Absorption, circular dichroism, magnetic circular dichroism and emission study of rat kidney Cd,Cu-metallothionein

Absorption, circular dichroism (CD), magnetic circular dichroism (MCD) and emission spectra of rat liver and rat kidney cadmium-, zinc- and copper-containing metallothioneins (MT) are reported. The absorption, CD and MCD data of native rat kidney Cd,Cu-MT protein closely resemble data recorded for the rat liver Cd,Zn-MT. This suggests that the major features in all three spectra of the native Cd,Cu-MT are dominated by cadmium-related bands. The CD spectrum of the Cd,Cu-MT recorded at pH 2.7 has the same band envelope that is observed for a Cd,Cu-MT formed in vitro by titration of Cd,Zn-MT with Cu(I), suggesting that the copper occupies the zinc sites in Cd,Cu-MT formed both in vivo and, at low molar ratios, in vitro. Remetallalion of the metallothionein from low pH in the presence of both copper and cadmium results in considerably less cadmium bound to the protein than was present in the native sample. It is suggested that this is due to the effect of the distribution of the copper amongst all available binding sites, thus inhibiting cluster formation by the cadmium. Emission spectra are reported for the first time for a cadmium- and copper-containing metallothionein. An emission band at 610 nm is shown to be a sensitive indicator of Cu(I) binding to metallothionein. Both the native Cd,Cu-MT and a Cd,Cu-MT formed in vitro exhibit an excitation spectrum with a band in the copper-thiolate charge-transfer region.     

Characterization of the cadmium(II) binding site in Cd,Zn-metallothionein by magnetic circular dichroism spectroscopy

Absorption and magnetic circular dichroism spectra of rat liver Cd, Zn-metallothionein, and the cadmium complexes of propanethiolate and 1,2 propanedithiolate are reported. Observation of the same derivative-like MCD signal in the 250 nm region of each of these species provides experimental evidence for the assignment of the 250 nm shoulder in the Cd, Zn-metallothionein absorption spectrum as a sulfur to cadmium charge transfer band.     

Spectroscopic studies on cadmium (II)- and cobalt(II)-substituted metallothionein from the crab Cancer pagurus. Evidence for one additional low-affinity metal-binding site

The binding of diamagnetic Cd(II) and paramagnetic Co(II) ions to the metal-free form of crab, Cancer pagurus, metallothionein (MT) was studied by various spectroscopic techniques. Both reconstituted and native Cd(II)-MT containing 6 mol Cd(II)/mol protein display electronic absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra which were indistinguishable. The stoichiometric replacement of Cd(II) ions in native Cd(II)6-MT by paramagnetic Co(II) ions enabled the geometry of the metal-binding sites to be probed. The electronic absorption and MCD spectra of Co(II)6-MT revealed features characteristic of distorted tetrahedral tetrathiolate Co(II) coordination for all six metal-binding sites. The stepwise incorporation of Cd(II) and Co(II) ions into this protein was monitored by electronic absorption and CD, and by electronic absorption and EPR spectroscopy, respectively. The results indicate that the metal-thiolate cluster structure is generated when more than four metal ions are bound. Below this titration point separate tetrahedral tetrathiolate complexes exist. This suggests that the cluster formation occurs in a two-step process. Furthermore, the spectroscopic features in both Cd(II)- and Co(II)-metal derivatives above the full metal occupancy of six suggest the existence of one additional metal-binding site. The subsequent loss of one Cd(II) ion from crab Cancer Cd(II)7-MT in the gel filtration studies demonstrate the low metal-binding affinity of the latter site. While the spectroscopic properties indicate an exclusively tetrahedral type of metal-thiolate sulfur coordination for the binding of the first six metal ions, they suggest that the seventh metal ion is coordinated in a different fashion.     

Cadmium binding to metallothioneins and the estimation of protein concentration using cadmium-saturation methods

The detailed spectral changes observed in the absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra upon addition of Cd²⁺ to rat liver Cd, Zn-metallothionein (MT) are reported. Results from dialysis experiments clearly demonstrate that up to 8.6 mole equivalents of Cd²⁺ can be bound to this protein. The excess Cd²⁺ ions bound appear to have lower binding constants than those of the first seven Cd²⁺ ions bound. Red blood cell hemolysate (RBC) can compete with the metallothionein for all Cd²⁺ bound in excess of seven mole equivalents. Thus the RBC hemolysate method of estimating protein concentrations is shown to be correct when based upon complete loading of all binding sites in MT with Cd²⁺.     

Cadmium binding to metallothioneins. Domain specificity in reactions of alpha and beta fragments, apometallothionein, and zinc metallothionein with Cd2+

The cadmium-binding properties of rabbit liver Zn7-metallothionein (MT) 2 and apo-MT, rat liver apo-alpha MT and Zn4-alpha MT, and calf liver apo-beta MT, have been studied using circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies. Both sets of spectra recorded during the titration of Zn7-MT 2 with Cd2+ exhibit a complicated pattern that is quite unexpected. Such behavior is not found at all in sets of spectra recorded during titrations of the apo-species (apo-MT, apo-alpha MT, and apo-beta MT), and is observed to a much lesser extent in the titration of Zn-alpha MT. Comparison between the band centers of the Cd-alpha MT and Cd-beta MT indicates that the CD spectrum of Cd7-MT is dominated by intensity from transitions that originate on Cd-S chromophores in the alpha domain, with little direct contribution from the beta domain. Analysis of the spectra recorded during titrations of Zn7-MT 2 with Cd2+ suggests: (i) that Cd2+ replaces Zn2+ in Zn7-MT isomorphously; (ii) that cadmium binds in a nonspecific, "distributed" manner across both domains; (iii) that cluster formation in the alpha domain only occurs after 4 mol eq of cadmium have been added and is indicated by the presence of a cluster-sensitive, CD spectral feature; (iv) that the characteristic derivative CD spectrum of native Cd4,Zn3-MT is only obtained from "synthetic" Cd4,Zn3-MT following a treatment cycle that allows the redistribution of cadmium into the alpha domain; warming the synthetic "native," Cd4,Zn3-MT, to 65 degrees C results in cadmium being preferentially bound in the alpha domain; and (v) Zn7-MT will bind Cd2+ quite normally at up to 65 degrees C but with greater specificity for the alpha domain compared with titrations carried out at 25 degrees C. These results suggest that the initial presence of zinc in both domains is an important factor in the lack of any domain specificity during cadmium binding to Zn-MT which contrasts the domain specific manner observed for cadmium binding to apo-MT.     

Evidence for noncooperative metal binding to the alpha domain of human metallothionein

In the present study, we investigated the metal-binding reactivity of the isolated alpha domain of human metallothionein isoform 1a, with specific emphasis on resolving the debate concerning the cooperative nature of the metal-binding mechanism. The metallation reaction of the metal-free alpha domain with Cd2+ was unequivocally shown to proceed by a noncooperative mechanism at physiologic pH by CD and UV absorption spectroscopy and ESI MS. The data clearly show the presence of intermediate partially metallated metallothionein species under limiting Cd2+ conditions. Titration with four molar equivalents of Cd2+ was required for the formation of the Cd4alpha species in 100% abundance. The implications of a noncooperative metal-binding mechanism are that the partially metallated and metal-free species are stable intermediates, and thus may have a potential role in the currently undefined function of metallothionein.     

Electrospray ionization mass spectrometry of zinc, cadmium, and copper metallothioneins: evidence for metal-binding cooperativity

Electrospray ionization (ESI) mass spectra of both well-characterized and novel metallothioneins (MTs) from various species were recorded to explore their metal-ion-binding modes and stoichiometries. The ESI mass spectra of the zinc- and cadmium-binding MTs showed a single main peak corresponding to metal-to-protein ratios of 4, 6, or 7. These findings combined with data obtained by other methods suggest that these MTs bind zinc or cadmium in a single predominant form and are consistent with the presence of three- and four-metal clusters. An unstable copper-specific MT isoform from Roman snails (Helix pomatia) could be isolated intact and was shown to preferentially bind 12 copper ions. To obtain additional information on the formation and relative stability of metal-thiolate clusters in MTs, a mass spectrometric titration study was conducted. One to seven molar equivalents of zinc or of cadmium were added to metal-free human MT-2 at neutral pH, and the resulting complexes were measured by ESI mass spectrometry. These experiments revealed that the formation of the four-metal cluster and of the thermodynamically less stable three-metal cluster is sequential and largely cooperative for both zinc and cadmium. Minor intermediate forms between metal-free MT, Me4MT, and fully reconstituted Me7MT were also observed. The addition of increasing amounts of cadmium to metal-free blue crab MT-I resulted in prominent peaks whose masses were consistent with apoMT, Cd3MT, and Cd6MT, reflecting the known structure of this MT with two Me3Cys9 centers. In a similar reconstitution experiment performed with Caenorhabditis elegans MT-II, a series of signals corresponding to apoMT and Cd3MT to Cd6MT species were observed.     

Peptide folding, metal-binding mechanisms, and binding site structures in metallothioneins

This minireview specifically focuses on recent studies carried out on structural aspects of metal-free metallothionein (MT), the mechanism of metal binding for copper and arsenic, structural studies using x-ray absorption spectroscopy and molecular mechanics modeling, and speciation studies of a novel cadmium and arsenic binding algal MT. Molecular mechanics-molecular dynamics calculations of apo-MT show that significant secondary structural features are retained by the polypeptide backbone upon sequential removal of the metal ions, which is stabilized by a possible H-bonding network. In addition, the cysteinyl sulfurs were shown to rotate from within the domain core, where they are found in the metallated state, to the exterior surface of the domain, suggesting an explanation for the rapid metallation reactions that were measured. Mixing Cu6beta-MT with Cd4alpha-MT and Cu6alpha-MT with Cd3beta-MT resulted in redistribution of the metal ions to mixed metal species in each domain; however, the Cu+ ions preferentially coordinated to the beta domain in each case. Reaction of As3+ with the individual metal-free beta and alpha domains of MT resulted in three As3+ ions coordinating to each of the domains, respectively, in a proposed distorted trigonal pyramid structure. Kinetic analysis provides parameters that allow simulation of the binding of each of the As3+ ions. X-ray absorption spectroscopy provides detailed information about the coordination environment of the absorbing element. We have combined measurement of x-ray absorption near edge structure (XANES) and extended x-ray absorption fine structure (EXAFS) data with extensive molecular dynamics calculations to determine accurate metal-thiolate structures. Simulation of the XANES data provides a powerful technique for probing the coordination structures of metals in metalloproteins. The metal binding properties of an algal MT, Fucus vesiculosus, has been investigated by UV absorption and circular dichroism spectroscopy and electrospray ionization-mass spectrometry. The 16 cysteine residues of this algal MT were found to coordinate six Cd2+ ions in two domains with stoichiometries of a novel Cd3S7 cluster and a beta-like Cd3S9 cluster.     

Mutation of invariant cysteines of mammalian metallothionein alters metal binding capacity, cadmium resistance, and 113Cd NMR spectrum.

Using a yeast expression vector system, we have expressed both wild type and six mutated Chinese hamster metallothionein coding sequences in a metal-sensitive yeast strain in which the endogenous metallothionein gene has been deleted. The mutant proteins have single or double cysteine to tyrosine replacements (C13Y, C50Y, and C13,50Y), single cysteine to serine replacements (C13S and C50S), or a single cysteine to alanine replacement (C50A). These proteins function in their yeast host in cadmium detoxification to differing extents. Metallothioneins which contain a cysteine mutation at position 50 (C50Y, C50S, C50A, and C13,50Y) conferred markedly less cadmium resistance than wild type metallothionein, or metallothionein with a single cysteine mutation at position 13 (C13Y and C13S). Wild type and three of the mutant Chinese hamster metallothioneins (C13Y, C50Y, and C13,50Y) were purified from yeast grown in subtoxic levels of either CdCl2 or 113CdCl2. All three of the mutant proteins bound less cadmium than the wild type protein when metal-binding stoichiometries were determined. The one-dimensional 113Cd NMR spectrum of the recombinant wild type Chinese hamster metallothionein was compared to the spectra of native rat and rabbit liver metallothioneins. The close correspondence between the 113Cd chemical shifts in these metallothioneins is consistent with the presence of two separate metal clusters, A and B, corresponding, respectively, to the alpha- and beta-domains, in the recombinant metallothionein. The one-dimensional 113Cd NMR spectra recorded on each of the three mutant metallothioneins, on the other hand, provide some indication as to the structural basis for the reduced, by one, metal stoichiometry of each of the mutant metallothioneins. For the C13Y mutant, it appears that the beta-domain now binds a total of two metal ions whereas with the C50Y mutant, the alpha-domain appears metal-deficient. For the double mutant, C13,50Y, the 113Cd resonances are indicative of major structural reorganizations in both domains.     

Identification of a cadmium-binding protein from a cadmium-resistant variant of human lymphoblastoid cells (WI-L2)

Cadmium-binding protein synthesis and induction by cadmium chloride were studied in the human lymphoblastoid cell line WI-L2. Lymphoblasts were adapted to growth in 5 microM cadmium chloride (Cdr) and these cells were 2.5-fold more resistant to cadmium than the parental line. There was no difference in the cellular protein profile between the parental line and lymphoblasts grown for a short period, less than 10 days, in cadmium chloride as measured by [35S]cysteine labelling and SDS-polyacrylamide gel electrophoresis. A basal level of cadmium binding protein was apparent, however, by gel filtration. The Cdr lymphoblasts were found to synthesize a substantial amount of cadmium-binding protein, approximately 25-fold more than the parental line. The cadmium-binding protein has the following properties which are consistent with its being a metallothionein: (1) [35S]Cysteine-labelled protein eluted at a Ve/Vo = 2.1 on a Sephadex G-75 column; (2) the molecular weight was estimated as 11 kDa on 7-17% SDS polyacrylamide gels; (3) the protein was heat-stable; (4) the unlabelled protein bound 109Cd2+.     

Spectroscopic characterization of rat kidney Hg,Cu-metallothionein

Absorption, circular dichroism (CD), magnetic circular dichroism (MCD) and emission spectra are reported for rat kidney Hg,Cu-metallothionein isoform 3 isolated following induction of the metallothionein with HgCl2. While the absorption spectrum is featureless, both the CD and MCD spectra show resolved bands that arise from the Cu-thiolate and Hg-thiolate groups. The emission spectrum at 77 K is much more complicated than would be expected for a copper (I)-containing metallothionein. It is suggested the emission only arises from the copper-thiolate groups but that the presence of the mercury results in copper ions in several different environments depending on the nature of the nearest neighbour.     

Metal-binding proteins of the Syrian hamster ovaries: apparent deficiency of metallothionein

A deficiency of metallothionein, a high-affinity metal-binding protein thought to detoxify cadmium, has been observed in rat and mouse testes, tissues that are highly susceptible to the necrotizing and carcinogenic effects of cadmium. Like the testes, the ovaries undergo a hemorrhagic necrosis when exposed to cadmium, and female Syrian hamsters have recently been shown to be highly susceptible to cadmium. However, the nature of cadmium-binding proteins in the ovary is unknown; thus, this study was undertaken to define the nature of any such proteins in the Syrian hamster ovary. A low molecular weight (Mr) zinc- and cadmium-binding protein was detected in cytosol derived from the ovaries after gel filtration that eluted with a relative elution volume similar to authentic metallothionein. This protein was extractable by heat-treatment and sequential acetone precipitation. When such extracts were further purified with a reverse phase high performance liquid chromatography (HPLC) technique developed for the isolation of metallothionein isoforms, two forms were separated. However, neither of these could be classified as metallothionein on the basis of amino acid composition, since both were particularly low in cysteine, a very common amino acid in metallothionein. The ovarian protein also contained significant amounts of aromatic amino acids, unlike metallothionein--which is devoid of aromatics, and contained much more glutamate than metallothionein. Hamsters were also made resistant to cadmium-induced ovarian necrosis by zinc treatment. Such zinc treatment, however, did not alter levels of this protein, yet caused a marked induction of hepatic metallothionein. Likewise, cadmium treatment did not increase the levels of the ovarian metal-binding protein yet markedly induced hepatic metallothionein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metal selectivity of clusters in rabbit liver metallothionein.

In mammalian metallothioneins the metals are organized in two adamantane-type clusters with three and four metal ions which are tetrahedrally coordinated by thiolate ligands. The metal selectivity of the metal-thiolate clusters in rabbit liver metallothionein has been studied by offering two ions, i.e. Co(II)/Cd(II), Zn(II)/Cd(II) or Co(II)/Zn(II), to the metal-free protein. The heterogeneous metal complexes thus formed were characterized by electronic absorption, magnetic circular dichroism. 113Cd-NMR and EPR spectroscopy. In the case of Co/Cd-metallothionein, homometallic cluster occupation occurs, with the Cd(II) ions bound exclusively to the four-metal cluster. In contrast, heterometallic clusters were formed for both Zn/Cd- and Co/Zn-metallothionein. Based on evidence from corresponding inorganic structures of adamantane metal-thiolate cages, it is suggested that the major factor governing the cluster type is the protein structure perturbation due to the cluster volume variations. Thus, while metal thiolate affinities are important in the folding process, size-match selectivity is the dominant factor in the metal-loaded protein.     

Properties of cadmium-binding proteins in rat testes. Characteristics unlike metallothionein.

Since the exposure of rats to cadmium causes zinc to accumulate in metallothionein in liver and kidney but not in a similar protein in the testes, the properties of the low-Mr cadmium-binding proteins were investigated in rat testes. Weanling rats that had been given dietary cadmium for 6 weeks were injected with 109CdCl2 and subsequently killed, and the 109Cd-labelled low-Mr proteins from testes were purified. The pooled low-Mr cadmium-containing fractions from the gel-filtration (Sephadex G-75) columns were eluted through DEAE-Sephacel columns, yielding two peaks. Each of the individual peaks from this Sephacel column was further purified by rechromatography on DEAE-Sephacel and on Bio-Gel P-10 columns. Amino acid analysis of the two purified proteins revealed a low cysteine (about 3%) content, with aspartate, glutamate and glycine as the predominant amino acids. Thus these low-Mr cadmium-binding proteins induced by cadmium in rat testes do not appear to be metallothionein.     

Characterization of cadmium-binding proteins detected in rat liver by the western blotting technique

Out of the three cadmium-binding proteins (CD-BPs) in rat liver parenchyma (40K, 29K, and 24K CdBPs), the 40K Cd-BP showed the highest affinity for cadmium (Cd), with a dissociation constant (K_D) of 1.2 × 10^?8M. This is in between the affinity of human serum albumin K_D = 3.8 × 10^?5M) and metallothionein (KD = < 10^?11). These Cd-BPs may be responsible for hepatic sequestration of cadmium.     

Metal exchange in metallothioneins: a novel structurally significant Cd(5) species in the alpha domain of human metallothionein 1a

Metallothioneins (MTs) are cysteine-rich, metal-binding proteins known to provide protection against cadmium toxicity in mammals. Metal exchange of Zn(2+) ions for Cd(2+) ions in metallothioneins is a critical process for which no mechanistic or structural information is currently available. The recombinant human alpha domain of metallothionein isoform 1a, which encompasses the metal-binding cysteines between Cys33 and Cys60 of the alpha domain of native human metallothionein 1a, was studied. Characteristically this fragment coordinates four Cd(2+) ions to the 11 cysteinyl sulfurs, and is shown to bind an additional Cd(2+) ion to form a novel Cd(5)alpha-MT species. This species is proposed here to represent an intermediate in the metal-exchange mechanism. The ESI mass spectrum shows the appearance of charge state peaks corresponding to a Cd(5)alpha species following addition of 5.0 molar equivalents of Cd(2+) to a solution of Cd(4)alpha-MT. Significantly, the structurally sensitive CD spectrum shows a sharp monophasic peak at 254 nm for the Cd(5)alpha species in contrast to the derivative-shaped spectrum of the Cd(4)alpha-MT species, with peak maxima at 260 nm (+) and 240 nm (-), indicating Cd-induced disruption of the exciton coupling between the original four Cd(2+) ions in the Cd(4)alpha species. The (113)Cd chemical shift of the fifth Cd(2+) is significantly shielded (approximately 400 p.p.m.) when compared with the data for the Cd(2+) ions in Cd(4)alpha-MT by both direct and indirect (113)Cd NMR spectroscopy. Three of the four original NMR peaks move significantly upon binding the fifth cadmium. Evidence from indirect (1)H-(113)Cd HSQC NMR spectra suggests that the coordination environment of the additional Cd(2+) is not tetrahedral to four thiolates, as is the case with the four Cd(2+) ions in the Cd(4)alpha-MT, but has two thiolate ligands as part of its ligand environment, with additional coordination to either water or anions in solution.     

Domain specificity in metal binding to metallothionein. A circular dichroism and magnetic circular dichroism study of cadmium and zinc binding at temperature extremes

Rabbit liver Zn metallothionein-(MT) will bind cadmium readily between -26 degrees C and 70 degrees C. The binding reaction was monitored by recording the circular dichroism and magnetic circular dichroism spectra, in the region of the RS(-)----Cd2+ charge transfer transition at 250 nm, at intervals as aliquots of cadmium were added. For all temperatures, these data can be analyzed in terms of a distributed mechanism for cadmium binding when Zn-MT is used, and a domain-specific mechanism when apo-MT is used. The CD spectrum measured at -26 degrees C for Cd,Zn-MT, which was made by adding excess cadmium directly to Zn7-MT at -26 degrees C, is not the same as the CD spectrum of Cd-MT prepared at room temperature from the same Zn7-MT. Measurements of the stoichiometry of the cadmium and zinc bound to MT in the presence of excess cadmium at different temperatures indicates that below 5 degrees C at least one zinc atom remains bound to the protein. The mixed metal metallothionein, Cd/Zn-MT, that always forms below 5 degrees C, is characterized by a single maximum near 250 nm in the CD spectrum, rather than the derivative-shaped CD envelope that is diagnostic of the (Cd4-S11)alpha cluster, which indicates that the zinc occupies a site in the alpha domain. Rearrangement of the bound metals to the domain-specific distribution takes place if Cd,Zn-MT, prepared at subzero temperatures, is warmed above 30 degrees C.     

Spectroscopic properties of the cobalt(II)-substituted alpha-fragment of rabbit liver metallothionein

The C-terminal segment of rabbit liver metallothionein 1 (alpha-fragment) containing four paramagnetic Co(II) ions was obtained by stoichiometric replacement of the originally bound diamagnetic Cd(II) ions. The latter form was prepared by limited proteolysis with subtilisin as described previously [Winge, D. R., & Miklossy, K. A. (1982) J. Biol. Chem. 257, 3471-3476]. Electronic absorption, magnetic circular dichroism (MCD), and electron paramagnetic resonance (EPR) measurements were employed to monitor the stepwise incorporation of Co(II) ions into the metal-free fragment. Absorption and MCD spectra of the apofragment containing the first 3 Co(II) equiv show the typical features of tetrahedral tetrathiolate Co(II) coordination. However, in the d-d region only small changes in the visible and no apparent change in the near-infrared region are discernible when the fourth Co(II) is bound. This unusual spectral behavior was not seen in Co(II) substitution of native metallothionein [Vasák, M., & K?gi, J. H. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6709-6713] and may indicate a different cluster geometry. In the charge-transfer region, the binding of all 4 Co(II) equiv is accompanied by characteristic increments of the thiolate S----Co(II) bands. As in the formation of Co(II)7-metallothionein, the development of the charge-transfer and EPR spectral properties upon binding of the first 2 Co(II) equiv to the apofragment is indicative of isolated, noninteracting tetrahedral tetrathiolate Co(II) complexes. The binding of the additional Co(II) ion is accompanied by a red shift in the charge-transfer region and by the dramatic loss of paramagnetism in the EPR spectra, both diagnostic of the formation of metal-thiolate cluster structures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cadmium binding studies to the earthworm Lumbricus rubellus metallothionein by electrospray mass spectrometry and circular dichroism spectroscopy

The earthworm Lumbricus rubellus has been found to inhabit cadmium-rich soils and accumulate cadmium within its tissues. Two metallothionein (MT) isoforms (1 and 2) have been identified and cloned from L. rubellus. In this study, we address the metalation status, metal coordination, and structure of recombinant MT-2 from L. rubellus using electrospray ionization mass spectrometry (ESI-MS), UV absorption, and circular dichroism (CD) spectroscopy. This is the first study to show the detailed mass and CD spectral properties for the important cadmium-containing earthworm MT. We report that the 20-cysteine L. rubellus MT-2 binds seven Cd(2+) ions. UV absorption and CD spectroscopy and ESI-MS pH titrations show a distinct biphasic demetalation reaction, which we propose results from the presence of two metal-thiolate binding domains. We propose stoichiometries of Cd(3)Cys(9) and Cd(4)Cys(11) based on the presence of 20 cysteines split into two isolated regions of the sequence with 11 cysteines in the N-terminal and 9 cysteines in the C-terminal. The CD spectrum reported is distinctly different from any other metallothionein known suggesting quite different binding site structure for the peptide.     

A unique low frequency Raman band associated with metal binding to metallothionein

We find that the low frequency Raman spectrum of Zn(II) metallothionein has a single prominent band at 138 cm-1 which is absent from the Raman spectrum of the metal-free protein. This feature is also found for Cd(II) binding to both of the independent metallothionein domains and the metallothionein from Neurospora crassa. TcO(III) coordination to metallothionein results in a similar Raman band which is also found for the complex (Ph4As)[ReO(SCH2CH2S)2]. By comparing these results to literature data for metal-thiolate complexes, this feature is identified as a bending vibration which appears to be characteristic of metal ion coordination by the metallothionein cysteines. Two likely assignments are a symmetric metal-centered mode (delta S-M-S) or a bending mode of the metal-coordinated cysteine thiolates (delta M-S-C).