New biochemical insight of conserved water molecules at catalytic and structural Zn<Superscript>2+</Superscript> ions in human matrix metalloproteinase-I: a study by MD-simulation

Human matrix metalloproteinase (MMP)-1 or collagenase–1 plays a significant role in embryonic development, tissue remodeling, and is also involved in several diseases like arthritis, metastasis, etc. Molecular dynamics simulation studies on hMMP-1 X-ray structures (PDB Id. 1CGE, 1CGF, 1CGL, 1HFC, and 2TCL) suggest that the three conserved water molecules (W_H/1, W_I, W_S) are coordinated with catalytic zinc (Zn_C), and one water molecule (W) is associated at structural zinc ion (Zn_S). Transition of the coordination geometry around Zn_C from tetrahedral to octahedral and tetrahedral to trigonal bipyramidal at Zn_S are also observed during the dynamics. Recognition of two zinc ions through water mediated bridges (Zn_C – W_H (W₁)…W₂….H₁₈₃ – Zn_S) and stabilization of secondary coordination zone around the metal ions indicates the possibility of Zn_C…Zn_S coupled catalytic mechanism in hMMP-I. This study not only reveals a functionally important role of conserved water molecules in hMMP-I but also highlights the involvement of other non catalytic residues, such as S172 and D170 in the catalytic mechanism. The results obtained in this study could be relevant for importance of conserved water mediated recognition site of the sequence residue id. 202(RWTNNFREY)210, interaction of W(tryptophan)203 to zinc bound histidine, their influence on the water molecules that are involved in bridging between Zn_C and Zn_S, and structure-based design of specific hMMP inhibitors.

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Graphical abstract Water mediated recognition of structural and catalytic zinc ions of hMMP-1 structure (MD simulatated conformation)

     

An insight to the conserved water mediated dynamics of catalytic His88 and its recognition to thyroxin and RBP binding residues in human transthyretin

Human transthyretin (hTTR) is a multifunctional protein involved in several amyloidogenic diseases. Besides transportation of thyroxin and vitamin-A, its role towards the catalysis of apolipoprotein-A1 and Aβ-peptide are also drawing interest. The role of water molecules in the catalytic mechanism is still unknown. Extensive analyses of 14 high-resolution X-ray structures of human transthyretin and MD simulation studies have revealed the presence of eight conserved hydrophilic centres near its catalytic zone which may be indispensable for the function, dynamics and stability of the protein. Three water molecules (W1, W2 and W3) form a cluster and play an important role in the recognition of the catalytic and RBP-binding residues. They also induce the reorganisation of the His88 for coupling with other catalytic residues (His90, Glu92). Another water molecule (W5) participate in inter-monomer recognition between the catalytic and thyroxin binding sites. The rest four water molecules (W6, W*, W^# and W^?) form a distorted tetrahedral cluster and impart stability to the catalytic core of hTTR. The conserved water mediated recognition dynamics of the different functional sites may provide some rational clues towards the understanding of the activity and mechanism of hTTR.     

Structural studies of β-carbonic anhydrase from the green alga Coccomyxa: inhibitor complexes with anions and acetazolamide

The β-class carbonic anhydrases (β-CAs) are widely distributed among lower eukaryotes, prokaryotes, archaea, and plants. Like all CAs, the β-enzymes catalyze an important physiological reaction, namely the interconversion between carbon dioxide and bicarbonate. In plants the enzyme plays an important role in carbon fixation and metabolism. To further explore the structure-function relationship of β-CA, we have determined the crystal structures of the photoautotroph unicellular green alga Coccomyxa β-CA in complex with five different inhibitors: acetazolamide, thiocyanate, azide, iodide, and phosphate ions. The tetrameric Coccomyxa β-CA structure is similar to other β-CAs but it has a 15 amino acid extension in the C-terminal end, which stabilizes the tetramer by strengthening the interface. Four of the five inhibitors bind in a manner similar to what is found in complexes with α-type CAs. Iodide ions, however, make contact to the zinc ion via a zinc-bound water molecule or hydroxide ion — a type of binding mode not previously observed in any CA. Binding of inhibitors to Coccomyxa β-CA is mediated by side-chain movements of the conserved residue Tyr-88, extending the width of the active site cavity with 1.5-1.8 Å. Structural analysis and comparisons with other α- and β-class members suggest a catalytic mechanism in which the movements of Tyr-88 are important for the CO₂-HCO₃ ^- interconversion, whereas a structurally conserved water molecule that bridges residues Tyr-88 and Gln-38, seems important for proton transfer, linking water molecules from the zinc-bound water to His-92 and buffer molecules.     

Synthesis and structural chemistry of novel heteropolymolybdates and -tungstates

The chemistry of polynuclear oxometalate anions is dominated by molybdenum and tungsten in their highest oxidation state. During the past twenty years this class of compounds has attracted much attention because of their variable applications, e.g. as reagents in analytical procedures, as industrial catalysts and as potential anticancer drugs.In order to obtain model systems for the investigation of the catalytic activity of heteropolyanions we have synthesized and structurally characterized some organo derivatives of polyoxoanions. We secondly focus on Sb(III) and Bi(III) heteropolytungstates to examine the important influence of the unshared electron pair on the resulting structures and properties. Some of these compounds may be regarded as supramolecular aggregates showing inclusion phenomena.In 9 two [SbW₉O₃₃]^9– anions are linked by a set of six sodium ions forming a nearly planar hexagon. The sodium ions are enveloped by an oxygen cage formed by terminal oxygen atoms of the polyanions and by water molecules. Furthermore, the four anions [Sb₂W₂₂O₇₄(OH)₂]^12–, [Sb₂W₂₀Fe₂O₇₀(H₂O)₆]^8–, [Sb₂W₂₀Co₂O₇₀(H₂O)₆]^10– and [Bi₂W₂₀Fe₂O₆₈(OH)₂(H₂O)₆]^6– (in10, 11, 12, 13) may be regarded as transition metal complexes of novel [Sb₂W₂₀O₇₀]^14– or [Bi₂W₂₀O₇₀]^14– anions which are serving as ligands. The octahedral coordination sphere of each transition metal is formed by three oxygen atoms of the anion and completed by three water molecules. The Sb(III) heteropolyanion, [Na₂Sb₈W₃₆O₁₃₂(H₂O)₄]^22– in (14) includes two sodium and four antimony ions besides four water molecules. Each anion consists of four -B-SbW₉-Keggin fragments linked together by four SbO₄-groups, incorporating two sodium and four water molecules effecting an additional connection of the subunits.     

The bacterial ribonuclease P holoenzyme requires specific, conserved residues for efficient catalysis and substrate positioning

RNase P is an RNA-based enzyme primarily responsible for 5′-end pre-tRNA processing. A structure of the bacterial RNase P holoenzyme in complex with tRNA^Phe revealed the structural basis for substrate recognition, identified the active site location, and showed how the protein component increases functionality. The active site includes at least two metal ions, a universal uridine (U52), and P RNA backbone moieties, but it is unclear whether an adjacent, bacterially conserved protein loop (residues 52–57) participates in catalysis. Here, mutagenesis combined with single-turnover reaction kinetics demonstrate that point mutations in this loop have either no or modest effects on catalytic efficiency. Similarly, amino acid changes in the ‘RNR’ region, which represent the most conserved region of bacterial RNase P proteins, exhibit negligible changes in catalytic efficiency. However, U52 and two bacterially conserved protein residues (F17 and R89) are essential for efficient Thermotoga maritima RNase P activity. The U52 nucleotide binds a metal ion at the active site, whereas F17 and R89 are positioned >20 Å from the cleavage site, probably making contacts with N₋₄ and N₋₅ nucleotides of the pre-tRNA 5′-leader. This suggests a synergistic coupling between transition state formation and substrate positioning via interactions with the leader.     

An insight to the dynamics of conserved water-mediated salt bridge interaction and interdomain recognition in hIMPDH isoforms

Inosine monophosphate dehydrogenase (IMPDH) is involved in de novo biosynthesis pathway of guanosine nucleotide. Type II isoform of this enzyme is selectively upregulated in lymphocytes and chronic myelogenous leukemia (CML) cells, and is an excellent target for antileukemic agent. The molecular dynamics simulation results (15?ns) of three unliganded 1B3O, 1JCN, and 1JR1 structures have clearly revealed that I_N, I_C (N- and C-terminal of catalytic domains) and C₁, C₂ (cystathionine-beta-synthase-1 and 2) domains of IMPDH enzyme have been stabilized by six conserved water (center) mediated salt bridge interactions. These conserved water molecules could be involved in interdomain or intradomain recognition, intradomain coupling, and charge transfer processes. The binding propensity of cystathionine-beta-synthase domain to catalytic domain (through conserved water-mediated salt bridges) has provided a new insight to the biochemistry of IMPDH. Stereospecific interaction of I_N with C₂ domain through conserved water molecule (K109–W^II ₁–D215/D216) is observed to be unique in the simulated structure of hIMPDH-II. The geometrical/structural consequences and topological feature around the W^II ₁ water center may be utilized for isoform specific inhibitor design for CML cancer.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:1     

172 Role of conserved water molecular triad in the recognition of IMP,NAD+ with Asp 274, Asn 303, Arg 322, and Asp 364 in both the isoform of hIMPDH

Inosine monophosphate dehydrogenase (IMPDH) plays an important role in the Guanosine monophosphate (GMP) biosynthesis pathway. As hIMPDH-II is involved in CML-Cancer, it is thought to be an active target for leukemic drug design. The importance of conserved water molecules in the salt-bridge-mediated interdomain recognition and loop-flap recognition of hIMPDH has already been indicated in some simulation studies (Bairagya et al., 2009, 2011a, 2011b, 2012; Mishra et al., 2012). In this work, the role of conserved water molecules in the recognition of Inosine monophosphate (IMP) and NAD⁺ (co-factor) to active site residues of both the isoforms has been investigated by all atoms MD-Simulation studies. During 25-ns dynamics of the solvated hIMPDH-II and I (1B3O and 1JCN PDB structures), the involvement of conserved water molecular triad (W _M, W _L and W _C) in the recognition of active site residues (Asp 274, Asn 303, Arg 322, and Asp 364), IMP and NAD⁺ has been observed (Figure 1). The H-bonding co-ordination of all three conserved water molecular centers is within 4–7 and their occupation frequency is 1.0. The H-bonding geometry and the electronic consequences of the water molecular interaction at the different residues (and also IMP and NAD⁺) may put forward some rational clues on antileukemic agent design.     

Zinc Ion-induced Domain Organization in Metallo-β-lactamases: A FLEXIBLE “ZINC ARM” FOR RAPID METAL ION TRANSFER?*

The reversible unfolding of metallo-β-lactamase from Chryseobacterium meningosepticum (BlaB) by guanidinium hydrochloride is best described by a three-state model including folded, intermediate, and unfolded states. The transformation of the folded apoenzyme into the intermediate state requires only very low denaturant concentrations, in contrast to the Zn₂-enzyme. Similarly, circular dichroism spectra of both BlaB and metallo-β-lactamase from Bacillus cereus 569/H/9 (BcII) display distinct differences between metal-free and Zn₂-enzymes, indicating that the zinc ions affect the folding of the proteins, giving a larger α-helix content. To identify the regions of the protein involved in this zinc ion-induced change, a hydrogen deuterium exchange study with matrix-assisted laser desorption ionization tandem time of flight mass spectrometry on metal-free and Zn₁- and Zn₂-BcII was carried out. The region spanning the metal binding metallo-β-lactamases (MBL) superfamily consensus sequence His-X-His-X-Asp motif and the loop connecting the N- and C-terminal domains of the protein undergoes a zinc ion-dependent structural change between intrinsically disordered and ordered states. The inherent flexibility even appears to allow for the formation of metal ion-bridged protein-protein complexes which may account for both electrospray ionization-mass spectroscopy results obtained upon variation of the zinc/protein ratio and stoichiometry-dependent variations of ^199mHg-perturbed angular correlation of γ-rays spectroscopic data. We suggest that this flexible “zinc arm” motif, present in all the MBL subclasses, is disordered in metal-free MBLs and may be involved in metal ion acquisition from zinc-carrying molecules different from MBL in an “activation on demand” regulation of enzyme activity.The production of metallo-β-lactamases (MBLs)² is one of the defense strategies of bacteria against β-lactam antibiotics. MBLs hydrolyze the C-N bond of the β-lactam ring of these compounds using protein-bound zinc ions as cofactors (1). Their emergence in pathogenic bacterial strains and their broad substrate profile make them clinically important (2). Whereas the overall structure of all known MBLs is very similar (3), distinct differences in the set of protein ligands for bound zinc ions led to the classification into subclasses B1–B3 (4). Here we have studied the two subclass B1 enzymes BcII and BlaB from Bacillus cereus strain 569/H/9 and Chryseobacterium meningosepticum, respectively, which show 35.2% identical residues (5). The very similar structure of these enzymes is organized in a αββα sandwich (6, 7). The N- and C-terminal domains are connected by an external loop, and the active site is located in a long channel between the two domains. The binuclear zinc binding site is composed of a 3-His (3H) site and a Asp-Cys-His (DCH) site. Three metal ion ligands are located on the N-terminal domain and constitute the HXHXD motif, which is strictly conserved in proteins of the MBL super family (8). The three remaining metal ligands are located on the C-terminal domain of the proteins. Both for the native and for the cadmium-substituted enzyme, it has been shown that a single metal ion, when bound to BcII, appears to be distributed between the metal binding sites (9–11).The metal ion requirement for catalytic activity of the three subclasses B1–B3 of MBLs is heavily debated. Although most crystal structures of subclass B1 enzymes show binuclear zinc sites (3), it was found that BcII from B. cereus 569/H/9, CcrA from Bacteroides fragilis, BlaB from C. meningosepticum, IMP-1 from Pseudomonas aeruginosa, and L1 from Stenotrophomonas maltophilia are both active as the mono- and di-zinc enzymes (9, 12–15). Recently a study with Co(II)-substituted BcII challenged this view in concluding that only the di-Co-enzyme might be catalytically active (16). The same authors came to the conclusion that also native BcII requires two bound zinc ions for activity (17). A very recent study on the Co(II)-substituted enzyme came to the conclusion that both the Co₁- and the Co₂-enzymes are catalytically active with the DCH site as the primary catalytic site (18).Variable metal loading states of zinc proteins are attracting increasing interest in the field of cellular regulation processes being a key to the understanding of physiological functions of zinc sensors and metallothioneins as well as regulatory functions of zinc ions. The coexistence of zinc proteins in the metal-loaded and the metal-free form, however, requires the regulation of “free” zinc ion concentrations in narrow limits, with nm to pm concentrations in eucaryotes (19) or even much lower concentration in procaryotes (20). The issue, however, that zinc enzymes in their natural environment might be regulated by reversible metal ion binding is infrequently considered.The impact of metal ion binding on structure and stability of MBL superfamily proteins has been studied in some detail. Zinc was found to be required for the folding of glyoxalase II (21) and arylsulfatases (22) into the native state. For CphA from Aeromonas hydrophila, differential scanning calorimetry and fluorescence spectroscopy demonstrated that zinc binding stabilizes the protein against denaturation with urea. The inactive Zn₂-CphA proved to be the most stabile species (23). Crystal structures of metal-free and metal-loaded BcII revealed minor structural changes in the active site of the protein (6). ¹H,¹⁵N heteronuclear single quantum coherence spectra of the backbones amides of BcII resulted in distinct signals for different metal ion/enzyme ratios which allowed discrimination of apoenzyme and metal-loaded states (24). Metal ion binding was considered to be essential for folding of L1 in vivo (25), and variable loading states were described in dependence of the bioavailability of various metal ions (26). We hypothesized earlier that metallo-β-lactamases are most likely in the metal-free apoenzyme state in the absence of substrates, which is because of the moderate affinity of the enzymes for zinc ions and the very low concentration of free zinc in cellular environments (14). We suggested that substrate availability might induce a spontaneous self-activation by direct transfer of zinc via ligand exchange reactions with delivery systems as substrate presence leads to a drastic increase of zinc affinity. The suggested self-activation mechanism, however, requires a direct interaction of the apoenzymes with other zinc carriers to allow a ligand exchange reaction to occur. Because such interactions might be considered as unspecific, it seemed reasonable to postulate a high structural flexibility to allow the transient formation of zinc-bridged complexes. To verify this prediction, we initiated an investigation on the role of zinc ions on folding and stabilization of MBLs with BlaB and BcII as test cases. Our results demonstrate that the systems, when unsaturated with metal ions, cannot be correctly described as being composed of variable fractions of the proteins in the loaded and unloaded state alone. We will present indications of the formation of labile, metal-bridged ternary complexes formed under such conditions. The latter may be considered as important intermediate states for metal ion transfer between identical or different zinc binding molecules in general.     

Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis

Alkaline exonuclease and single-strand DNA (ssDNA) annealing proteins (SSAPs) are key components of DNA recombination and repair systems within many prokaryotes, bacteriophages and virus-like genetic elements. The recently sequenced β-proteobacterium Laribacter hongkongensis (strain HLHK9) encodes putative homologs of alkaline exonuclease (LHK-Exo) and SSAP (LHK-Bet) proteins on its 3.17 Mb genome. Here, we report the biophysical, biochemical and structural characterization of recombinant LHK-Exo protein. LHK-Exo digests linear double-stranded DNA molecules from their 5′-termini in a highly processive manner. Exonuclease activities are optimum at pH 8.2 and essentially require Mg²⁺ or Mn²⁺ ions. 5′-phosphorylated DNA substrates are preferred over dephosphorylated ones. The crystal structure of LHK-Exo was resolved to 1.9 Å, revealing a ‘doughnut-shaped’ toroidal trimeric arrangement with a central tapered channel, analogous to that of λ-exonuclease (Exo) from bacteriophage-λ. Active sites containing two bound Mg²⁺ ions on each of the three monomers were located in clefts exposed to this central channel. Crystal structures of LHK-Exo in complex with dAMP and ssDNA were determined to elucidate the structural basis for substrate recognition and binding. Through structure-guided mutational analysis, we discuss the roles played by various active site residues. A conserved two metal ion catalytic mechanism is proposed for this class of alkaline exonucleases.     

A metal‐chelate affinity reverse micellar system for protein extraction

A new nonionic reverse micellar system is developed by blending two nonionic surfactants, Triton X‐45 and Span 80. At total surfactant concentrations lower than 60 mmol/L and molar fractions of Triton X‐45 less than 0.6, thermodynamically stable reverse micelles of water content (W₀) up to 30 are formed. Di(2‐ethylhexyl) phosphoric acid (HDEHP; 1–2 mmol/L) is introduced into the system for chelating transition metal ions that have binding affinity for histidine‐rich proteins. HDEHP exists in a dimeric form in organic solvents and a dimer associated with one transition metal ion, including copper, zinc, and nickel. The copper‐chelate reverse micelles (Cu‐RM) are characterized for their W₀, hydrodynamic radius (R_h), and aggregation number (N_ag). Similar with reverse micelles of bis‐2‐ethylhexyl sodium sulfosuccinate (AOT), R_h of the Cu‐RM is also linearly related to W₀. However, N_ag is determined to be 30–90 at W₀ of 5–30, only quarter to half of the AOT reverse micelles. Then, selective metal‐chelate extraction of histidine‐rich protein (myoglobin) by the Cu‐RM is successfully performed with pure and mixed protein systems (myoglobin and lysozyme). The solubilized protein can be recovered by stripping with imidazole or ethylinediaminetetraacetic acid (EDTA) solution. Because various transition metal ions can be chelated to the reverse micelles, it is convinced that the system would be useful for application in protein purification as well as simultaneous isolation and refolding of recombinant histidine‐tagged proteins expressed as inclusion bodies. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Cubane-like water clusters in a 3D supramolecular network of zinc with pyridine-2,6-dicarboxylic acid and 5-aminotetrazole

The reaction of zinc ions with pyridine-2,6-dicarboxylic acid (H₂PDA) and 5-aminotetrazole (HATZ) in hydrothermal condition gives rise to a 2D interdigitated network of composition [Zn₂(PDA)(ATZ)₂]·4H₂O (1). The two independent zinc(II) ions, both located on a crystallographic twofold axis, show a different coordination environment, namely a highly distorted trigonal bipyramidal and a tetrahedral geometry. An octameric cluster of lattice water molecules in the lattice voids produces a 3D supramolecular architecture through hydrogen bonding. Thermogravimetry, infrared spectra, and elemental analysis have also been applied to characterize 1. Fluorescence study indicates the intraligand π-π^∗ transition perturbed by the metal ion.     

NAR Breakthrough Article: Structure of human RNA N6-methyladenine demethylase ALKBH5 provides insights into its mechanisms of nucleic acid recognition and demethylation

ALKBH5 is a 2-oxoglutarate (2OG) and ferrous iron-dependent nucleic acid oxygenase (NAOX) that catalyzes the demethylation of N⁶-methyladenine in RNA. ALKBH5 is upregulated under hypoxia and plays a role in spermatogenesis. We describe a crystal structure of human ALKBH5 (residues 66–292) to 2.0 Å resolution. ALKBH5_66–292 has a double-stranded β-helix core fold as observed in other 2OG and iron-dependent oxygenase family members. The active site metal is octahedrally coordinated by an HXD…H motif (comprising residues His204, Asp206 and His266) and three water molecules. ALKBH5 shares a nucleotide recognition lid and conserved active site residues with other NAOXs. A large loop (βIV–V) in ALKBH5 occupies a similar region as the L1 loop of the fat mass and obesity-associated protein that is proposed to confer single-stranded RNA selectivity. Unexpectedly, a small molecule inhibitor, IOX3, was observed covalently attached to the side chain of Cys200 located outside of the active site. Modelling substrate into the active site based on other NAOX–nucleic acid complexes reveals conserved residues important for recognition and demethylation mechanisms. The structural insights will aid in the development of inhibitors selective for NAOXs, for use as functional probes and for therapeutic benefit.     

The influences of water solvent on the structures and stabilities of Na+–AD conformers

The influences of water solvent on the structures and stabilities of the complex ion conformers formed by the coordination of alanine dipeptide (AD) and Na⁺ have been investigated using supramolecular and polarizable continuum solvation models at the level of B3LYP/6-311++G**, respectively; 12 monohydrated and 12 dihydrated structures of Na⁺–AD complex ion were obtained after full geometrical optimization. The results showed that H₂O molecules easily bind with Na⁺ of Na⁺–AD complex ion, forming an ion-lone pair interaction with the Na–O bond length of 2.1–2.3 Å. Besides, H₂O molecules also can form hydrogen bonds O_W–H_W···O(1), O_W–H_W···O(2), N(1)–H(1)···O_W or N(2)–H(2)···O_W with O or N groups of the Na⁺–AD backbone. The most stable gaseous bidentate conformer C7AB of Na⁺–AD is still the most stable one in the solvent of water. However, the structure of the most unstable gaseous conformer α′B of Na⁺–AD collapses under the attack of H₂O molecules and changes into C7AB conformation. Computations with IEFPCM solvation model of self-consistent reaction field theory give that aqueous C5A is more stable than C7_eqB and that the stabilization energies of water solvent on monodentate conformers of Na⁺–AD complex ion (about 272–294 kJ/mol) are more than those on bidentate ones (about 243 kJ/mol).     

A chelate complex‐enhanced luminol system for selective determination of Co(II), Fe(II) and Cr(III)

A determination method for Co(II), Fe(II) and Cr(III) ions by luminol‐H₂O₂ system using chelating reagents is presented. A metal ion‐chelating ligand complex with a Co(II) ion and a chelating reagent like ethylenediaminetetraacetic acid (EDTA) produced highly enhanced chemiluminescence (CL) intensity as well as longer lifetime in the luminol‐H₂O₂ system compared to metals that exist as free ions. Whereas free Cu(II) and Pb(II) ions had a strong catalytic effect on the luminol‐H₂O₂ system, significantly, the complexes of Cu(II) and Pb(II) with chelating reagents lost their catalytic activity due to the chelating reagents acting as masking agents. Based on the observed phenomenon, it was possible to determine Co(II), Fe(II) and Cr(III) ions with enhanced sensitivity and selectivity using the chelating reagents of the luminol‐H₂O₂ system. The effects of ligand, H₂O₂ concentration, pH, buffer solution and concentrations of chelating reagents on CL intensity of the luminol‐H₂O₂ system were investigated and optimized for the determination of Co(II), Fe(II) and Cr(III) ions. Under optimized conditions, the calibration curve of metal ions was linear over the range of 2.0 × 10^‐8 to 2.0 × 10^‐5 M for Co(II), 1.0 × 10^‐7 to 2.0 × 10^‐5 M for Fe (II) and 2.0 × 10^‐7 to 1.0 × 10^‐4 M for Cr(III). Limits of detection (3σ/s) were 1.2 × 10^‐8, 4.0 × 10^‐8 and 1.2 × 10^‐7 M for Co(II), Fe(II) and Cr(III), respectively. Copyright © 2012 John Wiley & Sons, Ltd.     

Theoretical Study of the Water Exchange Reaction on Divalent Zinc Ion using Density Functional Theory

Recent ab initio studies reported in the literature have challenged the mechanistic assignments made on the basis of volume of activation data [1,2]. In addition to that ab initio molecular orbital calculations on hydrated zinc(II)-ions were used to elucidate the general role of this ion in metalloproteins [3]. Due to our interest in both inorganic reaction mechanisms and enzymatic catalysis we started a systematic investigation of solvent exchange processes on divalent zinc-ion using density functional calculations. Our investigations cover aqua complexes of the general form [Zn(H₂O)_n]²⁺·mH₂0 with n=3-6 and m=0-2, where n and m represent the number of water molecules in the coordination and solvation sphere, respectively.The complexes [Zn(H₂O)₅]²⁺·2H₂O and [Zn(H₂O)₄]²⁺·2H₂O turnend out to be the most stable zinc complexes with seven and six water molecules, respectively. This implies that a heptacoordinated zinc(II) complex, where all water molecules are located in the co-ordination sphere, should be energetically highly unfavorable and that [Zn(H₂O)₆]²⁺ can quite readily push two coordinated water molecules into the solvation sphere. For the pentaqua complex [Zn(H₂O)₅]²⁺ only one water molecule is easily lost to the solvation sphere, which makes the [Zn(H2O)₄]²⁺·H₂O complex the most favorable in order to consider the limiting dissociative and associative water exchange process of hexacoordinated zinc(II). The dehydration and hydration energies using the most stable zinc(II) complexes [Zn(H₂O)₄]²⁺·2H₂O, [Zn(H₂O)₅]²⁺·2H₂O and [Zn(H₂O)₄]²⁺·H₂O were calculated to be 24.1 and -21.0 kcal/mol, respectively.     

Iron prevents ascorbic acid (vitamin C) induced hydrogen peroxide accumulation in copper contaminated drinking water

Ascorbic acid (vitamin C) induced hydrogen peroxide (H₂O₂) formation was measured in household drinking water and metal supplemented Milli-Q water by using the FOX assay. Here we show that ascorbic acid readily induces H₂O₂ formation in Cu(II) supplemented Milli-Q water and poorly buffered household drinking water. In contrast to Cu(II), iron was not capable to support ascorbic acid induced H₂O₂ formation during acidic conditions (pH: 3.5–5). In 12 out of the 48 drinking water samples incubated with 2 mM ascorbic acid, the H₂O₂ concentration exceeded 400 μM. However, when trace amounts of Fe(III) (0.2 mg/l) was present during incubation, the ascorbic acid/Cu(II)-induced H₂O₂ accumulation was totally blocked. Of the other common divalent or trivalent metal ions tested, that are normally present in drinking water (calcium, magnesium, zinc, cobalt, manganese or aluminum), only calcium and magnesium displayed a modest inhibitory activity on the ascorbic acid/Cu(II)-induced H₂O₂ formation. Oxalic acid, one of the degradation products from ascorbic acid, was confirmed to actively participate in the iron induced degradation of H₂O₂. Ascorbic acid/Cu(II)-induced H₂O₂ formation during acidic conditions, as demonstrated here in poorly buffered drinking water, could be of importance in host defense against bacterial infections. In addition, our findings might explain the mechanism for the protective effect of iron against vitamin C induced cell toxicity.     

Kinetics of Energetic O<Superscript>−</Superscript> Ions in the Discharge Plasmas of Water Vapor and H<Subscript>2</Subscript>O-Containing Mixtures

The process of relaxation of energetic O^– ions formed via dissociative attachment of electrons to molecules in the discharge plasmas of water vapor and H₂O: O₂ mixtures in a strong electric field is studied by the Monte Carlo method. The probability of energetic ions being involved in threshold ion–molecular processes is calculated. It is shown that several percent of energetic O^– ions formed via electron attachment to H₂O molecules in the course of plasma thermalization transform into OH^– ions via charge exchange or are destroyed with the formation of free electrons. The probabilities of charge exchange of O^– ions and electron detachment from them increase significantly (up to 90%) when O^– ions are formed via electron attachment to O₂ molecules in water vapor with an oxygen additive. This effect decreases with increasing oxygen fraction in the mixture but remains appreciable even when the fraction of H₂O molecules in the H₂O: O₂ mixture does not exceed several percent.     

Recognition dynamics of dopamine to human Monoamine oxidase B: role of Leu171/Gln206 and conserved water molecules in the active site cavity

The human Monoamine oxidase (hMAO) metabolizes several biogenic amine neurotransmitters and is involved in different neurological disorders. Extensive MD simulation studies of dopamine-docked hMAO B structures have revealed the stabilization of amino-terminal of the substrate by a direct and water-mediated interaction of catalytic tyrosines, Gln206, and Leu171 residues. The catechol ring of the substrate is stabilized by Leu171(C–H)?π(Dop)?(H–C) Ile199 interaction. Several conserved water molecules are observed to play a role in the recognition of substrate to the enzyme, where W1 and W2 associate in dopamine– FAD interaction, reversible dynamics of W3 and W4 influenced the coupling of Tyr435 to Trp432 and FAD, and W5 and W8 stabilized the catalytic Tyr188/398 residues. The W6, W7, and W8 water centers are involved in the recognition of catalytic residues and FAD with the N⁺- site of dopamine through hydrogen bonding interaction. The recognition of substrate to gating residues is made through W9, W10, and W11 water centers. Beside the interplay of water molecules, the catalytic aromatic cage has also been stabilized by π?water, π?C–H, and π?π interactions. The topology of conserved water molecular sites along with the hydration dynamics of catalytic residues, FAD, and dopamine has added a new feature on the substrate binding chemistry in hMAO B which may be useful for substrate analog inhibitor design.     

Synthesis, structure and fluorescence of two novel manganese(II) and zinc(II)-1,3,5-benzene tricarboxylate coordination polymers: Extended 3D supramolecular architectures stabilised by hydrogen bonding

Two new coordination polymers {[Mn(H₂btc)(phen)(H₂O)₂]H₂btc · H₂O}_n (1) [H₃btc = 1,3,5-benzene tricarboxylic acid, phen = phenanthroline] and {[Zn₃(btc)₂(H₂O)₈](H₂O)₄}_n (2) have been synthesised and structurally characterised. Both the complexes crystallise as 1D chain, which further propagates through ligand-based hydrogen bonding interactions into a 3D supramolecular architecture. Supramolecular framework of 1 is constructed by [Mn(H₂btc)(phen)(H₂O)₂]⁺ as well as the constituent materials-uncoordinated H₂btc⁻ and water molecules. Complex 2 exists as a corrugated chain with both the bridging and terminal Zn²⁺ ions and each zinc centre is coordinated to four water molecules. Both 1 and 2 are stacked by mutual π-stacking of the ligands and exhibit strong fluorescence emission band at 414 and 400 nm, respectively.     

83 Application of differential scanning calorimetry to measure the differential binding of ions,water, and protons in the unfolding of DNA molecules

The overall stability of DNA molecules globally depends on base-pair stacking, base pairing, polyelectrolyte effect, and hydration contributions. In order to improve our understanding of the role of ions, water, and protons in the stability and melting behavior of DNA structures, we report an experimental approach to determine the differential binding of ions (Δn _ion), water (Δn _W), and protons (Δn _H+) in the helix-coil transition of DNA molecules. A combination of differential scanning calorimetry (DSC) and temperature-dependent UV and CD spectroscopic techniques to investigate the unfolding of a variety of DNA molecules: S.T. DNA, two dodecamers, one undecamer, nine short hairpins as a function of the GC content of their stem, and two triplexes. We determine complete thermodynamic profiles, including all the three linking numbers, for the unfolding of each molecule. The CD spectra indicated that all molecules adopted the B-conformation at low temperatures. Thermodynamic profiles obtained from the DSC curves indicate that the favorable folding of each molecule results from the typical compensation of favorable enthalpy and unfavorable entropy contributions, and negligible heat capacity effects. UV and DSC melting curves as a function of salt, osmolyte, and proton concentrations yielded releases of ions, water, and protons (for the triplex with C⁺GC base triplets). Therefore, the favorable folding of each DNA molecule results from the formation of base-pair stacks and uptake of water and counterions. The thermodynamic data will be discussed in terms of the effects of DNA length, loop contributions and type of water molecules.