A distinct Cu4-thiolate cluster of human metallothionein-3 is located in the N-terminal domain

Metallothionein-3 (MT-3), also known as neuronal growth inhibitory factor, is a metalloprotein expressed almost exclusively in the brain. Isolated MT-3 contains four Cu(I) and three Zn(II) ions organized in homometallic metal-thiolate clusters located in two independent protein domains. In this work a Cu(I) binding to metal-free MT-3 has been studied, aiming at the better understanding of the domain specificity for this metal ion. The cluster formation was followed by electronic absorption, circular dichroism, and by luminescence spectroscopy at room temperature and 77 K. The stepwise incorporation of Cu(I) into recombinant human apo-MT-3 revealed the cooperative formation of two Cu(4)S(9) clusters in succession, formed in both protein domains, i.e. Cu(4)- and Cu(8)-MT-3. Further binding of four Cu(I) caused an expansion of these Cu(I) cores, leading to fully metal-loaded Cu(12)-MT-3 containing Cu(6)S(9) and Cu(6)S(11) clusters in the beta- and alpha-domains of the protein, respectively. The location of the preferentially formed Cu(4) cluster in the protein was established by immunochemistry. Using domain-specific antibodies, in combination with limited tryptic digestion of a partially metal-occupied Cu(4)-MT-3, we could demonstrate that the Cu(4)S(9) cluster is located in the N-terminal beta-domain of the protein that contains a total of nine cysteine ligands.     

Redox silencing of copper in metal-linked neurodegenerative disorders: reaction of Zn7metallothionein-3 with Cu2+ ions

Dysregulation of copper and zinc homeostasis in the brain plays a critical role in Alzheimer disease (AD). Copper binding to amyloid-beta peptide (Abeta) is linked with the neurotoxicity of Abeta and free radical damage. Metallothionein-3 (MT-3) is a small cysteine- and metal-rich protein expressed in the brain and found down-regulated in AD. This protein occurs intra- and extracellularly, and it plays an important role in the metabolism of zinc and copper. In cell cultures Zn7MT-3, by an unknown mechanism, protects neurons from the toxicity of Abeta. We have, therefore, used a range of complementary spectroscopic and biochemical methods to characterize the interaction of Zn7MT-3 with free Cu2+ ions. We show that Zn7MT-3 scavenges free Cu2+ ions through their reduction to Cu+ and binding to the protein. In this reaction thiolate ligands are oxidized to disulfides concomitant with Zn2+ release. The binding of the first four Cu2+ is cooperative forming a Cu(I)4-thiolate cluster in the N-terminal domain of Cu4,Zn4MT-3 together with two disulfides bonds. The Cu4-thiolate cluster exhibits an unusual stability toward air oxygen. The results of UV-visible, CD, and Cu(I) phosphorescence at 77 K suggest the existence of metal-metal interactions in this cluster. We have demonstrated that Zn7MT-3 in the presence of ascorbate completely quenches the copper-catalyzed hydroxyl radical (OH.) production. Thus, zinc-thiolate clusters in Zn7MT-3 can efficiently silence the redox-active free Cu2+ ions. The biological implication of our studies as to the protective role of Zn7MT-3 from the Cu2+ toxicity in AD and other neurodegenerative disorders is discussed.     

Metal swap between Zn7-metallothionein-3 and amyloid-beta-Cu protects against amyloid-beta toxicity

Aberrant interactions of copper and zinc ions with the amyloid-beta peptide (Abeta) potentiate Alzheimer's disease (AD) by participating in the aggregation process of Abeta and in the generation of reactive oxygen species (ROS). The ROS production and the neurotoxicity of Abeta are associated with copper binding. Metallothionein-3 (Zn(7)MT-3), an intra- and extracellularly occurring metalloprotein, is highly expressed in the brain and downregulated in AD. This protein protects, by an unknown mechanism, cultured neurons from the toxicity of Abeta. Here, we show that a metal swap between Zn(7)MT-3 and soluble and aggregated Abeta(1-40)-Cu(II) abolishes the ROS production and the related cellular toxicity. In this process, copper is reduced by the protein thiolates forming Cu(I)(4)Zn(4)MT-3, in which an air-stable Cu(I)(4)-thiolate cluster and two disulfide bonds are present. The discovered protective effect of Zn(7)MT-3 from the copper-mediated Abeta(1-40) toxicity may lead to new therapeutic strategies for treating AD.     

Effect of the two conserved prolines of human growth inhibitory factor (metallothionein-3) on its biological activity and structure fluctuation: comparison with a mutant protein

Human neuronal growth inhibitory factor, a metalloprotein classified as metallothionein-3 (MT-3), impairs the survival and the neurite formation of cultured neurons. In these studies the double P7S/P9A mutant (mutMT-3) and single mutants P7S and P9A of human Zn(7)-MT-3 were generated, and their effects on the biological activity and the structure of the protein were examined. The biological results clearly established the necessity of both proline residues for the inhibitory activity, as even single mutants were found to be inactive. Using electronic absorption, circular dichroism (CD), magnetic CD (MCD), and (113)Cd NMR spectroscopy, the structural features of the metal-thiolate clusters in the double mutant Cd(7)-mutMT-3 were investigated and compared with those of wild-type Cd(7)-MT-3 [Faller, P., Hasler, D. W., Zerbe, O., Klauser, S., Winge, D. R., and Vasák, M. (1999) Biochemistry 38, 10158] and the well characterized Cd(7)-MT-2a from rabbit liver. Similarly to (113)Cd(7)-MT-3 the (113)Cd NMR spectrum of (113)Cd(7)-mutMT-3 at 298 K revealed four major and three minor resonances (approximately 20% of the major ones) between 590 and 680 ppm, originating from a Cd(4)S(11) cluster in the alpha-domain and a Cd(3)S(9) cluster in the beta-domain, respectively. Due to the presence of dynamic processes in the structure of MT-3 and mutMT-3, all resonances showed the absence of resolved homonuclear [(113)Cd-(113)Cd] couplings and large apparent line widths (between 140 and 350 Hz). However, whereas in (113)Cd(7)-mutMT-3 the temperature rise to 323 K resulted in a major recovery of the originally NMR nondetectable population of the Cd(3)S(9) cluster resonances, no such temperature effect was observed in (113)Cd(7)-MT-3. To account for the observed NMR features, a dynamic structural model for the beta-domain is proposed, which involves a folded and a partially unfolded state. It is suggested that in the partially unfolded state a slow cis/trans isomerization of Cys-Pro(7) or Cys-Pro(9) amide bonds in (113)Cd(7)-MT-3 takes place and that this process represents a rate-limiting step in a correct domain refolding. In addition, closely similar apparent stability constants of human MT-3, mutMT-3, and rabbit MT-2a with Cd(II) and Zn(II) ions were found. These results suggest that specific structural features dictated by the repetitive (Cys-Pro)(2) sequence in the beta-domain of MT-3 and not its altered metal binding affinity compared to MT-1/MT-2 isoforms are responsible for the biological activity of this protein.     

Coordination of three and four Cu(I) to the alpha- and beta-domain of vertebrate Zn-metallothionein-1, respectively, induces significant structural changes

Vertebrate metallothioneins are found to contain Zn(II) and variable amounts of Cu(I), in vivo, and are believed to be important for d10-metal control. To date, structural information is available for the Zn(II) and Cd(II) forms, but not for the Cu(I) or mixed metal forms. Cu(I) binding to metallothionein-1 has been investigated by circular dichroism, luminescence and 1H NMR using two synthetic fragments representing the alpha- and the beta-domain. The 1H NMR data and thus the structures of Zn4alpha metallothionein (MT)-1 and Zn3betaMT-1 were essentially the same as those already published for the corresponding domains of native Cd7MT-1. Cu(I) titration of the Zn(II)-reconstituted domains provided clear evidence of stable polypeptide folds of the three Cu(I)-containing alpha- and the four Cu(I)-containing beta-domains. The solution structures of these two species are grossly different from the structures of the starting Zn(II) complexes. Further addition of Cu(I) to the two single domains led to the loss of defined domain structures. Upon mixing of the separately prepared aqueous three and four Cu(I) loaded alpha- and beta-domains, no interaction was seen between the two species. There was neither any indication for a net transfer of Cu(I) between the two domains nor for the formation of one large single Cu(I) cluster involving both domains.     

Redox activity of α-synuclein-Cu is silenced by Zn₇-metallothionein-3

The aggregation of α-synuclein (α-Syn), the major component of intracellular Lewy body inclusions in dopaminergic neurons of the substantia nigra, plays a critical role in the etiology of Parkinson disease (PD). Long-term effects of redox-active transition metals (Cu, Fe) and oxidative chemical imbalance underlie the disease progression and neuronal death. In this work, we provide evidence that a brain metalloprotein, Zn₇-metallothionein-3 (Zn₇MT-3), possesses a dynamic role in controlling aberrant protein–copper interactions in PD. We examined the properties of the α-Syn–Cu(II) complex with regard to molecular oxygen, the biological reducing agent ascorbate, and the neurotransmitter dopamine. The results revealed that under aerobic conditions α-Syn–Cu(II) possesses catalytic oxidase activity. The observed metal-centered redox chemistry significantly promotes the production of hydroxyl radicals and α-Syn oxidation and oligomerization, processes considered critical for cellular toxicity. Moreover, we show that Zn₇MT-3, through Cu(II) removal from the α-Syn–Cu(II) complex, efficiently prevents its deleterious redox activity. We demonstrate that the Cu(II) reduction by thiolate ligands of Zn₇MT-3 and the formation of Cu(I)₄Zn₄MT-3, in which an unusual oxygen-stable Cu(I)₄–thiolate cluster is present, comprise the underlying molecular mechanism by which α-Syn and dopamine oxidation, α-Syn oligomerization, and ROS production are abolished. These studies provide new insights into the bioinorganic chemistry of PD.     

Rapid exchange of metal between Zn(7)-metallothionein-3 and amyloid-β peptide promotes amyloid-related structural changes

Metal ions, especially Zn(2+) and Cu(2+), are implemented in the neuropathogenesis of Alzheimer's disease (AD) by modulating the aggregation of amyloid-β peptides (Aβ). Also, Cu(2+) may promote AD neurotoxicity through production of reactive oxygen species (ROS). Impaired metal ion homeostasis is most likely the underlying cause of aberrant metal-Aβ interaction. Thus, focusing on the body's natural protective mechanisms is an attractive therapeutic strategy for AD. The metalloprotein metallothionein-3 (MT-3) prevents Cu-Aβ-mediated cytotoxicity by a Zn-Cu exchange that terminates ROS production. Key questions about the metal exchange mechanisms remain unanswered, e.g., whether an Aβ-metal-MT-3 complex is formed. We studied the exchange of metal between Aβ and Zn(7)-MT-3 by a combination of spectroscopy (absorption, fluorescence, thioflavin T assay, and nuclear magnetic resonance) and transmission electron microscopy. We found that the metal exchange occurs via free Cu(2+) and that an Aβ-metal-MT-3 complex is not formed. This means that the metal exchange does not require specific recognition between Aβ and Zn(7)-MT-3. Also, we found that the metal exchange caused amyloid-related structural and morphological changes in the resulting Zn-Aβ aggregates. A detailed model of the metal exchange mechanism is presented. This model could potentially be important in developing therapeutics with metal-protein attenuating properties in AD.     

Comparative insight into the Zn(II)-, Cd(II)- and Cu(I)-binding features of the protozoan Tetrahymena pyriformis MT1 metallothionein

Tetrahymena pyriformis MT1 (TpyMT1) is a model among ciliate metallothioneins (MTs). Here, we report on the analytic (ICP-AES, GC-FPD), spectroscopic (CD, UV-Vis, Raman) and spectrometric (ESI-MS) characterization of its recombinant Cd(II)-, Zn(II)- and Cu(I)-complexes, and of those formed during in vitro Zn/Cd and Zn/Cu replacement. In the presence of Cd(II), TpyMT1 renders a major Cd 11-TpyMT1 species, which is also the final step reached in the in vitro Zn/Cd exchange process in Zn 11-TpyMT1. Spectroscopic data supports a different folding of the isostoichiometric Cd 11- and Zn 11-TpyMT1 complexes. Unexpectedly, TpyMT1 biosynthesis in Zn(II)-rich cultures was sensitive to the aeration degree, so that high oxygenation rendered undermetalated, partially-oxidized, complexes (Zn9-TpyMT1). Biosynthesis in Cu(I)-rich media rendered extremely heterogeneous mixtures of CuxZny-species (x+y=8-20), where the higher the aeration, the higher the Zn(II) content. The complexity of these samples was reproduced during the Zn/Cu replacement, as the number of generated species increased gradually with the addition of copper to Zn(11)-TpyMT1. According to our results, a clear preference of TpyMT1 for Cd(II) binding, rather than for Zn(II), and especially Cu(I) can be postulated. This character is totally consistent with the induction pattern of the TpyMT1 gene and the postulated role of TpyMT1 in Cd-detoxification.     

(Cu,Zn)-metallothioneins from fetal bovine liver. Chemical and spectroscopic properties

Two metallothioneins (MTs) from bovine fetal liver were purified by a combination of gel filtration and ion-exchange chromatography. The primary structures of the isoproteins MT-1 and MT-2 were elucidated by peptide and amino acid sequence analysis. The amino-terminal part was deduced from automated Edman degradations of the pyridylethylated CNBr-cleaved derivatives. The remaining part of the sequence was established by a comparison of the carboxamidomethylated tryptic peptides to those from equine liver MT-1A and MT-2B. Peptides differing in either amino acid composition or retention time from high pressure liquid chromatography were further subjected to manual Edman degradations or carboxypeptidase Y digestion. The two isoproteins consist of 61 amino acids and show a sequence identity of 90%. When compared with the primary structures of other mammalian MTs, the 20 cysteinyl residues are totally conserved, in agreement with their function as metal ligands. The two isoproteins contain Cu and Zn at a ratio of 3:4. Spectroscopic data reveal absorption properties typical for both Cu- and Zn-thiolate transitions. The marked differences of MT-1 and MT-2 in the Cu-thiolate CD features can be attributed to the six amino acid substitutions occurring exclusively in the amino-terminal parts of the molecules. It is proposed that in bovine fetal MTs also the three copper ions are preferentially bound to the first 9 cysteinyl residues (cluster B) and the four zinc ions to the remaining 11 cysteinyl residues (cluster A) suggested previously by 113Cd NMR spectroscopy of calf liver MTs (Briggs, R. W., and Armitage, I. M. (1982) J. Biol. Chem. 257, 1259-1262).     

Engineering of metallothionein-3 neuroinhibitory activity into the inactive isoform metallothionein-1

The third isoform of mammalian metallothioneins (MT-3), mainly expressed in brain and down-regulated in Alzheimer's disease, exhibits neuroinhibitory activity in vitro and a highly flexible structure that distinguishes it from the widely expressed MT-1/-2 isoforms. Previously, we showed that two conserved prolyl residues of MT-3 are crucial for both the bioactivity and cluster dynamics of this isoform. We have now used genetic engineering to introduce these residues into mouse MT-1. The S6P,S8P MT-1 mutant is inactive in neuronal survival assays. However, the additional introduction of the unique Thr5 insert of MT-3 resulted in a bioactive MT-1 form. Temperature-dependent and saturation transfer (113)Cd NMR experiments performed on the (113)Cd-reconstituted wild-type and mutant Cd(7)-MT-1 forms revealed that the gain of MT-3-like neuronal inhibitory activity is paralleled by an increase in conformational flexibility and intersite metal exchange in the N-terminal Cd(3)-thiolate cluster. The observed correlation suggests that structure/cluster dynamics are critical for the biological activity of MT-3. We propose that the interplay between the specific Pro-induced conformational requirements and those of the metal-thiolate bonds gives rise to an alternate and highly fluctuating cluster ensemble kinetically trapped by the presence of the (5)TCPCP(9) motif. The functional significance of such heterogeneous cluster ensemble is discussed.     

Chemistry and biology of mammalian metallothioneins

Metallothioneins (MTs) are a class of ubiquitously occurring low molecular mass, cysteine- and metal-rich proteins containing sulfur-based metal clusters formed with Zn(II), Cd(II), and Cu(I) ions. In mammals, four distinct MT isoforms designated MT-1 through MT-4 exist. The first discovered MT-1/MT-2 are widely expressed isoforms, whose biosynthesis is inducible by a wide range of stimuli, including metals, drugs, and inflammatory mediators. In contrast, MT-3 and MT-4 are noninducible proteins, with their expression primarily confined to the central nervous system and certain squamous epithelia, respectively. MT-1 through MT-3 have been reported to be secreted, suggesting that they may play different biological roles in the intracellular and extracellular space. Recent reports established that these isoforms play an important protective role in brain injury and metal-linked neurodegenerative diseases. In the postgenomic era, it is becoming increasingly clear that MTs fulfill multiple functions, including the involvement in zinc and copper homeostasis, protection against heavy metal toxicity, and oxidative damage. All mammalian MTs are monomeric proteins, containing two metal–thiolate clusters. In this review, after a brief summary of the historical milestones of the MT-1/MT-2 research, the recent advances in the structure, chemistry, and biological function of MT-3 and MT-4 are discussed.     

Crystallographic analysis of the intact metal centres [3Fe-4S](1+/0) and [4Fe-4S](2+/1+) in a Zn(2+) -containing ferredoxin

Detailed structural models of di-cluster seven-iron ferredoxins constitute a valuable resource for folding and stability studies relating the metal cofactors' role in protein stability. The here reported, hemihedric twinned crystal structure at 2.0 A resolution from Acidianus ambivalens ferredoxin, shows an integral 103 residues, physiologically relevant native form composed by a N-terminal extension comprising a His/Asp Zn(2+) site and the ferredoxin (betaalphabeta)(2) core, which harbours intact clusters I and II, a [3Fe-4S](1+/0) and a [4Fe-4S](2+/1+) centres. This is in contrast with the previously available ferredoxin structure from Sulfolofus tokodai, which was obtained from an artificial oxidative conversion with two [3Fe-4S](1+/0) centres and poor definition around cluster II.     

Metal binding to brain-specific metallothionein-3 studied by electrospray ionization mass spectrometry.

Metallothionein-3 (MT-3) is a brain-specific isoform of metallothioneins, which is down-regulated in Alzheimer's disease (AD), inhibits the growth of neurons in vitro, and differs from common MTs also in gene regulation. To elucidate the differences in structure and function between MT-3 and common MTs, Zn2+ and Cd2+ binding to MT-3 and MT-1 were studied using electrospray ionization time of flight mass spectrometry (ESI TOF MS) at pH values between 7.5 and 2.7. The metal binding properties of MT-3 differ considerably from those of MT-1. After reconstitution with a metal excess, metallated MT-3 exists as a mixture of Zn7MT-3 (or Cd7MT-3, respectively) and several metalloforms with stoichiometries below and above seven. In contrast, MT-1 exists as a single Zn7MT-1 (or Cd7MT-1). Lowering of pH leads to a stepwise release of metals from metallated MT-3, first from extra sites, then from the 3-metal cluster and finally from the 4-metal cluster. At acidic pH values the 4-metal cluster of MT-3 is slightly more stable than that of MT-1. The results demonstrate higher structural plasticity, dynamics and metal binding capacity of MT-3 than of MT-1, which makes MT-3 suitable as a zinc buffer-transfer molecule in zinc-enriched neurons functioning at conditions of fluctuating zinc concentrations.     

The oxidation of 3-hydroxyanthranilic acid by Cu,Zn superoxide dismutase: mechanism and possible consequences

Cu,Zn SOD, but not Mn SOD, catalyzes the oxidation of 3-hydroxyanthranilic acid (3-HA) under aerobic conditions. In the absence of O2, the Cu(II) of the enzyme is reduced by 3-HA. One plausible mechanism involves the reduction of the active site Cu(II) to Cu(I), which is then reoxidized by the O2- generated by autoxidation of the anthranilyl or other radicals on the pathway to cinnabarinate. We may call this the superoxide reductase, or SOR, mechanism. Another possibility invokes direct reoxidation of the active site Cu(I) by the anthranilyl, or other organic radicals, or by the peroxyl radicals generated by addition of O2 to these organic radicals. Such oxidations catalyzed by Cu,Zn SOD could account for the deleterious effects of the mutant Cu,Zn SODs associated with familial amyotrophic lateral sclerosis and of the overproduction or overadministration of wild-type Cu,Zn SOD.     

Fourier transform IR and Fourier transform Raman spectroscopy studies of metallothionein-III: amide I band assignments and secondary structural comparison with metallothioneins-I and -II

The secondary structures of porcine brain Cu(4)Zn(3)-metallothionein (MT)-III and Cd(5)Zn(2)MT-I, Cd(5)Zn(2)MT-II, and Zn(7)MT-I from rabbit livers in the solid state are investigated by Fourier transform IR spectroscopy (FTIR) and Fourier transform Raman spectroscopy (FT-Raman). The Cu(4)Zn(3)MT-III contains 26-28% beta-turns and half-turns, 13-14% 3(10)-helices, 47-49% random coils, and 11-12% beta-extended chains. The structural comparison of porcine brain Cu(4)Zn(3)MT-III with rabbit liver Cd(5)Zn(2)MT-I (II) and Zn(7)MT-I shows that the contents of the random coil structure are obviously increased. The results indicate that the insert of an acidic hexapeptide in the alpha domain of Cu(4)Zn(3)MT-III possibly forms an alpha helix. However, because the bands assigned to the alpha-helix and random coil structures are overlapped in the spectra, the content of random coil structures in Cu(4)Zn(3)MT-III is therefore higher than those in Cd(5)Zn(2)MT-I, Cd(5)Zn(2)MT-II, and Zn(7)MT-I.     

Effect of Zn treatment on wild type and MT-null cell lines in relation to apoptotic and/or necrotic processes and on MT isoform gene expression

It has been shown in various systems that zinc is able to antagonize the catalytic properties of the redox-active transition metals iron and copper, although the process is still unclear. Probably, the protective effect of Zn against oxidative stress is mainly due to the induction of a scavenger metal binding protein such as metallothionein (MT), rather than a direct action. To support this hypothesis, in this study, the effects of Zn, Cu, Fe, Zn + Cu and Zn + Fe treatments were investigated in a fibroblast cell line corresponding to an SV40-transformed MT-1/-2 mutant (MT-/-), and in wild type (MT+/+), by valuing metal concentrations and apoptotic and/or necrotic processes. We also investigated the synthesis of MT and the levels of both MT-1 and MT-2 mRNAs. In MT+/+ cells, co-treatment with Zn + Fe caused a decrease in Fe content compared to treatment with Fe alone. After Zn and Zn + Cu exposure the expression of MT-1 and MT-2 isoforms increased with a concomitant increase in MT synthesis. Annexin V-FITC and propidium iodide staining revealed necrotic or apoptotic cells in terminal stages, especially after Fe treatments. Immunofluorescent staining with an anti-ssDNA Mab and annexin detected a lower signal in co-treated cells compared to the single treatments in both cell lines. The intensity and quantity of fluorescence resulting from anti-ssDNA and Annexin V staining of MT null cells was higher compared to wild type cells. These results suggest that Zn alone does not completely exert an anti-oxidant effect against Cu and Fe toxicity, but that induction of MT is necessary.     

Zinc and copper accumulation and isometallothionein induction in mouse ascites sarcoma S180A cells.

To investigate Zn and Cu accumulation and isometallothionein (iso-MT) induction in ascites-sarcoma S180A cells, 5 micrograms of Zn2+ or Cu2+/g body weight was administered to tumour-bearing mice intraperitoneally. In the tumour cells the Zn or Cu concentration increased more than in the host liver, which is the target organ for those metals; the maximum Zn or Cu level was about 2-3 times that in the host liver. The amounts of Zn-MT or Cu-MT accumulated in the tumour cells and host liver were proportional to such dose accumulation levels in the each cytosol; the maximum level of Zn-MT or Cu-MT was 4 or 2 times higher than in the host liver. MT accumulated in the tumour cells showed two subfractions (MT-1 and MT-2); the ratio of Zn (or Cu) bound to MT-1 to that bound to MT-2 in the host liver and tumour cells was 1.0 (or 1.0) and 0.7 (or 0.25) respectively, suggesting that the induction level of MT-2 in the tumour cells is more than that of MT-1. The h.p.l.c. profiles (using an anion-exchange column) of the isolated MT-1 and MT-2 subfractions from Zn-treated normal-mouse liver showed a single peak (MT-1-1) and two peaks (MT-2-1 and MT-2-2) respectively; mouse MTs were separated into three isoforms. In the ascites cells, the MT fraction obtained by a gel filtration was also separated into three isoforms; however, the amount of MT-2-1 isoform was 3 times that in the Zn-treated normal-mouse liver.     

Preparation and characterization of a (Cu,Zn)-pMMO from Methylococcus capsulatus (Bath)

We report the preparation of a (Cu,Zn)-particulate methane monooxygenase (pMMO) in which the bulk of the copper ions of the electron-transfer clusters (E-clusters) has been replaced by divalent Zn ions. The Cu and Zn contents in the (Cu,Zn)-pMMO were determined by both inductively coupled plasma mass spectroscopy (ICP-MS) and X-ray absorption K-edge spectroscopy. Further characterization of the (Cu,Zn)-pMMO was provided by pMMO-activity assays as well as low-temperature electron paramagnetic resonance (EPR) spectroscopy following reductive titration and incubation in air or air/propylene mixtures. The pMMO-activity assays indicated that the (Cu,Zn)-pMMO was no longer capable of supporting catalytic turnover of hydrocarbon substrates. However, the EPR studies revealed that the catalytic cluster (C-cluster) copper ions in the (Cu,Zn)-pMMO were still capable of supporting the activation of dioxygen when reduced, and that the 14N-superhyperfine features associated with one of the type 2 Cu(II) centers in the hydroxylation C-cluster remained unperturbed. The replacement of the E-cluster copper ions by Zn ions did compromise the ability of the protein to mediate the transfer of reducing equivalents from exogenous reductants to the C-clusters. These observations provide strong support for the electron transfer and catalytic roles for the E-cluster and C-cluster copper ions, respectively.     

Use of stable isotopically enriched proteins and directly coupled high-performance liquid chromatography inductively coupled plasma mass spectrometry for quantitatively monitoring the transfer of metals between proteins

Studies have shown that metallothionein (MT) may play an important role in modulating the activity of certain Zn-regulated enzymes under various oxidoreductive conditions by either donating or removing Zn. To better determine the role of MT in interprotein metal transfer, we describe a procedure that uses stable isotopically enriched (67)Zn(7) metallothionein 2 ((67)Zn(7)-MT-2) to quantitatively determine the stoichiometry of transfer of Zn from the protein to a recipient apo-metalloenzyme, apo-carbonic anhydrase (apo-CA) by directly coupled ion exchange high-performance liquid chromatography inductively coupled plasma mass spectrometry. Quantitatively, the transfer of (67)Zn was consistent with the enzymatic activation of the apo-enzyme as judged by its esterase activity and ability to cleave p-nitrophenyl acetate. Maximum enzyme activation occurred at an MT-2:apo-CA molar ratio of 1, implying the release of a single atom of Zn from MT-2. Preincubation of (67)Zn(7)-MT-2 with an excess of oxidized glutathione (GSSG) increased metal donation fourfold, whereas reduced glutathione (GSH) inhibited donation by approximately 50%. By using multiple recipient and donor proteins having different stable isotopic signatures, the technique has the potential for quantitatively studying the kinetic and thermodynamic aspects of Zn transfer between numerous competing ligands in vitro, an important first step toward understanding the regulatory role of this metal in protein functioning and cellular metabolism in vivo.