A new insight into the Ag+ and Cu+ binding sites in the metallothionein beta domain

The copper(I) and silver(I) binding properties of the beta fragment of recombinant mouse metallothionein I have been studied by electronic absorption and circular dichroism spectroscopy. When possible, the stoichiometry of the species formed was confirmed by electrospray mass spectrometry. The behaviour observed differs from that reported for the native protein. Titration of either Zn3-beta MT at pH 7 or apo-beta MT at pH 3 with Cu+ leads to the formation of species having the same stoichiometry and structure: Cu6-beta MT, Cu7-beta MT and Cu10-beta MT. In the first stage of the titration of Zn3-beta MT with Cu+ at pH 7 one additional species of formula Cu4Zn1-beta MT was detected. In contrast, the titration of Zn3-beta MT at pH 7.5 and of apo-beta MT at pH 2.5 with Ag+ proceeds through different reaction pathways, affording ZnxAg3-beta MT, Ag6-beta MT and Ag9-beta MT or Ag3-beta MT, Ag6-beta MT and Ag9-beta MT, respectively. The CD envelope corresponding to species with the same stoichiometric ratio, Ag6-beta MT and Ag9-beta MT, indicates that they have a different structure at each pH value. On the basis of the differences observed, the postulated similarity between copper and silver binding to metallothionein may be questioned.     

Distinct metal-binding configurations in metallothionein

In a study of the binding stoichiometry of various metals to rat liver metallothionein, the protein appears to coordinate metals in 2 distinct configurations. Ions of at least 18 different metals were shown to associate with the protein suggesting that there is little specificity in binding. Most metals exhibited saturation binding at 7 mol eq forming M7-metallothionein. These included Bi(III), Cd(II), Co(II), Hg(II), In(III), Ni(II), Pb(II), Sb(III), and Zn(II). Others metals including Os(III), Pd(II), Pt(IV), Re(V), Rh(III), and Tl(III) give a positive indication of binding, but stoichiometries were unclear. Ag(I) and Cu(I) bound in clusters as M12-metallothionein. This binding stoichiometry was determined in 3 ways: (a) by determining the equivalence point in Cu- and Ag-titrated samples where resistance to proteolysis is maximal; (b) by determining the point where Zn ions are completely displaced from Zn7-metallothionein; and (c) by direct binding studies. Ag-reconstituted protein, recovered from gel filtration, had an average Ag content of 11.5 g atoms/mol of protein. A similar stoichiometry for the Cu-protein resulted from displacement of Zn from Zn7-metallothionein by Cu(I). The M12-protein was converted to the M7-protein by displacement of Ag(I) or Cu(I) with 7 mol eq of Hg(II). Whereas the distribution of metals in the 2 domains of M7-metallothionein is M4 alpha and M3 beta, the arrangement in the M12-molecule is probably M6 alpha and M6 beta. We propose that metallothionein ligates Ag(I) and Cu(I) in a trigonal geometry by bridging thiolates. This is in contradistinction to a tetrahedral binding geometry in the M7-protein. Distinct binding configurations may result in different tertiary structures for M7- and M12-proteins which may relate to metabolic specificity of Zn-metallothionein and Cu-metallothionein, respectively.     

Chemical foundation of the attenuation of methylmercury(II) cytotoxicity by metallothioneins.

To elucidate the chemical interactions underlying the role of metallothioneins (MTs) in reducing the cytotoxicity caused by MeHg(II), we monitored in parallel by electronic absorption and CD spectroscopies the stepwise addition of MeHgCl stock solution to mammalian Zn(7)-MT1 and the isolated Zn(4)-alphaMT1 and Zn(3)-betaMT1 fragments. The incorporation of MeHg(+) into Zn(7)-MT and Zn(3)-betaMT entails total displacement of Zn(II) and unfolding of the protein. However, both features are only partial for Zn(4)-alphaMT. The different behavior observed for this fragment, whether isolated or constituting one of the two domains of Zn(7)-MT, indicates interdomain interactions in the whole protein. Overall, the binding properties of Zn(7)-MT, Zn(4)-alphaMT and Zn(3)-betaMT toward MeHg(+) are unprecedented. In addition, the sequestration of MeHg(+) by Zn(7)-MT and the concomitant release of Zn(II) are probably two of the main contributions in the detoxifying role of mammalian MT.     

The binding of copper(II) and zinc(II) to oxidized glutathione

1H and 13C NMR studies of Zn(II) binding to oxidized glutathione (GSSG) in aqueous solution over the pH range 4-11 show that it forms a complex with a 1:1 Zn:GSSG stoichiometry. At pH values between 6 and 11 the metal ligands are the COO- and NH2 groups of the glutamate residues. Below pH 5 the glycine end of the molecule also binds to the metal ions. EPR and visible absorption spectra of Cu(II) GSSG solutions suggest that similar complexes are formed with Cu(II). The solid products obtained from these solutions are shown by analysis and EPR to be primarily binuclear with Cu2GSSG stoichiometry, although the structures depend on the pH and stoichiometry of the solution from which they were obtained.     

Kinetic analysis of the effect of zinc ion on the inorganic pyrophosphatase reaction.

A kinetic study is presented in which the effect of Zn(II) on yeast inorganic pyrophosphatase was quantitatively determined. A dual role model for metal ion effect, previously determined for the Mg(II)-pyrophosphatase system (O. A. Moe and L. G. Butler, 1972, J. Biol. Chem.247, 7308–7315), was applied successfully to the analysis of the kinetics for Zn(II)-pyrophosphatase and Zn(II), Mg(II)-pyrophosphatase systems. The model, assigning an activator role to free Zn(II) ion and a substrate role to the Zn(II)-pyrophosphate complex, gave an excellent fit to the data. Inhibition of the Mg(II)-pyrophosphatase system by Zn(II) was analyzed by a model in which competitive binding of the Mg(II)-pyrophosphate and Zn(II)-pyrophosphate complexes occurred at the enzyme active site, with both complexes undergoing reaction at different rates. Relative maximal velocities and enzymeligand dissociation constants for the Zn(II)-pyrophosphate complex were determined for the cases where the metal ion activator role was fulfilled by Zn(II) and Mg(II), respectively. The maximal velocity parameter showed a dependence on the nature of the activator metal ion, demonstrating that the role of the latter is associated both with the process of substrate binding and with the mechanism of catalysis. Values for all kinetic parameters are reported for an ionic strength of 0.2, pH 7.0, and 25.0 °C.     

The nature of zinc in cytochrome c oxidase.

The zinc ion in bovine heart cytochrome c oxidase can be completely depleted from the enzyme with mercuric chloride without denaturing the protein. The metal atom stoichiometry of 5Cu/4Fe/0Zn/2Mg obtained for the enzyme following HgCl2 treatment indicates that this depletion is highly selective. Zinc depletion exposes one cysteine on subunit VIa and one cysteine on subunit VIb for N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylene-diamine (1,5-I-AEDANS) labelling, suggesting that the zinc plays a structural role in the protein by providing a bridge between these two subunits. Although the treatment of cytochrome c oxidase with mercuric chloride inhibits the steady-state activity of the enzyme, subsequent removal of the Hg2+ bound to cysteine residues by 1,5-I-AEDANS significantly reverses the inhibition. This latter result indicates that the removal of the zinc itself does not alter the steady-state activity of the enzyme.     

Effects of dissolved organic carbon and salinity on bioavailability of mercury.

Hypotheses that dissolved organic carbon (DOC) and electrochemical charge affect the rate of methylmercury [CH3Hg(I)] synthesis by modulating the availability of ionic mercury [Hg(II)] to bacteria were tested by using a mer-lux bioindicator (O. Selifonova, R. Burlage, and T. Barkay, Appl. Environ. Microbiol. 59:3083-3090, 1993). A decline in Hg(II)-dependent light production was observed in the presence of increasing concentrations of DOC, and this decline was more pronounced at pH 7 than at pH 5, suggesting that DOC is a factor controlling the bioavailability of Hg(II). A thermodynamic model (MINTEQA2) was used to select assay conditions that clearly distinguished among various Hg(II) species. By using this approach, it was shown that negatively charged forms of mercuric chloride (HgCl3-/HgCl(4)2-) induced less light production than the electrochemically neutral form (HgCl2), and no difference was observed between the two neutral forms, HgCl2 and Hg(OH)2. These results suggest that the negative charge of Hg(II) species reduces their availability to bacteria and may be one reason why accumulation of CH3Hg(I) is more often reported to occur in freshwater than in estuarine and marine biota.     

Coordination chemistry of vitamin C. Part II. Interaction of L-ascorbic acid with Zn(II), Cd(II), Hg(II), and Mn(II) ions in the solid state and in aqueous solution

The reaction of L-ascorbic acid with the zinc group and manganese ions has been investigated in aqueous solution at pH 6-7. The solid salts of the type M (L-ascorbate)2.2H2O, where M = Zn(II), Cd(II) and Mn(II) were isolated and characterized by 13C NMR and Fourier Transform infrared (FT-IR) spectroscopy. Spectroscopic evidence showed that in aqueous solution, the bindings of the Zn(II) and Mn(II) ions are through the ascorbate anion O-3 and O(2)-H groups (chelation), while the Cd(II) ion binding is via the O-3 atom only. In the solid state, the binding of these metal ions would be through two acid anions via O-3, O-2 of the first and O-1, O-3 of the second anion as well as to two H2O molecules, resulting in a six-coordinated metal ion. The Hg(II) ion interaction leads to the oxidation of the ascorbic acid in aqueous solution.     

Iron autoxidation and free radical generation: effects of buffers, ligands, and chelators.

The pH of the solution along with chelation and consequently coordination of iron regulate its reactivity. In this study we confirmed that, in general, the rate of Fe(II) autoxidation increases as the pH of the solution is increased, but chelators that provide oxygen ligands for the iron can override the affect of pH. Additionally, the stoichiometry of the Fe(II) autoxidation reaction varied from 2:1 to 4:1, dependent upon the rate of Fe(II) autoxidation, which is dependent upon the chelator. No partially reduced oxygen species were detected during the autoxidation of Fe(II) by ESR using DMPO as the spin trap. However, upon the addition of ethanol to the assay, the DMPO:hydroxyethyl radical adduct was detected. Additionally, the hydroxylation of terephthalic acid by various iron-chelator complexes during the autoxidation of Fe(II) was assessed by fluorometric techniques. The oxidant formed during the autoxidation of EDTA:Fe(II) was shown to have different reactivity than the hydroxyl radical, suggesting that some type of hypervalent iron complex was formed. Ferrous iron was shown to be able to directly reduce some quinones without the reduction of oxygen. In conclusion, this study demonstrates the complexity of iron chemistry, especially the chelation of iron and its subsequent reactivity.     

Resonance Raman characterization of Chromatium vinosum cytochrome c'. Effect of pH and comparison of equilibrium and photolyzed carbon monoxide species

Resonance Raman spectra of Chromatium vinosum cytochrome c' have been obtained for the five pH-dependent states of the protein [i.e., types I (pH 7), II (pH 10), and III (pH 12) of the ferric protein and type a (pH 7) and type n (pH 12) of the ferrous protein]. The raman spectra of type II and type a are consistent with those of high-spin, 5-coordinate heme proteins, such as deoxyhemoglobin, while spectra of type III and type n correspond more closely to those of low-spin, ferric and ferrous cytochrome c, respectively. Spectra of the CO-bound equilibrium species qualitatively resemble those of carbon monoxy human HbA. However, both the Fe-C and C = O stretching modes of the ligated species exhibit pH-dependent frequency shifts. Our data also indicate that CO photolysis is much more efficient at pH 7 than at pH 12. Moreover, the spectra of the photolytic transients suggest that unique, high-spin species are formed subsequent to CO photolysis from both type a and type n species.     

Kinetics and pH Dependence of Light-Induced Deprotonation of the Schiff Base of Rhodopsin: Possible Coupling to Proton Uptake and Formation of the Active Form of Meta II

In this paper we first review what is known about the kinetics of Meta II formation, the role and stoichiometry of protons in Meta II formation, the kinetics of the light-induced changes of proton concentration, and the site of proton uptake. We then go on to compare the processes that lead to the deprotonation of the Schiff base in bacteriorhodopsin with rhodopsin. We point out that the similarity of the signs of the light-induced electrical signals from the two kinds of oriented pigment molecules could be explained by bacteriorhodopsin releasing a proton from its extracellular side while rhodopsin taking up a proton on its cytoplasmic side. We then examined the pH dependence of both the absorption spectrum of the unphotolyzed state and the amplitude and kinetics of Meta II formation in bovine rhodopsin. We also measured the effect of deuteration and azide on Meta II formation. We concluded that the pK _a of the counter-ion to the Schiff base of bovine rhodopsin and of a surface residue that takes up a proton upon photolysis are both less than 4 in the unphotolyzed state. The data on pH dependence of Meta II formation indicated that the mechanisms involved are more complicated than just two sequential, isospectral forms of Meta II in the bleaching sequence. Finally we examined the evidence that, like in bacteriorhodopsin, the protonation of the Schiff bases's counter-ion (Glu113) is coupled to the changing of the pK _a of a protonatable surface group, called Z for rhodopsin and tentatively assigned to Glu134. We conclude that there probably is such a coupling, leading to the formation of the active form of Meta II.     

Effect of Zn（Ⅱ） on the Structure and Biological Activity of Natural β-NGF

Only β-NGF, the subunit of the 7S NGF complex, exhibits NGF activity, but the function ofthe zinc ion in native β-NGF has received little attention. Flameless atomic absorption spectroscopy (FAAS)measurements reveal that native β-NGF contains Zn(Ⅱ) with a Zn(Ⅱ)/β-NGF stoichiometry of 1 : 14.6.The presence of Zn(Ⅱ) in the native molecule results in significant changes of the secondary structure andlocal tertiary structure around Trp(s) with respect to those of apo β-NGF, as suggested by spectra offluorescence and circular dichrosim. Stopped-flow studies show that there are at least two steps during theinteraction of Zn(Ⅱ) with the apo form. In comparison with its apo form, the native β-NGF shows a higherability to trigger the proliferation of TF1 cells and mediate the survival of PC 12. Thus it is most likely that thestructural changes caused by the presence of Zn(II) directly lead to the increase in the biological activity of β-NGE All results indicate that Zn(Ⅱ) in native β-NGF plays an important role in the structure and thebiological activity of the protein.     

A ratiometric fluorescent detection of Zn(II) in aqueous solutions using pyrene-appended histidine

A fluorescent chemosensor, Py-His, based on histidine was easily synthesized in solid phase synthesis. Py-His displayed a highly sensitive ratiometric response to Zn(II) with potent binding affinity (K_a = 1.17 × 10¹³ M^?2) in aqueous solutions. The detection limit of Py-His for Zn(II) was calculated as 80.8 nM. Moreover, Py-His distinguished Zn(II) and Hg(II) by different ratiometric response type; the chemosensor showed a more enhanced increase of excimer emission intensity to Zn(II) than Hg(II). Upon addition of Ag(I) and Cu(II), Py-His showed a turn-off response mainly due to the quenching effect of these metal ions. The binding stoichiometry (2:1 or 1:1) of Py-His to target metal ions played a critical role in the fluorescent response type (ratiometric and turn off response) to target metal ions. The role of imidazole group of Py-His for ratiometric detection of Zn(II) was proposed by pH titration experiments.     

Physicochemical properties of cloned nucleocapsid protein from HIV. Interactions with metal ions

The nucleocapsid (NC) protein (p15) of the human immunodeficiency virus (HIV) has been cloned and overproduced (under the control of a phage T7 promoter) in soluble form in an Escherichia coli host. The soluble NC protein is a fusion protein containing 15 amino acids from the T7 gene 10 and 7 amino acids from the HIV p24 protein at the N-terminus to make a protein of 171 amino acids. The plasmid containing the fusion gene is designated p15DF. A homogeneous product has been isolated from the induced cells and, when isolated under aerobic conditions, contains 0.3-0.5 mol of Zn/mol of protein and has only 2 titratable SH groups. Reduction and refolding in the presence of Zn(II) yields a protein containing 2.0 mol of Zn/mol of protein and 6 titratable SH groups. On the other hand, if the cells are sonicated in 2 mM CdCl2 and purified at pH 5.0, an unoxidized protein containing 2 mol of Cd/mol of protein is obtained. The Cd(II) ions can be exchanged with Zn(II), Co(II), or 113Cd(II). The Co(II)2 NC protein shows d-d electronic transitions at 695 nm [epsilon = 675 M-1 cm-1 per Co(II)] and 640 nm [epsilon = 825 M-1 cm-1 per Co(II)] compatible with regular tetrahedral geometry around both Co(II) ions. The Co(II)2 and Cd(II)2 NC proteins show intense charge-transfer bands in the near-UV, at 355 nm (epsilon = approximately 4000 M-1 cm-1) and 310 nm (epsilon = approximately 8000 M-1 cm-1) for the Co(II) protein and 255 nm (epsilon = approximately 10(4) M-1 cm-1) for the Cd(II)2 NC protein, compatible with -S- coordination. 113Cd NMR of the 113Cd(II)2 NC protein shows two 113Cd NMR signals at 659 and 640 ppm, respectively, each integrating to approximately 1 Cd(II) ion. The downfield chemical shifts suggest coordination of each 113Cd(II) ion to 3 sulfur donor atoms. The spectroscopic data fully support the prediction that the NC protein binds metal ions to each of the tandem repeats of the -Cys-X2-Cys-X4-His-X4-Cys- sequence contained in the N-terminal half of the molecule. 113Cd NMR shows, however, that the sites are not identical. Isolation of the NC protein under standard aerobic conditions results in oxidation of the sulfhydryl groups and loss of the coordinated Zn(II) ions, while preparation of the NC protein as the Cd(II) derivative at low pH protects the sulfhydryl groups from oxidation.     

Biotoxicity of mercury as influenced by mercury(II) speciation.

Integration of physicochemical procedures for studying mercury(II) speciation with microbiological procedures for studying the effects of mercury on bacterial growth allows evaluation of ionic factors (e.g., pH and ligand species and concentration) which affect biotoxicity. A Pseudomonas fluorescens strain capable of methylating inorganic Hg(II) was isolated from sediment samples collected at Buffalo Pound Lake in Saskatchewan, Canada. The effect of pH and ligand species on the toxic response (i.e., 50% inhibitory concentration [IC50]) of the P. fluorescens isolated to mercury were determined and related to the aqueous speciation of Hg(II). It was determined that the toxicities of different mercury salts were influenced by the nature of the co-ion. At a given pH level, mercuric acetate and mercuric nitrate yielded essentially the same IC50s; mercuric chloride, on the other hand, always produced lower IC50s. For each Hg salt, toxicity was greatest at pH 6.0 and decreased significantly (P = 0.05) at pH 7.0. Increasing the pH to 8.0 had no effect on the toxicity of mercuric acetate or mercuric nitrate but significantly (P = 0.05) reduced the toxicity of mercuric chloride. The aqueous speciation of Hg(II) in the synthetic growth medium M-IIY (a minimal salts medium amended to contain 0.1% yeast extract and 0.1% glycerol) was calculated by using the computer program GEOCHEM-PC with a modified data base. Results of the speciation calculations indicated that complexes of Hg(II) with histidine [Hg(H-HIS)HIS+ and Hg(H-HIS)2(2+)], chloride (HgCl+, HgCl2(0), HgClOH0, and HgCl3-), phosphate (HgHPO4(0), ammonia (HgNH3(2+), glycine [Hg(GLY)+], alanine [Hg(ALA)+], and hydroxyl ion (HgOH+) were the Hg species primarily responsible for toxicity in the M-IIY medium. The toxicity of mercuric nitrate at pH 8.0 was unaffected by the addition of citrate, enhanced by the addition of chloride, and reduced by the addition of cysteine. In the chloride-amended system, HgCl+, HgCl2(0), and HgClOH0 were the species primarily responsible for observed increases in toxicity. In the cysteine-amended system, formation of Hg(CYS)2(2-) was responsible for detoxification effects that were observed. The formation of Hg-citrate complexes was insignificant and had no effect on Hg toxicity.     

Circular dichroism, kinetic and mass spectrometric studies of copper(I) and mercury(II) binding to metallothionein

The metalloprotein metallothionein (MT) is remarkable in its metal binding properties: for the mammalian protein, well-characterized species exist for metal to sulfur ratios of M7S20, M12S20, and M18S20, where M = Cd(II), Zn(II), Hg(II), Ag(I), Au(I), and Cu(I). Optical spectra in general, and circular dichroism (CD) and luminescence spectra in particular, provide rich detail of a complicated metal binding chemistry when metals are added directly to the metal-free or zinc-containing protein. CD spectral data unambiguously identify key metal to protein stoichiometric ratios that result in well-defined structures. Electrospray ionization-mass spectrometry data are reported for reactions in which Hg(II) binds to apo-MT 2A as previously described from CD data. Emission spectra in the 450-750 nm region have been reported for metallothioneins containing Ag(I), Au(I), and Cu(I). The luminescence of Cu-MT can also be detected directly from mammalian and yeast cells. We report both steady-state and new dynamic data for titrations of Zn-MT with Cu(I). Analysis of kinetic data for the addition of the first two Cu(I) atoms to Zn-MT indicates a first-order mechanism over a concentration range of 5-50 microM. Three-dimensional modeling was carried out using the results of the CD and EXAFS studies, model calculations for Zn7-MT, Hg7-MT, and Cu12-MT are described.     

The copper-enzyme dopamine beta-monooxygenase: studies on the ability of several metals to inhibit the enzyme activity and to replace the copper

The ability of several metals to inhibit dopamine beta-monooxygenase was measured and compared with their ability to compete with the binding of 64Cu to the water-soluble form of the bovine adrenal enzyme at pH 6.0. In the presence of an optimal concentration of copper (0.5 microM in the present assay system), an inhibition was observed upon addition of Hg(II), Zn(II), or Ni(II). Only a small fraction of the inhibition with these metals may be due to uncoupling of electron transport from hydroxylation. Preincubation of these metals with the Cu-depleted apoenzyme before addition of copper, revealed a stronger inhibition than if copper was added before the other metals. Hg(II), Zn(II), and Ni(II) also compete with the binding of 64Cu(II) to the protein. Hg(II) was the most effective and Ni(II) the least effective of these metals, both with respect to inhibition of the enzyme activity and to prevent the binding of 64Cu(II). Competition experiments on the binding of Zn(II) and 64Cu in the presence and absence of ascorbate, indicated i) a similar affinity of Cu(I) and Cu(II) to the native enzyme, and ii) a more rapid binding of Cu(I) than Cu(II) to the Cu-depleted and Zn-containing enzyme. Al(III), Fe(II), Mg(II), Mn(II), Co(II), Cd(II), and Pb(II) neither inhibited the enzyme activity nor competed with the binding of 64Cu(II) to the protein (Fe(II) was not tested for binding). Of those metals cited above only Cu(II)/Cu(I) was able to reactivate the apoenzyme.     

NMR study of a lymphocyte differentiating thymic factor. An investigation of the Zn(II)-nonapeptide complexes (thymulin)

Detailed investigations of a serum peptide (less than Glu1-Ala2-Lys3-Ser4-Gln5-Gly6-Gly7-Ser8-++ +Asn9) were carried out by 1H and 13C NMR spectroscopy to elucidate the structure of the complex formed with Zn(II), thymulin, which has been found to be active in vivo. These experiments were performed in dimethyl sulfoxide-d6 solution at different metal:peptide ratios. The results suggest the following conclusions. (i) The Zn(II) complexation corresponds to a fast exchange on the NMR time scale. (ii) The evolution of 1H and 13C NMR chemical shifts indicates the existence of two types of complexes: a 1:2 species associating two peptide molecules and one Zn(II) ion and a complex with 1:1 stoichiometry. The former is predominant for metal:peptide ratios below unity. (iii) In the 1:2 complex, Zn(II) is coordinated by the Ser4-O gamma H and Asn9-CO2- sites, while in the 1:1 complex, Ser8-O gamma H is the third ligand to the Zn(II) ion. The results are compared with those for the [Ala4] and [Ala8] analogues, and those for the complexes of thymulin with other metal ions (Cu2+ and Al3+) in terms of its biological activity. These comparative studies suggested that the 1:1 complex is the only conformation recognized by the antibodies.     

Thermodynamic model for the stabilization of trigonal thiolato mercury(II) in designed three-stranded coiled coils.

A thermodynamic model is presented that describes the binding of Hg(II) to de novo designed peptides, Tri L9C and Baby L9C, which were derived from the Tri family. The Tri peptides are based on the parent sequence Ac-NH-G(LKALEEK)(x)()G-CONH(2) and are known to form two-stranded coiled coils at low pH (pH <4) and three-stranded coiled coils at high pH (pH >7). Tri L9C (x = 4) contains a four heptad repeat sequence with cysteine in position 9 and leucines in the other a and d positions; Baby L9C (x = 3), which also has a cysteine in position 9 but is one heptad shorter than Tri L9C, was designed to form less stable helical coiled coils in solution. The free energies of coiled coil formation for Tri, Tri L9C, Baby Tri, and Baby L9C at pH 2.5 and 8.5 were determined by guanidinium denaturation titrations; Tri L9C was observed to be highly helical in the absence of denaturant at pH 8.5 while Baby L9C contained <20% helical content at pH 8.5, indicating a weakly associated or unassociated coiled coil. Size-exclusion chromatography (SEC) verified that Baby L9C was a monomer at pH 8.5. The helicity of Baby L9C was induced by addition of HgCl(2). The subsequent formation of a trigonal thiolato Hg(II) in the interior of a three-stranded coiled coil was verified by the presence of a characteristic HgS(3) UV band at 248 nm. Titrations of Tri L9C and Baby L9C into solutions of HgCl(2) at pH values between 7 and 9 were performed to extract binding constants. Global fits to the data employed a mechanism that involved initial binding of mercury to the peptides forming a two-stranded coiled coil with linear thiolato Hg(II) at [peptide]/[Hg] <2, followed by addition of a more weakly associated third helix to generate a three-stranded coiled coil. This mechanism would require the deprotonation of the third cysteine thiol to generate the trigonal thiolato Hg(II) at pH >7.5 [the pK(a) of the cysteine thiol in the presence of Hg(II)]. Support for this mechanism was given by the observation of a three-stranded coiled coil by SEC in a solution of Tri L9C at pH 7.0.