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Journal of the American Chemical Society 145(15), 8285-8290 (2023). doi:10.1021/jacs.3c00581, Published by American Chemical Society Publications, Washington, DC

MotivationIntrinsically disordered proteins (IDPs) play a vital role in various biological processes and have attracted increasing attention in the last decades. Predicting IDPs from primary structures of proteins provides a very useful tool for protein analysis. However, most of the existing prediction methods heavily rely on multiple sequence alignments (MSAs) of homologous sequences which are formed by evolution over billions of years. Obtaining such information requires searching against the whole protein databases to find similar sequences and since this process becomes increasingly time-consuming, especially in large-scale practical applications, the alternative method is needed.ResultsIn this paper, we proposed a novel IDP prediction method named IDP-PLM, based on the protein language model (PLM). The method does not rely on MSAs or MSA-based profiles but leverages only the protein sequences, thereby achieving state-of-the-art performance even compared with predictors using protein profiles. The proposed IDP-PLM is composed of stacked predictors designed for several different protein-related tasks: secondary structure prediction, linker prediction, and binding predictions. In addition, predictors for the single task also achieved the highest accuracy. All these are based on PLMs thus making IDP-PLM not rely on MSA-based profiles. The ablation study reveals that all these stacked predictors contribute positively to the IDP prediction performance of IDP-PLM.AvailabilityThe method is available athttp://github.com/xu-shi-jie.Contactakira.onoda@ees.hokudai.ac.jpSupplementary informationSupplementary data are available atBioinformaticsonline.

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.

The development of robust and reactive chiral catalysts is a fundamental aim in asymmetric catalysis, and crucial for providing efficient methods for synthesizing chiral molecules. Chiral paddle-wheel bimetallic complexes provide a highly tunable chiral environment in rhodium-catalysed asymmetric carbene/nitrene transfer reactions and Lewis acid-catalysed reactions. Chiral paddle-wheel complexes having other transition metals as the reactive metal centre, however, have not yet been identified in asymmetric catalysis. Here, we report the synthesis, structures and high catalytic performances of chiral paddle-wheel diruthenium complexes. The cationic chiral diruthenium complex [Ru2((S)-BPTPI)4]+ exhibited remarkable reactivity as a Lewis acid catalyst for asymmetric hetero-Diels–Alder reactions, achieving a turnover number of up to 1,880,000 with high enantioselectivity (>90% e.e.). The chiral diruthenium complexes also exhibited good reactivity and high enantioselectivity in C–H amination and cyclopropanation reactions under oxidizing conditions, indicating their high tolerance towards oxidation. Our results reveal the chiral paddle-wheel diruthenium scaffold as a promising platform for asymmetric catalysis. The development of chiral catalysts is of fundamental importance in asymmetric catalysis. Now, chiral paddle-wheel diruthenium complexes are reported that are stable under oxidizing conditions and effective in asymmetric C–C and C–N bond-forming reactions with turnover numbers of up to 1,880,000.

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 promo...

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.

International audience; 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.

Fe/N/C single-atom catalysts containing Fe-N

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.

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

International audience; 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–Nx active sites having precise structures is now required. We developed a strategy for thermally controlled construction of the Fe–Nx 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–Nx active sites along with the N-GNR structure in high-performance Fe/N/C catalysts for the ORR.

Directed evolution of Cp*RhIII‐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*RhIII‐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., More accurate library screening: A new high‐throughput screening platform including affinity purification has been developed based on an in vitro screening approach for MBP‐tagged nitrobindin having a Cp*RhIII complex as an artificial cofactor. Using this platform for directed evolution, a biohybrid catalyst with clearly enhanced activity toward the cycloaddition of acetophenone oximes with alkynes was obtained.

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 (H-2)-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 H-2 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 H-2 without the additional [Ru(bpy)(3)](2+) photosensitizer, suggesting efficient photoinduced electron transfer from the immobilized Ru1 to the immobilized FeFe1.

Incorporation of a synthetic metal catalyst into a protein scaffold yields a biohybrid catalyst, with a remarkable performance in aqueous media and the broad reaction scope of organometallic catalysts. A major challenge for efficient catalysis is the design of the interface between the protein scaffold and the metal catalyst. Until now, protein scaffolds have primarily been engineered by exchanging individual amino acids to anchor metal catalysts and alter their immediate environment. Here, cavity size engineering of the β-barrel protein nitrobindin was performed by duplicating multiple β-strands to generate an expanded variant. The approach of cavity size engineering enabled covalent incorporation of bulky catalysts at excellent coupling efficiencies and yielded excellent conversions in olefin metathesis, including ring-closing metathesis, ring-opening metathesis polymerization, and cross metathesis (conversions up to 99% and turnover numbers up to 10000).

Horseradish peroxidase apoprotein (apoHRP) was immobilized on a heme-modified polythiophene film, which was electropolymerized on an indium tin oxide surface to yield immobilized and reconstituted HRP. The HRP-immobilized electrode exhibits a 6-fold enhanced electrochemical response toward hydrogen peroxide reduction relative to having the protein randomly immobilized on the polythiophene electrode. This indicates that the insertion of the heme moiety into the heme pocket of HRP leads to an increase in the electrocatalytic current.

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 metal and nitrogen-containing carbon (M/N/C) catalysts with high ORR activity using π-expanded Fe(Salen) precursors. The Fe/N/C ORR catalysts were obtained by pyrolysis of a mixture of carbon support (Vulcan XC-72R) and the iron complex precursors. The Fe/N/C catalyst prepared from N,N´-bis(2-hydroxy-1-naphthylidene)-1,2-phenylenediamino-iron(III) chloride is found to have an onset potential of 860 mV, which is positively shifted by 60 mV from that of the catalyst prepared from the simple Fe(Salen) complex. The catalyst promotes efficient four-electron reduction in the ORR. XAFS and XPS studies reveal that the Fe/N/C catalysts have atomically-dispersed iron active sites and that the activity depends on high content of pyridinic nitrogen. This new methodology facilitates design of the nonprecious metal-carbon catalysts with excellent ORR activity.

Two polyamine derivatives of protoporphyrin IX (PPIX) were tested as photodynamic therapy (PDT) agents in HT29 colorectal cancer and HEP3B liver cancer cell lines. These compounds exhibit excellent singlet oxygen quantum yields and show strong in vitro PDT efficacy after 660 nm laser irradiation, whereas exogenous PPIX itself exhibits much weaker PDT effects. Confocal microscopy imaging studies reveal that a protoporphyrin derivative with eight amine moieties has excellent water solubility, and localizes mainly in the mitochondria of both HT29 and HEP3B cells, whereas the cellular distribution of a protoporphyrin derivative with four amine moieties is not as specific. This work demonstrates that polyamine moieties on macrocycles can enhance PDT efficacy by targeting mitochondria.

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.

A WO3 photoelectrode immobilizing a fibrous gold nanoparticle (AuNP) assembly using an amyloid-β (Aβ) peptide was constructed and its ability for photocurrent generation upon visible light irradiation was investigated. AuNPs attached using Aβ peptide (Aβ-AuNP) assemble with a fibrous structure at pH 4.5, which disperses at pH 11. The modified Aβ-AuNPs are immobilized on the surface of WO3 fabricated on a fluorine-doped tin oxide (FTO) electrode (Aβ-AuNPass@WO3/FTO or Aβ-AuNPdis@WO3/FTO, respectively). The photocurrent generation response of the Aβ-AuNPass@WO3/FTO electrode is clearly increased relative to that of Aβ-AuNPdis@WO3/FTO upon visible light irradiation (λ = 420–750 nm), indicating that the fibrous AuNP assembly enhances the visible light response of the WO3 photoelectrode.

ConspectusIn nature, heme cofactor-containing proteins participate not only in electron transfer and O2 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 st...

A biohybrid catalyst where a metal complex is embedded in a cavity of a protein can be regarded as an artificial metalloenzyme. In particular, incorporation of synthetic organometallic species in a protein matrix will promote catalytic reactions which cannot be seen in nature. This chapter focuses on olefin metathesis via C=C bond formation/cleavage catalyzed by biohybrid catalysts consisting of Hoveyda-Grubbs (HG) type Ru complex attached to protein. Robust β-barrel proteins, nitrobindin and FhuA, and α-chymotrypsin with a deep cleft are considered as the protein scaffold in this chapter. The HG-type complexes introduced into the scaffolds are found to catalyze ring-opening metathesis polymerization and ring-closing metathesis with good chemoselectivity. This strategy will serve as a new method to construct an attractive biohybrid catalyst for abiological transformations in aqueous media.

Journal of inorganic biochemistry 216, 111352 (2021). doi:10.1016/j.jinorgbio.2020.111352 special issue: "Celebrating Fifty Years of the Journal of Inorganic Biochemistry / Edited by Debbie Crans, Rachel Codd, Stephen Sligar, Takashi Hayashi, Martin Stillman, Angela Casini", Published by Elsevier, New York, NY [u.a.]

Photoinduced charge separation on a hybrid titanium dioxide (TiO2) surface with a gold nanoparticle (AuNP) assembly immobilized via a biotin-streptavidin interaction was visualized by Kelvin force microscopy (KFM). Biotinylated AuNP (Biot-AuNP) with a diameter of ca. 15 nm was assembled using streptavidin (STV) and the AuNP assembly was immobilized on a TiO2 surface modified with 1-(3-aminopropyl)silatrane. KFM reveals that the local contact potential difference (CPD) of dispersed AuNP, which is −17 ± 4.2 mV in the dark, undergoes a negative shift of −46 ± 3.0 mV upon UV-irradiation (λ=240-300 nm). In contrast, the AuNP assembly on the TiO2 surface exhibits a greater negative shift of −58 ± 6.0 mV of the CPD, although the CPD value of −16 ± 5.8 mV in the dark is similar to that of the dispersed AuNP. KFM visualization demonstrates that assembly of the AuNP on the TiO2 surface contributes to efficient storage of the negative charge generated by excited electrons in TiO2 during photoinduced charge separation.

Gold nanoparticles immobilized on a gold electrode surface were modified with a heme moiety through gold–sulfur bonds and then treated with apocytochrome b562 (apoCYT) to yield a reconstituted protein on the gold nanoparticles. Immobilization of apoCYT on the surface of the AuNP-modified electrode was confirmed by the charge-transfer resistance value RCT determined using alternating-current electrochemical impedance spectroscopy. The N 1s and Fe 2p peaks in the X-ray photoelectron spectrum support the reconstitution of the heme moiety on the surface of the AuNP electrode. The hemoprotein-immobilized electrode exhibits a ten-fold enhanced electrochemical response relative to the protein immobilized directly on the gold electrode.

A supramolecular conjugate of myoglobin (Mb) and water-soluble poly(acrylate), (PA5k and PA25k , where 5k and 25k represent the molecular weight of the polymers, respectively), is constructed on the basis of a noncovalent heme-heme pocket interaction. The modified heme with an amino group linked to the terminus of one of the heme-propionates is coupled to the side-chain carboxyl groups of poly(acrylate) activated by N-hydroxysuccinimide. The ratios of the heme-modified monomer unit and the unmodified monomer unit (m:n) in the polymer chains of Heme-PA5k and Heme-PA25k were determined to be 4.5:95.5 and 3.1:96.9, respectively. Subsequent addition of apoMb to the conjugated polymers provides Mb-connected fibrous nanostructures confirmed by atomic force microscopy. A mixture of the heme-modified polymer and dimeric apomyoglobin as a cross-linker forms a microgel in which the reconstituted myoglobin retains its native exogenous ligand binding activity.

A series of Grubbs–Hoveyda type catalyst precursors for olefin metathesis containing a maleimide moiety in the backbone of the NHC ligand was covalently incorporated in the cavity of the β-barrel protein nitrobindin. By using two protein mutants with different cavity sizes and choosing the suitable spacer length, an artificial metalloenzyme for olefin metathesis reactions in water in the absence of any organic cosolvents was obtained. High efficiencies reaching TON > 9000 in the ROMP of a water-soluble 7-oxanorbornene derivative and TON > 100 in ring-closing metathesis (RCM) of 4,4-bis(hydroxymethyl)-1,6-heptadiene in water under relatively mild conditions (pH 6, T = 25–40 °C) were observed.

Hydrogenase catalyses reversible transformation of H2 to H(+) using an active site which includes an iron or nickel atom. Synthetic model complexes and molecular catalysts inspired by nature have unveiled the structural and functional basis of the active site with remarkable accuracy and this has led to the discovery of active synthetic catalysts. To further improve the activity of such molecular catalysts, both the first and outer coordination spheres should be well-organized and harmonized for an efficient shuttling of H(+), electrons, and H2. This article reviews recent advances in the design and catalytic properties of artificial enzymes that mimic the hydrogenase active site and the outer coordination sphere in combination with a peptide or protein scaffold.

meso-Monobenzoporphycene (mMBPc) and meso-dibenzoporphycene (mDBPc), in which one or two benzene moieties are fused at ethylene-bridged positions (meso-positions) of porphycene, were prepared in an effort to further delocalize the π-electrons within the porphycene molecule. mMBPc and mDBPc were fully characterized by mass spectrometry, (1)H and (13)C NMR spectroscopy, and X-ray crystallography. The longest-wavelength Q-bands of mMBPc and mDBPc are red-shifted by 92 nm and 418 nm, respectively, compared to that of the unsubstituted porphycene (Pc). Electrochemical measurements indicate that the HOMO is destabilized and the LUMO is stabilized by the fused benzene moieties at the meso positions. Furthermore, both XPS and theoretical studies support the presence of a cis tautomeric form in the ground state of mDBPc, despite the fact that essentially all known porphycene derivatives adopt the trans tautomeric form.

A non-precious metal catalyst (NPMC) promoting a four-electron oxygen reduction reaction (ORR) was synthesized by heat treatment of myoglobin (Mb) containing a heme (iron protoporphyrin IX) as a source of iron, nitrogen, and carbon atoms. Samples of the mixture of Mb and carbon black (Vulcan XC72R: VC) were pyrolyzed at 740, 840, 940, 1040 or 1140°C under N2flow. The microstructures of the carbonized Mb catalysts were characterized by XRD, Raman spectroscopy, XPS, and TEM. Results indicate that the iron-containing active site is embedded within the surface structure in an amorphous domain of the carbon materials. The catalyst ink in a 0.05 wt% Nafion solution in isopropanol was coated onto a glassy carbon electrode and the ORR activity of Mb-based NPMCs was evaluated in a rotating disk electrode experiment in an O2-saturated 0.1 M HClO4solution at 25°C. The catalyst synthesized at 940°C has the highest ORR activity in terms of the onset potential and the current density. In contrast, pyrolytic temperatures above 940°C decrease the activity, suggesting that the active structure of the catalyst apparently decomposes at higher temperatures. The Koutecky–Levich plots indicate that the Mb-based catalyst prepared at 940°C catalyzes four-electron ORR (n = ca. 4). The catalysts prepared at other temperatures have n values of 3.6 at 740°C, 3.7 at 840°C, and 2.9 at 1040°C. The ORR of Mb/VC is diffusion-controlled at potentials lower than 0.3 V (vs. RHE) and the onset potential is 0.84 ± 0.01 V.

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 O2. This new methodology facilitates design of nonprecious bimetallic carbon catalysts with excellent ORR activity.

The O2 adduct of an aqueous synthetic heme/copper model system built on a porphyrin/cyclodextrin supramolecular complex has been characterized., In mitochondria, cytochrome c oxidase (CcO) catalyses the reduction of oxygen (O2) 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 CcO. A tridentate copper(ii) complex was fixed onto 5,10,15,20-tetrakis(4-sulfonatophenyl)porphinatoiron(iii) (FeIIITPPS) through supramolecular complexation between FeIIITPPS and a per-O-methylated β-cyclodextrin dimer linked by a (2,2′:6′,2′′-terpyridyl)copper(ii) complex (CuIITerpyCD2). The reduced FeIITPPS/CuITerpyCD2 complex reacted with O2 in an aqueous solution at pH 7 and 25 °C to form a superoxo-type FeIII–O2–/CuI complex in a manner similar to CcO. The pH-dependent autoxidation of the O2 complex suggests that water molecules gathered at the distal Cu site are possibly involved in the FeIII–O2–/CuI superoxo complex in an aqueous solution. Electrochemical analysis using a rotating disk electrode demonstrated the role of the FeTPPS/CuTerpyCD2 hetero-binuclear structure in the catalytic O2 reduction reaction.

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.

Heme can be removed from a number of native hemoproteins, thus forming corresponding apoproteins, each of which provides a site for binding of a metal complex. In one example, myoglobin, an O2 storage protein, can be reconstituted with iron porphycene to dramatically enhance the O2 affinity. Although it is known that myoglobin has poor enzymatic activity, the insertion of iron corrole or iron porphycene into apomyoglobin increases its H2O2-dependent peroxidase/peroxygenase activities. Furthermore, reconstitution with manganese porphycene promotes hydroxylation of an inert CH bond. It is also of interest to insert a non-porphyrinoid complex into an apoprotein. A cavity of apocytochrome c has been found to bind a diiron carbonyl complex, serving as a functional model of diiron hydrogenase. Aponitrobindin has a rigid β-barrel structure that provides an excellent cavity for covalently anchoring a metal complex. A rhodium complex embedded in the cavity of genetically modified nitrobindin has been found to promote stereoselective polymerization of phenylacetylene.

The hydrogen-evolving diiron complex, (μ-S)2Fe2(CO)6 with a tethered maleimide moiety was synthesized and covalently embedded within the cavity of a rigid β-barrel protein matrix by coupling a maleimide moiety to a cysteine residue within the β-barrel. The (μ-S)2Fe2(CO)6 core within the cavity was characterized by UV–vis absorption and a characteristic CO vibration determined by IR measurements. The diiron complex embedded within the cavity retains the necessary catalytic activity (TON up to 130 for 6 h) to evolve H2 via a photocatalytic cycle with a Ru photosensitizer in a solution of 100 mM ascorbate and 50 mM Tris/HCl at pH 4.0 and 25 °C.

A non-heme diiron active site in a 13-kDa hemerythrin-like domain of the bacterial chemotaxis protein, DcrH-Hr, contains an oxo bridge, two bridging carboxylate groups from Glu and Asp residues, and five terminally ligated His residues. We created a unique diiron coordination sphere containing five His and three Glu/Asp residues by replacing an Ile residue with Glu in DcrH-Hr. Direct coordination of the carboxylate group of E119 to Fe2 of the diiron site in the I119E variant was confirmed by X-ray crystallography. The substituted Glu is adjacent to an exogenous ligand-accessible tunnel. UV-vis absorption spectra indicate that the additional coordination of E119 inhibits the binding of the exogenous ligands, azide and phenol, to the diiron site. The extent of azide binding to the diiron site increases at pH ≤ 6, which is ascribed to protonation of the carboxylate ligand of E119. The diferrous state (deoxy form) of the engineered diiron site with the extra Glu residue is found to react more slowly than wild type with O2 to yield the diferric state (met form). The additional coordination of E119 to the diiron site also slows the rate of reduction from the met form. All these processes were found to be pH-dependent, which can be attributed to protonation state and coordination status of the E119 carboxylate. These results demonstrate that modifications of the endogenous coordination sphere can produce significant changes in the ligand binding and redox properties in a prototypical non-heme diiron-carboxylate protein active site.

Copper(I) and copper(II) complexes were covalently linked to an engineered variant of the transmembrane protein Ferric hydroxamate uptake protein component A (FhuA ΔCVFtev). Copper(I) was incorporated using an N-heterocyclic carbene (NHC) ligand equipped with a maleimide group on the side arm at the imidazole nitrogen. Copper(II) was attached by coordination to a terpyridyl ligand. The spacer length was varied in the back of the ligand framework. These biohybrid catalysts were shown to be active in the Diels–Alder reaction of a chalcone derivative with cyclopentadiene to preferentially give the endo product.

A method using biomolecules to precisely fabricate the morphology of metal nanoparticles immobilized on the surface of a semiconductor using biomolecules is described. A biotin moiety (Biot) is introduced onto the surface of a gold nanoparticle (AuNP) by covalent coupling with α-lipoic acid to assemble AuNPs in the presence of streptavidin (STV). The assembly of Biot-AuNP/STV is immobilized on the surface of TiO2 chemically modified with 1-(3-aminopropyl)silatrane (APS) to provide a positively charged surface. The Au content immobilized on the surface of TiO2 is clearly increased to 9.5 wt % (Au) as a result of the STV-biotin interaction and the electrostatic interaction between negatively charged Biot-AuNPs and the positively charged surface of APS/TiO2. Transmission electron microscopy (TEM) analysis reveals that the composite has an ordered surface geometry in which Biot-AuNPs are spread over the composite surface in two dimensions. The photocatalytic activity toward decomposition of methyl orange dye promoted by this composite is 55%, which is higher than that of the other composites. The Biot-AuNP/STV@APS/TiO2 composite efficiently reduces O2 molecules at Eonset = -0.23 V vs Ag|AgCl, which is more positive than that of other composites (Eonset = -0.40 to -0.32 V). The result suggests that an increased number of AuNPs immobilized in close contact with the TiO2 surface facilitates photoinduced charge transfer. This strategy, which takes advantage of the specific interactions provided by biomolecules and the chemical modification on the surface, has remarkable potential for efficient fabrication of metal nanoparticles on the surface of the semiconductor, which accelerates the reduction of oxygen molecules.

Linear assemblies of cytochrome b562 (cyt b562) containing a chemically-modified heme molecule on the protein surface were constructed by successive intermolecular heme–heme pocket interactions and then immobilized onto a gold electrode via a heme molecule anchored on the electrode. The accumulation of the cyt b562 units were confirmed by electrochemical analyses. The average number of proteins in each assembly was estimated to be about 6 units by quartz crystal microbalance and atomic force microscopy (AFM) analyses. The linear assemblies of the cyt b562 units were clearly visualized by AFM as a rod-like architecture with a vertical orientation to the electrode surface.

It is of particular interest to mimic the process of intramolecular electron relay at the active site of [FeFe]-hydrogenase in order to understand the mechanism of the catalytic activity of H(2) evolution. We have recently focused on using the native CXXCH peptide sequence of the C-terminal segment of cytochrome c(556) as a platform which holds a diiron carbonyl cluster via two cysteines and have attached a ruthenium photosensitizer via a histidine. The modified peptide with the two metal moieties is found to act as the photocatalyst for H(2) evolution with a turnover number of ~9 over 2h at pH 8.5 in the presence of ascorbate as a sacrificial reagent.

The bacterial chemotaxis protein of Desulfovibrio vulgaris DcrH (DcrH-Hr) functions as an O(2)-sensing protein. This protein has a hemerythrin-like domain that includes a nonheme diiron center analogous to the diiron center of the hemerythrin (Hr) family. Interestingly, the O(2) affinity of DcrH-Hr is 3.3 × 10(6) M(-1), a value 25-fold higher than that of the Pectinaria gouldii Hr. This high affinity arises from the fast association of the O(2) ligand with DcrH-Hr (k(on) = 5.3 × 10(8) M(-1) s(-1)), which is made possible by a hydrophobic tunnel that accelerates the passage of the O(2) ligand to the diiron site. Furthermore, the autoxidation kinetics indicate that the rate of autoxidation of DcrH-Hr is 54-fold higher than that of P. gouldii Hr, indicating that the oxy form of DcrH-Hr is not stable toward autoxidation. More importantly, a mixed-valent state, semimet(R), which was spectroscopically observed in previous Hr studies, was found to be stable for over 1 week and isolable in the case of DcrH-Hr. The high-resolution crystal structures of the semimet(R)- (1.8 Å) and met-DcrH-Hr (1.4 Å) indicate that the semimet(R)- and met-DcrH-Hr species have very similar coordination geometry at the diiron site.