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We report the unprecedented use of laccase to functionalize phenol-modified carbon nanotubes (CNTs). Enzymatically-generated metallopolyphenols or the laccase itself can be mildly and efficiently immobilized using the ability of laccase to generate phenoxyl radicals. Electrochemistry, XPS and EPR spectroscopy were used to assess the enzyme-catalyzed CNT functionalization. The efficient immobilization of laccase is confirmed by the high direct bioelectrocatalytic reduction of oxygen with a maximum current density of 1.95 mA cm−2. [ABSTRACT FROM AUTHOR]

This study investigates the site‐directed immobilization of a hybrid catalyst bearing a biquinoline‐based‐Pd(II) complex (1) and a robust laccase within cavities of a silica foam to favor veratryl alcohol oxidation. We performed the grafting of 1 at a unique surface located lysine of two laccase variants, either at closed (1⊂UNIK157) or opposite position (1⊂UNIK71) of the enzyme oxidation site. After immobilization into the cavities of silica monoliths bearing hierarchical porosity, we show that catalytic activity is dependent on the orientation and loading of each hybrid, 1⊂UNIK157 being twice as active than 1⊂UNIK71 (203 TON vs 100 TON) when operating under continuous flow. These systems can be reused 5 times, with an operational activity remaining as high as 40 %. We show that the synergy between 1 and laccase can be tuned within the foam. This work is a proof of concept for controlling the organization of a heterogeneous hybrid catalyst using a Pd/laccase/silica foam. [ABSTRACT FROM AUTHOR]

A maximization of a direct electron transfer (DET) between redox enzymes and electrodes can be obtained through the oriented immobilization of enzymes onto an electroactive surface. Here, a strategy for obtaining carbon nanotube (CNTs) based electrodes covalently modified with perfectly control‐oriented fungal laccases is presented. Modelizations of the laccase‐CNT interaction and of electron conduction pathways serve as a guide in choosing grafting positions. Homogeneous populations of alkyne‐modified laccases are obtained through the reductive amination of a unique surface‐accessible lysine residue selectively engineered near either one or the other of the two copper centers in enzyme variants. Immobilization of the site‐specific alkynated enzymes is achieved by copper‐catalyzed click reaction on azido‐modified CNTs. A highly efficient reduction of O2 at low overpotential and catalytic current densities over −3 mA cm−2 are obtained by minimizing the distance from the electrode surface to the trinuclear cluster. [ABSTRACT FROM AUTHOR]

Two model peptides that mimic the binding loop of the copper chaperone Atx1 were synthesized in order the analyze the coordination properties and selectivity of the amino acid sequence MxCxxC that is conserved in many soft-metal transporters that are involved in the control of the intercellular concentration of ions such Cu(II), Hg(II), Zn(II), Cd(II) and Pb(II). The results of the analysis demonstrate that the overall behavior of the two peptide complexes are similar, though the stabilities of the complexes obtained with cyclic Atx1are systematically higher than those observed with the linear one.

Research has been conducted on alpha-hydrazinoacetyl peptides. The authors describe the development of the peptide preparation procedure which is based on the solid-phase coupling of Boc-protected hydrazino acetic acid derivatives.

The mono-electronic oxidation of coniferyl alcohol leads to the formation of phenoxy radicals and ultimately to the synthesis of dimeric lignan and neo-lignan products. Coniferyl alcohol and dimeric products are all potential guests for cyclodextrins (CDs) to form noncovalent host–guest inclusion complexes. Herein, the influence of CDs with different cavity volumes (i.e., α, β or CD) on the laccase-driven oxidation of coniferyl alcohol is studied. We clearly show that βCD interacts with the dimeric products and selectively prevents their further oxidation by the enzyme. Moreover, amongst the three dimeric products generated, the system containing a laccase and βCD allows the selective enrichment of pinoresinol; this behaviour is similar to that of plant dirigent proteins. [ABSTRACT FROM AUTHOR]

Oxidation reactions are highly important chemical transformations that still require harsh reaction conditions and stoichiometric amounts of chemical oxidants that are often toxic. To circumvent these issues, olefins oxidation is achieved in mild conditions upon irradiation of an aqueous solution of the complex [Ru(bpy)3]2+ and the enzyme laccase. Epoxide formation is coupled to the light-driven reduction of O2 by [Ru(bpy)3]2+/laccase system. The reactivity can be explained by dioxygen acting both as an oxidative agent and as renewable electron acceptor, avoiding the use of a sacrificial electron acceptor. [ABSTRACT FROM AUTHOR]

A linear decapeptide containing three His and one Asp residues and a β-turn-inducing dProPro unit was synthesised. A detailed potentiometric, mass spectrometric and spectroscopic study showed that at a 1:1 ratio of CCu/Cpeptide this peptide formed a major [CuH(OdProAsp)]2+ species (pH range 5.5-7.0), in which the Cu2+ ion was bound to the His and Asp residues in square-planar or square-pyramidal geometries. The stability constant corrected for protonated species (log K* [ABSTRACT FROM AUTHOR]

Chemical reactions inside single crystals are quite rare because crystallinity is difficult to retain owing to atomic rearrangements. Protein crystals in general have a high solvent content. This allows for some molecular flexibility, which makes it possible to trap reaction intermediates of enzymatic reactions without disrupting the crystal lattice. A similar approach has not yet been fully implemented in the field of inorganic chemistry. Here, we have combined model chemistry and protein X-ray crystallography to study the intramolecular aromatic dihydroxylation by an arene-containing protein-bound iron complex. The bound complex was able to activate dioxygen in the presence of a reductant, leading to the formation of catechol as the sole product. The structure determination of four of the catalytic cycle intermediates and the end product showed that the hydroxylation reaction implicates an iron peroxo, generated by reductive O2 activation, an intermediate already observed in iron monooxygenases. This strategy also provided unexpected mechanistic details such as the rearrangement of the iron coordination sphere on metal reduction. [ABSTRACT FROM AUTHOR]

Abstract: In this study, we use HRMAS NMR as a non-invasive technique to monitor the in vivo metabolism of a xenobiotic. The antituberculosis Ethionamide is a pro-drug that has to be activated in mycobacteria before inhibiting its cellular target. The use of 1H HRMAS NMR has allowed to detect a metabolite (ETH*) of the drug directly in living bacteria, even with a spectrometer operating at the relatively low magnetic field of 300MHz. We show that metabolism monitoring of an unlabelled drug at a therapeutically relevant concentration as low as 5μg/ml is within reach of the technique. 1H HRMAS NMR in combination with diffusion filtering leads to the conclusion that the metabolite is located inside the intact cells. The comparison of the metabolite NMR signature with that of synthetic molecules proves the non-identity of ETH* with the ETH derivatives described previously in the literature. [Copyright &y& Elsevier]

A maximization of a direct electron transfer (DET) between redox enzymes and electrodes can be obtained through the oriented immobilization of enzymes onto an electroactive surface. Here, a strategy for obtaining carbon nanotube (CNTs) based electrodes covalently modified with perfectly control-oriented fungal laccases is presented. Modelizations of the laccase-CNT interaction and of electron conduction pathways serve as a guide in choosing grafting positions. Homogeneous populations of alkyne-modified laccases are obtained through the reductive amination of a unique surface-accessible lysine residue selectively engineered near either one or the other of the two copper centers in enzyme variants. Immobilization of the site-specific alkynated enzymes is achieved by copper-catalyzed click reaction on azido-modified CNTs. A highly efficient reduction of O 2 at low overpotential and catalytic current densities over -3 mA cm -2 are obtained by minimizing the distance from the electrode surface to the trinuclear cluster., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mercury(II) is an unphysiological soft ion with high binding affinity for thiolate ligands. Its toxicity lies in the interactions with low molecular weight thiols including glutathione and cysteine-containing proteins that disrupt the thiol balance and alter vital functions. However, mercury can also be detoxified via interactions with Hg(II)-responsive regulatory proteins such as MerR, which coordinates Hg(II) with three cysteine residues in a trigonal planar fashion (HgS 3 coordination). The model cyclodecapeptide P 3C , c(GCTCSGCSRP) was designed to promote Hg(II) chelation in a HgS 3 coordination environment through the parallel orientation of three cysteine side chains. The binding motif is derived from the dicysteine P 2C cyclodecapeptide validated previously as a model for d 10 metal transporters containing the binding sequence CxxC. The formation of the mononuclear HgP 3C complex with a HgS 3 coordination is demonstrated using electrospray ionization mass spectrometry, UV absorption, and 199 Hg NMR. Hg L III -edge extended X-ray absorption fine structure (EXAFS) spectroscopy indicates that the Hg(II) coordination environment is T-shaped with two short Hg-S distances at 2.45 Å and one longer distance at 2.60 Å. The solution structure of the HgP 3C complex was refined based on 1 H- 1 H NMR constraints and EXAFS results. The cyclic peptide scaffold has a rectangular shape with the three binding cysteine side chains pointing toward Hg(II). The HgP 3C H complex has a p K a of 4.3, indicating that the HgS 3 coordination mode is stable over a large range of pH. This low p K a value suggests that the preorientation of the three cysteine groups is particularly well-achieved for Hg(II) trithiolate coordination in P 3C .

Fungal laccases are robust multicopper oxidoreductases. Perfectly amenable to synthetic evolution, the fungal laccase scaffold is a potential generic for the production of tailored biocatalysts, which, in principle, can be secreted at substantial levels in industrially relevant organisms. In this chapter, the strategy we have developed for the rapid production of hundreds of milligram of laccase variants is detailed. It is based on the use of two heterologous expression hosts: the yeast Saccharomyces cerevisiae for a rapid upstream screening and the fungus Aspergillus niger for downstream production. Methods for screening active and nonactive laccase variants, convenient setups for enzyme production in both organisms as well as a methodology for efficient purification of large amounts of recombinant enzymes are given. The general procedure for developing new materials for artificial catalysis is also described., (© 2018 Elsevier Inc. All rights reserved.)

Oxidation reactions are highly important chemical transformations that still require harsh reaction conditions and stoichiometric amounts of chemical oxidants that are often toxic. To circumvent these issues, olefins oxidation is achieved in mild conditions upon irradiation of an aqueous solution of the complex [Ru(bpy)3 ](2+) and the enzyme laccase. Epoxide formation is coupled to the light-driven reduction of O2 by [Ru(bpy)3 ](2+) /laccase system. The reactivity can be explained by dioxygen acting both as an oxidative agent and as renewable electron acceptor, avoiding the use of a sacrificial electron acceptor., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A linear decapeptide containing three His and one Asp residues and a β-turn-inducing dProPro unit was synthesised. A detailed potentiometric, mass spectrometric and spectroscopic study showed that at a 1:1 ratio of CCu /Cpeptide this peptide formed a major [CuH(O(dPro)-Asp)](2+) species (pH range 5.5-7.0), in which the Cu(2+) ion was bound to the His and Asp residues in square-planar or square-pyramidal geometries. The stability constant corrected for protonated species (log K* CuH(O dPro-Asp)=9.33) is almost equal to the value obtained for the parent [CuH(OAsp)](2+) species (log K*CuH(O-Asp) =9.28), but lower than that obtained for the cyclic [CuH(C-Asp)](2+) complex (log K*CuH(C-Asp) =10.79) previously published. Thus, the replacement of the ProGly unit by the stronger β-turn-inducing dProPro unit did not generate a more stable copper(II) species, although the O(dPro)-Asp peptide was structured in solution, as shown by circular dichroism (CD) spectroscopy. Interestingly, the calculated value of Keff showed that this peptide behaved similarly to the O-Asp or C-Asp counterparts, depending on the pH value. The cyclic voltammetry data indicated that the most easily reducible species were [CuH(O-Asp)](2+) (E'(0) =262 mV versus a normal hydrogen electrode (NHE)) and [CuH(O(dPro)-Asp)](2+) (E'(0) =294 mV versus NHE) complexes, the peptidic scaffolds of which are open. A lower value was obtained for [CuH(C-Asp)](2+) (E'(0) =24 mV versus NHE). A different degree of non-reversibility was observed for the three copper(II) complexes; this could reflect a different degree of flexibility in their respective peptidic scaffolds., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chemical reactions inside single crystals are quite rare because crystallinity is difficult to retain owing to atomic rearrangements. Protein crystals in general have a high solvent content. This allows for some molecular flexibility, which makes it possible to trap reaction intermediates of enzymatic reactions without disrupting the crystal lattice. A similar approach has not yet been fully implemented in the field of inorganic chemistry. Here, we have combined model chemistry and protein X-ray crystallography to study the intramolecular aromatic dihydroxylation by an arene-containing protein-bound iron complex. The bound complex was able to activate dioxygen in the presence of a reductant, leading to the formation of catechol as the sole product. The structure determination of four of the catalytic cycle intermediates and the end product showed that the hydroxylation reaction implicates an iron peroxo, generated by reductive O(2) activation, an intermediate already observed in iron monooxygenases. This strategy also provided unexpected mechanistic details such as the rearrangement of the iron coordination sphere on metal reduction.

In this paper HRMAS NMR was used to investigate whether peptides on a peptidyl resin swollen in aqueous solution can adopt an intramolecular beta-sheet structure. A model peptide YQNPDGSQA, that was previously shown to adopt such a secondary structure in solution, (Blanco et al, J. Am. Chem. Soc., 1993) was grafted onto three different solid supports that swell in aqueous solution to examine the influence of the resin on the structure. Both parameters of resin loading and pH inside the swollen peptidyl resin proved to be important for the physicochemical behaviour of the peptide on the support.