm completely disappeared (Figure 3a,b). Thus, the formed species, assumed to be the Hg31MEY# complex, displayed an unordered structure. Since Hg(II) shows extreme affinity towards the cysteine thiolates, this was the expected outcome, independent of whether Zn(II) was completely displaced from the complex or a ternary species was formed. an intense positive band appeared around 215 nm, most probably due to a charge transfer band related to Hg(II) at ~ 8 eq Hg(II) per 1MEY# bs [57,62,63]. The sharp breakpoint in the plot of the CD intensity at 215 nm after adding one equivalent of Hg(II) per 1MEY# bs suggested that Hg(II) is indeed a much stronger competitor for ZF subunits than Cd(II) (Figure 3). The sharp breakpoint at one equivalent Hg(II) per ZF binding site also suggests that during the sequential exchange of Zn(II) for Hg(II), the unfolding of one subunit did not weaken the Zn(II) binding (and the secondary structure) of the remaining Zn(II)-bound subunit(s). This again indicated a high degree of independence of the subunits inside 1MEY#.

**Figure 3.** (**a**) CD spectra of 1MEY# ZFP in the presence of increasing equivalents of Hg(ClO₄)₂, starting from the Zn(II)-loaded protein (black full line). The black dashed spectrum belongs to the system containing 1 eq. Hg(II) per 1MEY# bs. (**b**) The plot of the ellipticity values (×) recorded at 215 nm vs. Hg(II) equivalents per 1MEY# bs. The breakpoint at 1 eq. is indicated by a vertical dashed grey line. *c*1MEY# = 16.4 µM. **Figure 3.** (**a**) CD spectra of 1MEY# ZFP in the presence of increasing equivalents of Hg(ClO<sup>4</sup> )2 , starting from the Zn(II)-loaded protein (black full line). The black dashed spectrum belongs to the system containing 1 eq. Hg(II) per 1MEY# bs. (**b**) The plot of the ellipticity values (×) recorded at 215 nm vs. Hg(II) equivalents per 1MEY# bs. The breakpoint at 1 eq. is indicated by a vertical dashed grey line. *c*1MEY# = 16.4 µM.

mono (HgA log*β* = 6.8 ± 0.1), bis (HgA₂ log*β* = 12.8 ± 0.3) and ternary complexes (HgZnA log*β* = 14.0 ± 0.1), although the chemical composition and coordination mode of such species remained ambiguous, but as it turned out there was no need for the use of these con-

As the next step, the Hg(II)–holo-1MEY# system was studied in the presence of FluoZin-3. As it turned out, Hg(II) bound very strongly to the 1MEY# ZFP, so that the first three equivalents (one equivalent per binding site) of Hg(II) displaced Zn(II) in the ZFP

Hg(II) interactions with 1MEY# ZFP was also investigated by fluorometric titrations. There are no published data characterising the solution equilibria in the Hg(II)–FluoZin-3 system. Therefore, we carried out competitive fluorometric measurements prior to the experiments without protein, titrating the Zn(II)–FluoZin-3 system at various ratios of

stants in further calculations (see below) (Figure S3b).

By the addition of further Hg(II), up to ~5 eq per 1MEY# bs, only small, but continuous, changes were observed in the CD spectra, indicating that further thermodynamic events took place. Furthermore, the shape of the CD spectrum changed completely and an intense positive band appeared around 215 nm, most probably due to a charge transfer band related to Hg(II) at ~8 eq Hg(II) per 1MEY# bs [57,62,63].

The sharp breakpoint in the plot of the CD intensity at 215 nm after adding one equivalent of Hg(II) per 1MEY# bs suggested that Hg(II) is indeed a much stronger competitor for ZF subunits than Cd(II) (Figure 3). The sharp breakpoint at one equivalent Hg(II) per ZF binding site also suggests that during the sequential exchange of Zn(II) for Hg(II), the unfolding of one subunit did not weaken the Zn(II) binding (and the secondary structure) of the remaining Zn(II)-bound subunit(s). This again indicated a high degree of independence of the subunits inside 1MEY#.

Hg(II) interactions with 1MEY# ZFP was also investigated by fluorometric titrations. There are no published data characterising the solution equilibria in the Hg(II)–FluoZin-3 system. Therefore, we carried out competitive fluorometric measurements prior to the experiments without protein, titrating the Zn(II)–FluoZin-3 system at various ratios of Hg(II). The best fit of these titration curves was achieved by assuming the formation of mono (HgA log*β* = 6.8 ± 0.1), bis (HgA<sup>2</sup> log*β* = 12.8 ± 0.3) and ternary complexes (HgZnA log*β* = 14.0 ± 0.1), although the chemical composition and coordination mode of such species remained ambiguous, but as it turned out there was no need for the use of these constants in further calculations (see below) (Figure S3b).

As the next step, the Hg(II)–holo-1MEY# system was studied in the presence of FluoZin-3. As it turned out, Hg(II) bound very strongly to the 1MEY# ZFP, so that the first three equivalents (one equivalent per binding site) of Hg(II) displaced Zn(II) in the ZFP almost quantitatively, and therefore, did not interact with the fluorophore (Figure 4a). This behaviour indicated that while Cd(II) could not compete efficiently with Zn(II) for 1MEY# ZFP, inorganic Hg(II) is an extremely strong competitor. Another interesting fact is that, contrary to expectations, no increase in fluorescence could be observed even after continuing the titration; the sample practically behaved as if it had been diluted with a buffer. This suggested that the extra added Hg(II) also bound to the 1MEY# ZFP with high affinity. This phenomenon provided an opportunity to estimate the lower limits of affinities of the ZF units as binding sites not only towards the first but also towards additional Hg(II) (Table 2). Based on the obtained stability constants, we could construct a distribution diagram, where the dashed section is based on the estimated limiting constants related to the binding of further Hg(II) (Figure 4b). Although this model may not be accurate in describing metal ion cluster formation, it could be successfully applied to describe the phenomena in this complicated system. Oligomerisation may also occur in these systems, but no species related to oligomers could be identified by ESI-MS.

**Table 2.** Estimated stability constants for Hg(II):1MEY# bs system based on the fluorometric method. The presented average constants were calculated for a single subunit of 1MEY# that was assumed to bind 1, 2, 3 and 4 Hg(II) in the 1MEY# protein binding 3, 6, 9 and 12 Hg(II), respectively.


Mass spectrometric measurements confirmed the fluorometric results. A gradual displacement of Zn(II) was detected through the Zn2Hg1MEY#, ZnHg21MEY# and Hg31MEY# complexes. In addition, it was possible to detect the presence of 1MEY# species with even 13 coordinated Hg(II) while increasing the metal ion excess up to 24 eqs (eight eqs per binding site); however, the exact mode of coordination remained unknown (Figure 4c). The stability constant of the monocomplex estimated from ESI-MS data was identical to the value obtained from fluorometric titration (Table 2); however, for the binding of the second Hg(II), a value three orders of magnitude lower was assigned, which is presumably due to the previously mentioned uncertainty of the mass spectrometry. It can be assumed that Hg(II) only coordinates to the cysteines [51]. Thus, in theory, the histidine residues remain available for the coordination of Zn(II). Despite this, no mixed metal species within the same ZF subunit could be seen under the conditions of the ESI-MS measurements (Scheme 2). In the Zn2Hg1MEY# and ZnHg21MEY# ternary complexes, the metal ions bind to different subunits and thus the binding events are independent. buffer. This suggested that the extra added Hg(II) also bound to the 1MEY# ZFP with high affinity. This phenomenon provided an opportunity to estimate the lower limits of affinities of the ZF units as binding sites not only towards the first but also towards additional Hg(II) (Table 2). Based on the obtained stability constants, we could construct a distribution diagram, where the dashed section is based on the estimated limiting constants related to the binding of further Hg(II) (Figure 4b). Although this model may not be accurate in describing metal ion cluster formation, it could be successfully applied to describe the phenomena in this complicated system. Oligomerisation may also occur in these systems, but no species related to oligomers could be identified by ESI-MS.

almost quantitatively, and therefore, did not interact with the fluorophore (Figure 4a). This behaviour indicated that while Cd(II) could not compete efficiently with Zn(II) for 1MEY# ZFP, inorganic Hg(II) is an extremely strong competitor. Another interesting fact is that, contrary to expectations, no increase in fluorescence could be observed even after continuing the titration; the sample practically behaved as if it had been diluted with a

*Inorganics* **2023**, *11*, x FOR PEER REVIEW 9 of 19

**Figure 4.** (**a**) Measured (symbols) and simulated (continuous line) relative fluorescence obtained during the titration of holo-1MEY# ZFP with Hg(II) (blue). Reference titrations were simultaneously performed to obtain data for the relative fluorescence of the system in the absence of 1MEY# (red) or in the presence of an amount of Zn(ClO₄)₂ equal to the Zn(II) content of 1MEY# (yellow). *c*FluoZin-3 = 6 µM and *c*1MEY# = 1 µM. (**b**) Distribution diagram obtained by the fitting of the fluorescence titration data. (**c**) Zn(II)-loaded 1MEY# in the presence of increasing amounts of Hg(II) followed by ESI-MS. Sample amount: 20 µL and *c*1MEY# = 2 µM. **Figure 4.** (**a**) Measured (symbols) and simulated (continuous line) relative fluorescence obtained during the titration of holo-1MEY# ZFP with Hg(II) (blue). Reference titrations were simultaneously performed to obtain data for the relative fluorescence of the system in the absence of 1MEY# (red) or in the presence of an amount of Zn(ClO<sup>4</sup> )<sup>2</sup> equal to the Zn(II) content of 1MEY# (yellow). *c*FluoZin-3 = 6 µM and *c*1MEY# = 1 µM. (**b**) Distribution diagram obtained by the fitting of the fluorescence titration data. (**c**) Zn(II)-loaded 1MEY# in the presence of increasing amounts of Hg(II) followed by ESI-MS. Sample amount: 20 µL and *c*1MEY# = 2 µM.

**Table 2.** Estimated stability constants for Hg(II):1MEY# bs system based on the fluorometric method. The presented average constants were calculated for a single subunit of 1MEY# that was assumed to bind 1, 2, 3 and 4 Hg(II) in the 1MEY# protein binding 3, 6, 9 and 12 Hg(II), respectively. **p***K***'** Hg₁1MEY# bs ≥16.7 Hg₂1MEY# bs ≥9.3 Hg₃1MEY# bs ≥8.5 Hg₄1MEY# bs ≥8.2 The obtained conditional stability constant for the binding of the first Hg(II) (log*β* 0 Hg(II)-1MEY# bs ≥ 16.7) is quite a high value on the scale of ZFP metal binding. Among the peptides containing the CXXC-amino acid sequence, only those had a similar or higher affinity for Hg(II), where the cysteines were in favourable position and the secondary structure was not completely unordered [64–67]. Although the CD spectra of 1MEY# in the presence of one Hg(II) eq. per binding site significantly differed from the unfolded structure (Figure S4), the contribution of the thiolate–Hg(II) charge transfer bands might be significant. Therefore, an accurate evaluation of the secondary structure composition of the Hg(II)-1MEY# complex is not possible [57,62,63]. Nevertheless, based on the NMR analysis of the similarly behaving Ros87 ZFP, it can be assumed that the structure is not completely disordered [51]. Sivo et. al. also determined the Hg(II) affinity of Ros87 using HgCl<sup>2</sup> (log*β* 0 Hg(II)-Ros87 = 6.1) [51].

HgCl<sup>2</sup> (log*β'*Hg(II)-Ros87 = 6.1) [51].

**Scheme 2.** Schematic representation of Zn(II)/Hg(II) exchange in 1MEY# ZFP. The extended scheme, including the microspecies that may form during the replacement of the first and second Zn(II), is included in Supplementary Scheme S2. **Scheme 2.** Schematic representation of Zn(II)/Hg(II) exchange in 1MEY# ZFP. The extended scheme, including the microspecies that may form during the replacement of the first and second Zn(II), is included in Supplementary Scheme S2.

Mass spectrometric measurements confirmed the fluorometric results. A gradual displacement of Zn(II) was detected through the Zn2Hg1MEY#, ZnHg21MEY# and Hg31MEY# complexes. In addition, it was possible to detect the presence of 1MEY# species with even 13 coordinated Hg(II) while increasing the metal ion excess up to 24 eqs (eight eqs per binding site); however, the exact mode of coordination remained unknown (Figure 4c). The stability constant of the monocomplex estimated from ESI-MS data was identical to the value obtained from fluorometric titration (Table 2); however, for the binding of the second Hg(II), a value three orders of magnitude lower was assigned, which is presumably due to the previously mentioned uncertainty of the mass spectrometry. It can be assumed that Hg(II) only coordinates to the cysteines [51]. Thus, in theory, the histidine residues remain available for the coordination of Zn(II). Despite this, no mixed metal species within the same ZF subunit could be seen under the conditions of the ESI-MS measurements (Scheme 2). In the Zn2Hg1MEY# and ZnHg21MEY# ternary complexes, the

metal ions bind to different subunits and thus the binding events are independent.

The obtained conditional stability constant for the binding of the first Hg(II) (log*β'*Hg(II)-1MEY# bs ≥ 16.7) is quite a high value on the scale of ZFP metal binding. Among the peptides containing the CXXC-amino acid sequence, only those had a similar or higher affinity for Hg(II), where the cysteines were in favourable position and the secondary structure was not completely unordered [64–67]. Although the CD spectra of 1MEY# in the presence of one Hg(II) eq. per binding site significantly differed from the unfolded structure (Figure S4), the contribution of the thiolate–Hg(II) charge transfer bands might be significant. Therefore, an accurate evaluation of the secondary structure composition of the Hg(II)-1MEY# complex is not possible [57,62,63]. Nevertheless, based on the NMR analysis of the similarly behaving Ros87 ZFP, it can be assumed that the structure is not completely disordered [51]. Sivo et. al. also determined the Hg(II) affinity of Ros87 using
