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Protein/peptide self-assembly into amyloid structures associates with major neurodegenerative disorders such as Alzheimer's disease (AD). Soluble assemblies (oligomers) of the Aβ peptide and their aggregates are perceived as neurotoxic species in AD. While screening for synthetic cleavage agents that could break down such aberrant assemblies through hydrolysis, we observed that the assemblies of Aβ oligopeptides, containing the nucleation sequence Aβ 14-24 (H 14 QKLVFFAEDV 24 ), could act as cleavage agents by themselves. Autohydrolysis showed a common fragment fingerprint among various mutated Aβ 14-24 oligopeptides, Aβ 12-25 -Gly and Aβ 1-28 , and full-length Aβ 1-40/42 , under physiologically relevant conditions. Primary endoproteolytic autocleavage at the Gln 15 -Lys 16 , Lys 16 -Leu 17 and Phe 19 -Phe 20 positions was followed by subsequent exopeptidase self-processing of the fragments. Control experiments with homologous d-amino acid enantiomers Aβ 12-25 -Gly and Aβ 16-25 -Gly showed the same autocleavage pattern under similar reaction conditions. The autohydrolytic cascade reaction (ACR) was resilient to a broad range of conditions (20-37 °C, 10-150 μM peptide concentration at pH 7.0-7.8). Evidently, assemblies of the primary autocleavage fragments acted as structural/compositional templates (autocatalysts) for self-propagating autohydrolytic processing at the Aβ 16-21 nucleation site, showing the potential for cross-catalytic seeding of the ACR in larger Aβ isoforms (Aβ 1-28 and Aβ 1-40/42 ). This result may shed new light on Aβ behaviour in solution and might be useful in the development of intervention strategies to decompose or inhibit neurotoxic Aβ assemblies in AD., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Lytic Polysaccharide Monooxygenases (LPMOs) oxidatively cleave recalcitrant polysaccharides. The mechanism involves (i) reduction of the Cu, (ii) polysaccharide binding, (iii) binding of different oxygen species, and (iv) glycosidic bond cleavage. However, the complete mechanism is poorly understood and may vary across different families and even within the same family. Here, we have investigated the protonation state of a secondary co-ordination sphere histidine, conserved across AA9 family LPMOs that has previously been proposed to be a potential proton donor. Partial unrestrained refinement of newly obtained higher resolution data for two AA9 LPMOs and re-refinement of four additional data sets deposited in the PDB were carried out, where the His was refined without restraints, followed by measurements of the His ring geometrical parameters. This allowed reliable assignment of the protonation state, as also validated by following the same procedure for the His brace, for which the protonation state is predictable. The study shows that this histidine is generally singly protonated at the Nε2 atom, which is close to the oxygen species binding site. Our results indicate robustness of the method. In view of this and other emerging evidence, a role as proton donor during catalysis is unlikely for this His.

Trace levels of copper can impact the flavor stability of beer. The main source of copper is malt, and the wort copper levels are established during mashing and lautering. This study focuses on sweet worts made from experimental roasted and caramel malts. Potentiometric titrations using ion-selective electrodes combined with electron paramagnetic resonance spectroscopy have been used to investigate Cu(II) binding in worts as well as the impact of Cu(II) ions on the wort oxidative stability. High-temperature treatment during malting decreased Cu(II) binding affinities in the worts, with roasted malt worts having lower affinities than caramel malt worts of similar color and pH. Electron paramagnetic resonance spectra indicated dipeptides as the main Cu(II) chelators. A positive correlation between Cu and free amino nitrogen levels in worts is demonstrated. In dark worts with high rates of radical formation, Cu(II) had pronounced antioxidative effects. In contrast, moderate prooxidative effects were observed when adding Cu(II) to pale worts with inherently low rates of oxidation.

Reepithelialisation is fundamental to wound healing, but our current understanding largely relies on cellular and animal studies. The aim of the present randomised double-blind three-arm controlled trial was to correlate genuine epidermal wound healing with key proteins and topical zinc treatment in humans. Sixty wounds were produced using deroofed suction blisters in 30 healthy volunteers and randomised to topical zinc sulphate (n = 20), placebo (n = 20), or control (n = 20) treatment for 4 days. All wounds with perilesional skin were processed for automatic immunostaining of paraffin tissue sections with monoclonal antibodies against Ki-67, metallothionein (MT) and matrix metalloproteinase (MMP)-1. Protein expression was quantified by automated digital image analysis. Epidermal Ki-67 and MT labelling indices were increased in keratinocytes in the neoepidermis (∼1.1 mm) and at the wound edge (0.5 mm) compared to normal skin. Increased MMP-1 immunostaining was restricted to the neoepidermis. MT was robustly upregulated in the upper dermis of the wounds. Zinc treatment enhanced MMP-1 expression beneath the neoepidermis via paracrine mechanisms and MT under the neoepidermis and in the nonepithelialised wound bed via direct actions of zinc as indicated by the induction of MT2A mRNA but not MMP-1 mRNA in cultured normal human dermal fibroblasts by zinc sulphate. The present human study demonstrates that quantitative immunohistochemistry can identify proteins involved in reepithelialisation and actions of external compounds. Increased dermal MT expression may contribute to the anti-inflammatory activities of zinc and increased MMP-1 levels to promote keratinocyte migration., (Copyright © 2020 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Catalytic breakdown of polysaccharides can be achieved more efficiently by means of the enzymes lytic polysaccharide monooxygenases (LPMOs). However, the LPMO mechanism has remained controversial, preventing full exploitation of their potential. One of the controversies has centered around an active site tyrosine, present in most LPMO classes. Recent investigations have for the first time obtained direct (spectroscopic) evidence for the possibility of chemical modification of this tyrosine. However, the spectroscopic features obtained in the different investigations are remarkably different, with absorption maximum at 420 and 490 nm, respectively. In this paper we use density functional theory (DFT) in a QM/MM formulation to reconcile these (apparently) conflicting results. By modeling the spectroscopy as well as the underlying reaction mechanism we can show how formation of two isomers (both involving deprotonation of tyrosine) explains the difference in the observed spectroscopic features. Both isomers have a [TyrO-Cu-OH] + moiety with the OH in either the cis - or trans -position to a deprotonated tyrosine. Although the cis -[TyrO-Cu-OH] + moiety is well positioned for oxidation of the substrate, preliminary calculations with the substrate reveal that the reactivity is at best moderate, making a protective role of tyrosine more likely., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Fluorescent, DNA-stabilized silver nanoclusters (DNA-AgNCs) are applied in a range of applications within nanoscience and nanotechnology. However, their diverse optical properties, mechanism of formation, and aspects of their composition remain unexplored, making the rational design of nanocluster probes challenging. Herein, a synthetic procedure is described for obtaining a high yield of emissive DNA-AgNCs with a C-loop hairpin DNA sequence, with subsequent purification by size-exclusion chromatography (SEC). Through a combination of optical spectroscopy, gel electrophoresis, inductively coupled plasma mass spectrometry (ICP-MS), and small-angle X-ray scattering (SAXS) in conjunction with the systematic study of various DNA sequences, the low-resolution structure and mechanism of the formation of AgNCs were investigated. Data indicate that fluorescent DNA-AgNCs self-assemble by a head-to-head binding of two DNA hairpins, bridged by a silver nanocluster, resulting in the modelling of a dimeric structure harboring an Ag 12 cluster., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Malodour from the axilla is commonly caused by specific microbes, and may be inhibited by zinc oxide. The aim of this study was to determine the effects of zinc oxide on the axillary microbiota, odour and pH in a randomized, double-blind, placebo-controlled trial in 30 healthy volunteers. In each participant 1 axilla was treated with zinc oxide and the other with a placebo for 13 days. The microbiota and pH were analysed before and during treatment. At the final visit, the participants judged their own axillary odour for comparison. With zinc oxide treatment total bacterial growth and, specifically, that of odour-producing Corynebacterium spp. and Staphylococcus hominis, decreased (p < 0.05), despite an increase (p < 0.0005) in skin-surface pH. Compared with the placebo, zinc oxide treatment reduced (p = 0.005) self-perceived malodour. In vitro, Corynebacterium spp. (19 isolated strains) survival was reduced (p < 0.0005) at pH 5.0 compared with pH 6.0; growth inhibition by zinc oxide occurred at ≤ 400 mg/l, and cell death occurred at ≤ 10,000 mg/l for 12 (63%) of the strains. In conclusion, application of zinc oxide reduced malodour and the counts of causative bacteria, but increased the pH of the axilla.

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes capable of oxidizing crystalline cellulose which have large practical application in the process of refining biomass. The catalytic mechanism of LPMOs still remains debated despite several proposed reaction mechanisms. Here, we report a long-lived intermediate (t 1/2 =6-8 minutes) observed in an LPMO from Thermoascus aurantiacus (TaLPMO9A). The intermediate with a strong absorption around 420 nm is formed when reduced LPMO-Cu I reacts with sub-equimolar amounts of H 2 O 2 . UV/Vis absorption spectroscopy, electron paramagnetic resonance, resonance Raman and stopped-flow spectroscopy suggest that the observed long-lived intermediate involves the copper center and a nearby tyrosine (Tyr175). Additionally, activity assays in the presence of sub-equimolar amounts of H 2 O 2 showed an increase in the LPMO oxidation of phosphoric acid swollen cellulose. Accordingly, this suggests that the long-lived copper-dependent intermediate could be part of the catalytic mechanism for LPMOs. The observed intermediate offers a new perspective into the oxidative reaction mechanism of TaLPMO9A and hence for the biomass oxidation and the reactivity of copper in biological systems., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Copper ion-catalyzed oxidation of yeast SOD1 (ySOD1) was examined to determine early oxidative modifications, including oxidation of a crucial disulfide bond, and the structural and functional repercussions of these events. The study used distinct oxidative conditions: Cu 2+ /H 2 O 2 , Cu 2+ /H 2 O 2 /AscH - and Cu 2+ /H 2 O 2 /glucose. Capillary electrophoresis experiments and quantification of protein carbonyls indicate that ySOD1 is highly susceptible to oxidative modification and that changes can be detected within 0.1 min of the initiation of the reaction. Oxidation-induced structural perturbations, characterized by circular dichroism, revealed the formation of partially-unfolded ySOD1 species in a dose-dependent manner. Consistent with these structural changes, pyrogallol assay indicates a partial loss of enzymatic activity. ESI-MS analyses showed seven distinct oxidized ySOD1 species under mild oxidation within 0.1 min. LC/MS analysis after proteolytic digestion demonstrated that the copper-coordinating active site histidine residues, His47 and His49, were converted into 2-oxo-histidine. Furthermore, the Cu and Zn bridging residue, His64 is converted into aspartate/asparagine. Importantly, the disulfide-bond Cys58-Cys147 which is critical for the structural and functional integrity of ySOD1 was detected as being oxidized at Cys147. We propose, based on LC/MS analyses, that disulfide-bond oxidation occurs without disulfide bond cleavage. Modifications were also detected at Met85 and five surface-exposed Lys residues. Based on these data we propose that the Cys58-Cys147 bond may act as a sacrificial target for oxidants and protect ySOD1 from oxidative inactivation arising from exposure to Cu 2+ /H 2 O 2 and auto-inactivation during extended enzymatic turnover., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Laccases of different biological origins have been widely investigated and these studies have elucidated fundamentals of the generic catalytic mechanism. However, other features such as surface properties and residues located away from the catalytic centres may also have impact on enzyme function. Here we present the crystal structure of laccase from Myceliophthora thermophila (MtL) to a resolution of 1.62 Å together with a thorough structural comparison with other members of the CAZy family AA1&#95;3 that comprises fungal laccases from ascomycetes. The recombinant protein produced in A. oryzae has a molecular mass of 75 kDa, a pI of 4.2 and carries 13.5 kDa N-linked glycans. In the crystal, MtL forms a dimer with the phenolic substrate binding pocket blocked, suggesting that the active form of the enzyme is monomeric. Overall, the MtL structure conforms with the canonical fold of fungal laccases as well as the features specific for the asco-laccases. However, the structural comparisons also reveal significant variations within this taxonomic subgroup. Notable differences in the T1-Cu active site topology and polar motifs imply molecular evolution to serve different functional roles. Very few surface residues are conserved and it is noticeable that they encompass residues that interact with the N-glycans and/or are located at domain interfaces. The N-glycosylation sites are surprisingly conserved among asco-laccases and in most cases the glycan displays extensive interactions with the protein. In particular, the glycans at Asn88 and Asn210 appear to have evolved as an integral part of the asco-laccase structure. An uneven distribution of the carbohydrates around the enzyme give unique properties to a distinct part of the surface of the asco-laccases which may have implication for laccase function-in particular towards large substrates., Competing Interests: Novozymes A/S is producing MtL laccase on a commercial basis. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Previous studies on metal-ion catalyzed oxidation of α-synuclein oxidation have mostly used conditions that result in extensive modification precluding an understanding of the early events in this process. In this study, we have examined time-dependent oxidative events related to α-synuclein modification using six different molar ratios of Cu 2+ /H 2 O 2 /protein and Cu 2+ /H 2 O 2 /ascorbate/protein resulting in mild to moderate extents of oxidation. For a Cu 2+ /H 2 O 2 /protein molar ratio of 2.3:7.8:1 only low levels of carbonyls were detected (0.078 carbonyls per protein), whereas a molar ratio of 4.7:15.6:1 gave 0.22 carbonyls per α-synuclein within 15 min. With the latter conditions, rapid conversion of 3 out of 4 methionines (Met) to methionine sulfoxide, and 2 out of 4 tyrosines (Tyr) were converted to products including inter- and intra-molecular dityrosine cross-links and protein oligomers, as determined by SDS-PAGE and Western blot analysis. Limited histidine (His) modification was observed. The rapid formation of dityrosine cross-links was confirmed by fluorescence and mass-spectrometry. These data indicate that Met and Tyr oxidation are early events in Cu 2+ /H 2 O 2 -mediated damage, with carbonyl formation being a minor process. With the Cu 2+ /H 2 O 2 /ascorbate system, rapid protein carbonyl formation was detected with the first 5 min, but after this time point, little additional carbonyl formation was detected. With this system, lower levels of Met and Tyr oxidation were detected (2 Met and 1 Tyr modified with a Cu 2+ /H 2 O 2 /ascorbate/protein ratio of 2.3:7.8:7.8:1), but greater His oxidation. Only low levels of intra- dityrosine cross-links and no inter- dityrosine oligomers were detected under these conditions, suggesting that ascorbate limits Cu 2+ /H 2 O 2 -induced α-synuclein modification., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A prominent current hypothesis is that impaired metal ion homeostasis may contribute to Alzheimer's disease (AD). We elucidate the interaction of Cu 2+ with wild-type (WT) Aβ 1-40 and the genetic variants A2T and A2V which display increasing pathogenicity as A2T2+ significantly extends the lag phase in aggregation kinetics, in particular for the pathogenic A2V variant. Additionally, a rapid, initial, low intensity ThT response is observed, possibly reflecting formation of Cu 2+ induced amorphous aggregates, as supported by atomic force microscopy (AFM) and circular dichroism (CD) spectroscopy, again most notably for the A2V variant. Electron paramagnetic resonance (EPR) spectroscopy gives pK a values for transition between two Cu 2+ coordination geometries (component I and II) of 7.4 (A2T), 7.9 (WT), and 8.4 (A2V), that is, component I is stabilized at physiological pH in the order A2T1 H NMR relaxation exhibits the same trend for the non-coordinating aromatic residues (A2T2+ exchange for the A2V variant than for WT and A2T. We therefore hypothesize that component I of the Cu-Aβ complex is related to pathogenicity, accounting for both the pathogenic nature of the A2V variant and the protective nature of the A2T variant., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

To meet the very specific requirements demanded by industry, proteins must be appropriately tailored. Engineering laccases, to improve the oxidation of small molecules, with applications in multiple fields, is, however, a difficult task. Most efforts have concentrated on increasing the redox potential of the enzyme, but in recent work, we have pursued an alternate strategy to engineering these biocatalysts. In particular, we have found that redesigning substrate binding at the T1 pocket, guided by in silico methodologies, to be a more consistent option. In this work, we evaluate the robustness of our computational approach to estimate activity, emphasizing the importance of the binding event in laccase reactivity. Strengths and weaknesses of the protocol are discussed along with its potential for scoring large numbers of protein sequences and thus its significance in protein engineering.

Here, we present a lipase mutant containing a biochemical switch allowing a controlled opening and closing of the lid independent of the environment. The closed form of the TlL mutant shows low binding to hydrophobic surfaces compared to the binding observed after activating the controlled switch inducing lid-opening. We directly show that lipid binding of this mutant is connected to an open lid conformation demonstrating the impact of the exposed amino acid residues and their participation in binding at the water-lipid interface. The switch was created by introducing two cysteine residues into the protein backbone at sites 86 and 255. The crystal structure of the mutant shows the successful formation of a disulfide bond between C86 and C255 which causes strained closure of the lid-domain. Control of enzymatic activity and binding was demonstrated on substrate emulsions and natural lipid layers. The locked form displayed low enzymatic activity (~10%) compared to wild-type. Upon release of the lock, enzymatic activity was fully restored. Only 10% binding to natural lipid substrates was observed for the locked lipase compared to wild-type, but binding was restored upon adding reducing agent. QCM-D measurements revealed a seven-fold increase in binding rate for the unlocked lipase. The TlL&#95;locked mutant shows structural changes across the protein important for understanding the mechanism of lid-opening and closing. Our experimental results reveal sites of interest for future mutagenesis studies aimed at altering the activation mechanism of TlL and create perspectives for generating tunable lipases that activate under controlled conditions., (Copyright © 2016. Published by Elsevier B.V.)

Thermomyces lanuginosus lipase (TlL) and related lipases become activated in low-polarity environments that exist at the water-lipid interface where a structural change of the "lid" region occurs. In this work, we have investigated the activation of TlL (Lipase&#95;W89) and certain lid mutants, containing either a single positive charge mutation, E87K (Lipase&#95;K87&#95;W89), within the lid region or a lid residue composition of both lipase and esterase character (Hybrid&#95;W89) as a function of solvent polarity. Activation differences between the variants and TlL were studied by a combination of biophysical and theoretical methods. To investigate the structural changes taking place in the lid region upon lipase activation, we used a fluorescence-based method measuring the efficiency of Trp89 in the lid to quench the fluorescence of a bimane molecule attached in front (C255) and behind (C61) the lid. These structural changes were compared to the enzymatic activity of each variant at the water-substrate interface and to theoretical calculations of the energies associated with lid opening as a function of the dielectric constant (ε) of the environment. Our results show that the lid in Lipase&#95;K87&#95;W89 undergoes a pronounced structural transition toward an open conformation around ε = 50, whereas only small changes are detected for Lipase&#95;W89 ascribed to the stabilizing effect of the positive charge mutation on the open lid conformation. Interestingly, Hybrid&#95;W89, with the same charge as Lipase&#95;W89, shows a stabilization of the open lid even more pronounced at high solvent polarities than that of Lipase&#95;K87&#95;W89, allowing activation at ε < 80. This is further indicated by measurement of the lipase activity for each variant showing that Hybrid&#95;W89 is more quickly activated at the water-lipid interface of a true, natural substrate. Combined, we show that a correlation exists between structural changes and enzymatic activities detected on one hand and theoretical calculations on lid opening energies on the other. These results highlight the key role that the lid plays in determining the polarity-dependent activation of lipases.

Triacylglycerol hydrolases (EC 3.1.1.3) are thought to become activated when they encounter the water-lipid interface causing a "lid" region to move and expose the catalytic site. Here, we tested this idea by looking for lid movements in Thermomyces lanuginosus lipase (TL lipase), and in variants with a mutated lid region of esterase (Esterase) and esterase/lipase (Hybrid) character. To measure lid movements, we employed the tryptophan-induced quenching (TrIQ) fluorescence method to measure how effectively a Trp residue on the lid of these mutants (at position 87 or 89) could quench a fluorescent probe (bimane) placed at nearby site 255 on the protein. To test if lid movement is induced when the enzyme detects a lower-polarity environment (such as at the water-lipid interface), we performed these studies in solvents with different dielectric constants (ε). The results show that lid movement is highly dependent on the particular lid residue composition and solvent polarity. The data suggest that in aqueous solution (ε = 80), the Esterase lid is in an "open" conformation, whereas for the TL lipase and Hybrid, the lid remains "closed". At lower solvent polarities (ε < 46), the lid region for all of the mutants is more "open". Interestingly, these behaviors mirror the structural changes thought to take place upon activation of the enzyme at the water-lipid interface. Together, these results support the idea that lipases are more active in low-polarity solvents because the lid adopts an "open" conformation and indicate that relatively small conformational changes in the lid region play a key role in the activation mechanism of these enzymes.

It is shown by rational site-directed mutagenesis of the lid region in Thermomyces lanuginosus lipase that it is possible to generate lipase variants with attractive features, e.g., high lipase activity, fast activation at the lipid interface, ability to act on water-soluble substrates, and enhanced calcium independence. The rational design was based on the lid residue composition in Aspergillus niger ferulic acid esterase (FAEA). Five constructs included lipase variants containing the full FAEA lid, a FAEA-like lid, an intermediate lid of FAEA and TlL character, and the entire lid region from Aspergillus terreus lipase (AtL). To investigate an altered activation mechanism for each variant compared to that of TlL, a combination of activity- and spectroscopic-based measurements were applied. The engineered variant with a lid from AtL displayed interfacial activation comparable to that of TlL, whereas variants with FAEA lid character showed interfacial activation independence with pronounced activity toward pNP-acetate and pNP-butyrate below the critical micelle concentration. For variants with lipase and esterase character, lipase activity measurements further indicated a faster activation at the lipid interface. Relative to their activity toward pNP-ester substrates in calcium-rich buffer, all lid variants retained between 15 and 100% activity in buffer containing 5 mM EDTA whereas TlL activity was reduced to less than 2%, demonstrating the lid's central role in governing calcium dependency. For FAEA-like lid variants, accessible hydrophobic surface area measurements showed an approximate 10-fold increase in the level of binding of extrinsic fluorophores to the protein surface relative to that of TlL accompanied by a blue shift in emission indicative of an open lid in aqueous solution. Together, these studies report on the successful alteration of the activation mechanism in TlL by rational design creating novel lipases with new, intriguing functionalities.

An on-line method, coupling reversed phase chromatography with static light scattering, was developed to determine the association state of freshly eluted proteins. Under downstream process conditions, human insulin desB30 and human insulin AspB28 were tested at concentrations up to 8.5mg/mL. The refractive index increment (dn/dc) for insulin was found to depend strongly on the solvent used. A refractive index increment of 0.184±0.003mL/g was found in an aqueous buffer, pH 7.4, whereas the value was 0.155±0.003mL/g in 30%, w/w ethanol. The methodology combines on-line SLS and UV measurements with the pre-determined refractive index increment values. The developed on-line method was verified by standard off-line measurements establishing the association state at concentrations between 0.2 and 6.0mg/mL. The equipment was calibrated utilizing insulin under conditions reported to ensure either monomer or hexamer forms. The self-association of human insulin desB30 was found to be strongly suppressed in 30%, w/w ethanol at pH 7.4 in which the monomer predominates. When stabilized by zinc ions in 30%, w/w ethanol at pH 7.4, an average association number of 3.7 was found. These data demonstrate the effect of ethanol to lower strongly the energy advantage by protein self-association. Potassium chloride and/or calcium chloride in the eluents were found to be of no consequence to the association state., (Copyright © 2013 Elsevier B.V. All rights reserved.)

(204m)Pb perturbed angular correlation of γ-rays (PAC) spectroscopy has been applied successfully for the first time to detect the nuclear quadrupole interaction in a lead(II) coordination compound in a molecular crystal [tetraphenylarsonium lead(II) isomaleonitriledithiolate ([AsPh(4)](4)[Pb(2)(i-mnt)(4)])]. The recorded parameters from a powder crystalline sample are ν(Q) = 0.178(1) GHz and η = 0.970(7). The electric field gradient (EFG) was determined at the PW91/QZ4P level including relativistic effects using the two-component zeroth-order regular approximation method for both the [Pb(i-mnt)(2)](2-) monomer and the [Pb(2)(i-mnt)(4)](4-) dimer. Only the EFG for the latter compares favorably with the experimental data, indicating that the picture of this complex as a prototypical hemidirected coordination geometry with a stereochemically active lone pair on lead(II) is inadequate. Advantages and limitations of (204m)Pb PAC spectroscopy as a novel technique to elucidate the electronic and molecular structures of lead-containing complexes and biomolecules are presented.

To enable utilization of the broad potential of copper isotopes in nuclear medicine, rapid and robust chelation of the copper is required. Bowl adamanzanes (bicyclic tetraaza ligands) can form kinetically stable copper complexes, but they are usually formed at low rates unless high pH values and high temperatures are applied. We have investigated the effects of the variation in the pH, different anions, and UV irradiation on the chelation rate. UV spectra of mixtures of Cu(2+) and [2(4).3(1)]adz in water show the existence of a long-lived two-coordinated copper(II) intermediate (only counting coordinated amine groups) at pH above 6. These findings are supported by pH titrations of mixtures of Cu(2+) and [2(4).3(1)]adz in water. Irradiation of this complex in the ligand-to-metal charge-transfer (LMCT) band by a diode-array spectrophotometer leads to photodeprotonation and subsequently to formation of the four-coordinated copper(II) complex at a rate up to 7800-fold higher at 25 °C than in the dark. Anions in the solution were found to have three major effects: competitive inhibition due to Cu(II) binding anions, inhibition of the photoinduced transchelation from UV-absorbing anions, and photoredox inhibition from acido ligands capable of acting as electron donors in LMCT reactions. Dissolved O(2) was also found to result in photoredox inhibition., (© 2011 American Chemical Society)

Capillary electrophoresis (CE) and electrospray ionisation (ESI) mass spectra of aqueous solutions of manganese(II) complexes of the monoanions of the pentadentate ligands N-methyl-N'-carboxymethyl-N,N'-bis(2-pyridylmethyl)ethane-1,2-diamine (mcbpen(-)) and N-benzyl-N'-carboxymethyl-N,N'-bis(2-pyridylmethyl)ethane-1,2-diamine (bcbpen(-)), show the presence of a mixture of closely related Mn(II) species, assigned to the mono, di-, tri- and poly-cationic complexes [Mn(II)(L)(H(2)O)](n)(n+), L = mcbpen(-) or bcbpen(-) with n = 1, 2, 3, etc. In solution, these complexes are reversibly oxidized by tert-butyl hydrogen peroxide (TBHP), (NH(4))(2)[Ce(NO(3))(6)], Ce(ClO(4))(4), oxone and [Ru(bipy)(3)](3+) to form metastable (t(½) = min to h) higher valent (hydr)oxide species, showing a collective maximum absorbance at 430 nm. The same species can be produced by [Ru(bipy)(3)](2+)-mediated photooxidization in the presence of an electron acceptor. TBHP oxidation of the complexes, in large excesses of the TBHP, is concurrent with an O(2) evolution with turnovers of up to 1.5 × 10(4) mol of O(2) per mol of [Mn] and calculated rate constants from two series of experiments of 0.039 and 0.026 mol[O(2)] s(-1) M(-2). A 1:1 reaction of TBHP with [Mn] is rate determining and the resultant species is proposed to be the mononuclear, catalytically competent, [Mn(IV)(O)(mcbpen)](+). At very close m/z values [Mn(III)(OH)(mcbpen)](+), [Mn(2)(III/IV)(O)(2)(mcbpen)(2)](+) and [Mn(IV)(2)(O)(2)(mcbpen)(2)](2+) are detected by ESI MS and CE when the concentration of TBHP is comparable to or lower than that of [Mn]. These are conditions that occur post catalysis and these species are derived from [Mn(IV)(O)(mcbpen)](+) through condensation reactions.

The specific activities of crude and purified Coprinus cinereus laccase preparations could be enhanced by a factor of 10-12 by activation with copper ions. The copper to protein contents of purified non-activated laccase were 2.3+/-0.1 compared to 3.3+/-0.1 in purified activated laccase indicating that only a fraction of the laccase can be activated. Purified laccase not activated with copper ions shows in isoelectric focusing four bands in order of decreasing pI in a ratio 1/5/3/1 where only bands I and II had laccase activity. Purified activated laccase showed only three bands (I, II and III) in the ratio 5/4/1 all with some laccase activity. The pH profile of the activity for activated and non-activated laccase showed identical behavior indicating that the active forms were the same. The change in UV-Vis around 330 nm following the depletion and reconstitution of the enzyme combined with activity measurements supports the reversibility of the selective removal and insertion of copper ions at the type 2 site. The circular dichroism spectrum of activated purified laccase has characteristic changes around 350 nm relative to non-activated laccase indicative of changes at the type 2/type 3 sites. The difference between the electron paramagnetic resonance spectra of non-activated and activated C. cinereus laccase indicates that a fraction of the non-activated purified laccase contained a copper(II) signal with a coupling constant between a type 1 and a type 2 copper(II). This electron paramagnetic resonance signal could be explained by an induced asymmetry in the type 3 site due to a missing type 2 copper ion., (Copyright 2010 Elsevier Inc. All rights reserved.)

In this paper we examine how the bacterial growth is influenced by the availability of copper ions in complex Mueller Hinton growth media. The data shows that the free copper concentration is seven to eight orders of magnitude lower the total copper concentration and that there seems to be a better correlation between the free copper concentration and bacterial growth, than for the total copper concentration and growth. Furthermore, it is shown that the initial pH influences the amount of free copper ions in the media and that this has a direct effect on the ability of bacterial cultures to grow. However, there still remains an effect of pH on bacterial growth which cannot be attributed to the influence of the Cu(2+) concentration alone. The study also shows that the sterilization treatment can have some effect on the availability of copper ions in the media over time. Freshly autoclaved and sterile filtered media contain the same level of free copper ions and perform equally well in the biological assay. However, storage in the refrigerator influences the free copper contents in media, which have been autoclaved, but not in media, which were sterile filtered. Therefore, the latter method is to be recommended, when working with bacterial copper testing.

The reactivity of the acido Ru(II) complexes cis-[RuCl(2)(LL)(2)], [RuCO(3)(LL)(2)], cis-[RuCO(3)-(bquin)(2)] (LL = 2,2'-bipyridine (bpy) and 1,10-phenanthroline (phen); bquin = 2,2'-biquinoline) and cyclometalated Ru(II) derivatives of 2-phenylpyridine and 4-(2-tolyl)pyridine [Ru(o-C(6)H(4)-2-py)(phen)(2)]PF(6) (1), [Ru(o-C(6)H(3)-p-R-2-py)(bpy)(MeCN)(2)]PF(6) (2), and [Ru(o-C(6)H(3)-p-R-2-py)(phen)(MeCN)(2)]PF(6) (3) (R = H (a), Me (b)) toward laccase from Coriolus hirsutus has been investigated by conventional UV-vis spectroscopy at pH 3-7 and 25 degrees C. The acido and cyclometalated complexes are readily oxidized into the corresponding Ru(III) species, but the two types of complexes differ substantially in reactivity and obey different rate laws. The acido complexes are oxidized more slowly and the second-order kinetics, first-order in laccase and Ru(II), holds with the rate constants around 5 x 10(4) M(-1) s(-1) at pH 4.5 and 25 degrees C. The cyclometalated complexes 1-3 react much faster and the hyperbolic Michaelis-Menten kinetics holds. However, it is not due to formation of an enzyme-substrate complex but rather because of the ping-pong mechanism of catalysis, viz. E(ox) + Ru(II) --> E(red) + Ru(III) (k(1)); E(red) + 1/4O(2) --> E(ox) (k(2)), with the rate constants k(1) in the range (2-9) x 10(7) M(-1) s(-1) under the same conditions. The huge values of k(1) move the enzymatic oxidation toward a kinetic regime when the dioxygen half-reaction becomes the rate-limiting step. Cyclometalated compounds 1-3 can therefore be used for routine estimation of k(2), that is, the rate constant for reoxidation for laccases by dioxygen. The mechanism proposed was confirmed by the direct stopped-flow measurements of the k(2) rate constant (8.1 x 10(5) M(-1) s(-1) at 26 degrees C) and supported by the theoretical modeling of interaction between the bpy analogue of 1 and Coriolus hirsutes laccase using Monte Carlo simulations.

A laccase from the fungus Trametes villosa (TviL) was investigated in order to elucidate the reaction mechanism of the reduction of dioxygen to water performed by this blue multi-copper oxidase. The ability of TviL to activate dioxygen was studied by stopped-flow experiments and under steady-state conditions. In the stopped-flow experiments TviL was reduced with a small excess of 4-hydroxyphenylacetic acid and afterwards the re-oxidation process was monitored by stopped-flow techniques by mixing with excess dioxygen. The reaction between reduced TviL and dioxygen was studied in the temperature range 10-35 degrees Celsius and with the concentration of dioxygen between 30 and 240microM. The observed rate constant k(obs) is found to be linear dependent on the dioxygen concentration and the observed second-order rate constant for the re-oxidation of reduced TviL is, at 25 degrees Celsius, determined to be 1.14x10(6)M(-1)s(-1). The activation energy, E(a), is from the same data determined to be 22kJmol(-1). Oxidation of different phenols (4-hydroxyphenylacetic acid, 4-hydroxybenzoic acid, guaiacolsulfonic acid and hydroquinone) under steady state conditions was investigated at concentrations of dioxygen ranging from 60 to 250microM. This line of experiments showed that TviL follows a ping-pong mechanism, and an observed second-order rate constant for the reaction with dioxygen of 7.1x10(5)M(-1)s(-1) at 25 degrees Celsius was found with 4-hydroxyphenylacetic acid as reducing substrate. The two kinetic methods resulted in observed rate constants of equal magnitudes for the reaction with dioxygen, which suggests that the rate limiting step(s) is (are) included in both the reactions studied by the two different techniques.

In order to create a heme environment that permits biomimicry of heme-containing peroxidases, a number of new hemin-peptide complexes--hemin-2(18)-glycyl-L-histidine methyl ester (HGH), hemin-2(18)-glycyl-glycyl-L-histidine methyl ester (HGGH), and hemin-2,18-bis(glycyl-glycyl-L-histidine methyl ester) (H2GGH)--have been prepared by condensation of glycyl-L-histidine methyl ester or glycyl-glycyl-L-histidine methyl ester with the propionic side chains of hemin. Characterization by means of UV/vis- and 1H NMR spectroscopy as well as cyclic- and differential pulse voltammetry indicates the formation of five-coordinate complexes in the case of HGH and HGGH, with histidine as an axial ligand. In the case of H2GGH, a six-coordinate complex with both imidazoles coordinated to the iron center appears to be formed. However, 1H NMR of H2GGH reveals the existence of an equilibrium between low-spin six-coordinate and high-spin five-coordinate species in solution. The catalytic activity of the hemin-peptide complexes towards several organic substrates, such as p-cresol, L-tyrosine methyl ester, and ABTS, has been investigated. It was found that not only the five-coordinate HGH and HGGH complexes, but also the six-coordinate H2GGH, catalyze the oxidation of substrates by H2O2. The longer and less strained peptide arm provides the HGGH complex with a slightly higher catalytic efficiency, as compared with HGH, due to formation of more stable intermediate complexes., (Copyright 2004 SBIC)

A method for the determination of stability constants of metal ion-protein binding, based on capillary electrophoresis, is presented. It utilizes the change in electrophoretic mobility of the protein upon binding of a metal ion. Taking advantage of edta(4-) as a controller of the free Ca(2+) concentration, a [Ca(2+)](free) as low as 10(-9) M has been attained in the solutions. We have found this method very useful for measuring binding of Ca(2+) to proteins, where the stability constant is in the range 10(5)-10(8) M(-1). The stability constants for the binding of Ca(2+) to proteinase K and bovine alpha-lactalbumin has by this method been measured at an ionic strength of 0.1 M, pH(c) 7.40 and 25 degrees C. For proteinase K a constant of 10(7.4) M(-1) is found, and for alpha-lactalbumin the constant has been found to be 10(9.2) M(-1). The structural stability of both proteins are found to be affected by the presence of Na(+) in the buffer solutions. From this observation, association constants for binding of Na(+) to the Ca(2+) sites have been calculated to 10(2.4) M(-1) for proteinase K and 10(3.5) M(-1) for alpha-lactalbumin. Less than 50 microg have been used of each protein in this study, an obvious advantage over other methods.