High-affinity Metal Research Articles

Biosorption of Cd(II) from aqueous solution using xanthated nano banana cellulose: Equilibrium and kinetic studies

Present study explored the biosorption capacity of xanthated nano banana cellulose (XNBC) for Cd(II) from aqueous solution. The biosorbent containing sulfur-bearing groups have a high affinity for heavy metals. Sulfur can be considered as a soft ligand group having strong affinity for cadmium. In the present study, the influence of various important parameters such as pH, time, biosorbent dose and initial Cd(II) concentration on the biosorption capacity were investigated. The maximum biosorption capacity of XNBC for Cd(II) was found to be 154.26mgg−1 at 298K. The Cd(II) sorption of XNBC was confirmed by SEM-EDS and XRF analysis. The isotherms such as Langmuir, Freundlich, Redlich–Peterson and Tempkin were studied. The Langmuir and the Redlich–Peterson isotherms had been well fitted the biosorption of Cd(II) with xanthated nano banana cellulose. The kinetics of Cd(II) removal using XNBC was well explained by second-order kinetic model. The thermodynamic parameters were also evaluated from the biosorption measurements. Among the various desorbing agents tested, the desorbing efficiency was found to be maximum with 0.1molL−1 HCl. It was found that XNBC is also suitable to be used under column operation.

Metallothionein I+II Expression as an Early Sign of Chronic Relapsing Experimental Autoimmune Encephalomyelitis in Rats

Metallothioneins (MTs) are small, cysteine-rich proteins which have been implicated in various forms of stress providing cytoprotective action against oxidative injury, DNA damage and apoptosis. Owing to their high affinity for physiological metals, such as zinc and copper MTs are also critical components of regulatory proteins involved in cell growth and multiplication, as well as in the maintenance of immune homeostasis. To elucidate the role of MTs in the pathomechanisms of autoimmune CNS disorders we estimated the expression of MT I+II proteins and the content of free Zn ions in the brain, spinal cord and in the liver early in the course of chronic relapsing experimental autoimmune encephalomyelitis (CR-EAE) pathogenesis, i.e. before the onset of any clinical symptoms. Disease was induced in the genetically susceptible Dark Agouti (DA) rats by subcutaneous injection of bovine brain homogenate in CFA. Control animals were treated with CFA alone. The data, obtained by immuno-histochemistry and in situ fluorescent labeling of free zinc ions, have shown that in the presymptomatic phase of CR-EAE (on the seventh postimmunization day) MTs I+II were markedly upregulated in the cells that form blood-brain and blood-cerebrospinal fluid barriers, as well as in the cerebellar parenchyma and hippocampal dentate gyri. Furthermore, we found that the liver also becomes a site of extensive MTs I+II synthesis shortly after immunization. Simultaneously, tissue content of free zinc ions increased at the sites of MTs induction, reflecting their antioxidative activity. The data, described in this paper point to regulatory and neuroprotective role of MTs in the pathogenesis of CR-EAE.

Modeling specific pH dependent sorption of divalent metals on montmorillonite surfaces. A review of pitfalls, recent achievements and current challenges

Within the context of the clay barrier concept for underground nuclear waste disposal, montmorillonite and bentonite have been widely used as reference materials for sorption. In some cases, accompanying modeling work aims at understanding and predicting sorption in complex natural systems where clays are assumed to be representative of the most reactive phases. This bottom-up approach relies heavily on good confidence in the mechanistic understanding of sorption phenomena. The present study aims at reviewing experimental and modeling work on montmorillonite with a focus on divalent metals experiencing pH dependent specific sorption. Current knowledge points out distinct sorption mechanisms on three types of sites: cation exchange on basal planes and surface complexation on edge surfaces with two types of sites: high energy (or strong) sites (HES) with high affinity for metals but low site density and low energy (or weak) sites (LES) with lower affinity for metals but high site density. Based on this current knowledge, criteria are given to select data relevant for surface complexation model calibration (especially ionic strength, pH, clay preparation and characterization, metal to clay ratio and solubility limits), with an emphasis on data uncertainties and reproducibility. Problematic experimental features are highlighted, especially those related to the reversibility of sorption and to the effect of the solid to liquid ratio (R~SL~) on sorption distribution coefficients. Guidelines for data acquisition and selection are proposed. Surface complexation models available in the literature are then tested in terms of efficiency (data fit) and mechanistic likelihood. None of the currently available models is able to satisfy both aspects. Models directly adapted from oxide surface complexation models fail in both aspects. The most efficient model (in terms of simplicity and accuracy) is a non-electrostatic model. It is the only one that reproduces pH dependent specific sorption data at a low metal clay ratio (\<0.001 mol/kg~clay~; HES) in all selected experimental conditions, as well as data obtained at medium metal to clay ratio (∼0.01-0.05 mol/kg~clay~; low energy sites). To account for physical mechanisms, an electrostatic surface complexation model has been developed. It takes into account the spill-over effect of negatively charged basal surfaces over edge surfaces, a typical feature of montmorillonite, and is able to reproduce sorption data for LES but not for HES. The reasons for this failure are explained through the mathematical derivations of model equations. This approach shows that it is impossible to reconcile HES properties with an oxide-like surface complexation electrostatic model. Amongst other alternatives, a successful electrostatic surface substitution model, which is compatible with current knowledge on HES structural properties, is proposed.

Cobalt speciation assay for human serum, Part I. Method for measuring large and small molecular cobalt and protein-binding capacity using size exclusion chromatography with inductively coupled plasma-mass spectroscopy detection

A method utilizing size exclusion liquid chromatography (SEC) was developed to separate and quantify large molecular cobalt (Co) (e.g., albumin-Co) from cyanocobalamin (vitamin B12) and small molecular Co (e.g., glutathione-Co and free Co) in human serum. Highly selective and sensitive detection using inductively coupled plasma–mass spectrometry was coupled with SEC to provide a method with reliable accuracy, precision, recoveries, stability, and a detection limit of 0.037 μg/L in undiluted serum. Other divalent metal cations known to compete with Co(II) for serum albumin-binding sites (such as iron, zinc, manganese, cadmium, copper, nickel, and lead) did not significantly alter Co(II) quantification. Co–protein binding capacity determination of individual serum samples indicated that addition of 2500 μg Co/L to undiluted human serum resulted in approximately 90% distribution to the large molecular Co peak, consistent with Co binding to high-affinity divalent metal binding sites on albumin. Since serum albumin binding partially sequesters biologically active Co(II) ions, this method provides an important tool for better understanding the kinetics and toxicology of Co compounds. Thus, the proposed method might play an important role in establishing Co dose–response relationships that affect the equilibrium concentrations of free ionic Co(II).

Hydroxamic Acids in Asymmetric Synthesis

Metal-catalyzed stereoselective reactions are a central theme in organic chemistry research. In these reactions, the stereoselection is achieved predominantly by introducing chiral ligands at the metal catalyst's center. For decades, researchers have sought better chiral ligands for asymmetric catalysis and have made great progress. Nevertheless, to achieve optimal stereoselectivity and to catalyze new reactions, new chiral ligands are needed. Because of their high metal affinity, hydroxamic acids play major roles across a broad spectrum of fields from biochemistry to metal extraction. Dr. K. Barry Sharpless first revealed their potential as chiral ligands for asymmetric synthesis in 1977: He published the chiral vanadium-hydroxamic-acid-catalyzed, enantioselective epoxidation of allylic alcohols before his discovery of Sharpless asymmetric epoxidation, which uses the titanium-tartrate complex as the chiral reagent. However, researchers have reported few highly enantioselective reactions using metal-hydroxamic acid as catalysts since then. This Account summarizes our research on metal-catalyzed asymmetric epoxidation using hydroxamic acids as chiral ligands. We designed and synthesized a series of new hydroxamic acids, most notably the C2-symmetric bis-hydroxamic acid (BHA) family. V-BHA-catalyzed epoxidation of allylic and homoallylic alcohols achieved higher activity and stereoselectivity than Sharpless asymmetric epoxidation in many cases. Changing the metal species led to a series of unprecedented asymmetric epoxidation reactions, such as (i) single olefins and sulfides with Mo-BHA, (ii) homoallylic and bishomoallylic alcohols with Zr- and Hf-BHA, and (iii) N-alkenyl sulfonamides and N-sulfonyl imines with Hf-BHA. These reactions produce uniquely functionalized chiral epoxides with good yields and enantioselectivities.

Structural changes during HCN channel gating defined by high affinity metal bridges

Hyperpolarization-activated cyclic nucleotide–sensitive nonselective cation (HCN) channels are activated by membrane hyperpolarization, in contrast to the vast majority of other voltage-gated channels that are activated by depolarization. The structural basis for this unique characteristic of HCN channels is unknown. Interactions between the S4–S5 linker and post-S6/C-linker region have been implicated previously in the gating mechanism of HCN channels. We therefore introduced pairs of cysteines into these regions within the sea urchin HCN channel and performed a Cd2+-bridging scan to resolve their spatial relationship. We show that high affinity metal bridges between the S4–S5 linker and post-S6/C-linker region can induce either a lock-open or lock-closed phenotype, depending on the position of the bridged cysteine pair. This suggests that interactions between these regions can occur in both the open and closed states, and that these regions move relative to each other during gating. Concatenated constructs reveal that interactions of the S4–S5 linker and post-S6/C-linker can occur between neighboring subunits. A structural model based on these interactions suggests a mechanism for HCN channel gating. We propose that during voltage-dependent activation the voltage sensors, together with the S4–S5 linkers, drive movement of the lower ends of the S5 helices around the central axis of the channel. This facilitates a movement of the pore-lining S6 helices, which results in opening of the channel. This mechanism may underlie the unique voltage dependence of HCN channel gating.

Nanoparticle Delivery of Transition-Metal Chelators to the Brain: Oxidative Stress will Never See it Coming!

The pathological lesions typical of Alzheimer disease (AD) are sites of significant and abnormal metal accumulation. Metal chelation therapy, therefore, provides a very attractive therapeutic measure for the neuronal deterioration of AD, though its institution suffers fundamental deficiencies. Namely, chelating agents, which bind to and remove excess transition metals from the body, must penetrate the blood-brain barrier to instill any real effect on the oxidative damages caused by the presence of the metals in the brain. Despite many advances in chelation administration, however, this vital requirement remains therapeutically out of reach: the most effective chelators-i.e., those that have high affinity and specificity for transition metals like iron and copper-are bulky and hydrophilic, making it difficult to reach their physiological place of action. Moreover, small, lipophilic chelators, which can pass through the brain's defensive wall, essentially suffer from their over-effectiveness. That is, they induce toxicity on proliferating cells by removing transition metals from vital RNA enzymes. Fortunately, research has provided a loophole. Nanoparticles, tiny, artificial or natural organic polymers, are capable of transporting metal chelating agents across the blood-brain barrier regardless of their size and hydrophilicity. The compounds can thereby sufficiently ameliorate the oxidative toxicity of excess metals in an AD brain without inducing any such toxicity themselves. We here discuss the current status of nanoparticle delivery systems as they relate to AD chelation therapy and elaborate on their mechanism of action. An exciting future for AD treatment lies ahead.

Preparation and application of poly(2-amino thiophenol)/MWCNTs nanocomposite for adsorption and separation of cadmium and lead ions via solid phase extraction

A new method which utilizes a multiwalled carbon nanotubes/poly(2-amino thiophenol) nanocomposites as an effective sorbents in solid-phase extraction has been developed for separation and preconcentration of Cd(II) and Pb(II) trace levels in environmental samples. The method is based on the adsorption of heavy metals on macromolecular chains due to sharing an electron pair of N and S groups of conducting polymer with cadmium and lead ions. The results indicate the novel nanocomposite presents a high affinity for heavy metals due to the presence of several good extractive sites (S and N) which are introduced to the synthesized nanocomposite. Some parameters including sample condition such as: pH, flow rate, sample volume, and eluent condition such as: type, volume, and concentration: moreover adsorption capacity of matrix ions was investigated on the recovery of Cd(II) and Pb(II). The maximum adsorption capacity of MWCNTs/P2AT nanocomposite as sorbents at optimum conditions for cadmium and lead ions was found to be 178.7 mg g −1 and 186.4 mg g −1, respectively. The detection limits of this method were 0.3 and 1 ng mL −1 for Cd(II) and Pb(II), respectively. The validation of the presented procedure was checked by reference material analysis. Finally, the presented procedure was applied for determination of Cd(II) and Pb(II) in some environmental samples.

On the mechanism and rate of gold incorporation into thiol-dependent flavoreductases

NADPH-dependent flavoreductases are important drug targets. During their enzymatic cycle thiolates and selenolates that have high affinity for transition metals are generated. Auranofin (AF), a gold-containing compound, is classified by the World Health Organization as an antirheumatic agent and it is indicated as the scaffold for the development of new anticancer and antiparasitic drugs. AF inhibits selenocysteine-containing flavoreductases (thioredoxin reductase and thioredoxin glutathione reductase) more effectively than non Se-containing ones (glutathione reductase); this preference has been ascribed to the high affinity of selenium for gold. We solved the 3D structure of the Se-containing Thioredoxin Glutathione Reductase from the human parasite Schistosoma mansoni complexed with Au and our results challenge this view: we believe that the relative velocity of the reaction rather than the relative affinity, depends on the presence of Sec residues, which appear to dictate AF selectivity.

Speciation and cysteine-simplified physiological-based extraction technique (SBET) bioaccesibility of heavy metals in biosolids

Cysteine residues on proteins have a high affinity for metals yet formulations used to determine bioaccessibility do not contain cysteine or thiol-containing molecules. As a result, we used a cysteine-simplified physiological-based extraction technique (SBET) and, the conventional glycine-SBET to determine bioaccesibility of selected heavy metals in biosolids and compared the data. We also determined speciation of the selected metals in the biosolids to assess further the health risk posed the use of biosolids as a soil amendment in agricultural soils. Samples, including a certified reference standard were analyzed using x-ray fluorescence and flame atomic absorption. Bioaccessibility was higher in cysteine-SBET than glycine-SBET, and regression data show that the two methods give different sets of results. We proposed a bioaccessibility model that involves cysteine and the hydrogen ion complementing each other to dissolve metals. The model also includes a three mode-bioavailability mechanism: absorption of free metal ions; ligand-mediated transport of metal ions from solution; and ligand-mediated transport of metal ions directly from the biosolids into the cell. Low pH in the gut increases bioaccessibility but reduces bioavailability due to protonation of receptor ligands. With the exception of Fe, bioaccessibility was directly correlated to the sequential extraction availability which followed the order: Mn(90.3 %)>Zn(50.3 %)>Cd(26.5 %)>Cu(24.9 %)>Fe(0.367 %). We calculated bioavailability from bioaccessibility using literature estimates of percent bioavailabilities. The order of abundance of the analyzed metals in the biosolids was as follows: Fe>Mn>Zn>Cu>Pb>Cd.

TroA of Streptococcus suis Is Required for Manganese Acquisition and Full Virulence

Streptococcus suis causes infections in pigs and occasionally in humans, resulting in manifestations as meningitis, sepsis, arthritis, and septic shock. For survival within the host, S. suis requires numerous nutrients including trace metals. Little is known about the specific proteins involved in metal scavenging in S. suis. In this study we evaluated the role of the putative high-affinity metal binding lipoprotein TroA in metal acquisition and virulence. A mutant strain deficient in the expression of TroA (ΔtroA mutant) was constructed. Growth of the ΔtroA mutant in Todd-Hewitt broth was similar to wild-type growth; however, growth of the ΔtroA mutant in cation-deprived Todd-Hewitt broth and in porcine serum was strongly reduced compared to growth of wild-type bacteria. Supplementing the medium with extra manganese but not with magnesium, zinc, copper, nickel, or iron restored growth to wild-type levels, indicating that TroA is specifically required for growth in environments low in manganese. The ΔtroA mutant also showed increased susceptibility to H2O2, suggesting that TroA is involved in counteracting oxidative stress. Furthermore, the expression of the troA gene was subject to environmental regulation at the transcript level. In a murine S. suis infection model, the ΔtroA mutant displayed a nonvirulent phenotype. These data indicate that S. suis TroA is involved in manganese acquisition and is required for full virulence in mice.

Multidentate Terephthalamidate and Hydroxypyridonate Ligands: Towards New Orally Active Chelators

The limitations of current therapies for the treatment of iron overload or radioisotope contamination have stimulated efforts to develop new orally bioavailable iron and actinide chelators. Siderophore-inspired tetradentate, hexadentate and octadentate terephthalamidate and hydroxypyridonate ligands were evaluated in vivo as selective and efficacious iron or actinide chelating agents, with several metal loading and ligand assessment procedures, using 59Fe, 238Pu, and 241Am as radioactive tracers. The compounds presented in this study were compared to commercially available therapeutic sequestering agents [deferoxamine (DFO) for iron and diethylenetriaminepentaacetic acid (DPTA) for actinides] and are unrivaled in terms of affinity, selectivity and decorporation efficacy, which attests to the fact that high metal affinity may overcome the low bioavailability properties commonly associated to multidenticity.

Manganese Neurotoxicity Presenting with Depression, Psychosis and Catatonia

Locura manganica or “manganese madness” is a discrete Parkinson-like syndrome documented in the last century that occurs with chronic exposure to manganese (Mn), an essential element abundant in the Earth’s crust. It is especially well known in Latin America, where the majority of Mn is mined. Acute Mn toxicity in the form of acute psychosis was documented as early as 1837 in the United States, produced by rapid inhalation of dust containing high concentrations of Mn. Neuropsychiatric symptoms are varied and include emotional lability, visual and auditory hallucinations, nervousness, irritability, and compulsive acts, which subside after several weeks and are replaced by muscular weakness and rigidity, postural tremor, dysarthria, gait disorders, and headaches. The primary route of toxic exposure is inhalation of Mn-containing aerosols or transport of particulate matter by the olfactory bulb. Onset is insidious, progression is chronic, and the severity of symptoms is modulated by individual susceptibilities as well as by the length and dose of exposure. Symptoms can continue to progress more than 10 years after exposure has stopped and Mn has been cleared from hair, skin and blood cells. Mn is normally absorbed in the GI tract and excreted in bile. It is utilized in trivalent and divalent oxidation reactions by a variety of enzymes. Most relevant neuropsychiatrically is Mn-containing superoxide dismutase (found in mitochondria) and glutamine synthetase (found in astrocytes). Transport occurs across the blood-brain barrier by a combination of active transport and facilitated diffusion via various highaffinity metal transporters. Divalent metal transporter 1 (DMT1) is of particular interest as the caudate, putamen, and substantia nigra pars reticulata stain densely for the transporter, where Mn tends to deposit. We report here a case of presumed manganism in a South American former welder who presented with fea-

Mechanism of Regulation of Native Cardiac Muscle Thin Filaments by Rigor Cardiac Myosin-S1 and Calcium

We have studied the mechanism of activation of native cardiac thin filaments by calcium and rigor myosin. The acceleration of the rate of 2'-deoxy-3'-O-(N-methylanthraniloyl)ADP (mdADP) dissociation from cardiac myosin-S1-mdADP-P(i) and cardiac myosin-S1-mdADP by native cardiac muscle thin filaments was measured using double mixing stopped-flow fluorescence. Relative to inhibited thin filaments (no bound calcium or rigor S1), fully activated thin filaments (with both calcium and rigor-S1 bound) increase the rate of product dissociation from the physiologically important pre-power stroke myosin-mdADP-P(i) by a factor of ∼75. This can be compared with only an ∼6-fold increase in the rate of nucleotide diphosphate dissociation from nonphysiological myosin-mdADP by the fully activated thin filaments relative to the fully inhibited thin filaments. These results show that physiological levels of regulation are not only dependent on the state of the thin filament but also on the conformation of the myosin. Less than 2-fold regulation is due to a change in affinity of myosin-ADP-P(i) for thin filaments such as would be expected by a simple "steric blocking" of the myosin-binding site of the thin filament by tropomyosin. Although maximal activation requires both calcium and rigor myosin-S1 bound to the cardiac filament, association with a single ligand produces ∼70% maximal activation. This can be contrasted with skeletal thin filaments in which calcium alone only activated the rate of product dissociation ∼20% of maximum, and rigor myosin produces ∼30% maximal activation.

Structural Determinants of the High Affinity Extracellular Zinc Binding Site on Cav3.2 T-type Calcium Channels

Ca(v)3.2 T-type channels contain a high affinity metal binding site for trace metals such as copper and zinc. This site is occupied at physiologically relevant concentrations of these metals, leading to decreased channel activity and pain transmission. A histidine at position 191 was recently identified as a critical determinant for both trace metal block of Ca(v)3.2 and modulation by redox agents. His(191) is found on the extracellular face of the Ca(v)3.2 channel on the IS3-S4 linker and is not conserved in other Ca(v)3 channels. Mutation of the corresponding residue in Ca(v)3.1 to histidine, Gln(172), significantly enhances trace metal inhibition, but not to the level observed in wild-type Ca(v)3.2, implying that other residues also contribute to the metal binding site. The goal of the present study is to identify these other residues using a series of chimeric channels. The key findings of the study are that the metal binding site is composed of a Asp-Gly-His motif in IS3-S4 and a second aspartate residue in IS2. These results suggest that metal binding stabilizes the closed conformation of the voltage-sensor paddle in repeat I, and thereby inhibits channel opening. These studies provide insight into the structure of T-type channels, and identify an extracellular motif that could be targeted for drug development.

Probing the Cu2+ and Zn2+ binding affinity of histidine-rich glycoprotein

The zinc(II) and copper(II) binding ability of two oligopeptide fragments, Ac-HHPHG-NH(2) and Ac-HHPHGHHPHG-NH(2), derived from the repeat-region of the His-Pro-rich domain of histidine-rich glycoprotein (HRG) and the structure of the formed complexes have been investigated by potentiometry, NMR-, UV-visible-, CD-, SRCD- and EPR spectroscopy. Exclusive coordination of the side-chain imidazoles of the peptides has been observed with both metal ions in the acidic and neutral pH range. While the three His units of the pentapeptide resulted in a modest stability of the ML complexes, the decapeptide with its increased number of His residues offered a high-affinity metal binding site for both metal ions with the participation of at least four nitrogen donors. Due to the high number of potential donor groups, the formation of binding isomers of the protonated and parent complexes is very likely. Both peptides show a synchrotron radiation (SR) CD-pattern resembling to that of the polyproline II structure, similarly to that of the His-Pro-rich domain of the HRG protein. The longer sequence was shown to bind a second metal ion in the slightly acidic pH-range. The determined stability data suggest a remarkable extra stabilization emerging in the decapeptide for the coordination of the second metal ions, as compared to the ML complexes of the pentapeptide. Whether the observed cooperativity has similarities to the cooperative metal binding feature of HRG or the two phenomena have different sources is a question yet to be clarified.

Zn(II)‐ and Cu(II)‐induced non‐fibrillar aggregates of amyloid‐β (1–42) peptide are transformed to amyloid fibrils, both spontaneously and under the influence of metal chelators

Aggregation of amyloid-beta (Abeta) peptides is a central phenomenon in Alzheimer's disease. Zn(II) and Cu(II) have profound effects on Abeta aggregation; however, their impact on amyloidogenesis is unclear. Here we show that Zn(II) and Cu(II) inhibit Abeta(42) fibrillization and initiate formation of non-fibrillar Abeta(42) aggregates, and that the inhibitory effect of Zn(II) (IC(50) = 1.8 micromol/L) is three times stronger than that of Cu(II). Medium and high-affinity metal chelators including metallothioneins prevented metal-induced Abeta(42) aggregation. Moreover, their addition to preformed aggregates initiated fast Abeta(42) fibrillization. Upon prolonged incubation the metal-induced aggregates also transformed spontaneously into fibrils, that appear to represent the most stable state of Abeta(42). H13A and H14A mutations in Abeta(42) reduced the inhibitory effect of metal ions, whereas an H6A mutation had no significant impact. We suggest that metal binding by H13 and H14 prevents the formation of a cross-beta core structure within region 10-23 of the amyloid fibril. Cu(II)-Abeta(42) aggregates were neurotoxic to neurons in vitro only in the presence of ascorbate, whereas monomers and Zn(II)-Abeta(42) aggregates were non-toxic. Disturbed metal homeostasis in the vicinity of zinc-enriched neurons might pre-dispose formation of metal-induced Abeta aggregates, subsequent fibrillization of which can lead to amyloid formation. The molecular background underlying metal-chelating therapies for Alzheimer's disease is discussed in this light.

Deciphering interactions of the aminoglycoside phosphotransferase(3′)‐IIIa with its ligands

Aminoglycoside phosphotransferase(3')-IIIa (APH) is the enzyme with broadest substrate range among the phosphotransferases that cause resistance to aminoglycoside antibiotics. In this study, the thermodynamic characterization of interactions of APH with its ligands are done by determining dissociation constants of enzyme-substrate complexes using electron paramagnetic resonance and fluorescence spectroscopy. Metal binding studies showed that three divalent cations bind to the apo-enzyme with low affinity. In the presence of AMPPCP, binding of the divalent cations occurs with 7-to-37-fold higher affinity to three additional sites dependent on the presence and absence of different aminoglycosides. Surprisingly, when both ligands, AMPPCP and aminoglycoside, are present, the number of high affinity metal binding sites is reduced to two with a 2-fold increase in binding affinity. The presence of divalent cations, with or without aminoglycoside present, shows only a small effect (<3-fold) on binding affinity of the nucleotide to the enzyme. The presence of metal-nucleotide, but not nucleotide alone, increases the binding affinity of aminoglycosides to APH. Replacement of magnesium (II) with manganese (II) lowered the catalytic rates significantly while affecting the substrate selectivity of the enzyme such that the aminoglycosides with 2'-NH(2) become better substrates (higher V(max)) than those with 2'-OH.

Role of Bound Zn(II) in the CadC Cd(II)/Pb(II)/Zn(II)-responsive Repressor

The Staphylococcus aureus plasmid pI258 cadCA operon encodes a P-type ATPase, CadA, that confers resistance to Cd(II)/Pb(II)/Zn(II). Expression is regulated by CadC, a homodimeric repressor that dissociates from the cad operator/promoter upon binding of Cd(II), Pb(II), or Zn(II). CadC is a member of the ArsR/SmtB family of metalloregulatory proteins. The crystal structure of CadC shows two types of metal binding sites, termed Site 1 and Site 2, and the homodimer has two of each. Site 1 is the physiological inducer binding site. The two Site 2 metal binding sites are formed at the dimerization interface. Site 2 is not regulatory in CadC but is regulatory in the homologue SmtB. Here the role of each site was investigated by mutagenesis. Both sites bind either Cd(II) or Zn(II). However, Site 1 has higher affinity for Cd(II) over Zn(II), and Site 2 prefers Zn(II) over Cd(II). Site 2 is not required for either derepression or dimerization. The crystal structure of the wild type with bound Zn(II) and of a mutant lacking Site 2 was compared with the SmtB structure with and without bound Zn(II). We propose that an arginine residue allows for Zn(II) regulation in SmtB and, conversely, a glycine results in a lack of regulation by Zn(II) in CadC. We propose that a glycine residue was ancestral whether the repressor binds Zn(II) at a Site 2 like CadC or has no Site 2 like the paralogous ArsR and implies that acquisition of regulatory ability in SmtB was a more recent evolutionary event.

Metal Preference At The Second Metal Binding Site Of E. coli NikR

Escherichia coli NikR (EcNikR) regulates cellular nickel uptake by binding free nickel ions at high affinity metal binding sites in the protein, which induces EcNikR binding to the nik operon - a process that leads to suppression of the gene encoding the nickel uptake transporter, NikABCDE. A structure of the EcNikR-DNA complex suggests that a second metal binding site is present in addition to the high affinity sites, and raises the question of which metal occupies this second site under physiologic conditions: K+, which is present in the crystal structure, or Ni+2. To determine which ion is preferred at the second metal binding site, and the reason for any preference of one ion over another, we calculated the electrostatic free energy of EcNikR binding to DNA with either K+ or Ni+2 in the second site. While the interaction between EcNikR and DNA is more favorable when the second site contains Ni+2, the large desolvation penalty associated with moving Ni+2 from solution to the relatively buried second site offsets this favorable interaction. Consequently, our data suggest that EcNikR binding to DNA is more favorable when the second site contains a K+ ion. Moreover, additional calculations indicate that the second site is best suited for an ion having the size of K+ and not Ni+2, suggesting that the second site is optimized for K+. Taken together, our data suggest that the second metal binding site contains K+ and explain why K+ is preferred over Ni+2 at this site.