The effect of nitric oxide on metal release from metallothionein-3: gradual unfolding of the protein

Metallothionein-3 (MT-3), or neuronal growth inhibitory factor, which exhibits growth inhibitory activity, is a brain-specific metallothionein. In this study, the effect of nitric oxide (NO) on metal release (using Cd²⁺ as a probe) from MT-3 was examined by ¹¹³Cd and 2D [¹H–¹⁵N] heteronuclear single-quantum coherence NMR spectroscopy. The exposure of human MT-3 to NO leads to a nonselective release of the three metals from the β-domain. In contrast to metallothionein-1 and metallothionein-2, two of the bound metals in the α-domain were also partially released, with the domain structure remaining almost unchanged. Further addition of NO resulted in the complete release of metals and concomitant unfolding of the protein. The preference of release of the two metals in the α-domain was attributed to the presence of two slightly different coordination environments for the four cadmium/zinc atoms.     

Important roles of the conserved linker-KKS in human neuronal growth inhibitory factor

Metallothinein-3 (MT3), also named neuronal growth inhibitory factor (GIF), is attractive by its distinct neuronal growth inhibitory activity, which is not shared by other MT isoforms. The polypeptide chain of GIF is folded into two individual domains, which are connected by a highly conserved linker, KKS. In order to figure out the significance of the conserved segment, we constructed several mutants of human GIF (hGIF), including the K31/32A mutant, the K31/32E mutant and the KKS-SP mutant by site-directed mutagenesis. pH titration and DTNB reaction exhibited that all the three mutations made the β-domain lower in stability and looser. More significantly, change of KKS to SP also altered the general backbone conformation and metal–thiolate cluster geometry. Notably, bioassay results showed that the bioactivity of the K31/32A mutant and the K31/32E mutant decreased obviously, while the KKS-SP mutant lost inhibitory activity completely. Based on these results, we proposed that the KKS linker was a crucial factor in modulating the stability and the solvent accessibility of the Cd₃S₉ cluster in the β-domain through domain–domain interactions, thus was indispensable to the biological activity of hGIF.     

The metal-binding properties of the blue crab copper specific CuMT-2: a crustacean metallothionein with two cysteine triplets

Most crustacean metallothioneins (MTs) contain 18 Cys residues and bind six divalent metal ions. The copper-specific CuMT-2 (MTC) of the blue crab Callinectes sapidus with 21 Cys residues, of which six are organized in two uncommon Cys-Cys-Cys sequences, represents an exception. However, its metal-binding properties are unknown. By spectroscopic and spectrometric techniques we show that all 21 Cys residues of recombinant MTC participate in the binding of Cu(I), Zn(II), and Cd(II) ions, indicating that both Cys triplets act as ligands. The fully metallated M₈ ^II–MTC (M is Zn, Cd) form possesses high- and low-affinity metal binding sites, as evidenced by the formation of Zn₆–MTC and Cd₇–MTC species from M₈ ^II–MTC after treatment with Chelex 100. The NMR characterization of Cd₇–MTC suggests the presence of a two-domain structure, each domain containing one Cys triplet and encompassing either the three-metal or the four-metal thiolate cluster. Whereas the metal–Cys connectivities in the three-metal cluster located in the N-terminal domain (residues 1–31) reveal a Cd₃Cys₉ cyclohexane-like structure, the presence of dynamic processes in the C-terminal domain (residues 32–64) precluded the determination of the organization of the four-metal cluster. Absorption and circular dichroism features accompanying the stepwise binding of Cu(I) to MTC suggest that all 21 Cys are involved in the binding of eight to nine Cu(I) ions (Cu_8–9–MTC). The subsequent generation of Cu₁₂–MTC involves structural changes consistent with a decrease in the Cu(I) coordination number. Overall, the metal-binding properties of MTC reported here contribute to a better understanding of the role of Cys triplets in MTs.     

Effect of α-domain substitution on the structure, property and function of human neuronal growth inhibitory factor

Human metallothionein-3 (hMT3), also named human neuronal growth inhibitory factor (hGIF), is attractive due to its distinct neuronal growth inhibitory activity, which is not shown by other human MT isoforms. It has been reported that the neuronal growth inhibitory activity arises from the N-terminal β-domain rather than its C-terminal α-domain. However, previous bioassay results have shown that the single β-domain is less effective at inhibiting the neuron growth than that in intact hMT3 on a molar basis, which suggests that the α-domain is indispensable to the neuronal growth inhibitory activity of hMT3. In order to confirm this assumption, we constructed two domain-hybrid mutants, the β(MT3)–β(MT3) mutant and the β(MT3)–α(MT1) mutant, and investigated their structural and metal binding properties by UV-vis spectroscopy, CD spectroscopy, pH titration, DTNB reaction, EDTA reaction, etc. The results showed that stability of the Cd₃S₉ cluster of the β(MT3)–β(MT3) mutant decreased significantly while the Cd₃S₉ cluster of the β(MT3)–α(MT1) mutant had a similar stability and solvent accessibility to that of hMT3. Interestingly, the bioassay results showed that the neuronal growth inhibitory activity of the β(MT3)–β(MT3) mutant decreased significantly, while the β(MT3)–α(MT1) mutant showed similar inhibitory activity to hMT3. Based on these results, we conclude that the α-domain is indispensable and plays an important role in modulating the stability of the metal cluster in the β-domain by domain–domain interactions, thus influencing the bioactivity of hMT3. Z.-C. Ding and Q. Zheng contributed equally to this work.     

The Properties of the Metal–Thiolate Clusters in Recombinant Mouse Metallothionein-4

Metallothioneins (MTs) are metal-binding proteins with low molecular weight and conservative cysteine residues. Metallothionein-4 (MT-4), one of MT isoforms, is first reported to be distributed in a tissue-specific manner, mainly in stratified squamous epithelia. Here, we compare the properties of metal–thiolate clusters in MT-4 to those in MT-1 and MT-3, including the stabilities toward both pH change and EDTA, as well as the exposure of thiolates to solvent. The metal–thiolate clusters in MT-3 show different property and activity to the reactions compared with MT-4 and MT-1. The structure of metal–thiolate clusters in MT-4 is similar to that of MT-1 from the UV and CD spectra. During pH titration and DTNB reaction, MT-4 and MT-1 exhibit comparable behavior. But while reacting with EDTA, the metal–thiolate clusters in MT-4 are more stable than those of MT-1. We suppose the negative charge of the β-domain of MT-4 prevents the EDTA attack to MT-4.     

Reaction of human metallothionein-3 with cisplatin and transplatin

Human metallothioneins, small cysteine- and metal-rich proteins, play an important role in the acquired resistance to platinum-based anticancer drugs. These proteins contain a M(II)₄(CysS)₁₁ cluster and a M(II)₃(CysS)₉ cluster localized in the α-domain and the β-domain, respectively. The noninducible isoform metallothionein-3 (Zn₇MT-3) is mainly expressed in the brain, but was found overexpressed in a number of cancer tissues. Since the structural properties of this isoform substantially differ from those of the ubiquitously occurring Zn₇MT-1/Zn₇MT-2 isoforms, the reactions of cis-diamminedichloridoplatinum(II) (cisplatin) and trans-diamminedichloridoplatinum(II) (transplatin) with human Zn₇MT-3 were investigated and the products characterized. A comparison of the reaction kinetics revealed that transplatin reacts with cysteine ligands of Zn₇MT-3 faster than cisplatin. In both binding processes, stoichiometric amounts of Zn(II) were released from the protein. Marked differences between the reaction rates of cisplatin and transplatin binding to Zn₇MT-3 and the formation of the Pt–S bonds suggest that the binding of both Pt(II) compounds is a complex process, involving at least two subsequent binding steps. The electrospray ionization mass spectrometry characterization of the products showed that whereas all ligands in cisplatin were replaced by cysteine thiolates, transplatin retained its carrier ammine ligands. The ¹¹³Cd NMR studies of Pt₁ ¹¹³Cd₆MT-3 revealed that cisplatin binds preferentially to the β-domain of the protein. The rates of reaction of cisplatin and transplatin with Zn₇MT-3 were much faster than those of cisplatin and transplatin with Zn₇MT-2. The biological consequences of a substantially higher reactivity of cisplatin toward Zn₇MT-3 than Zn₇MT-2 in the acquired resistance to platinum-based drugs are discussed.     

Characterization of the cadmium complex of peptide 49–61: a putative nucleation center for cadmium-induced folding in rabbit liver metallothionein IIA

 The synthetic peptide fragment containing residues 49–61 of rabbit liver metallothionein II (MT-II) (Ac-Ile-Cys-Lys-Gly-Ala-Ser-Asp-Lys-Cys-Ser-Cys-Cys-Ala-COOH), which includes the only sequential four cysteines bound to the same metal ion in Cd₇MT, forms a stable, monomeric Cd-peptide complex with 1 : 1 stoichiometry (Cd:peptide) via Cd-thiolate interactions. This represents the first synthesis of a single metal-binding site of MT independent of the domains. The ¹¹¹Cd NMR chemical shift at 716 ppm indicates that the ¹¹¹Cd²⁺ in the metal site is terminally coordinated to four side-chain thiolates of the cysteine residues. The pH of half dissociation for this Cd-peptide derivative, ∼3.3, demonstrates an affinity similar to that for Cd₇MT. Molecular mechanics calculations show that the thermodynamically most stable folding for this isolated Cd²⁺ center has the same counterclockwise chirality (Λ or S) observed in the native holo-protein. These properties are consistent with its proposed role as a nucleation center for cadmium-induced protein folding. However, the kinetic reactivity of the CdS₄ structure toward 5,5′-dithiobis(5-nitrobenzoate) (DTNB) and EDTA is greatly increased compared to the complete cluster (α-domain or holo-protein). The rate law for the reaction with DTNB is rate=(k _uf +k _1,f +k _2,f [DTNB])[peptide], where k _uf=0.15 s^–1, k _1,f=2.59×10^–3s^–1, and k _2,f=0.88 M^–1s^–1. The ultrafast step (uf), observable only by stopped-flow measurement, is unprecedented for mammalian (M₇MT) and crustacean (M₆MT) holo-proteins or the isolated domains. The accommodation of other metal ions by the peptide indicates a rich coordination chemistry, including stoichiometries of M-peptide for Hg²⁺, Cd²⁺, and Zn²⁺, M₂-peptide for Hg²⁺ and Au⁺, and (Et₃PAu)₂-peptide. Received: 9 December 1998 / Accepted: 20 May 1999     

Nickel(II)-substituted azurin I from Alcaligenes xylosoxidans as characterized by resonance Raman spectroscopy at cryogenic temperature

Metal-substituted blue copper proteins (cupredoxins) have been successfully used to study the effect of metal-ion identity on their active-site properties, specifically the coordination geometry and metal–ligand bond strengths. In this work, low-temperature (77 K) resonance Raman (RR) spectra of the blue copper protein Alcaligenes xylosoxidans azurin I and its Ni(II) derivative are reported. A detailed analysis of all observed bands is presented and responsiveness to metal substitution is discussed in terms of structural and bonding changes. The native cupric site exhibits a RR spectrum characteristic of a primarily trigonal planar (type 1) coordination geometry, identified by the ν(Cu–S)_Cys markers at 373, 399, 409, and 430 cm⁻¹. Replacement of Cu(II) with Ni(II) results in optical and RR spectra that reveal (1) a large hypsochromic shift in the main (Cys)S → M(II) charge-transfer absorption from 622 to 440 nm, (2) greatly reduced metal–thiolate bonding interaction, indicated by substantially lower ν(Ni–S)_Cys stretching frequencies, (3) elevation of the cysteine ν(C _β –S) stretching, amide III, and ρ _s(C _β H₂) scissors vibrational modes, and (4) primarily four-coordinated, trigonally distorted tetrahedral geometry of the Ni(II) site that is marked by characteristic ν(Ni–S)_Cys stretching RR bands at 347, 364, and 391 cm⁻¹. Comparisons of the electronic and vibrational properties between A. xylosoxidans azurin I and its closely structurally related azurin from Pseudomonas aeruginosa are made and discussed. For cupric azurins, the intensity-weighted average M(II)–S(Cys) stretching frequencies are calculated to be ν(Cu–S)_iwa = 406.3 and 407.6 cm⁻¹, respectively. These values decreased to ν(Ni–S)_iwa = 359.3 and 365.5 cm⁻¹, respectively, after Ni(II) → Cu(II) exchange, suggesting that the metal–thiolate interactions are similar in the two native proteins but are much less alike in their Ni(II)-substituted forms.     

Kinetic and structural studies on roles of the serine ligand and a strictly conserved tyrosine residue in nitrile hydratase

Nitrile hydratases (NHase), which catalyze the hydration of nitriles to amides, have an unusual Fe³⁺ or Co³⁺ center with two modified Cys ligands: cysteine sulfininate (Cys-SO₂ ⁻) and either cysteine sulfenic acid or cysteine sulfenate [Cys-SO(H)]. Two catalytic mechanisms have been proposed. One is that the sulfenyl oxygen activates a water molecule, enabling nucleophilic attack on the nitrile carbon. The other is that the Ser ligand ionizes the strictly conserved Tyr, activating a water molecule. Here, we characterized mutants of Fe-type NHase from Rhodococcus erythropolis N771, replacing the Ser and Tyr residues, αS113A and βY72F. The αS113A mutation partially affected catalytic activity and did not change the pH profiles of the kinetic parameters. UV–vis absorption spectra indicated that the electronic state of the Fe center was altered by the αS113A mutation, but the changes could be prevented by a competitive inhibitor, n-butyric acid. The overall structure of the αS113A mutant was similar to that of the wild type, but significant changes were observed around the catalytic cavity. Like the UV–vis spectra, the changes were compensated by the substrate or product. The Ser ligand is important for the structure around the catalytic cavity, but is not essential for catalysis. The βY72F mutant exhibited no activity. The structure of the βY72F mutant was highly conserved but was found to be the inactivated state, with αCys114-SO(H) oxidized to Cys-SO₂ ⁻, suggesting that βTyr72 affected the electronic state of the Fe center. The catalytic mechanism is discussed on the basis of the results obtained.     

Simultaneous α/β spin-state selection for <Superscript>13</Superscript>C and <Superscript>15</Superscript>N from a time-shared HSQC-IPAP experiment

Two novel HSQC-IPAP approaches are proposed to achieve α/β spin-state editing simultaneously for ¹³C and ¹⁵N in a single NMR experiment. The pulse schemes are based on a time-shared (TS) 2D ¹H,¹³C/¹H,¹⁵N-HSQC correlation experiment that combines concatenated echo elements for simultaneous J(CH) and J(NH) coupling constants evolution, TS evolution of ¹³C and ¹⁵N chemical shifts in the indirect dimension and heteronuclear α/β-spin-state selection by means of the IPAP principle. Heteronuclear α/β-editing for all CH _n (n = 1–3) and NH _n (1–2) multiplicities can be achieved in the detected F2 dimension of a single TS-HSQC-F2-IPAP experiment. On the other hand, an alternative TS-HSQC-F1-IPAP experiment is also proposed to achieve α/β-editing in the indirect F1 dimension. Experimental and simulated data is provided to evaluate these principles in terms of sensitivity and performance simultaneously on backbone and side-chain CH, CH₂, CH₃, NH, and NH₂ spin systems in uniformly ¹³C/¹⁵N-labeled proteins and in small natural-abundance peptides.     

Influence of {\text{NH - }}{{\text{S}}^\gamma } bonding interactions on the structure and dynamics of metallothioneins

Mammalian metallothioneins ( \textM₇^\textIIMTs {\text{M}}_7^{\text{IIMTs}} ) show a clustered arrangement of the metal ions and a nonregular protein structure. The solution structures of Cd₃-thiolate cluster containing β-domain of mouse β-MT-1 and rat β-MT-2 show high structural similarities, but widely differing structure dynamics. Molecular dynamics simulations revealed a substantially increased number of \textNH - \textS^g {\text{NH - }}{{\text{S}}^\gamma } hydrogen bonds in β-MT-2, features likely responsible for the increased stability of the Cd₃-thiolate cluster and the enfolding protein domain. Alterations in the \textNH - \textS^g {\text{NH - }}{{\text{S}}^\gamma } hydrogen-bonding network may provide a rationale for the differences in dynamic properties encountered in the β-domains of MT-1, -2, and -3 isoforms, believed to be essential for their different biological function.     

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.     

Structural and immunochemical characterization of β-1,2-linked mannobiosyl phosphate residue in the cell wall mannan of Candida glabrata

A mannan of Candida glabrata IFO 0622 digested by Arthrobacter exo-α-mannosidase and a β-1,2-linked mannobiose obtained from the parent mannan by acid treatment was analyzed using ¹³C nuclear magnetic resonance spectroscopy. The results show that the β-1,2-linked mannobiosyl residue is esterified to a phosphate group through position C-1 in the α-configuration, Manβ1– 2Manα1–HPO₃–. The results of immunochemical assays of these mannans using the commercial antigenic factor sera of the genus Candida (Candida Check, Iatron) indicate that the main recognition site of serum no. 6 in this kit is the mannotetraosyl side-chain Manβ1–2Manα1– 2Manα1–2Man in C. glabrata mannan and also suggest that the phosphate-containing unit (such as Manβ1– 2Manα1–HPO₃– in this mannan) behaves as one of the antigenic determinants of serum no. 6, but not of serum no. 5. Therefore, the present and previous findings indicate that serum no. 5 recognizes relatively longer β-1,2-linked oligomannosyl side-chains, Manβ1–[2Manβ1–]_n 2Man (n = 1–6), attached to the phosphate groups previously observed in the cell wall mannans of Candida albicans, Candida stellatoidea, and Candida tropicalis. Received: 18 March 1997 / Accepted: 16 September 1997     

Relationship between tolerance and accumulation characteristics of cadmium in higher plants

Ten plant species belonging to 5 families,i.e., Cruciferae, Cucurbitaceae, Gramineae, Leguminosae, and Solanaceae, were grown in a sand soil at two pH levels. The soil was subjected to an application of CdCl₂ at rates of 0 to 700 mgCd·kg⁻¹ soil. The relationship between Cd concentration in the shoots (tc) and soil (sc; NH₄NO₃ extractable) was expressed by the equation: log (tc)=α+βlog (sc). The coefficients α and β were estimated for each species at each level of soil pH. Plottings of the scores on α and β axes showed that the Cd accumulation characteristics in the plants appeared to depend on the families irrespective of soil pH. Based on theupper critical concentration of Cd in the tops (C_t), the Cruciferae and Leguminosae species were found to be the most and the least tolerant to Cd, respectively. The C_t values correlated exceedingly well with the values of α.     

Resonance assignments and secondary structure of a phytocystatin from Ananas comosus

A cDNA encoding a cysteine protease inhibitor, cystatin was cloned from pineapple (Ananas comosus L.) stem. This clone was constructed into an expression vector and expressed in E. coli and purified to homogeneous. The recombinant pineapple cystatins (AcCYS) showed effectively inhibitory activity toward cysteine proteases including papain, bromelain, and cathepsin B. In order to unravel its inhibitory action from structural point of view, multidimensional heteronuclear NMR techniques were used to characterize the structure of AcCYS. The full ¹H, ¹⁵N, and ¹³C resonance assignments of AcCYS were determined. The secondary structure of AcCYS was identified by using the assigned chemical shift of ¹Hα, ¹³Cα, ¹³Cβ, and ¹³CO through the consensus chemical shift index (CSI). The results of CSI analysis suggest 5 β–strands (residues 45–47, 84–91, 94–104, 106–117, and 123–130) and one α–helix (residues 55–73).     

Preparation and reactivity of a tetranuclear Fe(II) core in the metallothionein α-domain

Metallothioneins (MTs) are small cysteine-rich proteins which exhibit high affinities for various metal ions and play roles in storage of essential metals and detoxification of toxic metals. Studies on the redox properties of MTs have been quite limited. Recently, we focused on the α-domain of MT (MTα) as a protein matrix and incorporated a tetranuclear metal cluster as a reductant. UV-visible, CD and MS data indicate the formation of the stable tetranuclear metal-cysteine cluster in the MTα matrix with Fe^II₄-MTα and Co^II₄-MTα species existing in water. Furthermore, the Fe^II₄-MTα species was found to promote the reduction of met-myoglobin and azobenzene derivatives under mild conditions. Particularly, the stoichiometric reduction of methyl red with Fe^II₄-MTα (1:1) was found to proceed with a conversion of 98% over a period of 6 h at 25 °C. This indicates that all of the four Fe(II) cores contribute to the reduction. In this paper, we describe the preparation and reactivity of the tetranuclear iron cluster in the protein matrix.     

Direct metal ion substitution at the [M(SCys)4]2– site of rubredoxin

 The single Fe(II) in reduced rubredoxin from Clostridium pasteurianum was found to be quantitatively displaced by either Cd²⁺ or Zn²⁺ when a modest molar excess of the substituting metal salt was anaerobically incubated with the reduced rubredoxin under mild conditions, namely, room temperature, pH 5.4–8.4, and no protein denaturants. Under the same conditions, cadmium-for-zinc substitution was also achieved upon aerobic incubation of the zinc-substituted rubredoxin with a modest molar excess of Cd²⁺. Displacements of Fe(II) from the reduced rubredoxin were not observed upon anaerobic incubation with Ni²⁺, Co²⁺, or VO²⁺ salts, and no reaction with any of the divalent metal ions was observed for the oxidized [Fe(III)] rubredoxin. Fe(II) could not be re-inserted into the Zn- or Cd-substituted rubredoxins without resorting to protein denaturation. ¹H and ¹¹³Cd NMR experiments showed that the cadmium-substituted rubredoxin prepared by the non-denaturing substitution method retained the pseudotetrahedral M(SCys)₄ coordination geometry and secondary structural elements characteristic of the native rubredoxin, and that "unzipping" of the β-sheet did not occur during metal substitution. Rates of Fe(II) displacement by M²⁺ (M=Cd or Zn) increased with increasing M²⁺/rubredoxin ratio, decreasing pH, and lower ionic strength. The substitution rates were faster for M=Cd than for M=Zn. Rates of Cd²⁺ substitution into a V8A-mutated rubredoxin were significantly faster than for the wild-type protein. The side-chain of V8 is on the protein surface and close to the metal-ligating Cys42Sγ at the M(SCys)₄ site. Therefore, the rate-limiting step in the substitution process is suggested to involve direct attack of the [M(SCys)₄]^2– site by the incoming M²⁺, without global unfolding of the protein. Implications of these results for metal ion incorporation into rubredoxins in vivo are discussed. Received: 29 May 1998 / Accepted: 11 August 1998     

Hemoglobin types in saanen goats and Barbary sheep: Genetic and comparative aspects

By the use of the Immobiline technique at pH ranges 7.0–7.6 and 6.9–7.9, 16 different hemoglobin (Hb) phenotypes were observed in 61 English Saanen goats. They are explained in this breed by a genetic theory of five β-globin genes (A ₄,A ₆,A ₈,E, andD) and two closely linked α-globin loci (′α and ″α) of which the ″α has a variant allele, provisionally called ″α ^X . Family data together with observed and expected Hb frequencies were in agreement with the genetic theory. Among six Barbary sheep there were three Hb phenotypes explained by the occurrence of the β-chain allelesB andC ^na.     

Enhanced cadmium accumulation in maize roots—the impact of organic acids

Low molecular weight organic acids are important components of root exudates and therefore, knowledge regarding the mechanisms of cadmium (Cd) uptake and distribution within plants under the influence of organic acids, is necessary for a better understanding of Cd behavior in the plant–soil system. In this study, acetic and malic acids increased the uptake of Cd by maize (Zea mays L. cv. TY2) roots and enhanced Cd accumulation in shoots under hydroponic conditions. Concentration-dependent net Cd influx in the presence and absence of organic acids could be resolved into linear and saturable components. The saturable component followed Michaelis–Menten kinetics, which indicated that Cd uptake across the plasma membrane was transporter-mediated. While the K _m values were similar, the V _max values in the presence of acetic and malic acids were respectively 6.0 and 3.0 times that of the control. Zinc transporters were the most probable pathways for Cd accumulation. It was hypothesized that Cd(II)–organic acid complexes associated with the root zone, could decompose and liberate Cd²⁺ for subsequent absorption by maize roots; and that in the layer of the roots or within the root free space, depletion of Cd²⁺ was buffered by the presence of Cd(II)–organic acid complexes. Plant response to elevated Cd levels involved overproduction of organic acids in maize roots as a resistance mechanism to alleviate Cd toxicity.     

Ab initio model studies of copper binding to peptides containing a His–His sequence: relevance to the β-amyloid peptide of Alzheimer’s disease

Two of the defining hallmarks of Alzheimer’s disease (AD) are deposits of the β-amyloid peptide, Aβ, and the generation of reactive oxygen species, both of which may be due to the Aβ peptide coordinating metal ions. The Cu²⁺ concentrations in cores of senile plaques are significantly elevated in AD patients. Experimental results indicate that Aβ1–42 in particular has a very high affinity for Cu²⁺, and that His13 and His14 are the two most firmly established ligands in the coordination sphere of the copper ion. Quantum chemical calculations using the unrestricted B3LYP hybrid density functional method with the 6–31G(d) basis set were performed for geometries, zero point energies and thermochemistry. The effects of solvation were accommodated using the CPCM method. The enthalpies were calculated with the 6–311+G(2df,2p) basis set. Calculations show that when Cu(H₂O)₄²⁺ combines with the model compound 1 (3-(1H-imidazol-5-yl)-N-[2-(1H-imidazol-5-yl)ethyl] propanamide) in the aqueous phase, the most stable binding site involves the Nπ atoms of His13 and His14 as well as the carbonyl of the intervening backbone amide group. These structures are fairly rigid and the implications for conformational changes to the Aβ backbone are discussed. In solution at pH=7, Cu²⁺ promotes the deprotonation and involvement in the binding of the backbone amide nitrogen in a β-sheet like structure. This geometry does not induce strain in the peptide backbone, making it the most likely representation of that portion of the Cu²⁺–Aβ complex monomer in aqueous solution.