Asymmetric Michael addition catalysed by copper-amyloid complexes.

We developed self-assembled peptides containing a partial amyloid ß protein sequence and a metal-coordination site. The amyloid fibril-copper complexes exhibited excellent reactivity and moderate enantioselectivity in Michael addition reactions with 2-azachalcone and dimethylmalonate. The catalytic amyloids were characterized using various measurements to confirm their amyloid-like nanofibre structures.

An artificial metallolyase with pliable 2-His-1-carboxylate facial triad for stereoselective Michael addition.

We repurposed the metal-binding site of a cupin superfamily protein into the 2-His-1-carboxylate facial triad, which is one of the common motifs in natural non-heme enzymes, to construct artificial metalloenzymes that can catalyze new-to-nature reactions. The Cu2+-H52A/H58E variant catalyzed the stereoselective Michael addition reaction and was found to bear a flexible metal-binding site in the high-resolution crystal structure. Furthermore, the H52A/H58E/F104W mutant accommodated a water molecule, which was supported by Glu58 and Trp104 residues via hydrogen bonding, presumably leading to high stereoselectivity. Thus, the 2-His-1-carboxylate facial triad was confirmed to be a versatile and promising metal-binding motif for abiological and canonical biological reactions.

Copper-Oxygen Dynamics in the Tyrosinase Mechanism.

The dinuclear copper enzyme, tyrosinase, activates O2 to form a (µ-η2 :η2 -peroxido)dicopper(II) species, which hydroxylates phenols to catechols. However, the exact mechanism of phenolase reaction in the catalytic site of tyrosinase is still under debate. We herein report the near atomic resolution X-ray crystal structures of the active tyrosinases with substrate l-tyrosine. At their catalytic sites, CuA moved toward l-tyrosine (CuA1 → CuA2), whose phenol oxygen directly coordinates to CuA2, involving the movement of CuB (CuB1 → CuB2). The crystal structures and spectroscopic analyses of the dioxygen-bound tyrosinases demonstrated that the peroxide ligand rotated, spontaneously weakening its O-O bond. Thus, the copper migration induced by the substrate-binding is accompanied by rearrangement of the bound peroxide species so as to provide one of the peroxide oxygen atoms with access to the phenol substrate's ϵ carbon atom.

Cupin Variants as a Macromolecular Ligand Library for Stereoselective Michael Addition of Nitroalkanes.

Cupin superfamily proteins (TM1459) work as a macromolecular ligand framework with a double-stranded ß-barrel structure ligating to a Cu ion through histidine side chains. Variegating the first coordination sphere of TM1459 revealed that H52A and H54A/H58A mutants effectively catalyzed the diastereo- and enantioselective Michael addition reaction of nitroalkanes to an α,ß-unsaturated ketone. Moreover, calculated substrate docking signified C106N and F104W single-point mutations, which inverted the diastereoselectivity of H52A and further improved the stereoselectivity of H54A/H58A, respectively.

His-Cys and Trp-Cys cross-links generated by post-translational chemical modification.

Galactose oxidase and amine oxidase contain a cofactor which is generated by post-translational chemical modification to the corresponding amino acid side chains near the copper active center. Such cofactors provide proteins unusual catalytic ability that canonical amino acids cannot exert as well as their structural stability, and thereby are called as protein-derived cofactors. These cofactors and modifications are mostly derived from aromatic amino acid residues, especially Tyr, Trp, and His. Current information about unusual cofactors derived from two of those, heteroaromatic residues (Trp and His) is summarized, especially chemical properties and maturation process of the cross-links between cysteine and heteroaromatic amino acids (His-Cys and Trp-Cys cross-links).Abbreviations: FMN: flavin mononucleotide; FAD: flavin adenine nucleotide; RNA: ribonucleic acid; PDC: protein-derived cofactor; GFP: green fluorescent protein; MIO: 3,5-dihydro-5-methylidene-4-imidazol-4-one; LTQ: lysyl tyrosylquinone; CTQ: cysteine tryptophylquinone; TTQ: tryptophan tryptophylquinone; E.coli: Escherichia coli; WT: wild type.

Geometric effects on OO bond scission of copper(II)-alkylperoxide complexes.

Copper(II) complexes supported by N3-tridentate ligands, consisting of a rigid cyclic diamine (8-membered cyclic-diamine; L8 or 7-membered cyclic-diamine; L7) and a 2-(2-pyridyl)ethyl (-CH2CH2Py) group, were synthesized and structurally characterized. Reaction of the copper(II) complexes and cumene hydroperoxide (CmOOH) in the presence of triethylamine in CH3CN gave the corresponding cumylperoxide complexes L8CuIIOOCm and L7CuIIOOCm. The UV-vis and EPR spectra suggested that L8CuIIOOCm takes a tetrahedrally distorted structure, whereas L7CuIIOOCm has a planar geometry in solution. Resonance Raman spectra of these alkylperoxide complexes indicated that the O-O stretching vibration energy of L8CuIIOOCm (νO-O=878cm-1) is somewhat lower than that of L7CuIIOOCm (νO-O=881cm-1). Such a difference in O-O bond strength is reflected to the reactivity difference of these two alkylperoxide complexes. Namely, the reactivity L8CuIIOOCm toward CHD (1,4-cyclohexadiene) as well as solvent molecule (CH3CN) is higher than that of L7CuIIOOCm due to the weaker O-O bond of the former complex as compared to that of the latter complex. Geometric effects on the reactivity induced by the supporting ligands are discussed.

Tetrahedral Copper(II) Complexes with a Labile Coordination Site Supported by a Tris-tetramethylguanidinato Ligand.

A new tridentate N3 ligand (TMG3tach) consisting of cis,cis-1,3,5-triaminocyclohexane (tach) and three N,N,N',N'-tetramethylguanidino (TMG) groups has been developed to prepare copper complexes with a tetrahedral geometry and a labile coordination site. Treatment of the ligand with CuIIX2 (X = Cl and Br) gave copper(II)-halide complexes, [CuII(TMG3tach)Cl]+ (2Cl) and [CuII(TMG3tach)Br]+ (2Br), the structures of which have been determined by X-ray crystallographic analysis. The complexes exhibit a four-coordinate structure with C3v symmetry, where the labile halide ligand (X) occupies a position on the trigonal axis. 2Br was converted to a methoxido-copper(II) complex [CuII(TMG3tach)(OMe)](OTf) (2OMe), also having a similar four-coordinate geometry, by treating it with an equimolar amount of tetrabutylammonium hydroxide in methanol. The methoxido-complex 2OMe was further converted to the corresponding phenolato-copper(II) (2OAr) and thiophenolato-copper(II) (2SAr) complexes by ligand exchange reactions with the neutral phenol and thiophenol derivatives, respectively. The electronic structures of the copper(II) complexes with different axial ligands are discussed on the basis of EPR spectroscopy and DFT calculations.

Generation and characterisation of a stable nickel(ii)-aminoxyl radical complex.

A stable nickel(ii)-aminoxyl radical complex was generated by the reaction of a nickel(ii) complex supported by a tren ligand (tris(2-aminoethyl)amine) having bulky m-terphenyl substituents (TIPT: 3,5-bis(2,6-diisopropylphenyl)phenyl) and m-CPBA (m-chloroperoxybenzoic acid). The formation mechanism of the nickel(ii)-aminoxyl radical complex was examined.

A Well-Defined Osmium-Cupin Complex: Hyperstable Artificial Osmium Peroxygenase.

Thermally stable TM1459 cupin superfamily protein from Thermotoga maritima was repurposed as an osmium (Os) peroxygenase by metal-substitution strategy employing the metal-binding promiscuity. This novel artificial metalloenzyme bears a datively bound Os ion supported by the 4-histidine motif. The well-defined Os center is responsible for not only the catalytic activity but also the thermodynamic stability of the protein folding, leading to the robust biocatalyst (Tm ≈ 120 °C). The spectroscopic analysis and atomic resolution X-ray crystal structures of Os-bound TM1459 revealed two types of donor sets to Os center with octahedral coordination geometry. One includes trans-dioxide, OH, and mer-three histidine imidazoles (O3N3 donor set), whereas another one has four histidine imidazoles plus OH and water molecule in a cis position (O2N4 donor set). The Os-bound TM1459 having the latter donor set (O2N4 donor set) was evaluated as a peroxygenase, which was able to catalyze cis-dihydroxylation of several alkenes efficiently. With the low catalyst loading (0.01% mol), up to 9100 turnover number was achieved for the dihydroxylation of 2-methoxy-6-vinyl-naphthalene (50 mM) using an equivalent of H2O2 as oxidant at 70 °C for 12 h. When octene isomers were dihydroxylated in a preparative scale for 5 h (2% mol cat.), the terminal alkene octene isomers was converted to the corresponding diols in a higher yield as compared with the internal alkenes. The result indicates that the protein scaffold can control the regioselectivity by the steric hindrance. This protein scaffold enhances the efficiency of the reaction by suppressing disproportionation of H2O2 on Os reaction center. Moreover, upon a simple site-directed mutagenesis, the catalytic activity was enhanced by about 3-fold, indicating that Os-TM1459 is evolvable nascent osmium peroxygenase.

Generation, Characterization, and Reactivity of a Cu(II)-Alkylperoxide/Anilino Radical Complex: Insight into the O-O Bond Cleavage Mechanism.

The reaction of [Cu(I)(TIPT3tren) (CH3CN)]ClO4 (1) and cumene hydroperoxide (C6H5C(CH3)2OOH, ROOH) at -60 °C in CH2Cl2 gave a Cu(II)-alkylperoxide/anilino radical complex 2, the formation of which was confirmed by UV-vis, resonance Raman, EPR, and CSI-mass spectroscopy. The mechanism of formation of 2, as well as its reactivity, has been explored.

Direct hydroxylation of benzene to phenol using hydrogen peroxide catalyzed by nickel complexes supported by pyridylalkylamine ligands.

Selective hydroxylation of benzene to phenol has been achieved using H2O2 in the presence of a catalytic amount of the nickel complex [Ni(II)(tepa)](2+) (2) (tepa = tris[2-(pyridin-2-yl)ethyl]amine) at 60 °C. The maximum yield of phenol was 21% based on benzene without the formation of quinone or diphenol. In an endurance test of the catalyst, complex 2 showed a turnover number (TON) of 749, which is the highest value reported to date for molecular catalysts in benzene hydroxylation with H2O2. When toluene was employed as a substrate instead of benzene, cresol was obtained as the major product with 90% selectivity. When H2(18)O2 was utilized as the oxidant, (18)O-labeled phenol was predominantly obtained. The reaction rate for fully deuterated benzene was nearly identical to that of benzene (kinetic isotope effect = 1.0). On the basis of these results, the reaction mechanism is discussed.

Enzyme repurposing of a hydrolase as an emergent peroxidase upon metal binding.

As an alternative to Darwinian evolution relying on catalytic promiscuity, a protein may acquire auxiliary function upon metal binding, thus providing it with a novel catalytic machinery. Here we show that addition of cupric ions to a 6-phosphogluconolactonase 6-PGLac bearing a putative metal binding site leads to the emergence of peroxidase activity (kcat 7.8 × 10-2 s-1, KM 1.1 × 10-5 M). Both X-ray crystallographic and EPR data of the copper-loaded enzyme Cu·6-PGLac reveal a bis-histidine coordination site, located within a shallow binding pocket capable of accommodating the o-dianisidine substrate.

Geometric control of nuclearity in copper(I)/dioxygen chemistry.

Copper(I) complexes supported by a series of N3-tridentate ligands bearing a rigid cyclic diamine framework such as 1,5-diazacyclooctane (L8, eight-membered ring), 1,4-diazacycloheptane (L7, seven-membered ring), or 1,4-diazacyclohexane (L6, six-membered ring) with a common 2-(2-pyridyl)ethyl side arm were synthesized and their reactivity toward O2 were compared. The copper(I) complex of L8 preferentially provided a mononuclear copper(II) end-on superoxide complex S as reported previously [Itoh, S., et al. J. Am. Chem. Soc. 2009, 131, 2788-2789], whereas a copper(I) complex of L7 gave a bis(µ-oxido)dicopper(III) complex O at a low temperature (-85 °C) in acetone. On the other hand, no such active-oxygen complex was detected in the oxygenation reaction of the copper(I) complex of L6 under the same conditions. In addition, O2-reactivity of the copper(I) complex supported by an acyclic version of the tridentate ligand (LA, PyCH2CH2N(CH3)CH2CH2CH2N(CH3)2; Py = 2-pyridyl) was examined to obtain a mixture of a (µ-η(2):η(2)-peroxido)dicopper(II) complex (S)P and a bis(µ-oxido)dicopper(III) complex O. Careful inspection of the crystal structures of copper(I) and copper(II) complexes and the redox potentials of copper(I) complexes has revealed important geometric effects of the supporting ligands on controlling nuclearity of the generated copper active-oxygen complexes.

Redox chemistry of nickel(II) complexes supported by a series of noninnocent ß-diketiminate ligands.

Nickel complexes of a series of ß-diketiminate ligands ((R)L(-), deprotonated form of 2-substituted N-[3-(phenylamino)allylidene]aniline derivatives (R)LH, R = Me, H, Br, CN, and NO2) have been synthesized and structurally characterized. One-electron oxidation of the neutral complexes [Ni(II)((R)L(-))2] by AgSbF6 or [Ru(III)(bpy)3](PF6)3 (bpy = 2,2'-bipyridine) gave the corresponding metastable cationic complexes, which exhibit an EPR spectrum due to a doublet species (S = 1/2) and a characteristic absorption band in near IR region ascribable to a ligand-to-ligand intervalence charge-transfer (LLIVCT) transition. DFT calculations have indicated that the divalent oxidation state of nickel ion (Ni(II)) is retained, whereas one of the ß-diketiminate ligands is oxidized to give formally a mixed-valence complex, [Ni(II)((R)L(-))((R)L(•))](+). Thus, the doublet spin state of the oxidized cationic complex can be explained by taking account of the antiferromagnetic interaction between the high-spin nickel(II) ion (S = 1) and the organic radical (S = 1/2) of supporting ligand. A single-crystal structure of one of the cationic complexes (R = H) has been successfully determined to show that both ligands in the cationic complex are structurally equivalent. On the basis of theoretical analysis of the LLIVCT band and DFT calculations as well as the crystal structure, the mixed-valence complexes have been assigned to Robin-Day class III species, where the radical spin is equally delocalized between the two ligands to give the cationic complex, which is best described as [Ni(II)((R)L(0.5•-))2](+). One-electron reduction of the neutral complexes with decamethylcobaltocene gave the anionic complexes when the ligand has the electron-withdrawing substituent (R = CN, NO2, Br). The generated anionic complexes exhibited EPR spectra due to a doublet species (S = 1/2) but showed no LLIVCT band in the near-IR region. Thus, the reduced complexes are best described as the d(9) nickel(I) complexes supported by two anionic ß-diketiminate ligands, [Ni(I)((R)L(-))2](-). This conclusion was also supported by DFT calculations. Substituent effects on the electronic structures of the three oxidation states (neutral, cationic, and anionic) of the complexes are systematically evaluated on the basis of DFT calculations.

Redox properties of a mononuclear copper(II)-superoxide complex.

Redox properties of a mononuclear copper(II) superoxide complex, (L)Cu(II)-OO(•), supported by a tridentate ligand (L = 1-(2-phenethyl)-5-[2-(2-pyridyl)ethyl]-1,5-diazacyclooctane) have been examined as a model compound of the putative reactive intermediate of peptidylglycine α-hydroxylating monooxygenase (PHM) and dopamine ß-monooxygenase (DßM) (Kunishita et al. J. Am. Chem. Soc. 2009, 131, 2788-2789; Inorg. Chem. 2012, 51, 9465-9480). On the basis of the reactivity toward a series of one-electron reductants, the reduction potential of (L)Cu(II)-OO(•) was estimated to be 0.19 ± 0.07 V vs SCE in acetone at 298 K (cf. Tahsini et al. Chem.-Eur. J. 2012, 18, 1084-1093). In the reaction of TEMPO-H (2,2,6,6-tetramethylpiperidine-N-hydroxide), a simple HAT (hydrogen atom transfer) reaction took place to give the corresponding hydroperoxide complex LCu(II)-OOH, whereas the reaction with phenol derivatives ((X)ArOH) gave the corresponding phenolate adducts, LCu(II)-O(X)Ar, presumably via an acid-base reaction between the superoxide ligand and the phenols. The reaction of (L)Cu(II)-OO(•) with a series of triphenylphosphine derivatives gave the corresponding triphenylphosphine oxides via an electrophilic ionic substitution mechanism with a Hammett ρ value as -4.3, whereas the reaction with thioanisole (sulfide) only gave a copper(I) complex. These reactivities of (L)Cu(II)-OO(•) are different from those of a similar end-on superoxide copper(II) complex supported by a tetradentate TMG3tren ligand (1,1,1-Tris{2-[N(2)-(1,1,3,3-tetramethylguanidino)]ethyl}amine (Maiti et al. Angew. Chem., Int. Ed. 2008, 47, 82-85).

Crystal structures of copper-depleted and copper-bound fungal pro-tyrosinase: insights into endogenous cysteine-dependent copper incorporation.

Tyrosinase, a dinuclear copper monooxygenase/oxidase, plays a crucial role in the melanin pigment biosynthesis. The structure and functions of tyrosinase have so far been studied extensively, but the post-translational maturation process from the pro-form to the active form has been less explored. In this study, we provide the crystal structures of Aspergillus oryzae full-length pro-tyrosinase in the holo- and the apo-forms at 1.39 and 2.05 Å resolution, respectively, revealing that Phe(513) on the C-terminal domain is accommodated in the substrate-binding site as a substrate analog to protect the dicopper active site from substrate access (proteolytic cleavage of the C-terminal domain or deformation of the C-terminal domain by acid treatment transforms the pro-tyrosinase to the active enzyme (Fujieda, N., Murata, M., Yabuta, S., Ikeda, T., Shimokawa, C., Nakamura, Y., Hata, Y., and Itoh, S. (2012) ChemBioChem. 13, 193-201 and Fujieda, N., Murata, M., Yabuta, S., Ikeda, T., Shimokawa, C., Nakamura, Y., Hata, Yl, and Itoh, S. (2013) J. Biol. Inorg. Chem. 18, 19-26). Detailed crystallographic analysis and structure-based mutational studies have shown that the copper incorporation into the active site is governed by three cysteines as follows: Cys(92), which is covalently bound to His(94) via an unusual thioether linkage in the holo-form, and Cys(522) and Cys(525) of the CXXC motif located on the C-terminal domain. Molecular mechanisms of the maturation processes of fungal tyrosinase involving the accommodation of the dinuclear copper unit, the post-translational His-Cys thioether cross-linkage formation, and the proteolytic C-terminal cleavage to produce the active tyrosinase have been discussed on the basis of the detailed structural information.

Activation mechanism of melB tyrosinase from Aspergillus oryzae by acidic treatment.

The pro form of recombinant tyrosinase from Aspergillus oryzae (melB) shows no catalytic activity, but acid treatment (around pH 3.5) of protyrosinase activates it to induce tyrosinase activity. Circular dichroism spectra, gel filtration analysis, and colorimetric assay have indicated that acid treatment around pH 3.5 induced the disruption of the conformation of the C-terminal domain covering the enzyme active site. These structural changes induced by the acid treatment may open the entrance to the enzyme active site for substrate incorporation. To compare the mechanism of hydroxylation by the acid-treated tyrosinase with that by trypsin-treated tyrosinase, a detailed steady-state kinetic analysis of the phenolase activity was performed by monitoring the O(2)-consumption rate using a Clark-type oxygen electrode. The results clearly show that the phenolase activity (phenol hydroxylation) of the activated tyrosinase involves an electrophilic aromatic substitution mechanism as in the case of mushroom tyrosinase (Yamazaki and Itoh in J. Am. Chem. Soc. 125:13034-13035, 2003) and activated hemocyanin with urea (Morioka et al. in J. Am. Chem. Soc. 128:6788-6789, 2006).

Copper complexes of the non-innocent ß-diketiminate ligand containing phenol groups.

A new type of non-innocent ß-diketiminate ligand having redox active phenol groups (LH(3), fully protonated form) has been developed, and the structure, physical properties and reactivity of the supported copper(II) complex [Cu(II)(L(3-))](-) (L(3-), fully deprotonated tri-anionic form) as well as the one-electron and two-electron oxidised complexes, [Cu(II)(L˙(2-))] and [Cu(II)(L(-))](+), have been examined in detail. The two-electron oxidised form [Cu(II)(L(-))](+) exhibited hydrogen atom abstraction ability from 1,4-cyclohexadiene (CHD), whereas the one-electron oxidised form [Cu(II)(L˙(2-))] was found to disproportionate into [Cu(II)(L(3-))](-) and [Cu(II)(L(-))](+) during the course of the reaction with CHD.

Active site models for the Cu(A) site of peptidylglycine α-hydroxylating monooxygenase and dopamine ß-monooxygenase.

A mononuclear copper(II) superoxo species has been invoked as the key reactive intermediate in aliphatic substrate hydroxylation by copper monooxygenases such as peptidylglycine α-hydroxylating monooxygenase (PHM), dopamine ß-monooxygenase (DßM), and tyramine ß-monooxygenase (TßM). We have recently developed a mononuclear copper(II) end-on superoxo complex using a N-[2-(2-pyridyl)ethyl]-1,5-diazacyclooctane tridentate ligand, the structure of which is similar to the four-coordinate distorted tetrahedral geometry of the copper-dioxygen adduct found in the oxy-form of PHM (Prigge, S. T.; Eipper, B. A.; Mains, R. E.; Amzel, L. M. Science2004, 304, 864-867). In this study, structures and physicochemical properties as well as reactivity of the copper(I) and copper(II) complexes supported by a series of tridentate ligands having the same N-[2-(2-pyridyl)ethyl]-1,5-diazacyclooctane framework have been examined in detail to shed light on the chemistry dictated in the active sites of mononuclear copper monooxygenases. The ligand exhibits unique feature to stabilize the copper(I) complexes in a T-shape geometry and the copper(II) complexes in a distorted tetrahedral geometry. Low temperature oxygenation of the copper(I) complexes generated the mononuclear copper(II) end-on superoxo complexes, the structure and spin state of which have been further characterized by density functional theory (DFT) calculations. Detailed kinetic analysis on the O(2)-adduct formation reaction gave the kinetic and thermodynamic parameters providing mechanistic insights into the association and dissociation processes of O(2) to the copper complexes. The copper(II) end-on superoxo complex thus generated gradually decomposed to induce aliphatic ligand hydroxylation. Kinetic and DFT studies on the decomposition reaction have suggested that C-H bond abstraction occurs unimolecularly from the superoxo complex with subsequent rebound of the copper hydroperoxo species to generate the oxygenated product. The present results have indicated that a superoxo species having a four-coordinate distorted tetrahedral geometry could be reactive enough to induce the direct C-H bond activation of aliphatic substrates in the enzymatic systems.