metallothionein and 4-(2-pyridylazo)resorcinol

Metallothioneins (MTs) are among others involved in the cellular regulation of essential Zn(II) and Cu(I) ions. However, the high binding affinity of these proteins requires additional factors to promote metal ion release under physiological conditions. The mechanisms and efficiencies of these processes leave many open questions. We report here a comprehensive analysis of the Zn(II)-release properties of various MTs with special focus on members of the four main subfamilies of plant MTs. Zn(II) competition experiments with the metal ion chelator 4-(2-pyridylazo)resorcinol (PAR) in the presence of the cellular redox pair glutathione (GSH)/glutathione disulfide (GSSG) show that plant MTs from the subfamilies MT1, MT2, and MT3 are remarkably more affected by oxidative stress than those from the Ec subfamily and the well-characterized human MT2 form. In addition, we evaluated proteolytic digestion with trypsin and proteinase K as an alternative mechanism for selective promotion of metal ion release from MTs. Also here the observed percentage of liberated metal ions depends strongly on the MT form evaluated. Closer evaluation of the data additionally allowed deducing the thermodynamic and kinetic properties of the Zn(II) release processes. The Cu(I)-form of chickpea MT2 was used to exemplify that both oxidation and proteolysis are also effective ways to increase the transfer of copper ions to other molecules. Zn(II) release experiments with the individual metal-binding domains of Ec-1 from wheat grain reveal distinct differences from the full-length protein. This triggers the question about the roles of the long cysteine-free peptide stretches typical for plant MTs.

Topics: Amino Acid Sequence; Binding, Competitive; Copper; Glutathione; Glutathione Disulfide; Ions; Kinetics; Metallothionein; Metals; Molecular Sequence Data; Oxidation-Reduction; Plant Proteins; Protein Binding; Proteolysis; Resorcinols; Spectrophotometry; Zinc

Fish and mammalian metallothioneins (MTs) differ in the amino acid residues placed between their conserved cysteines. We have expressed the MT of an Antarctic fish, Notothenia coriiceps, and characterized it by means of multinuclear NMR spectroscopy. Overall, the architecture of the fish MT is very similar to that of mammalian MTs. However, NMR spectroscopy shows that the dynamic behaviour of the two domains is markedly different. With the aid of absorption and CD spectroscopies, we studied the conformational and electronic features of fish and mouse recombinant Cd-MT and the changes produced in these proteins by heating. When the temperature was increased from 20 to 90 degrees C, the Cd-thiolate chromophore absorbance at 254 nm of mouse MT was not modified up to 60 degrees C, whereas the absorbance of fish MT decreased significantly starting from 30 degrees C. The CD spectra also changed quite considerably with temperature, with a gradual decrease of the positive band at 260 nm that was more pronounced for fish than for mouse MT. The differential effect of temperature on fish and mouse MTs may reflect a different stability of metal-thiolate clusters of the two proteins. Such a conclusion is also corroborated by results showing differences in metal mobility between fish and mouse Zn-MT.

Topics: Amino Acid Sequence; Animals; Cadmium; Circular Dichroism; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Kinetics; Magnetic Resonance Spectroscopy; Metallothionein; Mice; Molecular Sequence Data; Perciformes; Protein Conformation; Recombinant Proteins; Resorcinols; Sequence Alignment; Spectrophotometry, Atomic; Temperature; Zinc

The alpha- and beta-polypeptides of human metallothionein (isoform 2), obtained by chemical synthesis, were converted into their respective zinc/thiolate clusters, and each domain was investigated separately. Proton titration data for the N-terminal beta-domain fit a simple model with three ionizations of the same apparent pK(a) value of 4.9 and a collective binding constant for zinc of 5 x 10(-12) M at pH 7.0. The zinc cluster in the C-terminal alpha-domain is more stable than that in the beta-domain. Its pH titration is also more complex, indicating at least two classes of zinc sites with different affinities. The whole molecule is stabilized with regard to the individual domains. Chemical modification implicates lysine side chains in both the stabilization of the beta-domain cluster and the mutual stabilization of the domains in the whole molecule. The two zinc clusters also differ in the reactivity of their cysteine sulfurs and their potential to donate zinc to an acceptor molecule dependent on its type and characteristics. The isolated beta-domain cluster reacts faster with Ellman's reagent and is a better zinc donor toward zinc-depleted sorbitol dehydrogenase than is the isolated alpha-domain cluster, whereas the reverse is observed when a chelating agent is the zinc acceptor. Thus, although each cluster assembles independently of the other, the cumulative properties of the individual domains do not suffice to describe metallothionein either structurally or functionally. The two-domain structure of the whole molecule is important for its interaction with ligands and for control of its reactivity and overall conformation.

Topics: Cadmium; Circular Dichroism; Dithionitrobenzoic Acid; Dose-Response Relationship, Drug; Humans; Hydrogen-Ion Concentration; Kinetics; Lysine; Metallothionein; Oxidation-Reduction; Protein Binding; Protein Structure, Tertiary; Resorcinols; Spectrophotometry; Sulfhydryl Reagents; Time Factors; Zinc

The Synechococcus PCC7942 SmtB is a zinc-responsive transcriptional repressor and a member of the ArsR superfamily of prokaryotic metalloregulatory transcription factors. The mechanism of negative regulation by Zn(II) and other metals as well as the coordination chemistry (stoichiometry, affinity, and specificity) of SmtB is poorly understood. In contrast to previous results [Kar, S. R., Adams, A. C., Lebowitz, J., Taylor, K. B., and Hall, L. M. (1997) Biochemistry 36, 15343-15348], we find that fully reduced SmtB binds 1 mol equiv of Zn(II) with a very high affinity, K(Zn) in excess of 10(11) M(-1) (pH 7.4, 0.15 M KCl, 22 degrees C). Optical spectroscopic experiments reveal that SmtB binds 1 mol equiv of Co(II) in a tetrahedral or distorted tetrahedral environment with one or two cysteine thiolate ligands in the first coordination shell. Zn(II) and Co(II) EXAFS studies are consistent with the optical spectroscopic data, and further suggest the presence of a mixture of carboxylate and imidazole-containing ligands. K(Co) was determined to be 1.7 (+/-0.1) x 10(9) M(-1) in a chelator (EGTA) competition assay; 1 equiv of Zn(II) results in complete displacement of the bound Co(II). SmtB also binds 1 mol equiv of Ni(II), which, when formed at low Ni(II):SmtB molar ratios, adopts a non-native, six-coordinate complex characterized by at least two histidine and no thiolate ligands. The hierarchy of metal binding affinities is Zn(II) >> Co(II) >> Ni(II).

Topics: Bacterial Proteins; Binding, Competitive; Cobalt; DNA-Binding Proteins; Fluorescent Dyes; Fura-2; Metallothionein; Nickel; Protein Binding; Repressor Proteins; Resorcinols; Solutions; Spectrophotometry, Atomic; Spectrophotometry, Ultraviolet; Spectrum Analysis; Titrimetry; X-Rays; Zinc

Selenium has been increasingly recognized as an essential element in biology and medicine. Its biochemistry resembles that of sulfur, yet differs from it by virtue of both redox potentials and stabilities of its oxidation states. Selenium can substitute for the more ubiquitous sulfur of cysteine and as such plays an important role in more than a dozen selenoproteins. We have chosen to examine zinc-sulfur centers as possible targets of selenium redox biochemistry. Selenium compounds release zinc from zinc/thiolate-coordination environments, thereby affecting the cellular thiol redox state and the distribution of zinc and likely of other metal ions. Aromatic selenium compounds are excellent spectroscopic probes of the otherwise relatively unstable functional selenium groups. Zinc-coordinated thiolates, e.g., metallothionein (MT), and uncoordinated thiolates, e.g., glutathione, react with benzeneseleninic acid (oxidation state +2), benzeneselenenyl chloride (oxidation state 0) and selenocystamine (oxidation state -1). Benzeneseleninic acid and benzeneselenenyl chloride react very rapidly with MT and titrate substoichiometrically and with a 1:1 stoichiometry, respectively. Selenium compounds also catalyze the release of zinc from MT in peroxidation and thiol/disulfide-interchange reactions. The selenoenzyme glutathione peroxidase catalytically oxidizes MT and releases zinc in the presence of t-butyl hydroperoxide, suggesting that this type of redox chemistry may be employed in biology for the control of metal metabolism. Moreover, selenium compounds are likely targets for zinc/thiolate coordination centers in vivo, because the reactions are only partially suppressed by excess glutathione. This specificity and the potential to undergo catalytic reactions at low concentrations suggests that zinc release is a significant aspect of the therapeutic antioxidant actions of selenium compounds in antiinflammatory and anticarcinogenic agents.

Topics: Animals; Binding Sites; Glutathione; Glutathione Peroxidase; Kinetics; Metallothionein; Models, Chemical; Oxidation-Reduction; Rabbits; Resorcinols; Selenium; Selenium Compounds; Spectrophotometry; Sulfhydryl Compounds; Zinc