Order of metal binding in metallothionein

Purified isoforms of rat liver apometallothionein were reconstituted in vitro with Cd and Zn ions to study the order of binding of the 7 metal sites in the two separate metal clusters, one containing four metal ions (cluster A) and the other containing three (cluster B). Reconstitution with 7 Cd ions resulted in a metalloprotein similar to induced Cd,Zn-metallothionein by the criteria of electrophoretic mobility, insensitivity to proteolysis by subtilisin, and the pH-dependent release of Cd. Proteolytic digestion of metallothionein reconstituted with suboptimal quantities of Cd followed by separation of Cd-containing polypeptide fragments by electrophoresis and chromatography revealed metal ion binding initially occurs in the 4-metal center, cluster A. Upon saturation of the 4 sites in cluster A, binding occurs in the 3-metal center, cluster B. Samples reconstituted with 1 to 4 Cd ions per protein molecule, followed by digestion with subtilisin, yielded increasing amounts of a proteolytically stable polypeptide fragment identical with the alpha fragment domain that is known to encompass the 4-metal center. Samples renatured with 5 to 7 Cd ions per metallothionein molecule showed decreasing quantities of alpha fragment and increasing amounts of native-like metallothionein. Similar results were obtained in reconstitution studies with Zn ions. Samples reconstituted with 7 Cd eq followed by incubation with EDTA revealed that cluster B Cd ions were removed initially. The binding process in each domain is cooperative. Reconstitution of apometallothionein with 2 Cd ions followed by proteolysis yields a 50% recovery of saturated Cd4 alpha cluster. Likewise, when Cd5-renatured metallothionein was digested with subtilisin, 30% of the molecules were identified as Cd7 metallothionein with the remainder as Cd4 alpha fragment.     

Comparative 113Cd-n.m.r. studies on rabbit 113Cd7-, (Zn1,Cd6)- and partially metal-depleted 113Cd6-metallothionein-2a.

Rabbit 113Cd7-metallothionein-2a (MT) contains two metal-thiolate clusters of three (cluster B) and four (cluster A) metal ions. The 113Cd-n.m.r. spectrum of 113Cd6-MT, isolated from 113Cd7-MT upon treatment with EDTA, is similar to that of 113Cd7-MT, but the cluster B resonances are lower in intensity, suggesting its co-operative metal depletion. (Zn1,113Cd6)-MT, formed upon addition of the Zn(II) ions to 113Cd6-MT, shows 113Cd-n.m.r. features characteristic of cluster B populations containing both Cd(II) and Zn(II) ions. The overall intensity gain of the mixed cluster B resonances per Cd as to those in 113Cd6- and 113Cd7-MT suggests a stabilization effect of the bound Zn(II) ions upon the previously established intramolecular 113Cd exchange within this cluster.     

113Cd NMR studies on metal-thiolate cluster formation in rabbit Cd(II)-metallothionein: evidence for a pH dependence

The formation of two metal-thiolate clusters in rabbit liver metallothionein 2 (MT) has been examined by 113Cd NMR spectroscopy at pH 7.2 and 8.6. The chemical shifts of the 113Cd resonances developing in the course of apoMT titration with 113Cd(II) ions have been compared with those of fully metal occupied 113Cd7-MT. At pH 7.2 and at low metal occupancy (less than 4), a cooperative formation of the four-metal cluster (cluster A) occurs. Further addition of 113Cd(II) ions generates all the resonances of the three-metal cluster (cluster B) in succession, suggesting cooperative metal binding to this cluster also. In contrast, similar studies at pH 8.6, at low metal occupancy (less than 4), reveal a broad NMR signal centered at 688 ppm. This observation indicates that an entirely different protein structure exists. When exactly 4 equiv of 113Cd(II) are bound to apoMT, the 113Cd NMR spectrum changes to the characteristic spectrum of cluster A. Further addition of 113Cd(II) ions again leads to the cooperative formation of cluster B. These results stress the determining role of the cluster A domain on the overall protein fold. The observed pH dependence of the cluster formation in MT can be rationalized by the different degree of deprotonation of the cysteine residues (pKa approximately 8.9), i.e., by the difference in the Gibbs free energy required to bind Cd(II) ions to the thiolate ligands at both pH values.     

Evidence that conserved residues Cys-62 and Cys-154 within the Azotobacter vinelandii nitrogenase MoFe protein α-subunit are essential for nitrogenase activity but conserved residues His-83 and Cys-88 are not

Metallocluster extrusion requirements, interspecies MoFe-protein primary sequence comparisons and comparison of the primary sequences of the MoFe-protein subunits with each other have been used to assign potential P-cluster (Fe-S cluster) domains within the MoFe protein. In each alpha-beta unit of the MoFe protein, alpha-subunit domains, which include potential Fe-S cluster ligands Cys-62, His-83, Cys-88 and Cys-154, and beta-subunit domains, which include potential Fe-S cluster ligands Cys-70, His-90, Cys-95 and Cys-153, are proposed to comprise nearly equivalent P-cluster environments located adjacent to each other in the native protein. As an approach to test this model and to probe the functional properties of the P clusters, amino acid residue substitutions were placed at the alpha-subunit Cys-62, His-83, Cys-88 and Cys-154 positions by site-directed mutagenesis of the Azotobacter vinelandii nifD gene. The diazotrophic growth rates, MoFe-protein acetylene-reduction activities, and whole-cell S = 3/2 electron paramagnetic resonance spectra of these mutants were examined. Results of these experiments show that MoFe-protein alpha-subunit residues, Cys-62 and Cys-154, are probably essential for MoFe-protein activity but that His-83 and Cys-88 residues are not. These results indicate either that His-83 and Cys-88 do not provide essential P-cluster ligands or that a new cluster-ligand arrangement is formed in their absence.     

Identification of the [Fe-S] cluster-binding residues of Escherichia coli biotin synthase

The gene encoding Escherichia coli biotin synthase (bioB) has been expressed as a histidine fusion protein, and the protein was purified in a single step using immobilized metal affinity chromatography. The His(6)-tagged protein was fully functional in in vitro and in vivo biotin production assays. Analysis of all the published bioB sequences identified a number of conserved residues. Single point mutations, to either serine or threonine, were carried out on the four conserved (Cys-53, Cys-57, Cys-60, and Cys-188) and one non-conserved (Cys-288) cysteine residues, and the purified mutant proteins were tested both for ability to reconstitute the [2Fe-2S] clusters of the native (oxidized) dimer and enzymatic activity. The C188S mutant was insoluble. The wild-type and four of the mutant proteins were characterized by UV-visible spectroscopy, metal and sulfide analysis, and both in vitro and in vivo biotin production assays. The molecular masses of all proteins were verified using electrospray mass spectrometry. The results indicate that the His(6) tag and the C288T mutation have no effect on the activity of biotin synthase when compared with the wild-type protein. The C53S, C57S, and C60S mutant proteins, both as prepared and reconstituted, were unable to covert dethiobiotin to biotin in vitro and in vivo. We conclude that three of the conserved cysteine residues (Cys-53, Cys-57, and Cys-60), all of which lie in the highly conserved "cysteine box" motif, are crucial for [Fe-S] cluster binding, whereas Cys-188 plays a hitherto unknown structural role in biotin synthase.     

Sequential resonance assignments of oxidized high-potential iron-sulfur protein from Chromatium vinosum.

2D NMR spectra of the high-potential iron-sulfur protein (HiPIP) from Chromatium vinosum have been used to obtain partial resonance assignments for the oxidized paramagnetic redox state of the protein. Sequence-specific assignments were made using NOESY and COSY spectra in H2O and D2O of the following backbone segments: Asn-5-Arg-33, Glu-39-Asp-45, Gly-55-Cys-63, Gly-68-Ala-78, and Leu-82-Gly-85. NOESY spectra with a spectral width wide enough to include the hyperfine-shifted resonances revealed numerous NOE contacts between these signals and those in the main envelope of the proton spectrum. With the aid of the X-ray crystal structure [Carter, C.W., Kraut, J., Freer, S. T., Xuong, N. H., Alden, R. A., & Bartsch, R. G. (1974) J. Biol. Chem. 249, 4212], these NOEs permitted seven of the nine hyperfine-shifted signals to be assigned to three of the cysteine residues liganded to the metal cluster (Cys-43, Cys-46, and Cys-77). The other two hyperfine-shifted signals produced no detectable NOEs to other resonances in the spectrum and were tentatively assigned to the remaining cysteinyl ligand (Cys-63). These assignments, in conjunction with recent theoretical models of the electronic structure of the Fe4S4 cluster [Noodleman, L. (1988) Inorg. Chem. 27, 3677; Bertini, I., Briganti, F., Luchinat, C., Scozzafava, A., & Sola, M. (1991) J. Am. Chem. Soc. 113, 1237], indicate that the iron atoms coordinated to Cys-63 and Cys-77 are those of the mixed-valence Fe(3+)-Fe2+ pair whereas Cys-43 and Cys-46 are ligands to the Fe(3+)-Fe3+ metal pair.     

Cadmium-109 as a probe of the metal binding sites in horse liver alcohol dehydrogenase.

The noncatalytic and catalytic zinc atoms of horse liver alcohol dehydrogenase, [(LADH)Zn2Zn2] or LADH, have been replaced differentially with 109Cd by equilibrium dialysis, resulting in two new enzymatically active species, [(LADH)109Cd2Zn2] and [(LADH)109Cd2109Cd2]. The UV difference spectra of the cadmium enzymes vs. native [(LADH)Zn2Zn2] reveal maxima at 240 nm with molar absorptivities, delta epsilon 240, of 1.6 X 10(4) M-1 cm-1 per noncatalytic 109Cd atom and 0.9 X 10(4) M-1 cm-1 per catalytic 109Cd atom, consistent with coordination of the metals by four and two thiolate ligands, respectively, strikingly similar to the 250-nm charge-transfer absorbance in metallothionein. Carboxymethylation of the Cys-46 ligand to the catalytic metal in LADH presumably lowers the overall stability constant of the coordination complex and results in loss of catalytic 109Cd or catalytic cobalt but not catalytic zinc from the enzyme.     

Facilitated transfer of IscU-[2Fe2S] clusters by chaperone-mediated ligand exchange

The scaffold protein IscU and molecular chaperones HscA and HscB play central roles in biological assembly of iron-sulfur clusters and maturation of iron-sulfur proteins. However, the structure of IscU-FeS complexes and the molecular mechanism whereby the chaperones facilitate cluster transfer to acceptor proteins are not well understood. We have prepared amino acid substitution mutants of Escherichia coli IscU in which potential ligands to the FeS cluster (Cys-37, Cys-63, His-105, and Cys-106) were individually replaced with alanine. The properties of the IscU-FeS complexes formed were investigated by measuring both their ability to transfer preformed FeS clusters to apo-ferredoxin and the activity of the IscU proteins in catalyzing cluster assembly on apo-ferredoxin using inorganic iron with inorganic sulfide or with IscS and cysteine as a sulfur source. The ability of the HscA/HscB chaperone system to accelerate ATP-dependent cluster transfer from each IscU substitution mutant to apo-ferredoxin was also determined. All of the mutants formed FeS complexes with a stoichiometry similar to the wild-type holo-protein, i.e., IscU(2)[2Fe2S], raising the possibility that different cluster ligation states may occur during iron-sulfur protein maturation. Spectroscopic properties of the mutants and the kinetics of transfer of performed IscU-FeS clusters to apo-ferredoxin indicate that the most stable form of holo-IscU involves iron coordination by Cys-63 and Cys-106. Results of studies on the ability of mutants to catalyze formation of holo-ferredoxin using iron and different sulfur sources were consistent with proposed roles for Cys-63 and Cys-106 in FeS cluster binding and also indicated an essential role for Cys-106 in sulfide transfer to IscU from IscS. Measurements of the ability of the chaperones HscA and HscB to facilitate cluster transfer from holo-IscU to apo-ferredoxin showed that only IscU(H105A) behaved similarly to wild-type IscU in exhibiting ATP-dependent stimulation of cluster transfer. IscU(C63A) and IscU(C106A) displayed elevated rates of cluster transfer in the ±ATP whereas IscU(C37A) exhibited low rates of cluster transfer ±ATP. In interpreting these findings, we propose that IscU(2)[2Fe2S] is able undergo structural isomerization to yield conformers having different cysteine residues bound to the cluster. On the basis of the crystal structure of HscA complexed with an IscU-derived peptide, we propose that the chaperone binds and stabilizes an isomer of IscU(2)[2Fe2S] in which the cluster is bound by cysteine residues 37 and 63 and that the [2Fe2S] cluster, being held less tightly than that coordinated by Cys-63 and Cys-106 in free IscU(2)[2Fe2S], is more readily transferred to acceptor proteins such as apo-ferredoxin.     

Molecular cloning and characterization of a phytochelatin synthase gene, PvPCS1, from Pteris vittata L.

Pteris vittata L. is a staggeringly efficient arsenic hyperaccumulator that has been shown to be capable of accumulating up to 23,000 microg arsenic g(-1), and thus represents a species that may fully exploit the adaptive potential of plants to toxic metals. However, the molecular mechanisms of adaptation to toxic metal tolerance and hyperaccumulation remain unknown, and P. vittata genes related to metal detoxification have not yet been identified. Here, we report the isolation of a full-length cDNA sequence encoding a phytochelatin synthase (PCS) from P. vittata. The cDNA, designated PvPCS1, predicts a protein of 512 amino acids with a molecular weight of 56.9 kDa. Homology analysis of the PvPCS1 nucleotide sequence revealed that it has low identity with most known plant PCS genes except AyPCS1, and the homology is largely confined to two highly conserved regions near the 5'-end, where the similarity is as high as 85-95%. The amino acid sequence of PvPCS1 contains two Cys-Cys motifs and 12 single Cys, only 4 of which (Cys-56, Cys-90/91, and Cys-109) in the N-terminal half of the protein are conserved in other known PCS polypeptides. When expressed in Saccharomyces cerevisae, PvPCS1 mediated increased Cd tolerance. Cloning of the PCS gene from an arsenic hyperaccumulator may provide information that will help further our understanding of the genetic basis underlying toxic metal tolerance and hyperaccumulation.     

Independence of the domains of metallothionein in metal binding

Mammalian metallothionein is a low molecular weight protein with two metal-binding domains. To determine if metal binding in one domain affects binding in the other, we prepared peptides corresponding to the regions that enfold the two metal-thiolate clusters. Metal reconstitution studies of these peptides revealed stoichiometries of metal binding similar to those observed within the intact molecule. Thus, the alpha domain coordinates 4 Cd(II), 6 Cu(I), or 6 Ag(I) ions regardless of whether the domain is part of the total protein or is studied as a separate peptide. Likewise, the beta domain binds 3 Cd(II), 6 Cu(I), or 6 Ag(I) ions in both the intact protein and as a separate peptide. If cluster B in intact metallothionein is preformed with Cu(I) or Ag(I), cluster A saturates with either 4 mol eq of Cd(II) or 6 mol eq of Ag(I). Similarly, preformation of the A cluster with Cd(II) does not affect the binding of 6 Cu(I) ions in the B cluster. Therefore, the metal-dependent folding of the protein to create one cluster occurs independent of constraints or influences from the other domain. Formation of the protein with a tetrahedrally coordinated metal in one cluster and a trigonally coordinated metal in the other center is possible.     

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.     

Cadmium binding and metal cluster formation in metallothionein: a differential modification study

Mammalian metallothioneins (MT) contain 20 Cys in a total of 61 amino acid residues and bind 7 Cd and/or Zn ions. The metal is localized in two clusters made up of three and four metal-thiolate complexes in the NH2- and COOH-terminal half of the chain, respectively [Otvos, J.D., & Armitage, I. M. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7094-7098]. The formation of these oligonuclear complexes designated as Cd4 and Cd3 clusters has now been monitored in MT reconstituted with varying amounts of Cd by using differential chemical modification of Cys with [14C]iodoacetamide. At ratios below 2-3 mol of Cd/mol of MT bound, no differential protection of Cys by the metal, and hence no preferred binding, is detectable. At Cd-to-protein ratios between 3 and 5 mol of Cd/mol of MT, the modification profiles reveal preferred and cooperative binding in the COOH-terminal half of the chain, indicating formation of the Cd4 cluster. At still higher ratios, formation of the Cd3 cluster is initiated in the NH2-terminal section of the polypeptide chain. Comparison of the differential modification data of Cd6-MT and Cd7-MT suggests that the last Cd to be bound is coordinated to Cys ligands located mainly between positions 20 and 30 of the sequence. The extent of labeling of the different Cys in Cd7-MT indicates that the ligands of the Cd3 cluster are 3 times as accessible to iodoacetamide than those of the Cd4 cluster, suggesting a greater thermodynamic or kinetic stability of the latter.     

Products of metal exchange reactions of metallothionein

Hepatic metallothionein (MT) isolated from Cd-exposed animals always contains Zn (2-3 mol/mol of protein) in addition to Cd (4-5 mol/mol of protein), and the two metals are distributed in a nonuniform, but reproducible, manner among the seven binding sites of the protein's two metal-thiolate clusters. Different methodologies of preparing rabbit liver Cd, Zn-MT in vitro were investigated to provide insight into why such a distinct mixture of mixed-metal clusters is produced in vivo and by what mechanism they form. 113Cd NMR spectra of the products of stepwise displacement of Zn2+ from Zn7-MT by 113Cd2+ show that Cd binding to the clusters is not cooperative (i.e., clusters containing exclusively Cd are not formed in preference to mixed-metal Cd, Zn clusters), there is no selective occupancy of one cluster before the other, and many clusters are produced with a nonnative metal distribution indicating that this pathway is probably not followed in vivo. In contrast, the surprising discovery was made that the native cluster compositions and their relative concentrations could be reproduced exactly by simply mixing together the appropriate amounts of Cd7-MT and Zn7-MT and allowing intermolecular metal exchange to occur. This heretofore unknown metal interchange reaction occurs readily, and the driving force appears to be the relative thermodynamic instability of three-metal clusters containing Cd. With this new insight into how Cd,Zn-MT is likely to be formed in vivo we are able for the first time to postulate rational explanations for previous observations regarding the response of hepatic Zn and metallothionein levels to Cd administration.     

Metal co-ordination in rat liver metallothionein-2 prepared with or without reconstitution of the metal clusters, and comparison with rabbit liver metallothionein-2

Possible origins of the different metal co-ordination topologies in the recently determined structures of rat metallothionein-2 (MT2) in single crystals and rabbit MT2 in solution were investigated. A complete structure determination for rat MT2 in solution by nuclear magnetic resonance (n.m.r.) showed that the differences in the spatial structures cannot be attributed to the different primary structures of the two species. Comparison of [113Cd7]MT2 obtained by reconstitution of the apoprotein in vitro with preparations using a different procedure showed, moreover, that the metal co-ordination observed in solution by n.m.r. is not an artefact of the protein reconstitution. Solutions of high-pressure liquid chromatographically homogeneous biosynthetic preparations of [113Cd, Zn]MT2 were obtained from rat liver following injection of 113Cd into rats in vivo, without further metal exchange after protein isolation. They contain a mixture of several forms of MT2 with different relative metal compositions, giving rise to an increased number of 113Cd resonances. For the components of the four-metal cluster, the major one of these different forms exhibits patterns in the two-dimensional [1H, 113Cd]-correlated spectra that are indistinguishable from those of [113Cd7]MT2, thereby implying identity of cluster coordination and topology. These results are discussed with regard to continued investigations into the differences between the solution structure and crystal structure of MT2.     

Organization and assembly of metal-thiolate clusters in epithelium-specific metallothionein-4

Mammalian metallothionein-4 (MT-4) was found to be specifically expressed in stratified squamous epithelia where it plays an essential but poorly defined role in regulating zinc or copper metabolism. Here we report on the organization, stability, and the pathway of metal-thiolate cluster assembly in MT-4 reconstituted with Cd(2+) and Co(2+) ions. Both the (113)Cd NMR studies of (113)Cd(7)MT-4 and the spectroscopic characterization of Co(7)MT-4 showed that, similar to the classical MT-1 and MT-2 proteins, metal ions are organized in two independent Cd(4)Cys(11) and Cd(3)Cys(9) clusters with each metal ion tetrahedrally coordinated by terminal and bridging cysteine ligands. Moreover, we have demonstrated that the cluster formation in Cd(7)MT-4 is cooperative and sequential, with the Cd(4)Cys(11) cluster being formed first, and that a distinct single-metal nucleation intermediate Cd(1)MT-4 is required in the cluster formation process. Conversely, the absorption and circular dichroism features of metal-thiolate clusters in Cd(7)MT-4 indicate that marked differences in the cluster geometry exist when compared with those in Cd(7)MT-1/2. The biological implication of our studies as to the role of MT-4 in zinc metabolism of stratified epithelia is discussed.     

Spectroscopic studies on metal distribution in Co(II)/Zn(II) mixed-metal clusters in rabbit liver metallothionein 2.

Metal selectivity of metal-thiolate clusters in rabbit liver metallothionein (MT) 2 has been studied by examining the metal distribution of two similarly sized divalent metal ions, cobalt and zinc, which have different thiolate affinity. The forms of mixed-metal cluster species in (Co/Zn)7-MT generated with different ratios of both metal ions offered to the metal-free protein were investigated using EPR, ultraviolet/visible absorption and MCD spectroscopy. The results demonstrated that the distribution of these metals between the two metal-thiolate clusters is not random. Thus, the EPR absorption intensities of the bound Co(II) ions in the Zn-cluster matrix increased linearly up to a ratio of Co(II)/Zn(II) equivalents of 3:4, with the final EPR intensity of three non-interacting Co(II)-binding sites. This EPR behaviour is consistent with a binding scheme in which one Co(II) ion occupies a metal-binding site within the three-metal cluster and the remaining two Co(II) ions occupy two distinctly separate sites in the four-metal cluster. With four or more Co(II) ions in the cluster matrix, magnetic coupling between adjacent, sulphur-bridged Co(II) ions was observed. In previous studies on mixed-metal clusters in MT formed with Co(II)/Cd(II), Zn(II)/Cd(II) and Cd(II)/Fe(II), changes in the respective cluster volumes were shown to be a significant factor dictating the widely differing metal distributions in these systems. Based on the results of the current study, it is suggested that both the sizes of the two metal ions and their relative affinities towards the cysteine-thiolate ligands are important in the formation of mixed-metal clusters in MT.