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The O2-binding carboxylate-bridged diiron site in DcrH-Hr was engineered in an effort to perform the H2O2-dependent oxidation of external substrates. A His residue was introduced near the diiron site in place of a conserved residue, Ile119. The I119H variant promotes the oxidation of guaiacol and 1,4-cyclohexadiene upon addition of H2O2. [ABSTRACT FROM AUTHOR]

Interaction of apohemoprotein with a covalently immobilized heme moiety onto a gold nanoparticle surface resulted in supramolecular hemoprotein–gold nanoparticle conjugates. The addition of an apohemoprotein dimer further led to a densely-packed hemoprotein–gold nanoparticle assembly, which was visualized by TEM and AFM measurements. [ABSTRACT FROM AUTHOR]

The title compound, {[Tb(C11H11N2O4)3(H2O)3]·H2O} n, has a Tb ion coordinated by bulky benzoate ligands. The Tb ion coordination sphere is occupied by nine O atoms, five from benzoate groups, three from water mol­ecules and one from an amide carbonyl group of a neighboring ligand. Five amide NH groups of the three ligands are involved in intramolecular N—H⋯O hydrogen bonds. [ABSTRACT FROM AUTHOR]

The peptide ligand, Cys-Pro-Leu-Cys, was covalently immobilized on the surface of quartz via (3-aminopropyl) triethoxysilane (APTES) by the general peptide-synthesis method; then, Ni2+ was introduced into this surface to construct the [Ni(S-Cys)4]2- unit contained in the active site of hydrogenases. [ABSTRACT FROM AUTHOR]

A novel poly(vinyl phosphate) ligand was synthesized as a model of biopolymer ligands containing the OH...O hydrogen bonds and CaHPO4⋅2H2O (brushite) composites with the polymer ligands were also prepared. Successful detection of the polymer ligand binding to the CaHPO4⋅2H2O crystals is achieved by 31 P MAS NMR with 1 pda (single pulse with decoupling and phase cycling) sequence, which enhances 31P signals of the phosphate polymer having shorter T1 value. [ABSTRACT FROM AUTHOR]

A hybrid biocatalyst containing a metal terpyridine (tpy) complex within a rigid β-barrel protein nitrobindin (NB) is constructed. A tpy ligand with a maleimide group, N -[2-([2,2′:6′,2′′-terpyridin]-4′-yloxy)ethyl]maleimide ( 1 ), was covalently linked to Cys96 inside the cavity of NB to prepare a conjugate NB– 1 . Binding of Cu 2 + , Zn 2 + , or Co 2 + ion to the tpy ligand in NB– 1 was confirmed by UV–vis spectroscopy and ESI–TOF MS measurements. Cu 2 + -bound NB– 1 is found to catalyze a Diels–Alder reaction between azachalcone and cyclopentadiene in 22% yield, which is higher than that of the Cu 2 + –tpy complex without the NB matrix. The results suggest that the hydrophobic cavity close to the copper active site within the NB scaffold supports the binding of the two substrates, dienophile and diene, to promote the reaction. [ABSTRACT FROM AUTHOR]

Fe/N/C single‐atom catalysts containing Fe−Nx sites prepared by pyrolysis are promising cathode materials for fuel cells and metal‐air batteries due to their high oxygen reduction reaction (ORR) activities. We have developed iron complexes containing N2‐ or N3‐chelating coordination structures with preorganized aromatic rings in a 1,12‐diazatriphenylene framework tethering bromo substituents as precursors to precisely construct Fe−N4 sites in an Fe/N/C catalyst. One‐step pyrolysis of the iron complex with carbon black forms atomically dispersed Fe−N4 sites without iron aggregates. X‐ray absorption spectroscopy (XAS) and electrochemical measurements revealed that the iron complex with N3‐coordination is more effectively converted to Fe−N4 sites catalyzing ORR with a TOF value of 0.21 e site−1 s−1 at 0.8 V vs. RHE. This indicates that the formation of Fe−N4 sites is controlled by precise tuning of the chemical structure of the iron complex precursor. [ABSTRACT FROM AUTHOR]

A diiron [FeFe]‐hydrogenase model complex tethered with a maleimide group, FeFe1, was covalently grafted on the pore surface of a periodic mesoporous organosilica with thiol groups (SH‐PMO) to form an efficient heterogeneous hydrogen (H2)‐evolution catalyst FeFe1@PMO. The coordination structure of the FeFe1 complex and the ordered pore structure were almost completely preserved even after immobilization of FeFe1 on SH‐PMO. The FeFe1@PMO promoted photocatalysis for H2 evolution in water containing a photosensitizer [Ru(bpy)3]2+, with a turnover number (TON) of 310 over 120 min. The TON was greater than those of an analogous homogeneous FeFe1 catalyst (TON=180) and conventional diiron complexes immobilized on solid supports (TON=6–18). The increased TON for FeFe1@PMO compared to the homogeneous FeFe1 was attributed to the improvement in the stability of the FeFe1 complex by immobilization on the pore surface of SH‐PMO. A [Ru(bpy)3]2+ photosensitizer tethered with a maleimide (Ru1) was prepared and co‐immobilized on FeFe1@PMO to form an all‐solid‐state photocatalyst FeFe1‐Ru1@PMO. FeFe1‐Ru1@PMO evolved H2 without the additional [Ru(bpy)3]2+ photosensitizer, suggesting efficient photoinduced electron transfer from the immobilized Ru1 to the immobilized FeFe1. Holding tight: An [FeFe]‐hydrogenase model complex was covalently anchored to a periodic mesoporous organosilica. The resulting FeFe1@PMO promoted photoinduced hydrogen evolution in water containing a photosensitizer [Ru(bpy)3]2+, with a turnover number of 310 over 120 min. The TON was greater than those of an analogous homogeneous FeFe1 catalyst and conventional diiron complexes immobilized on solid supports. [ABSTRACT FROM AUTHOR]

A unique π-expanded reaction cavity tethering a polycyclic moiety which provides a platform for substrate binding was constructed within the robust β-barrel structure of nitrobindin (NB). NB variants with cavities of different sizes and shapes are coupled with N-(1-pyrenyl)maleimide ( Pyr) to prepare a series of NB- Pyr conjugates. The orientation of the pyrene moiety is fixed within the cavity by the coupling reaction. The fluorescent quenching analysis of NB- Pyr indicates that azachalcone ( aza), which is a dienophile for a Diels-Alder (DA) reaction, is efficiently incorporated within the pyrene-linked reaction cavity by the aromatic interaction. The DA reaction between aza and cyclopentadiene proceeds within the reaction cavity of NB- Pyr in the presence of CuII ion in high yield and high enantio- and regioselectivity. [ABSTRACT FROM AUTHOR]

Supramolecular assembly of an engineered hemoprotein with an externally-attached heme moiety via an azobenzene or stilbene linker demonstrates drastic structural transitions between two distinct forms: the thermodynamically stable fiber-type assembly and the kinetically trapped metastable micelle-type assembly induced by transient thermal stimulus. [ABSTRACT FROM AUTHOR]

The application of low-flux sunlight begins with the synthesis of effective antenna systems. This requires the development of dye integrates with optimized dye orientation for effective energy transfer. We here report a series of peptide-linked porphyrin arrays, denoted by Boc-(PorZn,S)n-OBut (n = 2, 4, and 8), that change their dye orientation to increase fluorescence responsively to additive reagents. The B-band absorption (AB) regions of the arrays show blue shifts (dimer, 407.6 nm; tetramer, 408.2 nm; octamer, 407.8 nm) in organic solvents as compared to that of Boc-PorZn,S-OBut (monomer, 422.6 nm) and the fluorescence yield Φ′ of the arrays decreases with increasing n, obeying the relationship Φ′ = 0.03/n1.5. however, the arrays are timed up in fluorescence emission by the addition of 1 ,2-diaminoethane (en). The addition of a sufficient amount of en increases the fluorescence of the porphyrins in monomer, dimer, tetramer, and octamer by ~5, ~12, ~12, and >730 times, respectively, when compared with that observed in the absence of en. This also causes asymptotic red shifts in absorption (AB) bands (B-band Amax: 410 to 429-430 nm), as well as changes in circular dichroism (CD) spectra, and makes porphyrins approach new mutual asymmetric orientations. Our results show the potentiality of the tunable dye polymers that are a posteriorioptimized in dye orientation and fluorescence emission by additive reagents for the development of effective light-harvesting materials. [ABSTRACT FROM AUTHOR]

Novel poly(allylaminocarboxylate) ligands, which have a carboxylate and the neighboring amide group in the same side-chain, were synthesized as model ligands for crystalline CaCO3 biominerals. Poly{N-allyl-malonamate}, poly{N-allyl-succinamate} and poly{4-allylcarbamoyl-butyrate} form 6-, 7- or 8-membered ring intra-side-chain NH⋅O hydrogen bonds, respectively, between the carboxylate and the neighboring amide NH in the carboxylate anion state, although the formation of each intra-side-chain NH⋅O hydrogen bond is independent on the stereoisomers of the polymer main-chain. In the polymer ligand–CaCO3 composites, strong binding of polymer ligands to CaCO3 crystals is caused by stabilizing a Ca–O (carboxylate) bond due to the pKa shift of carboxylic acid by the NH⋅O hydrogen bond. Furthermore, the strong Ca binding in CaCO3 composites stabilizes the meta-stable morphology of CaCO3 vaterite crystals. [Copyright &y& Elsevier]

Ein Pyren‐verknüpfter Proteinhohlraum zieht zwei Substratmoleküle an – ungefähr so wie der Duft einer Blume Schmetterlinge anlockt. A. Onoda, T. Hayashi und Mitarbeiter demonstrieren in ihrer Zuschrift auf S. 13806, dass eine polycyclische Pyrengruppe, die an das starre Proteingerüst der β‐Fass‐Struktur von Nitrobindin geknüpft ist, aromatische Wechselwirkungen mit einem Substrat bereitstellt. Eine asymmetrische Diels‐Alder‐Reaktion zwischen Azachalkon und Cyclopentadien verläuft glatt mit hoher Stereoselektivität innerhalb des Reaktionsgerüsts. [ABSTRACT FROM AUTHOR]

A pyrene‐linked protein cavity attracts two substrate molecules like the scent of a flower attracts butterflies. In their Communication on page 13618 ff., A. Onoda, T. Hayashi, and co‐workers demonstrate that a polycyclic pyrene moiety linked within the rigid protein scaffold of the β‐barrel of nitrobindin acts as a platform to provide an aromatic interaction with a substrate. An asymmetric Diels–Alder reaction between azachalcone and cyclopentadiene proceeds smoothly with high stereoselectivity within the reaction scaffold. [ABSTRACT FROM AUTHOR]

Intrinsically disordered proteins (IDPs) play a vital role in various biological processes and have attracted increasing attention in the past few decades. Predicting IDPs from the primary structures of proteins offers a rapid and facile means of protein analysis without necessitating crystal structures. In particular, machine learning methods have demonstrated their potential in this field. Recently, protein language models (PLMs) are emerging as a promising approach to extracting essential information from protein sequences and have been employed in protein modeling to utilize their advantages of precision and efficiency. In this article, we developed a novel IDP prediction method named IDP-ELM to predict the intrinsically disordered regions (IDRs) as well as their functions including disordered flexible linkers and disordered protein binding. This method utilizes high-dimensional representations extracted from several state-of-the-art PLMs and predicts IDRs by ensemble learning based on bidirectional recurrent neural networks. The performance of the method was evaluated on two independent test data sets from CAID (critical assessment of protein intrinsic disorder prediction) and CAID2, indicating notable improvements in terms of area under the receiver operating characteristic (AUC), Matthew's correlation coefficient (MCC), and F1 score. Moreover, IDP-ELM requires solely protein sequences as inputs and does not entail a time-consuming process of protein profile generation, which is a prerequisite for most existing state-of-the-art methods, enabling an accurate, fast, and convenient tool for proteome-level analysis. The corresponding reproducible source code and model weights are available at https://github.com/xu-shi-jie/idp-elm.

Evolutionary engineering of our previously reported Cp*Rh(III)-linked artificial metalloenzyme was performed based on a DNA recombination strategy to improve its catalytic activity toward C(sp 2 )-H bond functionalization. Improved scaffold design was achieved with α-helical cap domains of fatty acid binding protein (FABP) embedded within the β-barrel structure of nitrobindin (NB) as a chimeric protein scaffold for the artificial metalloenzyme. After optimization of the amino acid sequence by directed evolution methodology, an engineered variant, designated NB HLH1 (Y119A/G149P) with enhanced performance and enhanced stability was obtained. Additional rounds of metalloenzyme evolution provided a Cp*Rh(III)-linked NB HLH1 (Y119A/G149P) variant with a >35-fold increase in catalytic efficiency ( k cat / K M ) for cycloaddition of oxime and alkyne. Kinetic studies and MD simulations revealed that aromatic amino acid residues in the confined active-site form a hydrophobic core which binds to aromatic substrates adjacent to the Cp*Rh(III) complex. The metalloenzyme engineering process based on this DNA recombination strategy will serve as a powerful method for extensive optimization of the active-sites of artificial metalloenzymes.

Fe/N/C single-atom catalysts containing Fe-N x sites prepared by pyrolysis are promising cathode materials for fuel cells and metal-air batteries due to their high oxygen reduction reaction (ORR) activities. We have developed iron complexes containing N2- or N3-chelating coordination structures with preorganized aromatic rings in a 1,12-diazatriphenylene framework tethering bromo substituents as precursors to precisely construct Fe-N 4 sites in an Fe/N/C catalyst. One-step pyrolysis of the iron complex with carbon black forms atomically dispersed Fe-N 4 sites without iron aggregates. X-ray absorption spectroscopy (XAS) and electrochemical measurements revealed that the iron complex with N3-coordination is more effectively converted to Fe-N 4 sites catalyzing ORR with a TOF value of 0.21 e site -1  s -1 at 0.8 V vs. RHE. This indicates that the formation of Fe-N 4 sites is controlled by precise tuning of the chemical structure of the iron complex precursor., (© 2021 Wiley-VCH GmbH.)

Pyrolytically prepared iron and nitrogen codoped carbon (Fe/N/C) catalysts are promising nonprecious metal electrocatalysts for the oxygen reduction reaction (ORR) in fuel cell applications. Fabrication of the Fe/N/C catalysts with Fe-N x active sites having precise structures is now required. We developed a strategy for thermally controlled construction of the Fe-N x structure in Fe/N/C catalysts by applying a bottom-up synthetic methodology based on a N-doped graphene nanoribbon (N-GNR). The preorganized aromatic rings within the precursors assist graphitization during generation of the N-GNR structure with iron-coordinating sites. The Fe/N/C catalyst prepared from the N-GNR precursor, iron ion, and the carbon support Vulcan XC-72R provides a high onset potential of 0.88 V (vs reversible hydrogen electrode (RHE)) and promotes efficient four-electron ORR. X-ray absorption fine structure (XAFS) and X-ray photoelectron spectroscopy (XPS) studies reveal that the N-GNR precursor induces the formation of iron-coordinating nitrogen species during pyrolysis. The details of the graphitization process of the precursor were further investigated by analyzing the precursors pyrolyzed at various temperatures using MgO particles as a sacrificial template, with the results indicating that the graphitized structure was obtained at 700 °C. The preorganized N-GNR precursors and its pyrolysis conditions for graphitization are found to be important factors for generation of the Fe-N x active sites along with the N-GNR structure in high-performance Fe/N/C catalysts for the ORR.

A whole-cell biohybrid catalyst where a (pentamethylcyclopentadienyl)rhodium(III) (Cp*Rh(III)) complex was covalently incorporated into the cavity of nitrobindin (NB), a β-barrel protein, was prepared on an E. coli cell surface to produce isoquinolines via C(sp 2 )-H bond activation. In this whole-cell biohybrid system, the Cp*Rh(III)-dithiophosphate complex with latent catalytic activity was utilized as a precursor of the metal cofactor. Strong chelation of the dithiophosphate ligands protects the rhodium complex from being deactivated by abundant nucleophiles in cellular environments during conjugation of the cofactor with the protein scaffold. The whole-cell biohybrid catalyst was then activated upon addition of Ag + ion to dissociate the dithiophosphate ligands and promoted cycloaddition of acetophenone oxime with diphenylacetylene. Furthermore, the activity of the Cp*Rh(III)-linked whole-cell biohybrid catalyst was enhanced 2.1-fold by introducing glutamate residues at positions adjacent to the Cp*Rh(III) cofactor. These results indicate that the use of the Cp*Rh(III)-dithiophosphate complex with switchable activity from a "latent" form to an "active" form provides a new strategy for generating whole-cell biohybrid catalysts., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Directed evolution of Cp*Rh III -linked nitrobindin (NB), a biohybrid catalyst, was performed based on an in vitro screening approach. A key aspect of this effort was the establishment of a high-throughput screening (HTS) platform that involves an affinity purification step employing a starch-agarose resin for a maltose binding protein (MBP) tag. The HTS platform enables efficient preparation of the purified MBP-tagged biohybrid catalysts in a 96-well format and eliminates background influence of the host E. coli cells. Three rounds of directed evolution and screening of more than 4000 clones yielded a Cp*Rh III -linked NB(T98H/L100K/K127E) variant with a 4.9-fold enhanced activity for the cycloaddition of acetophenone oximes with alkynes. It is confirmed that this HTS platform for directed evolution provides an efficient strategy for generating highly active biohybrid catalysts incorporating a synthetic metal cofactor., (© 2020 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

A Cp*Rh(III)-dithiophosphate cofactor with "latent" catalytic activity was developed to construct an artificial metalloenzyme representing a new type of biohybrid catalyst which is capable of promoting C(sp 2 )-H bond functionalization within the β-barrel structure of nitrobindin (NB). To covalently conjugate the Cp*Rh(III) cofactor into a specific position of the hydrophobic cavity of NB via a maleimide-Cys linkage, strong chelation of the dithiophosphate ligand is employed to protect the rhodium metal center against attack by nucleophilic amino acid residues in the protein. It is found that subsequent addition of the Ag + ion induces dissociation of the dithiophosphate ligands, thereby activating the catalytic activity of the Cp*Rh(III) cofactor. The resulting "active" biohybrid catalyst promotes cycloaddition of acetophenone oxime with diphenylacetylene via C(sp 2 )-H bond activation. This catalytic activity is enhanced 2.3-fold with the introduction of two glutamate residues (A100E/L125E) adjacent to the Cp*Rh(III) cofactor. The Cp*Rh(III) cofactor with switchable activity from a "latent" form to an "active" form provides a new strategy for generating biohybrid catalysts incorporating a variety of highly reactive transition metal complexes specifically within its protein scaffolds.

Site-specific modification of peptides and proteins is a key aspect of protein engineering. We developed a method for modification of the N terminus of proteins using 1H-1,2,3-triazole-4-carbaldehyde (TA4C) derivatives, which can be prepared in one step. The N-terminal specific labeling of bioactive peptides and proteins with the TA4C derivatives proceeds under mild reaction conditions in excellent conversion (angiotensin I: 92 %, ribonuclease A: 90 %). This method enables site-specific conjugation of various functional molecules such as fluorophores, biotin, and polyethylene glycol attached to the triazole ring to the N terminus. Furthermore, a functional molecule modified with a primary amine moiety can be directly converted into a TA4C derivative through a Dimroth rearrangement reaction with 1-(4-nitrophenyl)-1H-1,2,3-triazole-4-carbaldehyde. This method can be used to obtain N-terminal-modified proteins via only two steps: 1) convenient preparation of a TA4C derivative with a functional group and 2) modification of the N terminus of the protein with the TA4C derivative., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Site-specific modification of peptides and proteins is an important method for introducing an artificial function to the protein surface. Recently, we found that new bioconjugation reagents, 6-(azidomethyl)-2-pyridinecarbaldehyde (6AMPC) derivatives, allow specific N-terminal modification and enhance the reaction rate of the subsequent bioconjugation in a chelation-assisted CuAAC reaction. The N-terminal specific azide-labeling of bioactive peptides and proteins occurs under mild reaction conditions with 6AMPC derivatives (angiotensin I: 90%, ribonuclease A: 90%). Kinetic analysis of the CuAAC reaction with azide-labeled proteins reveals that the ligation is promoted in the presence of a copper-chelating pyridine moiety. Importantly, the introduction of an electron-donating methoxy group to the pyridine moiety further accelerates the CuAAC ligation. We demonstrate that this method enables site-specific conjugation of various functional molecules such as fluorophores, biotin, and polyethylene glycol.

In nature, heme cofactor-containing proteins participate not only in electron transfer and O 2 storage and transport but also in biosynthesis and degradation. The simplest and representative cofactor, heme b, is bound within the heme pocket via noncovalent interaction in many hemoproteins, suggesting that the cofactor is removable from the protein, leaving a unique cavity. Since the cavity functions as a coordination sphere for heme, it is of particular interest to investigate replacement of native heme with an artificial metal complex, because the substituted metal complex will be stabilized in the heme pocket while providing alternative chemical properties. Thus, cofactor substitution has great potential for engineering of hemoproteins with alternative functions. For these studies, myoglobin has been a focus of our investigations, because it is a well-known oxygen storage hemoprotein. However, the heme pocket of myoglobin has been only arranged for stabilizing the heme-bound dioxygen, so the structure is not suitable for activation of small molecules such as H 2 O 2 and O 2 as well as for binding an external substrate. Thus, the conversion of myoglobin to an enzyme-like biocatalyst has presented significant challenges. The results of our investigations have provided useful information for chemists and biologists. Our own efforts to develop functionalized myoglobin have focused on the incorporation of a chemically modified cofactor into apomyoglobin in order to (1) construct an artificial substrate-binding site near the heme pocket, (2) increase cofactor reactivity, or (3) promote a new reaction that has never before been catalyzed by a native heme enzyme. In pursuing these objectives, we first found that myoglobin reconstituted with heme having a chemically modified heme-propionate side chain at the exit of the heme pocket has peroxidase activity with respect to oxidation of phenol derivatives. Our recent investigations have succeeded in enhancing oxidation and oxygenation activities of myoglobin as well as promoting new reactions by reconstitution of myoglobin with new porphyrinoid metal complexes. Incorporation of suitable metal porphyrinoids into the heme pocket has produced artificial enzymes capable of efficiently generating reactive high valent metal-oxo and metallocarbene intermediates to achieve the catalytic hydroxylation of C(sp 3 )-H bonds and cyclopropanation of olefin molecules, respectively. In other efforts, we have focused on nitrobindin, an NO-binding hemoprotein, because aponitrobindin includes a β-barrel cavity, which provides a robust structure highly similar to that of the native holoprotein. It was expected that the aponitrobindin would be suitable for development as a protein scaffold for a metal complex. Recently, it was confirmed that several organometallic complexes can bind to this scaffold and function as catalysts promoting hydrogen evolution or C-C bond formation. The hydrophobic β-barrel structure plays a significant role in substrate binding as well as controlling the stereoselectivity of the reactions. Furthermore, these catalytic activities and stereoselectivities are remarkably improved by mutation-dependent modifications of the cavity structure for the artificial cofactor. This Account demonstrates how apoproteins of hemoproteins can provide useful protein scaffolds for metal complexes. Further development of these concepts will provide a useful strategy for generation of robust and useful artificial metalloenzymes.

In mitochondria, cytochrome c oxidase (C c O) catalyses the reduction of oxygen (O 2 ) to water by using a heme/copper hetero-binuclear active site. Here we report a highly efficient supramolecular approach for the construction of a water-soluble biomimetic model for the active site of C c O. A tridentate copper(ii) complex was fixed onto 5,10,15,20-tetrakis(4-sulfonatophenyl)porphinatoiron(iii) (Fe III TPPS) through supramolecular complexation between Fe III TPPS and a per- O -methylated β-cyclodextrin dimer linked by a (2,2':6',2''-terpyridyl)copper(ii) complex (Cu II TerpyCD 2 ). The reduced Fe II TPPS/Cu I TerpyCD 2 complex reacted with O 2 in an aqueous solution at pH 7 and 25 °C to form a superoxo-type Fe III -O 2 - /Cu I complex in a manner similar to C c O. The pH-dependent autoxidation of the O 2 complex suggests that water molecules gathered at the distal Cu site are possibly involved in the Fe III -O 2 - /Cu I superoxo complex in an aqueous solution. Electrochemical analysis using a rotating disk electrode demonstrated the role of the FeTPPS/CuTerpyCD 2 hetero-binuclear structure in the catalytic O 2 reduction reaction.

Nonprecious metal electrocatalysts are being explored as alternatives to platinum-group metal electrocatalysts for the oxygen reduction reaction (ORR) which is required for cathode materials in fuel cells. Herein, we describe a new method for preparing bimetallic nitrogen-containing carbon catalysts with high ORR activity using π-expanded M(salen) precursors. The M/N/C and bimetallic FeM/N/C ORR catalysts were obtained by pyrolysis of a mixture of a carbon support (Vulcan XC-72R) and the metal complex as a precursor. The bimetallic FeCu catalyst prepared from Fe and Cu complexes with the N , N '-bis(2-hydroxy-1-naphthylidene)-1,2-phenylenediamine ligand (2NAPD) is found to have an onset potential of 0.87 V, which is positively shifted by 50 mV from that of the catalyst prepared from the monometallic Fe(2NAPD) complex. The FeCu/N/C catalyst promotes efficient four-electron reduction in the ORR. High-resolution transmission electron microscopy studies reveal that both Fe and Cu metals together with pyridinic nitrogen species are highly dispersed within the carbonaceous structure in FeCu/2NAPD@VC, suggesting that the N-coordinated Fe and Cu sites promote efficient four-electron reduction of O 2 . This new methodology facilitates design of nonprecious bimetallic carbon catalysts with excellent ORR activity., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Metallothioneins (MTs) are small cysteine-rich proteins which exhibit high affinities for various metal ions and play roles in storage of essential metals and detoxification of toxic metals. Studies on the redox properties of MTs have been quite limited. Recently, we focused on the α-domain of MT (MTα) as a protein matrix and incorporated a tetranuclear metal cluster as a reductant. UV-visible, CD and MS data indicate the formation of the stable tetranuclear metal-cysteine cluster in the MTα matrix with Fe(II)(4)-MTα and Co(II)(4)-MTα species existing in water. Furthermore, the Fe(II)(4)-MTα species was found to promote the reduction of met-myoglobin and azobenzene derivatives under mild conditions. Particularly, the stoichiometric reduction of methyl red with Fe(II)(4)-MTα (1:1) was found to proceed with a conversion of 98% over a period of 6h at 25°C. This indicates that all of the four Fe(II) cores contribute to the reduction. In this paper, we describe the preparation and reactivity of the tetranuclear iron cluster in the protein matrix., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Zinc finger fusion proteins, having a Ca-binding site from troponin C, were created to develop Ca-responsive regulation of DNA binding. The typical zinc finger folding of a novel fusion protein with a single finger, F2-Tn, was investigated using UV-vis spectroscopy of the Co-substituted form and CD experiments. Detailed structural analyses of F2-Tn/Zn2+ using NMR experiments and structural calculations clarify that our fusion protein gives a native zinc finger folding with the artificial Ca-binding domain intervening two helices. The Ca-responsive DNA-binding affinity of troponin-fused protein with two fingers (using F1F2-Tn) was investigated by electrophoretic mobility shift assay (EMSA). EMSA analyses of F1F2-Tn were performed under the conditions of various concentrations of the Ca ion. F1F2-Tn has a Kd value of 5.8 nM in the absence of Ca ion and shows a higher Kd value of 13 nM in the presence of 100 equiv of Ca ion. The artificially designed fusion zinc finger protein with a Ca-binding domain has Ca-responsive DNA-binding affinity. It is leading to a better understanding of the construction of zinc finger-based artificial transcriptional factors with a Ca switch.

Aspartic acid protease model peptides Z-Phe-Asp(COOH)-Thr-Gly-Ser-Ala-NHCy (1) and AdCO-Asp(COOH)-Val-Gly-NHBzl (3), and their aspartate anions (NEt4)[Z-Phe-Asp(COO-)-Thr-Gly-Ser-Ala-NHCy] (2) and (NEt4)[AdCO-Asp(COO-)-Val-Gly-NHBzl] (4), having an invariant primary sequence of the Asp-X(Thr,Ser)-Gly fragment, were synthesized and characterized by 1H-NMR, CD, and infrared (IR) spectroscopies. NMR structure analyses indicate that the Asp O(delta) atoms of the aspartate peptide 2 are intramolecularly hydrogen-bonded with Gly, Ser, Ala NH, and Ser OH, supporting the rigid beta-turn-like conformation in acetonitrile solution. The tripeptide in the aspartic acid 3 forms an inverse gamma-turn structure, which is converted to a beta-turn-like conformation because of the formation of the intramolecular NH . . . O- hydrogen bonds with the Asp O(delta) in 4. Such a conformational change is not detected between dipeptides AdCO-Asp(COOH)-Va-NHAd (5) and (NEt4)[AdCO-Asp(COO-)-Val-NHAd] (6). The pK(a) value of side-chain carboxylic acid (5.0) for 3 exhibits a lower shift (0.3 unit) from that of 5 in aqueous polyethyleneglycol lauryl ether micellar solution. NMR structure analyses for 3 in an aqueous micellar solution indicate that the preorganized turn structure, which readily forms the NH . . . O- hydrogen bonds, lowers the pK(a) value and that resulting hydrogen bonds stabilize the rigid conformation in the aspartate anion state. We found that the formation of the NH . . . O- hydrogen bonds involved in the hairpin turn is correlated with the protonation and deprotonation state of the Asp side chain in the conserved amino acid fragments., (Copyright 2005 Wiley Periodicals, Inc)

Two new mononuclear Ca(II) complexes with aryl dihydrogen phosphate ligands having two strategically oriented bulky amide groups, 2,6-(Ph3CCONH)2C6H3OPO3H2 (1), including one with a phosphate monoanion, (NMe4)[CaII[O2P(OH)OC6H3-2,6-(NHCOCPh3)2]3(NCMe)3] (3), and one with a phosphate dianion, [CaII[O3POC6H3-2,6-(NHCOCPh3)2](H2O)3(MeOH)2] (4). Both are analogues for the NH...O hydrogen bonds in the active site of Ca(II)-containing phosphotransferase. Crystallographic studies of these Ca(II) complexes revealed that the amide NHs are directed to uncoordinated O atoms of the phosphates, and the IR and 1H NMR spectra indicate that strong NH...O hydrogen bonds are formed only in the phosphate dianion state. The ligand exchange reaction of 3 with a non-hydrogen-bonded phosphate ligand shows that the NH...O hydrogen bonds prevent the Ca-O bond from dissociation. A scatter plot analysis comparing the distance of a Ca-O bond with the Ca-O-P angle, the Fourier density analysis, and DFT calculations reveal that a partial degree of covalency in the Ca-O(phosphate) bonds is present.

Three new polynuclear Ca(II)- and Na(I) phosphate complexes with two strategically oriented bulky amide groups, 2,6-(PhCONH)(2)C(6)H(3)OPO(3)H(2), were synthesized, including one with a zigzag-chain, [Ca(II)[O(3)POC(6)H(3)-2,6-(NHCOPh)(2)](H(2)O)(4)(EtOH)](n), a cyclic-octanuclear form, [Ca(II)(8)[O(3)POC(6)H(3)-2,6-(NHCOPh)(2)](8)(O=CHNMe(2))(8)(H(2)O)(12)], and a hexanuclear complex, (NHEt(3))[Na(3)[O(3)POC(6)H(3)-2,6-(NHCOPh)(2)](2)(H(2)O)(MeOH)(7)]. X-ray crystallography revealed that all have an unsymmetric ligand position due to the bulky amide groups. A dynamic transformation of the Ca(II) zigzag-chain structure to the cyclic-octanuclear complex was induced by changing coordination of DMF molecules, which caused a reorganization of the intermolecular/intramolecular hydrogen bond network.

A novel dinuclear calcium complex, [Ca(2){(2-OCO-3-CH(3)C(6)H(3)NHCO)(2)C(CH(3))(2)}(2)(CH(3)OH)(6)] (1), was synthesized as a structural model of 8-coordinated Ca(II) ions in the double calcium-binding site of thermolysin. The complex has four NH.O hydrogen bonds between the amide NH and the carboxylate oxygen anion. Two types of bridging coordination of the carboxylate ligand to Ca(II) were found in 1. The amide NH forms a strong NH.O hydrogen bond with the anionic oxygen of the two carboxylate oxygens. A ligand-exchange reaction between the dinuclear calcium complex and eight equimolar amounts of 2,4,6-trimethylbenzoic acid or 2-CH(3)-6-t-BuCONHC(6)H(3)COOH indicates that the NH.O hydrogen bond prevents the dissociation of the Ca-O bond.