Functional comparison of metallothioneins MTT1 and MTT2 from Tetrahymena thermophila

Metallothioneins MTT1 and MTT2 from Tetrahymena thermophila have been characterized. The MTT1 contains mainly characteristic Cys-Cys-Cys and Cys-Cys clusters, but MTT2 contains mainly Cys-X-Cys cluster. Cd₁₆-MTT1 mainly consists of α-helix and β-turns, in contrast, Cd₁₁-MTT2 mainly consists of random coils. Reaction of Cd₁₆-MTT1 and Cd₁₁-MTT2 with nitric oxide leads to intramolecular disulfide bond formation, respectively. Binding stabilities of Cd²⁺, Hg²⁺ and Zn²⁺ to MTT1 are stronger than those to MTT2. Cu²⁺ can not replace Cd²⁺ from Cd₁₆-MTT1 complex, but can replace Cd²⁺ from Cd₁₁-MTT2 complex. The analysis of qRT-PCR revealed MTT2 mRNA levels were 31-fold higher than those of MTT1 under basal conditions. These results further suggest MTT1 possibly play a role in the detoxification of heavy metal ions, and MTT2 may be involved in the homeostasis of copper ions.     

Functional comparision between truncated MTT1 and truncated MTT2 from Tetrahyemna thermophila

Metallothioneins (MTs) are low-molecular-weight proteins with high Cys content and high metal-chelating ability. CdMT and CuMT subfamilies present different characteristics in Tetrahymena. To explore the effect of the cysteine arrangement and sequence length of MTs for binding different metal ions, MTT1, truncated MTT1 (TM1), MTT2, and truncated MTT2 (TM2) were expressed in E. coli. The half-maximal inhibiting concentrations (IC₅₀) of Cd²⁺ and Cu⁺ for the recombinant strains were different. Furthermore, E. coli cells expressing MTT1 and TM1 exhibited higher accumulating ability for Cd²⁺ than cells expressing MTT2 and TM2. However, the opposite is true for Cu⁺. The binding ability of the different recombinant proteins to Cd²⁺ and Cu⁺ were also different. MTT1 and truncated mutant TM1 were the preference for Cd²⁺, whereas MTT2 and truncated mutant TM2 were the preference for Cu⁺ coordination. These results showed that metal ion tolerance and accumulation ability not only depended on cysteine arrangement pattern but also on sequence length of MT in Tetrahymena.     

Heterologous Expression and Metal-Binding Characterization of a Type 1 Metallothionein Isoform (OsMTI-1b) from Rice (Oryza sativa)

Metallothioneins (MTs) are ubiquitous, low molecular mass and cysteine-rich proteins that play important roles in maintaining intracellular metal homeostasis, eliminating metal toxification and protecting the cells against oxidative damages. MTs are able to bind metal ions through the thiol groups of their cysteine residues. Plants have several MT isoforms which are classified into four types based on the arrangement of cysteine residues. In the present study, a rice (Oryza sativa) gene encoding type 1 MT isoform, OsMTI-1b, was inserted in vector pET41a and overexpressed in Escherichia coli as carboxy-terminal extensions of glutathione-S-transferase (GST). The recombinant protein GST-OsMTI-1b was purified using affinity chromatography and its ability to bind with Ni²⁺, Cd²⁺, Zn²⁺ and Cu²⁺ ions was analyzed. The results demonstrated that this isoform has ability to bind Ni²⁺, Cd²⁺ and Zn²⁺ ions in vitro, whereas it has no substantial ability to bind Cu²⁺ ions. From competitive reaction with 5,5′-dithiobis(2-nitrobenzoic acid), DTNB, the affinity of metal ions for recombinant form of GST-OsMTI-1b was as follows: Ni²⁺/Cd²⁺ > Zn²⁺ > Cu²⁺     

Metal binding properties and structure of a type III metallothionein from the metal hyperaccumulator plant Noccaea caerulescens

Metallothioneins (MT) are low molecular weight proteins with cysteine-rich sequences that bind heavy metals with remarkably high affinities. Plant MTs differ from animal ones by a peculiar amino acid sequence organization consisting of two short Cys-rich terminal domains (containing from 4 to 8 Cys each) linked by a Cys free region of about 30 residues. In contrast with the current knowledge on the 3D structure of animal MTs, there is a striking lack of structural data on plant MTs. We have expressed and purified a type III MT from Noccaea caerulescens (previously Thlaspi caerulescens). This protein is able to bind a variety of cations including Cd²⁺, Cu²⁺, Zn²⁺ and Pb²⁺, with different stoichiometries as shown by mass spectrometry. The protein displays a complete absence of periodic secondary structures as measured by far-UV circular dichroism, infrared spectroscopy and hydrogen/deuterium exchange kinetics. When attached onto a BIA-ATR biosensor, no significant structural change was observed upon removing the metal ions.     

The βE-Domain of Wheat Ec-1 Metallothionein: A Metal-Binding Domain with a Distinctive Structure

Metallothioneins (MTs) are ubiquitous cysteine-rich proteins with a high affinity for divalent metal ions such as Zn^II, Cu^I, and Cd^II that are involved in metal ion homeostasis and detoxification, as well as protection against reactive oxygen species. Here we show the NMR solution structure of the β_E-domain of the early cysteine-labeled protein (E_c-1) from wheat (β_E-E_c-1), which represents the first three-dimensional structure of a plant MT. The β_E-domain comprises the 51 C-terminal residues of E_c-1 and exhibits a distinctive unprecedented structure with two separate metal-binding centers, a mononuclear Zn^II binding site constituted by two cysteine and two highly conserved histidine residues as found in certain zinc-finger motifs, and a cluster formed by three Zn^II ions coordinated by nine Cys residues that resembles the cluster in the β-domain of vertebrate MTs. Cys-metal ion connectivities were determined by exhaustive structure calculations for all 7560 possible configurations of the three-metal cluster. Backbone dynamics investigated by ¹⁵N relaxation experiments support the results of the structure determination in that β_E-E_c-1 is a rigidly folded polypeptide. To further investigate the influence of metal ion binding on the stability of the structure, we replaced Zn^II with Cd^II ions and examined the effects of metal ion release on incubation with a metal ion chelator.     

The interaction of zinc, nickel and cadmium with serum albumin and histidine-rich glycoprotein assessed by equilibrium dialysis and immunoadsorbent chromatography

Human serum albumin (HSA) has been shown to bind 2–3 mol of Zn²⁺, Ni²⁺, or Cd²⁺ per mole of protein with apparent dissociation constants (K_d) in the range of 10 μm. Rabbit histidine-rich glycoprotein (HRG) binds 13, 9, and 6 mol of Zn²⁺, Ni²⁺, and Cd²⁺ per mole of protein, respectively, with apparent K_ds also near 10 μm. However, the binding of metals by HRG exhibits positive cooperativity, so that the apparent K_ds may underestimate HRGs true affinity for metal ions. The relative affinities of HSA and HRG for metal ions were found to be Zn²⁺ > Ni²⁺ > Cd²⁺. In addition, histidine (a serum metal chelator) affected the binding of Ni²⁺ by both proteins but not that of Zn²⁺ or Cd²⁺. At physiological concentrations of HSA (250 μm), HRG (2.5 μm), and histidine (100 μm), HRG bound 36% of the Zn²⁺, 9% of the Ni²⁺, and 13% of the Cd²⁺ at a total metal concentration of 25 μm. Under the same conditions HSA held 37% of the Zn²⁺, 14% of the Ni²⁺, and 56% of the Cd²⁺. Thus, HSA appears to have a lower intrinsic affinity for the three metals than HRG but would be expected to bind a higher proportion of these metals in serum. A specific immunoadsorbent column was prepared and used to study the metal binding by HRG in serum directly. Both ⁶⁵Zn²⁺ and ⁶³Ni²⁺ were associated with HRG in aliquots of rabbit serum after incubation with the corresponding metal ion. This evidence indicates that HRG must be considered as a metal binding component of serum.     

Inhibition of nitrogen fixation in alfalfa by arsenate, heavy metals, fluoride, and simulated Acid rain

The acute effects of aqueous solutions of As, Cd, Cu, Pb, F, and Zn ions at concentrations from 0.01 to 100 micrograms per milliliter and solutions adjusted to pH 2 to 6 with nitric or sulfuric acid were studied with respect to acetylene reduction, net photosynthesis, respiration rate, and chlorophyll content in Vernal alfalfa (Medicago sativa L. cv. Vernal). The effects of the various treatments on acetylene reduction varied from no demonstrable effect by any concentration of F⁻ and 42% inhibition by 100 micrograms Pb²⁺ per milliliter, to 100% inhibition by 10 micrograms Cd²⁺ per milliliter and 100 micrograms per milliliter As, Cu²⁺, and Zn²⁺ ions. Zn²⁺ showed statistically significant inhibition of activity at 0.1 micrograms per milliliter. Acid treatments were not inhibitory above pH 2, at which pH nitric acid inhibited acetylene reduction activity more than did sulfuric acid. The inhibition of acetylene reduction by these ions was Zn²⁺ > Cd²⁺ > Cu²⁺ > AsO₃⁻ > Pb²⁺ > F⁻. The sensitivity of acetylene reduction to the ions was roughly equal to the sensitivity of photosynthesis, respiration, and chlorophyll content when Pb²⁺ was applied, but was 1,000 times more sensitive to Zn²⁺. The relationship of the data to field conditions and industrial pollution is discussed.     

Breaking-off tissue specific activity of the oil palm metallothionein-like gene promoter in T1 seedlings of tomato exposed to metal ions

Metallothioneins (MTs) are cysteine-rich metal-binding proteins that are involved in cell growth regulation, transportation of metal ions and detoxification of heavy metals. A mesocarp-specific metallothionein-like gene (MT3-A) promoter was isolated from the oil palm (Elaeis guineensis Jacq). A vector construct containing the MT3-A promoter fused to the β-glucuronidase (GUS) gene in the pCAMBIA 1304 vector was produced and used in Agrobacterium-mediated transformation of tomato. Histochemical GUS assay of different tissues of transgenic tomato showed that the MT3-A promoter only drove GUS expression in the reproductive tissues and organs, including the anther, fruit and seed coat. Competitive RT-PCR and GUS fluorometric assay showed changes in the level of GUS mRNA and enzyme activity in the transgenic tomato (T₀). No GUS mRNA was found in roots and leaves of transgenic tomato. In contrast, the leaves of transgenic tomato seedlings (T₁) produced the highest GUS activity when treated with 150 μM Cu²⁺ compared to the control (without Cu²⁺). However, Zn²⁺ and Fe²⁺ treatments did not show GUS expression in the leaves of the transgenic tomato seedlings. Interestingly, the results showed a breaking-off tissue-specific activity of the oil palm MT3-A promoter in T₁ seedlings of tomato when subjected to Cu²⁺ ions.     

Topochemical factors in potentiation of contraction by heavy metal cations

In addition to the previously studied Zn²⁺, low concentrations (about 0.5 mM) of Be²⁺, Ba²⁺, Cd²⁺, Ni²⁺, Cu²⁺, Pt⁴⁺ and, outstandingly, 0.5 µM of UO₂ ²⁺, potentiate the twitch of frog sartorius and toe muscles by prolonging the active state of contraction. The degree of potentiation is a roughly S-shaped function of p(metal²⁺), suggesting that each metal binds to a ligand of the muscle fiber, representative apparent affinity constants being: UO₂ ²⁺, 5 x 10⁶; Zn²⁺, 2.8 x 10⁵; and Cd²⁺, 2 x 10⁴. UO₂ ²⁺ potentiation effects are rapidly reversed by PO₄, and Zn²⁺ and Cd²⁺ effects by EDTA, PO₄, and cysteine. The rapidity of these reversals by the nonpenetrating EDTA and PO₄, and the fact that heavy metal ions evidently potentiate by prolonging the action potential, indicate that the metal potentiators exert their primary action at readily accessible (i.e. plasma and T tubular) membrane sites. The relatively slow kinetics of development of potentiation, and the even slower reversal of it in pure Ringer''s solution, indicate that the metal ions are bound to connective tissue, as well as to muscle fibers. The binding effects at the readily accessible membrane sites evidently impairs delayed rectification and thus modifies the action potential and excitation-contraction coupling so as to cause potentiation. SH is excluded, and PO₄ and imidazole are possibilities, as the membrane ligand binding the potentiating metal ions.     

Modeling the Zn and Cd metalation mechanism in mammalian metallothionein 1a

Mammalian metallothioneins (MTs) are a family of small cysteine rich proteins believed to have a number of physiological functions, including both metal ion homeostasis and toxic metal detoxification. Mammalian MTs bind 7 Zn²⁺ or Cd²⁺ ions into two distinct domains: an N-terminal β-domain that binds 3 Zn²⁺ or Cd²⁺, and a C-terminal α-domain that binds 4 Zn²⁺ or Cd²⁺. Although stepwise metalation to the saturated M₇-MT (where M = Zn²⁺ or Cd²⁺) species would be expected to take place via a noncooperative mechanism involving the 20 cysteine thiolate ligands, literature reports suggest a cooperative mechanism involving cluster formation prior to saturation of the protein. Electrospray ionization mass spectrometry (ESI-MS) provides this sensitivity through delineation of all species (M_n-MT, n = 0-7) coexisting at each step in the metalation process. We report modeled ESI-mass spectral data for the stepwise metalation of human recombinant MT 1a (rhMT) and its two isolated fractions for three mechanistic conditions: cooperative (where the binding affinities are: K₁ < K₂ < K₃ < ··· < K₇), weakly cooperative (where K₁ = K₂ = K₃ = ··· = K₇), and noncooperative, (where K₁ > K₂ > K₃ > ··· > K₇). Detailed ESI-MS metalation data of human recombinant MT 1a by Zn²⁺ and Cd²⁺ are also reported. Comparison of the experimental data with the predicted mass spectral data provides conclusive evidence that metalation occurs in a noncooperative fashion for Zn²⁺ and Cd²⁺ binding to rhMT 1a.     

Heavy metal ions affect the activity of DNA glycosylases of the Fpg family

Prokaryotic enzymes formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease VIII (Nei) and their eukaryotic homologs NEIL1, NEIL2, and NEIL3 define the Fpg family of DNA glycosylases, which initiate the process of repair of oxidized DNA bases. The repair of oxidative DNA lesions is known to be impaired in vivo in the presence of ions of some heavy metals. We have studied the effect of salts of several alkaline earth and transition metals on the activity of Fpg-family DNA glycosylases in the reaction of excision of 5,6-dihydrouracil, a typical DNA oxidation product. The reaction catalyzed by NEIL1 was characterized by values K _m = 150 nM and k _cat = 1.2 min⁻¹, which were in the range of these constants for excision of other damaged bases by this enzyme. NEIL1 was inhibited by Al³⁺, Ni²⁺, Co²⁺, Cd²⁺, Cu²⁺, Zn²⁺, and Fe²⁺ in Tris-HCl buffer and by Cd²⁺, Zn²⁺, Cu²⁺, and Fe²⁺ in potassium phosphate buffer. Fpg and Nei, the prokaryotic homologs of NEIL1, were inhibited by the same metal ions as NEIL1. The values of I₅₀ for NEIL1 inhibition were 7 μM for Cd²⁺, 16 μM for Zn²⁺, and 400 μM for Cu²⁺. The inhibition of NEIL1 by Cd²⁺, Zn²⁺, and Cu²⁺ was at least partly due to the formation of metal-DNA complexes. In the case of Cd²⁺ and Cu²⁺, which preferentially bind to DNA bases rather than phosphates, the presence of metal ions caused the enzyme to lose the ability for preferential binding to damaged DNA. Therefore, the inhibition of NEIL1 activity in removal of oxidative lesions by heavy metal ions may be a reason for their comutagenicity under oxidative stress.     

重金属铜、锌、镉复合胁迫对麻疯树幼苗生理生化的影响

该研究以Cu~(2+)、Zn~(2+)、Cd~(2+)单一胁迫为对照,探讨不同浓度的Cu~(2+)、Zn~(2+)、Cd~(2+)复合胁迫对麻疯树幼苗生理生化指标的影响。结果表明:随着Cu~(2+)、Zn~(2+)、Cd~(2+)浓度的增加,麻疯树幼苗叶片中的蛋白质(Pro)、丙二醛(MDA)含量均逐渐增加,其叶片叶绿素含量随着Zn~(2+)胁迫浓度的增加呈现出先降后升的趋势,在中等浓度(100 mg·L-1)的Zn~(2+)胁迫时含量最低、随着Cu~(2+)胁迫浓度的增加叶绿素含量先升高后降低,在Cu~(2+)浓度为200 mg·L-1时含量最高,达到1 200 mg·g-1FW; Cd~(2+)胁迫对叶绿素含量和根系活力无明显影响。根系活力在Zn~(2+)浓度为100 mg·L~(-1)时最强,随着Cu~(2+)浓度的增加而减弱。低浓度的Cu~(2+)、Zn~(2+)、Cd~(2+)对过氧化物酶活性和可溶性糖含量都具有促进作用。Cu~(2+)、Zn~(2+)、Cd~(2+)复合胁迫时对可溶性蛋白、叶绿素和丙二醛含量均无明显影响,随着复合胁迫时浓度的增加,可溶性糖含量和根系活力先增后减。这表明麻疯树对三种重金属的胁迫具有一定的抗性,过高浓度的胁迫会影响麻疯树幼苗生理生化的一些指标,但是麻疯树可以通过自身的防御系统使伤害降到最小。此外,重金属复合胁迫可以在一定程度上减轻单一胁迫对麻疯树幼苗造成的毒害作用。     

Isolation of GIF from porcine brain and studies of its zinc transfer kinetics with apo-carbonic anhydrase

Neuronal growth inhibitory factor (GIF) of porcine brain, was isolated and purified by a similar procedure which was used on the isolation of human and bovine GIF. The native porcine protein with stoichiometry of 4Cu⁺, 3Zn²⁺ was obtained for the first time. The kinetics of zinc transfer from Cu₄Zn₃MT-3 to apo-carbonic anhydrase were studied, and zinc transfer rate constants and thermodynamic parameters were obtained. It is found that like other MTs, porcine Cu₄Zn₃MT-3 can also transfer its zinc atom to apoCA, even much easier than other MTs. A possible association mechanism has been proposed, the formation of Cu₄Zn₃MT3-apoCA complex may be the rate-determining step. The obtained data indicate besides its neuronal growth inhibitory function, GIF might play a role in cellular Zn homeostasis in brain.     

重金属Cd2+和Cu2+胁迫下泥蚶消化酶活性的变化

为了探讨重金属Cd²⁺和Cu²⁺胁迫对泥蚶消化酶活性的影响,运用酶学分析的方法,按《渔业水质标准》（GB 11607）规定的Cd²⁺、Cu²⁺最高限量值的1、2、5、10倍设置重金属离子Cd²⁺、Cu²⁺浓度及其组合,研究了在重金属Cd²⁺、Cu²⁺胁迫下,30d内泥蚶3种消化酶活性的变化规律。结果表明：与空白对照组相比,在重金属Cd²⁺、Cu²⁺或其组合的胁迫下,较低浓度组泥蚶的淀粉酶活性实验前期增强（即被诱导）,实验后期减弱（即被抑制）,较高浓度组泥蚶的淀粉酶活性从实验一开始就减弱,并保持在较低水平,毒性比较,同一重金属高浓度 > 低浓度,不同重金属及其组合Cu²⁺ > （Cd²⁺+Cu²⁺）组合 > Cd²⁺;泥蚶脂肪酶的活性实验前期增强,实验后期转为微减弱或减弱,毒性比较,同一重金属高浓度 > 低浓度,不同重金属及其组合（Cd²⁺+Cu²⁺）组合 > Cu²⁺ > Cd²⁺;泥蚶胃蛋白酶的活性实验前期增强,且活性呈现升高-降低-再升高-再降低的变化,实验后期分别表现微增强、微减弱和减弱,毒性比较,同一重金属高浓度 > 低浓度,不同重金属及其组合（Cd²⁺+Cu²⁺）组合 > Cu²⁺ > Cd²⁺。可见：环境中的Cd²⁺和Cu²⁺对泥蚶的消化酶活性起着明显的影响作用。     

Action of Heavy Metals on Hill Activity and O(2) Evolution in Anacystis nidulans

Addition of 5 micromolar Cu²⁺, Cd²⁺, and Zn²⁺ was inhibitory to 10 micromolar H₂O₂-supported Hill activity (dichlorophenolindophenol reduction) and O₂ evolution in membrane preparation from Anacystis nidulans. The reversal of Cd²⁺ and Zn²⁺ inhibition, in contrast to Cu²⁺, by exogenously added catalase (EC 1.11.1.6) suggested that the former cations were inhibitory to H₂O₂ degradation. Ascorbic acid (20 micromolar) supported 27% of the Hill activity which was insensitive to DCMU (10 micromolar) and the remaining activity, attributable to the DCMU sensitive process, was sensitive to inhibition by Cu²⁺ only. It is suggestive that the action site of Cd²⁺ and Zn²⁺ is located between the electron donation sites of H₂O₂ and ascorbic acid, while that of Cu²⁺ is located beyond it. Electron donation by reduced glutathione was insensitive to DCMU and Cu²⁺, indicating that the action site of Cu²⁺ is prior to its electron donation site. Further, the phenanthroline (10 micromolar) reversal of Cu²⁺ inhibition of Hill activity suggested a tentative action site of Cu²⁺ at the level of cytochrome.     

Solid phase speciation of Zn and Cd in zinc smelter effluent-irrigated soils

Solubility of metal in contaminated soils is a key factor which controls the phytoavailability and toxic effects of metals on soil environment. The chemical equilibria of metal ions between soil solution and solid phases govern the solubility of metals in soil. Hence, an attempt was made to identify the probable solid phases (minerals), which govern the solubility of Zn²⁺ and Cd²⁺ in zinc smelter effluent-irrigated soils. Estimation of free ion activities of Zn²⁺ (pZn²⁺) and Cd²⁺ (pCd²⁺) by Baker soil test indicated that metal ion activities were higher in smelter effluent-irrigated soils as compared to that in tubewell water-irrigated soils. Identification of solid phases further reveals that free ion activity of Zn²⁺ and Cd²⁺ in soil highly contaminated with Zn and Cd due to long-term irrigation with zinc smelter effluent is limited by the solubility of willemite (Zn₂SiO₄) in equilibrium with quartz and octavite (CdCO₃), respectively. However, in case of tubewell water-irrigated soil, franklinite (ZnFe₂O₄) in equilibrium with soil-Fe and exchangeable Cd are likely to govern the activity of Zn²⁺ and Cd²⁺ in soil solution, respectively. Formation of highly soluble minerals namely, willemite and octavite indicates the potential ecological risk of Zn and Cd, respectively in smelter effluent irrigated soil.     

Tamarix hispida metallothionein-like ThMT3, a reactive oxygen species scavenger, increases tolerance against Cd2+, Zn2+, Cu2+, and NaCl in transgenic yeast

A metallothionein-like gene, ThMT3, encoding a type 3 metallothionein, was isolated from a Tamarix hispida leaf cDNA library. Expression analysis revealed that mRNA of ThMT3 was upregulated by high salinity as well as by heavy metal ions, and that ThMT3 was predominantly expressed in the leaf. Transgenic yeast (Saccharomyces cerevisiae) expressing ThMT3 showed increased tolerance to Cd²⁺, Zn²⁺, Cu²⁺, and NaCl stress. Transgenic yeast also accumulated more Cd²⁺, Zn²⁺, and NaCl, but not Cu²⁺. Analysis of the expression of four genes (GLR1, GTT2, GSH1, and YCF1) that aid in transporting heavy metal (Cd²⁺) from the cytoplasm to the vacuole demonstrated that none of these genes were induced under Cd²⁺, Zn²⁺, Cu²⁺, and NaCl stress in ThMT3-transgenic yeast. H₂O₂ levels in transgenic yeast under such stress conditions were less than half those in control yeast under the same conditions. Three antioxidant genes (SOD1, CAT1, and GPX1) were specifically expressed under Cd²⁺, Zn²⁺, Cu²⁺, and NaCl stress in the transgenic yeast. Cd²⁺, Zn²⁺, and Cu²⁺ increased the expression levels of SOD1, CAT1, and GPX1, respectively, whereas NaCl induced the expression of SOD1 and GPX1.     

Metal ion complexes of d-biotin in solution. Stability of the stereoselective thioether coordination

By spectrophotometry and ¹H nmr, several of the stability constants of the thioether complexes between Mg²⁺, Ca²⁺, Mn²⁺, Cu²⁺, Zn²⁺, Cd²⁺, or Ag⁺ and d-biotin (Bio), tetrahydrothiophene (Tht), and dimethyl sulfide (Dms) have been measured in 50% aqueous ethanol, 96% N,N-dimethylformamide (DMF), 98% d₆-dimethyl sulfoxide, or in D₂O. With decreasing concentration of water, the thioether interaction increases with the biologically important metal ions, whereas, e.g., Ag⁺ behaves in the opposite way. The stability of these complexes is, in general, quite small: for example, with d-biotin in 96% DMF (I = 1.0; 25°C) log K_M(Bio)^M = 0.03 and 1.64 for Cu²⁺ and Ag⁺, respectively; in D₂O (I = 0.5 for Ag⁺, all others 2–5; 27°C) log K_M(Bio)^M ? ?1.0, ?1.4, ?1.2, ?0.9, or 4.20 for Mg²⁺, Ca²⁺, Zn²⁺, Cd²⁺, or Ag⁺. In those cases where the difference log K_M(Tht)^M ? log K_M(Bio)^M can be calculated, it is in the order of 0.3 log units; this observation, as well as the chemical shifts measured, confirm the earlier suggestion that the interaction at the sulfur of biotin is stereoselective: the metal ion coordinates from “below” the tetrahydrothiophene ring of biotin to the sulfur atom, i.e., trans to the urea ring. It is emphasized that despite the low stability of these complexes with the biologically meaningful metal ions, the extent of the interaction is enough to create specific structures.     

Spectroscopic characterization of a fruit-specific metallothionein: M. acuminata MT3

Metallothioneins (MTs) are ubiquitous low molecular mass, cysteine-rich proteins with the ability to bind d¹⁰ metal ions in the form of metal-thiolate clusters. In contrast to the vertebrate forms, knowledge about the properties of members of the plant metallothionein family is still scarce. The amino acid sequences of plant MTs are distinctively different to the sequences of other MT species. The protein under investigation, Musa acuminata (banana) MT3, belongs to the plant MT fruit-specific p3 subfamily. With a total of 10 cysteine residues, MT3 features a cysteine content and percentage that is more comparable to fungal and prokaryotic MTs than to the well characterized mammalian iso-forms. The gene sequence encoding MT3 was cloned into a suitable vector and the protein was recombinantly overexpressed in Escherichia coli. MT3 is able to coordinate a maximum of four divalent d¹⁰ metal ions under the formation of metal-thiolate clusters. The hitherto unknown spectroscopic behavior of MT3 in combination with the metal ions Zn²⁺, Cd²⁺, Pb²⁺, and Hg²⁺ will be presented and gives rise to the existence of a weaker metal ion coordination site. The pH stability of the investigated zinc and cadmium clusters is comparable to the values found for other plant metallothioneins though significantly lower than for the mammalian iso-forms. Possible metal-thiolate cluster structures will additionally be discussed.