Domain-specific fluorescence resonance energy transfer (FRET) sensors of metallothionein/thionein.

Each of the two domains of mammalian metallothioneins contains a zinc-thiolate cluster. Employing site-directed mutagenesis and chemical modification, fluorescent probes were introduced into human metallothionein (isoform 2) with minimal perturbations of the structures of these clusters. The resulting FRET (fluorescence resonance energy transfer) sensors are specific for each domain. The design and construction of a sensor for the alpha-domain cluster is based on a FRET pair where a C-terminally added tryptophan serves as the donor for a fluorescence acceptor attached to a free cysteine in the linker region between the two domains. Molecular modeling studies and steady-state fluorescence polarization anisotropy measurements suggest unrestricted motion of the tryptophan donor, but limited motion of the AEDANS ([[(amino)ethyl]amino]naphthalene-1-sulfonic acid) acceptor, putting constraints on the use of the alpha-domain sensor with this FRET pair as a spectroscopic ruler. The fluorescent metallothioneins allow distance measurements during binding and removal of metals in the individual domains. The overall dimensions of the apoprotein, thionein, for which no structural information is available, do not seem to be significantly different from those of the holoprotein. The single- and double-labeled fluorescent metallothioneins overcome a longstanding impediment in studies of the function of this protein, namely its lack of intrinsic probe characteristics.

Catalytic oxidation of zinc/sulfur coordination sites in proteins by selenium compounds.

Zinc/thiolate (cysteine) coordination occurs in a very large number of proteins. These coordination sites are thermodynamically quite stable. Yet the redox chemistry of thiolate ligands confers extraordinary reactivities on these sites. The significance of such ligand-centered reactions is that they affect the binding and release of zinc, thus helping to distribute zinc, and perhaps controlling zinc-dependent cellular events. One new aspect focuses on the thiolate ligands of zinc as targets for the redox action of selenium compounds. A distinctive feature of this chemistry is the capacity of selenols to catalyze the oxidation of zinc/thiolate sites. We here use a chromophoric compound, 2-nitrophenylselenocyanate, to investigate its reaction mechanism with the zinc/thiolate clusters of metallothionein, a protein that is a cellular reservoir for zinc and together with its apoprotein, thionein, is involved in zinc distribution as a zinc donor/acceptor pair. The reaction is particularly revealing as it occurs in two steps. A selenenylsulfide intermediate is formed in the fast oxidative step, followed by the generation of 2-nitrophenylselenol that initiates the second, catalytic step. The findings demonstrate the high reactivity of selenium compounds with zinc/thiolate coordination sites and the potent catalytic roles that selenoproteins and selenium redox drugs may have in affecting gene expression via modulation of the zinc content of zinc finger proteins.

Catalytic selenols couple the redox cycles of metallothionein and glutathione.

Co-ordination of zinc to the thiol group of cysteine allows mobilization of zinc through oxidation of its ligand. This molecular property links the binding and release of zinc in metallothionein (MT) to the cellular redox state [Maret W. & Vallee B.L. (1998) Proc. Natl Acad. Sci. USA 95, 3483-3488]. Biological disulfides such as glutathione disulfide (GSSG) oxidize MT with concomitant release of zinc, while glutathione (GSH) reduces the oxidized protein to thionein, which then binds to available zinc. Neither of these two redox processes is very efficient, even at high concentrations of GSSG or GSH. However, the GSH/GSSG redox pair can efficiently couple with the MT/thionein system in the presence of a selenium compound that has the capacity to form a catalytic selenol(ate). This coupling provides a very effective means of modulating oxidation and reduction. Remarkably, selenium compounds catalyze the oxidation of MT even under overall reducing conditions such as those prevailing in the cytosol. In this manner, the binding and release of zinc from zinc-thiolate co-ordination sites is linked to redox catalysis by selenium compounds, changes in the glutathione redox state, and the availability of either a zinc donor or a zinc acceptor. The results also suggest that the pharmacological actions of selenium compounds in cancer prevention and other antiviral and anti-inflammatory therapeutic applications, as well as unknown functions of selenium-containing proteins, may relate to coupling between the thiol redox state and the zinc state.

Differential fluorescence labeling of cysteinyl clusters uncovers high tissue levels of thionein.

The isolation of thionein (T) from tissues has not been reported heretofore. T contains 20 cysteinyl residues that react with 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide to form fluorescent adducts. In metallothionein (MT) the cysteinyl residues, which are bound to zinc, do not react. However, they do react in the presence of a chelating agent such as EDTA. The resultant difference in chemical reactivity provides a means to measure T in the absence of EDTA, (MT + T) in its presence, and, of course, MT by difference. The 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide derivative of T can be isolated from tissue homogenates by HPLC and quantified fluorimetrically with a detection limit in the femtomolar range and a linear response over 3 orders of magnitude. Analysis of liver, kidney, and brain of rats reveals almost as much T as MT. Moreover, in contrast to earlier views, MT in tissue extracts appears to be less stable than T. The existence of T in tissues under normal physiological conditions has important implications for its function both in zinc metabolism and the redox balance of the cell.

Enzyme regulation by reversible zinc inhibition: glycerol phosphate dehydrogenase as an example.

Since cellular zinc is not freely available as the inorganic ion, zinc proteins must acquire their metal from some other source. But how, when, and where they acquire it is unknown. Metallothionein can participate in the controlled delivery of zinc by binding it with high stability and by mobilizing it through a novel biochemical mechanism that critically depends on the redox activity of the zinc-sulfur bond. Thus, metallothionein activates zinc-depleted alcohol (sorbitol) dehydrogenases by glutathione-modulated zinc transfer. In addition to its catalytic, co-catalytic, and/or structural roles in a myriad of enzymes, zinc also inhibits some enzymes that are not necessarily zinc enzymes, e.g. glyceraldehyde and glycerol phosphate dehydrogenases, and aldehyde dehydrogenase. Zinc inhibits glycerol phosphate dehydrogenase with an IC(50) value of 100 nM. Zinc binding is slow at low pH, but instantaneous at high pH. Thionein, the apoprotein of metallothionein, re-activates the zinc-inhibited enzyme. Tight inhibition by zinc and activation of glycerol phosphate dehydrogenase by thionein, a biological chelating agent, provide further support that modulation of zinc binding by metallothionein and thionein is a physiological mechanism of enzyme regulation. Since glycerol phosphate dehydrogenase is a key enzyme in energy metabolism, the effect of zinc is expected to elicit significant physiological responses.

Zinc metallothionein imported into liver mitochondria modulates respiration.

Metallothionein (MT) localizes in the intermembrane space of liver mitochondria as well as in the cytosol and nucleus. Incubation of intact liver mitochondria with physiological, micromolar concentrations of MT leads to the import of MT into the mitochondria where it inhibits respiration. This activity is caused by the N-terminal beta-domain of MT; in this system, the isolated C-terminal alpha-domain is inactive. Free zinc inhibits respiration at concentrations commensurate with the zinc content of either MT or the isolated beta-domain, indicating that MT inhibition involves zinc delivery to mitochondria. Respiratory inhibition of uncoupled mitochondria identifies the electron transfer chain as the primary site of inhibition. The apoform of MT, thionein, is an endogenous chelating agent and activates zinc-inhibited respiration with a 1:1 stoichiometry ([zinc binding sites]/[zinc]). Carbamoylation of the lysines of MT significantly attenuates the inhibitory effect, suggesting that these residues are critical for the passage of MT through the outer mitochondrial membrane. Such an import pathway has been proposed for other proteins that also lack a mitochondrial targeting sequence, e.g., apocytochrome c, and possibly Cox17, a mitochondrial copper chaperone that is the only protein known so far to exhibit significant primary sequence homology to MT. The presence and respiratory inhibition of MT in liver, but not heart, mitochondria suggest a hitherto unknown biological modulating activity of MT in cellular respiration and energy metabolism in a tissue-specific manner.

High yield expression and single step purification of human thionein/metallothionein.

Human metallothionein (MT), isoform 2, was expressed in Escherichia coli as an intein (protein splicing element) fusion protein in the absence of added metals and purified by intein-mediated purification with an affinity chitin-binding tag (IMPACT system). This procedure constitutes a novel and simple strategy to prepare thionein (T), the metal-free form, or MT when reconstituting T with metals in vitro. The yield was 8 mg of T or 6 mg of pure Cd(7)- or Zn(7)-MT from a 1-L culture, significantly higher than yields from any other expression system. Purified recombinant protein is indistinguishable from the native protein on the basis of its metal-binding ability, titration of its sulfhydryls, and UV and CD spectra. The MALDI-TOF mass spectrum is consistent with that of T with a free N-terminus.

The function of zinc metallothionein: a link between cellular zinc and redox state.

A chemical and biochemical mechanism of action of the metallothionein (MT)/thionein (T) couple has been proposed. The mechanism emphasizes the importance of zinc/sulfur cluster bonding in MT and the significance of the two cluster networks as redox units that confer mobility on otherwise tightly bound and redox-inert zinc in MT. In this article, it is further explored how this redox mechanism controls the metabolically active cellular zinc pool. The low redox potential of the sulfur donor atoms in the clusters readily allows oxidation by mild cellular oxidants with concomitant release of zinc. Such a release by oxidants and the preservation of zinc binding by antioxidants place MT under the control of the cellular redox state and, consequently, energy metabolism. The binding of effectors, e.g., ATP, elicits conformational changes and alters zinc binding in MT. The glutathione/glutathione disulfide redox couple as well as selenium compounds effect zinc delivery from MT to the apoforms of zinc enzymes. This novel action of selenium on zinc/sulfur coordination sites has significant implications for the interaction between these essential elements. Tight binding and kinetic lability, modulation of MT by cellular ligands and the redox state, control of MT gene expression by zinc and many other inducers all support a critical function of the MT/T system in cellular homeostasis and distribution of zinc.

Zinc transfer potentials of the alpha - and beta-clusters of metallothionein are affected by domain interactions in the whole molecule.

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.

Selenium redox biochemistry of zinc-sulfur coordination sites in proteins and enzymes.

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.

Inhibitory sites in enzymes: zinc removal and reactivation by thionein.

Thionein (T) has not been isolated previously from biological material. However, it is generated transiently in situ by removal of zinc from metallothionein under oxidoreductive conditions, particularly in the presence of selenium compounds. T very rapidly activates a group of enzymes in which zinc is bound at an inhibitory site. The reaction is selective, as is apparent from the fact that T does not remove zinc from the catalytic sites of zinc metalloenzymes. T instantaneously reverses the zinc inhibition with a stoichiometry commensurate with its known capacity to bind seven zinc atoms in the form of clusters in metallothionein. The zinc inhibition is much more pronounced than was previously reported, with dissociation constants in the low nanomolar range. Thus, T is an effective, endogenous chelating agent, suggesting the existence of a hitherto unknown and unrecognized biological regulatory system. T removes the metal from an inhibitory zinc-specific enzymatic site with a resultant marked increase of activity. The potential significance of this system is supported by the demonstration of its operations in enzymes involved in glycolysis and signal transduction.

Ebselen, a selenium-containing redox drug, releases zinc from metallothionein.

Selenium compounds oxidize the thiolate ligands in the zinc clusters of metallothionein and release zinc. This chemistry defines new cellular targets for biological forms of selenium and suggests important interactions between zinc and selenium, two biologically essential elements. In the course of delineating the redox chemistry of biological zinc complexes with thiolate ligands, we have found that the non-toxic experimental drug ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) releases zinc from metallothionein. The reaction follows a 1:1 stoichiometry for thiols, is very rapid (t1/2 < 1 min), and proceeds through the opening of the isoselenazol ring and formation of a selenodisulfide with metallothionein. Despite the fast reaction of ebselen with glutathione (t1/2 < 1 s), which proceeds past the stage of the selenodisulfide adduct to the selenol and diselenide derivatives, ebselen reacts with MT even in the presence of glutathione, suggesting that it can also react with MT in vivo. These findings reveal a new mode of action for ebselen and therefore suggest therapeutic applications in zinc-related medical disorders as well as a possible role of biological selenium compounds in zinc metabolism.

The ATP-metallothionein complex.

We have previously shown that glutathione (GSH) and glutathione disulfide interact with metallothionein (MT) and modulate its capacity to donate and transfer zinc. In this paper, we show that ATP also forms a 1:1 complex with MT (Kd = 176 +/- 33 microM, pH 7. 4) that enhances the transfer of zinc to zinc-depleted sorbitol dehydrogenase, increases the rate of thiol-disulfide interchange with Ellman's reagent [5,5'-dithiobis (Z-nitrobenzoic acid)], and changes the apparent shape of the protein. GTP produces almost identical effects. The corresponding di- or monophosphates and pyrimidine nucleotides, however, neither bind as strongly as ATP nor enhance zinc transfer. Carbamoylation of MT lysines abolishes ATP binding, indicating that these highly conserved residues are part of the binding site. GSH decreases, whereas glutathione disulfide increases, ATP binding. The interaction of MT with two critical cellular ligands, i.e., GSH and ATP, and ensuing effects on zinc transfer and reactivity suggest that MT is not merely a cellular zinc buffer but, rather, actively participates in zinc distribution. Apparently, when isolated, MT lacks two important effectors that affect its redox behavior and function. The magnitude of the binding constant and the cellular concentration of ATP indicate that in the cell MT could be essentially saturated with ATP at low concentrations of GSH. Both the redox and energy states of the cell seem to control zinc distribution from MT, but their relative contributions require further studies.

Thiolate ligands in metallothionein confer redox activity on zinc clusters.

We postulate a novel and general mechanism in which the redox-active sulfur donor group of cyst(e)ine confers oxidoreductive characteristics on stable zinc sites in proteins. Thus, the present, an earlier, and accompanying manuscripts [Maret, W., Larsen, K. S. & Vallee, B. L. (1997) Proc. Natl. Acad. Sci. USA 94, 2233-2237; Jiang, L.-J., Maret, W. & Vallee, B. L. (1998) Proc. Natl. Acad. Sci. USA 95, 3483-3488; and Jacob, C., Maret, W. & Vallee, B. L. (1998) Proc. Natl. Acad. Sci. USA 95, 3489-3494] demonstrate that the interactive network featuring multiple zinc/sulfur bonds as found in the clusters of metallothionein (MT) constitutes a coordination unit critical for the concurrent oxidation of cysteine ligands and the ensuing release of zinc. The low position of MT (<-366 mV) on a scale of redox reagents allows its effective oxidation by relatively mild cellular oxidants, in particular disulfides. When MT is exposed to an excess of dithiodipyridine, all of its 20 cysteines are oxidized within 1 hr with the concomitant release of all 7 zinc atoms; similarly, the thiol/disulfide oxidoreductase DsbA reacts stoichiometrically with MT to release zinc. Zinc and sulfur ligands in the clusters are in a spatial arrangement that seemingly favors disulfide bond formation. Jointly, this and the above-mentioned manuscripts conclude that the control of cellular zinc distribution as a function of the energy state of the cell is the long sought role of MT. This specific MT function renders dubious the widely held belief that MT primarily scavenges radicals or detoxifies metals and is consistent with the frequent use of cysteine as a zinc ligand in proteins as a means of both tight and weak zinc binding of thiols and disulfides, respectively. Thus, we relate changes in the reducing power of the cell to the stability of the zinc/sulfur network in MT and the relative mobility of zinc and its control.

The glutathione redox couple modulates zinc transfer from metallothionein to zinc-depleted sorbitol dehydrogenase.

The release and transfer of zinc from metallothionein (MT) to zinc-depleted sorbitol dehydrogenase (EC 1.1.1.14) in vitro has been used to explore the role of MT in cellular zinc distribution. A 1:1 molar ratio of MT to sorbitol dehydrogenase is required for full reactivation, indicating that only one of the seven zinc atoms of MT is transferred in this process. Reduced glutathione (GSH) and glutathione disulfide (GSSG) are critical modulators of both the rate of zinc transfer and the ultimate number of zinc atoms transferred. GSSG increases the rate of zinc transfer 3-fold, and its concentration is the major determinant for efficient zinc transfer. GSH has a dual function. In the absence of GSSG, it inhibits zinc transfer from MT, indicating that MT is in a latent state under the relatively high cellular concentrations of GSH. In addition, it primes MT for the reaction with GSSG by enhancing the rate of zinc transfer 10-fold and by increasing the number of zinc atoms transferred to four. 65Zn-labeling experiments confirm the release of one zinc from MT in the absence of glutathione and the more effective release of zinc in the presence of GSH and GSSG. In vivo, MT may keep the cellular concentrations of free zinc very low and, acting as a temporary cellular reservoir, release zinc in a process that is dynamically controlled by its interactions with both GSH and GSSG. These results suggest that a change of the redox state of the cell could serve as a driving force and signal for zinc distribution from MT.

Control of zinc transfer between thionein, metallothionein, and zinc proteins.

Metallothionein (MT), despite its high metal binding constant (KZn = 3.2 x 10(13) M-1 at pH 7.4), can transfer zinc to the apoforms of zinc enzymes that have inherently lower stability constants. To gain insight into this paradox, we have studied zinc transfer between zinc enzymes and MT. Zinc can be transferred in both directions-i.e., from the enzymes to thionein (the apoform of MT) and from MT to the apoenzymes. Agents that mediate or enhance zinc transfer have been identified that provide kinetic pathways in either direction. MT does not transfer all of its seven zinc atoms to an apoenzyme, but apparently contains at least one that is more prone to transfer than the others. Modification of thiol ligands in MT zinc clusters increases the total number of zinc ions released and, hence, the extent of transfer. Aside from disulfide reagents, we show that selenium compounds are potential cellular enhancers of zinc transfer from MT to apoenzymes. Zinc transfer from zinc enzymes to thionein, on the other hand, is mediated by zinc-chelating agents such as Tris buffer, citrate, or glutathione. Redox agents are asymmetrically involved in both directions of zinc transfer. For example, reduced glutathione mediates zinc transfer from enzymes to thionein, whereas glutathione disulfide oxidizes MT with enhanced release of zinc and transfer of zinc to apoenzymes. Therefore, the cellular redox state as well as the concentration of other biological chelating agents might well determine the direction of zinc transfer and ultimately affect zinc distribution.

Coordination dynamics of biological zinc "clusters" in metallothioneins and in the DNA-binding domain of the transcription factor Gal4.

The almost universal appreciation for the importance of zinc in metabolism has been offset by the considerable uncertainty regarding the proteins that store and distribute cellular zinc. We propose that some zinc proteins with so-called zinc cluster motifs have a central role in zinc distribution, since they exhibit the rather exquisite properties of binding zinc tightly while remaining remarkably reactive as zinc donors. We have used zinc isotope exchange both to probe the coordination dynamics of zinc clusters in metallothionein, the small protein that has the highest known zinc content, and to investigate the potential function of zinc clusters in cellular zinc distribution. When mixed and incubated, metallothionein isoproteins-1 and -2 rapidly exchange zinc, as demonstrated by fast chromatographic separation and radiometric analysis. Exchange kinetics exhibit two distinct phases (k(fast) approximately 5000 min(-1) x M(-1); k(slow) approximately 200 min(-1) x M(-1), pH 8.6, 25 degrees C) that are thought to reflect exchange between the three-zinc clusters and between the four-zinc clusters, respectively. Moreover, we have observed and examined zinc exchange between metallothionein-2 and the Gal4 protein (k approximately 800 min(-1) x M(-1), pH 8.0, 25 degrees C), which is a prototype of transcription factors with a two-zinc cluster. This reaction constitutes the first experimental example of intermolecular zinc exchange between heterologous proteins. Such kinetic reactivity distinguishes zinc in biological clusters from zinc in the coordination environment of zinc enzymes, where the metal does not exchange over several days with free zinc in solution. The molecular organization of these clusters allows zinc exchange to proceed through a ligand exchange mechanism, involving molecular contact between the reactants.