Dinuclear Center Research Articles

Bioinspired CO2 Reduction with Iron Porphyrin Catalysts in High Efficiency

Conversion of CO2 to useful carbon resources as well as water splitting is the important ways to store renewable energy as chemical energy. Both reactions are typical multi-electron ones, thus, they tend to require large overpotentials (or activation energies) unless one uses proper catalysts. Particulary, CO2 reduction potentially gives several reduced products, CO, HCO2H, (CO2H)2, CH3OH, etc. CO2 reduction also competes proton reduction. Thus, the product selectivity is another important factor on its reaction. Following issues are keys on the development of CO2 reduction catalysts: (1) low overpotential, (2) high turnover frequency, (3) robustness, (4) product selectivity, (5) easy separation of product(s), and (6) use of base metal (complexes) on the elemental strategy aspect. Carbon monoxide dehydrogenase (CODH), an enzyme in bacteria, catalyzes the mutual conversions between CO2 and CO. The active site of the enzyme is consisted of Ni-Fe cluster and this enzyme realizes selective CO2 reduction to CO at very low overpotential (η < 100 mV). We develop new catalysts to fulfill the above criteria referring the enzyme structure. We developed iron porphyrin dimers as bio-inspired catalysts, which realized electrochemical CO2 reduction at the lowest overpotentials and high TOF in high selectivity and robustness by choosing suitable functional groups. In a homogeneous solution, continuous electrocatalytic reduction of CO2 for 12 h gave a good Faradaic efficiency as well as a good product selectivity by keeping a constant current through the electrolysis and any decrease of the catalyst activity is not observed. We will discuss the importance of the dinuclear center in the CO2 reduction.

The unique serine/threonine phosphatase from the minimal bacterium Mycoplasma synoviae: biochemical characterization and metal dependence.

Serine/threonine protein phosphatases have been described in many pathogenic bacteria as essential enzymes involved in phosphorylation-dependent signal transduction pathways and frequently associated with the virulence of these organisms. An inspection of Mycoplasma synoviae genome revealed the presence of a gene (prpC) encoding a putative protein phosphatase of the protein phosphatase 2C (PP2C) subfamily. Here, we report a complete biochemical characterization of M. synoviae phosphatase (PrpC) and the particular role of metal ions in the structure-function relationship of this enzyme. PrpC amino acid sequence analysis revealed that all the residues involved in the dinuclear metal center and the putative third metal ion-coordinating residues, conserved in PP2C phosphatases, are present in PrpC. PrpC is a monomeric protein able to dephosphorylate phospho-substrates with Mn(2+) ions' dependence. Thermal stability analysis demonstrated the enzyme stability at mild temperatures and the influence of Mn(2+) ions in this property. Mass spectrometry analysis suggested that three metal ions bind to PrpC, two of which with an apparent high-affinity constant. Mutational analysis of the putative third metal-coordinating residues, Asp122 and Arg164, revealed that these variants exhibited a weaker binding of manganese ions, and that both mutations affected PrpC phosphatase activity. According to these results, PrpC is a metal-dependent protein phosphatase member with an improved stability in the holo form and with Asp122, possibly implicated in the third metal-binding site, essential to catalytic activity.

Tetracarboxylate Ligands as New Chelates Supporting Copper(II) Paddlewheel-Like Structures

Two new ligands N,N,N',N'-tetrakis(2-methylbenzoic acid)-1,4-diaminomethylbenzene, 5H4, and N,N,N',N'-tetrakis(2-methylbenzoic acid)-4,4'-diaminomethyldiphenyl, 6H4, carrying four carboxylate groups suitable for bridging dinuclear centers have been prepared and their paddlewheel complexes with copper(II) prepared. The phenyl-bridged ligand 5H4 gives a cyclic octanuclear species [(Cu2)4(5)4], while the diphenyl-bridged ligand 6H4 gives a lantern-like tetranuclear species [(Cu2)2(6)2]; both were characterized by X-ray crystallography. If the amine functions of 5 are protonated, intramolecular hydrogen bonds position the four carboxylates in such a way as to allow formation of the unusual compound [Cu4(5H2)2Cl](3+) in which a Cu4 square centered by a chloro ligand is sandwiched between two (5H2)(2-) ligands. The magnetic properties of this compound have been studied and show antiferromagnetic coupling between adjacent coppers (J = -33.7 cm(-1)).

Density Functional Study for the Bridged Dinuclear Center Based on a High-Resolution X-ray Crystal Structure of ba3 Cytochrome c Oxidase from Thermus thermophilus

Strong electron density for a peroxide type dioxygen species bridging the Fea3 and CuB dinuclear center (DNC) was observed in the high-resolution (1.8 Å) X-ray crystal structures (PDB entries 3S8G and 3S8F) of ba3 cytochrome c oxidase (CcO) from Thermus thermophilus. The crystals represent the as-isolated X-ray photoreduced CcO structures. The bridging peroxide was proposed to arise from the recombination of two radiation-produced HO(•) radicals formed either very near to or even in the space between the two metals of the DNC. It is unclear whether this peroxide species is in the O2(2-), O2(•)(-), HO2(-), or the H2O2 form and what is the detailed electronic structure and binding geometry including the DNC. In order to answer what form of this dioxygen species was observed in the DNC of the 1.8 Å X-ray CcO crystal structure (3S8G), we have applied broken-symmetry density functional theory (BS-DFT) geometric and energetic calculations (using OLYP potential) on large DNC cluster models with different Fea3-CuB oxidation and spin states and with O2(2-), O2(•)(-), HO2(-), or H2O2 in the bridging position. By comparing the DFT optimized geometries with the X-ray crystal structure (3S8G), we propose that the bridging peroxide is HO2(-). The X-ray crystal structure is likely to represent the superposition of the Fea3(2+)-(HO2(-))-CuB(+) DNC's in different states (Fe(2+) in low spin (LS), intermediate spin (IS), or high spin (HS)) with the majority species having the proton of the HO2(-) residing on the oxygen atom (O1) which is closer to the Fea3(2+) site in the Fea3(2+)-(HO-O)(-)-CuB(+) conformation. Our calculations show that the side chain of Tyr237 is likely trapped in the deprotonated Tyr237(-) anion form in the 3S8G X-ray crystal structure.

Dinuclear gold(I) and gold(III) complexes involving di(N-heterocyclic carbene) ligands – Synthesis, characterization and DFT studies

Some new dinuclear gold(I) and gold(III) complexes containing di(NHC) ligands have been synthesized and characterized. The dinuclear centers were coordinated by methylene, ethylene or propylene bridged di(NHC) ligands bearing alcohol functionalized side arms. The oxidation of the gold(I) to gold(III) have been carried out using bromine. X-ray structure analyses of two dinuclear gold(I) and three dinuclear gold(III) complexes show the solid state structures of these compounds. Thermodynamic aspects of the oxidative additions of bromine to the gold(I) species leading to gold(III) compounds passing by unstable gold(II) intermediates have been studied by DFT calculations.

Detection of the hyponitrite species (HO‐N=N‐O) in denitrification: Reactivity of NO with the heme Fe‐Cu center of cytochrome caa3 and the heme Fe –Fe center of Nitric oxide reductase

Nitric oxide reductase (NOR) is the third of the four enzymes of bacterial denitrification that is responsible for the catalytic formation of laughing gas (N2O). Here we report on the formation of the hyponitrite (HO‐N=N‐O−) species (νN‐N=1332 cm−1) in the heme b3 Fe/non‐heme FeB dinuclear center of NOR from Paracoccus denitrificans. With the identification of the hyponitrite species the mechanism for the 2e−/2H+ reduction of NO to N2O can be described with more certainty.This work was financially supported by the Cyprus Research Promotion Foundation grant TEXNOLOGIA/THEPIS/0609(BE)/05.

Two coordination modes around the Cu(II) cations in complexes with benzo[b]furancarboxylic acids

Three Cu(II) complexes with derivatives of the benzo[b]furancarboxylic acid have been synthesized and characterized by the elemental and thermal analyses, and IR spectroscopy. The geometry of metal–ligand interaction for all compounds has been described using X-ray absorption spectroscopy and for one of them by X-ray crystallography. Two mononuclear Cu(II) complexes, with 7-acetyl-5-bromo-6-hydroxy-3-methylbenzo[b]furan-2-carboxylic and 6-acetyl-5-hydroxy-2-methylbenzo[b]furan-3-carboxylic acids, exhibit a tetra-fold coordination, CuO4. The Cu(II) cation in crystals with 7-acetyl-6-methoxy-3-methyl-benzo[b]furan-2-carboxylic acid is penta-coordinated; the bridging COO− groups and ethanol molecule stabilize the dinuclear center Cu2O10. The powdered form of this complex is based on the Cu2O8 units, indicating the absence of the ethanol molecules.

Binding of oxo-Cu2 clusters to ferric ion-binding protein A from Neisseria gonorrhoeae: a structural insight

The ferric ion-binding protein A (FbpA), a member of transferrin superfamily, is a periplasmic iron transporter employed by many Gram-negative pathogens. Our experiments indicated copper(ii) could bind with Neisseria gonorrhoeae FbpA (NgFbpA), and the binding constant reached up to (8.7 ± 0.2) × 10(8) M(-1)via UV-vis titration. The crystal structure of recombinant Cu-NgFbpA at 2.1 Å revealed that the oxo-Cu2 clusters (dinuclear centres) assembled in the iron binding cleft and were bound to the two adjacent tyrosine residues (Y195 and Y196) of the protein, two Cu ions coordinated with two tyrosines, Y195 and Y196, respectively, which was different from the binding model of Fe ion with FbpA, in which Y195 and Y196 coordinated together with one Fe ion. While this was similar to the binding of Zr and Hf ion clusters, Y195 and Y196 coordinated with two metal ions and the μ-oxo-bridges linking the metal ions. Structural superimposition demonstrated that oxo-Cu2-NgFbpA still keeping an open conformation, similar to the apo-form of NgFbpA. The structure presented additional information towards an understanding of the function of FbpA, and provided a detailed binding model for FbpA protein with the possible metal ions in a biological system.

3P002 二核フェロキシダーゼ中心をもつピロリ菌好中球活性化タンパク質の構造(01A.蛋白質:構造,ポスター,日本生物物理学会年会第51回(2013年度))

3P002 二核フェロキシダーゼ中心をもつピロリ菌好中球活性化タンパク質の構造(01A.蛋白質:構造,ポスター,日本生物物理学会年会第51回(2013年度))

Spectroscopic and Catalytic Characterization of a Functional FeIIIFeII Biomimetic for the Active Site of Uteroferrin and Protein Cleavage

A mixed-valence complex, [Fe(III)Fe(II)L1(μ-OAc)(2)]BF(4)·H(2)O, where the ligand H(2)L1 = 2-{[[3-[((bis(pyridin-2-ylmethyl)amino)methyl)-2-hydroxy-5-methylbenzyl](pyridin-2-ylmethyl)amino]methyl]phenol}, has been studied with a range of techniques, and, where possible, its properties have been compared to those of the corresponding enzyme system purple acid phosphatase. The Fe(III)Fe(II) and Fe(III)(2) oxidized species were studied spectroelectrochemically. The temperature-dependent population of the S = 3/2 spin states of the heterovalent system, observed using magnetic circular dichroism, confirmed that the dinuclear center is weakly antiferromagnetically coupled (H = -2JS(1)·S(2), where J = -5.6 cm(-1)) in a frozen solution. The ligand-to-metal charge-transfer transitions are correlated with density functional theory calculations. The Fe(III)Fe(II) complex is electron paramagnetic resonance (EPR)-silent, except at very low temperatures (<2 K), because of the broadening caused by the exchange coupling and zero-field-splitting parameters being of comparable magnitude and rapid spin-lattice relaxation. However, a phosphate-bound Fe(III)(2) complex showed an EPR spectrum due to population of the S(tot) = 3 state (J= -3.5 cm(-1)). The phosphatase activity of the Fe(III)Fe(II) complex in hydrolysis of bis(2,4-dinitrophenyl)phosphate (k(cat.) = 1.88 × 10(-3) s(-1); K(m) = 4.63 × 10(-3) mol L(-1)) is similar to that of other bimetallic heterovalent complexes with the same ligand. Analysis of the kinetic data supports a mechanism where the initiating nucleophile in the phosphatase reaction is a hydroxide, terminally bound to Fe(III). It is interesting to note that aqueous solutions of [Fe(III)Fe(II)L1(μ-OAc)(2)](+) are also capable of protein cleavage, at mild temperature and pH conditions, thus further expanding the scope of this complex's catalytic promiscuity.

Poly[[(2,2′-bipyridine)(μ3-7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylato)cadmium] monohydrate

The title compound, {[Cd(C8H8O5)(C10H8N2)]·H2O}n, was obtained by the reaction of cadmium acetate with 2,2′-bi­pyridine and 7-oxabicyclo­(2.2.1)heptane-2,3-dicarb­oxy­lic anhydride. The CdII atom is seven-coordinated in a distorted penta­gonal–bipyramidal configuration, defined by five O atoms from the carboxyl­ate groups of three 7-oxabicyclo­[2.2.1]heptane-2,3-dicarboxyl­ato ligands and two N atoms from the 2,2′-bipyridine ligand. Two O atoms link two CdII atoms, forming a dinuclear center: the Cd—O—Cd bridging angle is 110.19 (6)°. The polymeric structure extends along [100] and is linked by inter­molecular O—H⋯O hydrogen bonds involving the solvent water molecule. Extensive π–π stacking exists between 2,2-bypiridine ligands along [010] with centroid-centroid distance of 3.650 (2) Å

Electron transfer to the active site of the bacterial nitric oxide reductase is controlled by ligand binding to heme b3

The active site of the bacterial nitric oxide reductase from Paracoccus denitrificans contains a dinuclear centre comprising heme b 3 and non heme iron (Fe B). These metal centres are shown to be at isopotential with midpoint reduction potentials of E m ≈ + 80 mV. The midpoint reduction potentials of the other two metal centres in the enzyme, heme c and heme b, are greater than the dinuclear centre suggesting that they act as an electron receiving/storage module. Reduction of the low-spin heme b causes structural changes at the dinuclear centre which allow access to substrate molecules. In the presence of the substrate analogue, CO, the midpoint reduction potential of heme b 3 is raised to a region similar to that of heme c and heme b. This leads us to suggest that reduction of the electron transfer hemes leads to an opening of the active site which allows substrate to bind and in turn raises the reduction potential of the active site such that electrons are only delivered to the active site following substrate binding.

M2δ to ligand π-conjugation: testbeds for current theories of mixed valence in ground and photoexcited states of molecular systems

Redox active quadruply bonded units, M(2), can be combined so that they either (i) are bridged by an organic linker or (ii) function as a bridge between two identical organic ligands. When two M(2) units are linked together by an organic group that affords M(2)δ-bridge π-conjugation the electronic structure of each M(2) unit is perturbed by the other in the ground state, the photoexcited states, and the mixed valence oxidized form. Similarly when a M(2) center links two organic π systems represented by L, the two organic units are coupled by Lπ*-M(2)δ-Lπ* interactions in their ground state, their photoexcited states, and the mixed valence reduced state. The photoexcited states of the neutral complexes (both case i and ii) provide examples of excited state mixed valence. In case (i), the positive charge may be localized on one dinuclear center or may be delocalized over both M(2) units. Similarly in (ii), the electron may be localized on one ligand or delocalized over both. In this tutorial review, spectroscopic studies (UV-vis-NIR absorption, steady state emission, EPR, and time resolved infrared) of these mixed valence systems employing carboxylate tethers are described and the data are discussed in terms of contemporary theories of mixed valence ions.

Role of H-1 and H-2 Subunits of Soybean Seed Ferritin in Oxidative Deposition of Iron in Protein

Naturally occurring phytoferritin is a heteropolymer consisting of two different H-type subunits, H-1 and H-2. Prior to this study, however, the function of the two subunits in oxidative deposition of iron in ferritin was unknown. The data show that, upon aerobic addition of 48-200 Fe(2+)/shell to apoferritin, iron oxidation occurs only at the diiron ferroxidase center of recombinant H1 (rH-1). In addition to the diiron ferroxidase mechanism, such oxidation is catalyzed by the extension peptide (a specific domain found in phytoferritin) of rH-2, because the H-1 subunit is able to remove Fe(3+) from the center to the inner cavity better than the H-2 subunit. These findings support the idea that the H-1 and H-2 subunits play different roles in iron mineralization in protein. Interestingly, at medium iron loading (200 irons/shell), wild-type (WT) soybean seed ferritin (SSF) exhibits a stronger activity in catalyzing iron oxidation (1.10 ± 0.13 μm iron/subunit/s) than rH-1 (0.59 ± 0.07 μm iron/subunit/s) and rH-2 (0.48 ± 0.04 μm iron/subunit/s), demonstrating that a synergistic interaction exists between the H-1 and H-2 subunits in SSF during iron mineralization. Such synergistic interaction becomes considerably stronger at high iron loading (400 irons/shell) as indicated by the observation that the iron oxidation activity of WT SSF is ∼10 times larger than those of rH-1 and rH-2. This helps elucidate the widespread occurrence of heteropolymeric ferritins in plants.

Investigation of Streptomyces antibioticus tyrosinase reactivity toward chlorophenols

Tyrosinase (Ty) is a copper-containing enzyme ubiquitously distributed in nature. In recent years, Ty has attracted interest as a potential detoxifying agent for xenobiotic compounds with phenolic structure. Among these, chlorophenols are particularly relevant pollutants, commonly found in waste waters. The activity of Streptomyces antibioticus tyrosinase toward isomeric monochlorophenols was studied. Tyrosinase oxidizes both 3- and 4-chlorophenol to the same product, 4-chloro-1,2- ortho-quinone, which subsequently undergoes a nucleophilic substitution reaction at the chlorine atom by excess phenol to give the corresponding phenol-quinone adduct. By contrast, 2-chlorophenol is not reactive and acts as a competitive inhibitor. Docking calculations suggest that the substrates point to one of the copper atoms of the dinuclear center (copper B) and appear to interact preferentially with one of the two coordinated oxygen atoms. The approach of the substrate toward the active site is favored by a π-stacking interaction with one of the copper-coordinated histidines (His194) and by a hydrogen bonding interaction with the O1 oxygen. With this study, we provide the first characterization of the early intermediates in the biotechnologically relevant reaction of Ty with chlorophenols. Additionally, combining experimental evidences with molecular modeling simulations, we propose a detailed reaction scheme for Ty-mediated oxidation of monochlorophenols.

Spectroscopic signature of a ubiquitous metal binding site in the metallo-β-lactamase superfamily

The metallo-β-lactamase (MβL) superfamily is a functionally diverse group of metalloproteins sharing a distinctive αβ/αβ fold and a characteristic metal binding motif. A large number of open reading frames identified in genomic sequencing efforts have been annotated as members of this superfamily through sequence comparisons. However, structural and functional studies performed on purified proteins are normally needed to unequivocally include a newly discovered protein in the MβL superfamily. Here we report the spectroscopic characterization of recombinant YcbL, a gene product annotated as a member of the MβL superfamily whose function in vivo remains unknown. By taking advantage of the structural features characterizing the MβL superfamily metal binding motif, we performed spectroscopic studies on Zn(II)- and Co(II)-substituted YcbL to structurally interrogate the metal binding site. The dinuclear center in Co(II)-YcbL was shown to display characteristic electronic absorption features in the visible region, which were also observed in an engineered MβL aimed at mimicking this metal site. Thus, the spectroscopic features reported herein can be employed as a signature to readily identify and characterize the presence of these ubiquitous metal binding sites.

ChemInform Abstract: Structure, Properties and Reactivity of the FeIIFeIII and ZnIIFeIII Purple Acid Phosphatases

This microreview describes the structure, properties and mechanisms of the purple acid phosphatases (PAP). The enzyme is isolated from mammalian, plant and bacterial sources. X-ray structural information is now available for the enzyme from pig (uteroferrin), rat and kidney beans. Features of the mechanism are the concerted action of a labile MII centre (FeII or ZnII) alongside a more inert FeIII. The latter is effective as a conjugate-base FeOH2+, which initiates hydrolysis at the MII-bound phosphate ester by a process involving OH− replacement of OR− at the PV. Histidine residues near to the active site help bind the phosphate and are involved in the release of OR−. Effects of replacement of the FeII by MnII, CoII, NiII, CuII and ZnII, and of FeIII by GaIII,AlIII and InIII have been studied. The mechanistic role of the ZnIIZnII combination in alkaline phosphatases, and other related dinuclear centres is also considered.

Electronic Structure Analysis of the Dinuclear Metal Center in the Bioremediator Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes

The glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a promiscuous, dinuclear metallohydrolase that has potential application in the remediation of organophosphate nerve agents and pesticides. GpdQ employs an unusual reaction mechanism in which the enzyme is predominantly mononuclear in the resting state, and substrate binding induces the formation of the catalytically competent dinuclear center (Hadler et al. J. Am. Chem. Soc. 2008, 130, 14129). Reactivity is further modulated by the coordination flexibility of Asn80, a ligand that binds to the second, loosely bound metal ion (Hadler et al. J. Am. Chem. Soc. 2009, 131, 11900). It is proposed that hydrolysis is initiated by a terminal, metal-bound hydroxide molecule which is activated at unusually low pH by electrostatic/hydrogen bonding interactions with a bridging hydroxide species. In this study, electronic structure analysis of the dinuclear center is employed to study the coordination environment of the dinuclear center at the resting and product-bound stage of catalysis. This is achieved through the use of variable temperature, variable field magnetic circular dichroism experiments involving the Co(II)-substituted wild type enzyme and its Asn80Asp variant. The data support the above model for the catalytic mechanism whereby the metal ion-bridging hydroxide molecule activates a terminally bound hydroxide nucleophile. Replacement of Asn80 by an aspartate residue does prevent coordination flexibility but also leads to cleavage of the mu-hydroxide bridge and reduced reactivity. This is the first study to investigate the electronic structure of an enzyme with a mu-1,1-carboxylate bridged dicobalt(II) center.

Crystal structure of the conserved hypothetical protein TTHA1606 from Thermus thermophilus HB8

Crystal structure of the conserved hypothetical protein TTHA1606 from <i>Thermus thermophilus</i> HB8

Combined Microspectrophotometric and Crystallographic Examination of Chemically Reduced and X-ray Radiation-Reduced Forms of Cytochrome ba3 Oxidase from Thermus thermophilus: Structure of the Reduced Form of the Enzyme

Three paths for obtaining crystals of reduced (II-E4Q/I-K258R) cytochrome ba(3) are described, and the structures of these are reported at approximately 2.8-3.0 A resolution. Microspectrophotometry of single crystals of Thermus ba(3) oxidase at 100 K was used to show that crystals of the oxidized enzyme are reduced in an intense X-ray (beam line 7-1 at the Stanford Synchrotron Radiation Laboratory), being nearly complete in 1 min. The previously reported structures of ba(3) (Protein Data Bank entries 1EHK and 1XME ), having a crystallographically detectable water between the Cu(B) and Fe(a3) metals of the dinuclear center, actually represent the X-ray radiation-reduced enzyme. Dithionite-reduced crystals or crystals formed from dithionite-reduced enzyme revealed the absence of the above-mentioned water and an increase in the Cu(B)-Fe(a3) distance of approximately 0.3 A. The new structures are discussed in terms of enzyme function. An unexpected optical absorption envelope at approximately 590 nm is also reported. This spectral feature is tentatively thought to arise from a five-coordinate, low-spin, ferrous heme a(3) that is trapped in the frozen crystals.