Bilirubin oxidase from Myrothecium verrucaria: X-ray determination of the complete crystal structure and a rational surface modification for enhanced electrocatalytic O2 reduction

The blue multi-copper oxidase bilirubin oxidase (BOx) from the ascomycete plant pathogen Myrothecium verrucaria (Mv) efficiently catalyses the oxidation of bilirubin to biliverdin, with the concomitant reduction of O(2) to water, a reaction of considerable interest for low-temperature bio-fuel cell applications. We have solved the complete X-ray determined structure of Mv BOx at 2.4 ? resolution, using molecular replacement with the Spore Coat Protein A (CotA) enzyme from Bacillus subtilis (PDB code 1GSK) as a template. The structure reveals an unusual environment around the blue type 1 copper (T1 Cu) that includes two non-coordinating hydrophilic amino acids, asparagine and threonine. The presence of a long, narrow and hydrophilic pocket near the T1 Cu suggests that structure of the substrate-binding site is dynamically determined in vivo. We show that the interaction between the binding pocket of Mv BOx and its highly conjugated natural organic substrate, bilirubin, can be used to stabilise the enzyme on a pyrolytic graphite electrode, more than doubling its electrocatalytic activity relative to the current obtained by simple adsorption of the protein to the carbon surface.     

Role of the electronic properties of azurin active site in the electron‐transfer process

Electron transfer proteins, such as azurin (a blue copper protein), are promising candidates for the implementation of biomolecular nanoelectronic devices. To understand the details of electron transfer in redox active azurin molecules, we performed plane‐wave pseudo‐potential density functional theory (DFT) calculations of the protein active site in the two possible oxidation states Cu(I) and Cu(II). The ab initio results are used to discuss how the electronic spectrum and wavefunctions may mediate the shuttling of electrons through the copper ion. We find that the Cu‐ligand hybridization is very similar in the two charge states of the metal center, but the energy spectrum changes substantially. This result might indicate important effects of electronic correlations in the redox activity and consequent electron transfer through the Cu site. © 2004 Wiley Periodicals, Inc. Int J Quantum Chem, 2005     

Characterisation of [Cu4S], the catalytic site in nitrous oxide reductase, by EPR spectroscopy

The enzyme nitrous oxide reductase (N(2)OR) has a unique tetranuclear copper centre [Cu(4)S], called Cu(Z), at the catalytic site for the two-electron reduction of N(2)O to N(2). The X- and Q-band EPR spectra have been recorded from two forms of the catalytic site of the enzyme N(2)OR from Paracoccus pantotrophus, namely, a form prepared anaerobically, Cu(Z), that undergoes a one-electron redox cycle and Cu(Z)*, prepared aerobically, which cannot be redox cycled. The spectra of both species are axial with that of Cu(Z) showing a rich hyperfine splitting in the g||-region at X-band. DFT calculations were performed to gain insight into the electronic configuration and ground-state properties of Cu(Z) and to calculate EPR parameters. The results for the oxidation state [Cu(+1)(3)Cu(+2)(1)S](3+) are in good agreement with values obtained from the fitting of experimental spectra, confirming the absolute oxidation state of Cu(Z). The unpaired spin density in this configuration is delocalised over four copper ions, thus, Cu(I) 20.1%, Cu(II) 9.5%, Cu(III) 4.8% and Cu(IV) 9.2%, the mu(4)-sulfide ion and oxygen ligand. The three copper ions carrying the highest spin density plus the sulfide ion lie approximately in the same plane while the fourth copper ion is perpendicular to this plane and carries only 4.8% spin density. It is suggested that the atoms in this plane represent the catalytic core of Cu(Z), allowing electron redistribution within the plane during interaction with the substrate, N(2)O.     

Nature of the intermediate formed in the reduction of O(2) to H(2)O at the trinuclear copper cluster active site in native laccase

The multicopper oxidases contain at least four copper atoms and catalyze the four-electron reduction of O(2) to H(2)O at a trinuclear copper cluster. An intermediate, termed native intermediate, has been trapped by a rapid freeze-quench technique from Rhus vernicifera laccase when the fully reduced form reacts with dioxygen. This intermediate had been described as an oxygen-radical bound to the trinuclear copper cluster with one Cu site reduced. XAS, however, shows that all copper atoms are oxidized in this intermediate. A combination of EXAFS, multifrequency EPR, and VTVH MCD has been used to understand how this fully oxidized trinuclear Cu cluster relates to the fully oxidized resting form of the enzyme. It is determined that in the native intermediate all copper atoms of the cluster are bridged by the product of full O(2) reduction. In contrast, the resting form has one copper atom of the cluster (the T2 Cu) magnetically isolated from the others. The native intermediate decays to the resting oxidized form with a rate that is too slow to be in the catalytic cycle. Thus, the native intermediate appears to be the catalytically relevant fully oxidized form of the enzyme, and its role in catalysis is considered.     

Spectroscopic and kinetic studies of perturbed trinuclear copper clusters: the role of protons in reductive cleavage of the O-O bond in the multicopper oxidase Fet3p

The multicopper oxidase Fet3p couples four 1e(-) oxidations of substrate to the 4e(-) reduction of O2 to H2O. Fet3p uses four Cu atoms to accomplish this reaction: the type 1, type 2, and coupled binuclear type 3 sites. The type 2 and type 3 sites together form a trinuclear Cu cluster (TNC) which is the site of O2 reduction. This study focuses on mutants of two residues, E487 and D94, which lie in the second coordination sphere of the TNC and defines the role that each plays in the structural integrity of the TNC, its reactivity with O2, and in the directional movement of protons during reductive cleavage of the O-O bond. The E487D, E487A, and D94E mutants have been studied in the holo and type 1 depleted (T1D) forms. Residue E487, located near the T3 center, is found to be responsible for donation of a proton during the reductive cleavage of the O-O bond in the peroxide intermediate and an inverse kinetic solvent isotope effect, which indicates that this proton is already transferred when the O-O bond is cleaved. Residue D94, near the T2 site, plays a key role in the reaction of the reduced TNC with O2 and drives electron transfer from the T2 Cu to cleave the O-O bond by deprotonating the T2 Cu water ligand. A mechanism is developed where these second sphere residues participate in the proton assisted reductive cleavage of the O-O bond at the TNC.     

Pyrolytic Carbon‐coated Cu‐Fe Alloy Nanoparticles with High Catalytic Performance for Oxygen Electroreduction

Well‐dispersed carbon‐coated or nitrogen‐doped carbon‐coated copper‐iron alloy nanoparticles (FeCu@C or FeCu@C?N) in carbon‐based supports are obtained using a bimetallic metal‐organic framework (Cu/Fe‐MOF‐74) or a mixture of Cu/Fe‐MOF‐74 and melamine as sacrificial templates and an active‐component precursor by using a pyrolysis method. The investigation results attest formation of Cu?Fe alloy nanoparticles. The obtained FeCu@C catalyst exhibits a catalytic activity with a half‐wave potential of 0.83 V for oxygen reduction reaction (ORR) in alkaline medium, comparable to that on commercial Pt/C catalyst (0.84 V). The catalytic activity of FeCu@C?N for ORR (E_half‐wave=0.87 V) outshines all reported analogues. The excellent performance of FeCu@C?N should be attributed to a change in the energy of the d‐band center of Cu resulting from the formation of the copper–iron alloy, the interaction between alloy nanoparticles and supports and N‐doping in the carbon matrix. Moreover, FeCu@C and FeCu@C?N show better electrochemical stability and methanol tolerance than commercial Pt/C and are expected to be widely used in practical applications.     

The Resting Oxidized State of Small Laccase Analyzed with Paramagnetic NMR Spectroscopy

The enzyme laccase catalyzes the reduction of dioxygen to water at the trinuclear copper center (TNC). The TNC comprises a type-3 (T3) and a type-2 (T2) copper site. The paramagnetic NMR spectrum of the small laccase from Streptomyces coelicolor (SLAC) without the substrate shows a mixture of two catalytic states, the resting oxidized (RO) state and the native intermediate (NI) state. An analysis of the resonances of the RO state is reported. In this state, hydrogen resonances only of the T3 copper ligands can be found, in the region of 12–22 ppm. Signals from all six histidine ligands are found and can be attributed to Hδ1, Hβ or backbone amide H^N nuclei. Two sequence-specific assignments are proposed on the basis of a second-coordination shell variant that also lacks the copper ion at the T1 site, SLAC−T1D/Q291E. This double mutant is found to be exclusively in the RO state, revealing a subtle balance between the RO and the NI states.     

Platinum–Copper Nanoframes: One‐Pot Synthesis and Enhanced Electrocatalytic Activity

Platinum–copper nanoframes were produced from copper nanoparticles by a one‐pot synthesis method. The growth mechanism was thoroughly studied by experiment and theoretical calculations. Owing to the unique structure, Pt‐Cu nanoframes exhibited significantly enhanced catalytic activity toward the electro‐oxidation of methanol compared to commercial Pt black.     

Isolated Square‐Planar Copper Center in Boron Imidazolate Nanocages for Photocatalytic Reduction of CO2 to CO

Photocatalytic reduction of CO₂ to value‐added fuel has been considered to be a promising strategy to reduce global warming and shortage of energy. Rational design and synthesis of catalysts to maximumly expose the active sites is the key to activate CO₂ molecules and determine the reaction selectivity. Herein, we synthesize a well‐defined copper‐based boron imidazolate cage (BIF‐29) with six exposed mononuclear copper centers for the photocatalytic reduction of CO₂. Theoretical calculations show a single Cu site including weak coordinated water delivers a new state in the conduction band near the Fermi level and stabilizes the *COOH intermediate. Steady‐state and time‐resolved fluorescence spectra show these Cu sites promote the separation of electron–hole pairs and electron transfer. As a result, the cage achieves solar‐driven reduction of CO₂ to CO with an evolution rate of 3334 μmol g^?1 h^?1 and a high selectivity of 82.6 %.     

A Crystalline Copper(II) Coordination Polymer for the Efficient Visible‐Light‐Driven Generation of Hydrogen

A crystalline coordination polymer (CP) photocatalyst (Cu‐RSH) which combines redox‐active copper centers with photoactive rhodamine‐derived ligands remains stable in acid and basic solutions from pH 2 to 14, and efficiently catalyzes dihydrogen evolution at a maximum rate of 7.88 mmol g^?1 h^?1 in the absence of a mediator and a co‐catalyst. Cyclic voltammetry, control experiments, and DFT calculations established that copper nodes with open coordination sites and favorable redox potentials, aided by spatially ordered stacking of rhodamine‐based linkers, account for the high catalytic performance of Cu‐RSH. Emission quenching, time‐resolved fluorescence decay, and transient photocurrent experiments disclosed the charge separation and transfer process in the catalytic system. The present study demonstrates the potential of crystalline copper CPs for the practical utilization of light.     

Using direct electrochemistry to probe rate limiting events during nitrate reductase turnover

Protein film voltammetry of NarGH catalysing nitrate reduction under steady state conditions provides information on events occurring within the enzyme during the catalytic cycle. In this discussion we have focused on exploring the ability of two simple catalytic schemes to reproduce the voltammetric response of NarGH; electron transfer to the enzyme's active site being described either by interfacial electron exchange (Scheme 1) or intramolecular electron delivery via the operation of an electron relay centre (Scheme 2). When the two electron reduced, catalytically competent active site of the enzyme is generated from the oxidised form in 'rapid', non-rate limiting steps of the catalytic cycle, the voltammetric behaviour of NarGH cannot be reproduced. Rather under all the conditions investigated, one electron reduction of the active site from a semi-reduced to a fully-reduced state is found to be crucial to progression through the enzyme's catalytic cycle. The catalytically relevant semi- and fully-reduced oxidation states of the NarGH active site are most likely to correspond to the Mo(V) and Mo(IV) states of the Mo(MGD)2 centre, respectively, although it is not possible to rule out the possibility that they correspond to molybdopterin based oxidation states as observed in other enzymes. We suggest that the rate of either conformational rearrangement within the semi-reduced active site or intramolecular electron delivery to the active site constitutes a defining feature in the catalytic cycle of NarGH and results in the napp approximately 1 appearance of the catalytic waveform.     

Reaction mechanism and the role of copper in the photooxidation of alcohol over Cu/Nb2O5

Loading of a small amount of copper on Nb(2)O(5) significantly enhances the activity of alcohol photooxidation without organic solvents. Alcohol is adsorbed on the Lewis acid site (Nb(V)) to form an alkoxide species. Photogenerated holes and electrons on Cu/Nb(2)O(5) are trapped by the adsorbed alkoxide and Cu(II) species to form the alkoxide carbon radical and Cu(I) species. The formed alkoxide carbon radical is converted to a carbonyl compound and then desorbed. Finally, the reduced Cu(I) sites are reoxidized by reaction with O(2). The alcohol photooxidation over Nb(2)O(5) takes place under not only UV irradiation but also under visible light irradiation up to 450 nm, although the band gap of Nb(2)O(5) is 390 nm (3.2 eV). DFT calculations reveal that 1) the surface donor level derived from the adsorbed alkoxide species is located in the forbidden band, 2) direct electron transition from the surface donor level to the conduction band takes place by absorbing a photon, 3) the excitation energy from surface donor level to the Nb 4d conduction band is lower than that from the O 2p valence band to Nb 4d. The kinetic study and FT/IR spectra suggest that Cu(I) acts as an effective desorption site for the products. Based on these results, we conclude that copper functions as an effective redox promoter and desorption site for the product.     

具有丰富介孔Cu-SAPO-34催化剂制备及其低温氨气选择性催化还原反应(英文)

氮氧化物(NO_x)是主要的大气污染物之一,与光化学烟雾、全球气候变暖等环境问题密切相关.随着汽车产业的高速发展,柴油车排放尾气中的NOx脱除成为国内外尾气催化净化领域最突出的难点之一.其中氨气选择性催化还原技术(NH_3-SCR)由于其高效率、低成本的特征已成为主要的移动源脱硝技术.目前,实际应用中最广泛的是V_2O_5-WO_3(MoO_3)/TiO_2催化剂,然而一些不可避免的因素仍然存在,比如V具有较强的毒性,较高的操作温度,较窄的活性温度窗口以及易将SO_2氧化为SO_3导致催化剂表面会有大量的硫酸盐沉积而失活等.因此很有必要开发一种无钒SCR催化剂.近年来,分子筛负载过渡金属作为催化剂引起了研究者的广泛兴趣,其中Cu-CHA分子筛催化剂因其高SCR活性,高N2选择性,较宽的温度窗口以及优异的稳定性引起研究者的广泛关注.就Cu/SAPO-34而言,传统的制备方法是利用离子交换法将Cu离子引入到SAPO-34微孔孔道中,然而由于微孔会限制Cu离子的分布,导致绝大多数Cu优先分布在分子筛外表面,从而限制了其活性发挥.Martínez-Franco课题组利用双模板一步法成功制备了Cu-SAPO-34催化剂,提高了分子筛中活性Cu离子数目.Peden课题组发现在NOx的NH_3-SCR反应中Cu-SAPO-34催化剂存在低温动力学限制.因此开发一种具有丰富介孔的多级孔Cu-SAPO-34催化剂势在必行.我们利用一步水热晶化法成功制备了一系列具有丰富介孔的Cu-SAPO-34催化剂.采用扫描电子显微镜(SEM)、透射电子显微镜(TEM)、氮气吸附-脱附、X射线衍射(XRD)、~(27)Al核磁共振(Al-NMR)、紫外可见漫反射光谱(UV-Vis DRS)、电感耦合等离子体-原子发射光谱(ICP-AES)、X射线光电子能谱(XPS)、氢气程序升温还原(H2-TPR)和电子顺磁共振(EPR)等表征手段研究了Cu-SAPO-34多级孔催化剂的物理化学性质.XRD测试结果证实,H-Cu-SAPO-34催化剂具有典型的CHA结构.TEM和N2吸附-脱附测试结果表明,H-Cu-SAPO-34催化剂具有丰富的介孔结构.Al-NMR测试结果表明,多种配位的Al物种存在于H-Cu-SAPO-34中.UV-Vis DRS测试结果证实了孤立Cu~(2+)和高分散的CuO的存在,没有观察到(Cu-O-Cu)2+和CuAl2O4物种的存在.ICP-AES和XPS测试结果表明,H-Cu-SAPO-34催化剂具有相似的Cu含量,并且H-Cu-SAPO-34-20催化剂具有最高的Cu~(2+)含量.H2-TPR测试结果表明,H-Cu-SAPO-34-20催化剂具有最低的孤立Cu~(2+)还原温度以及最高的孤立Cu~(2+)含量.这可能有利于其NH3-SCR活性提高.同时H2-TPR还表明,H-Cu-SAPO-34催化剂中存在含量不等的孤立Cu+,并且孤立Cu~(2+)是NH3-SCR反应的主要活性中心.EPR测试结果进一步表明,位于SAPO-34椭球腔内(Site(I))的孤立Cu~(2+)是该反应的主要活性位.由NO的NH3-SCR反应测试结果来看,相比于普通的Cu/SAPO-34催化剂,具有丰富介孔结构的H-Cu-SAPO-34催化剂呈现出更高的低温催化活性,同时H-Cu-SAPO-34-20催化剂具有最高的低温NH3-SCR催化活性,这与其较高的活性Cu~(2+)含量以及较低的孤立Cu~(2+)还原温度密切相关.动力学测试结果表明,所合成的H-Cu-SAPO-34多级孔催化剂具有相似的活化能(Ea=98 kJ/mol),并且该值远大于普通CHA基SCR催化剂,这意味着介孔的存在确实大大降低了反应物分子在H-Cu-SAPO-34孔道内的扩散阻力,提高了反应物分子与活性位的接触概率,从而提高了其低温NH3-SCR催化性能.     

A molecular orbital analysis of Cu(I)-catalysed cyclopropanation using diazoalkanes with CH3 and CF3 substituents

The mechanism of cyclopropanation catalysed by Cu(I) complexes has been investigated by calculation using a series of diazoalkanes containing inductive electron donating (methyl) and withdrawing (CF3) substituents and a range of metal fragments (Cu+, [(DAB)Cu]+, ClCu and (triflate)Cu). Copper-diazoalkane complexes exist as an equilibrium of C- and N-bonded isomers. Catalysis occurs through lowering of the activation energy for rate determining C-N bond cleavage of the C-bonded isomer; this is most marked for (triflate)Cu. Direct reaction of the copper-carbene complex occurs to yield stable cupracyclobutanes in all but one case. Associative substitution of the cupracyclobutane by diazoalkane completes the catalytic cycle.     

Spectroscopic and density functional studies of the red copper site in nitrosocyanin: role of the protein in determining active site geometric and electronic structure

The electronic structure of the red copper site in nitrosocyanin is defined relative to that of the well understood blue copper site of plastocyanin by using low-temperature absorption, circular dichroism, magnetic circular dichroism, resonance Raman, EPR and X-ray absorption spectroscopies, combined with DFT calculations. These studies indicate that the principal electronic structure change in the red copper site is the sigma rather than the pi donor interaction of the cysteine sulfur with the Cu 3d(x2-y2) redox active molecular orbital (RAMO). Further, MCD data show that there is an increase in ligand field strength due to an increase in coordination number, whereas resonance Raman spectra indicate a weaker Cu-S bond. The latter is supported by the S K-edge data, which demonstrate a less covalent thiolate interaction with the RAMO of nitrosocyanin at 20% relative to plastocyanin at 38%. EXAFS results give a longer Cu-S(Cys) bond distance in nitrosocyanin (2.28 A) compared to plastocyanin (2.08 A) and also show a large change in structure with reduction of the red copper site. The red copper site is the only presently known blue copper-related site with an exogenous water coordinated to the copper. Density functional calculations reproduce the experimental properties and are used to determine the specific protein structure contributions to exogenous ligand binding in red copper. The relative orientation of the CuNNS and the CuSC(beta) planes (determined by the protein sequence) is found to be key in generating an exchangeable coordination position at the red copper active site. The exogenous water ligation at the red copper active site greatly increases the reorganization energy (by approximately 1.0 eV) relative to that of the blue copper protein site, making the red site unfavorable for fast outer-sphere electron transfer, while providing an exchangeable coordination position for inner-sphere electron transfer.     

Elucidating the mechanism for the reduction of nitrite by copper nitrite reductase--a contribution from quantum chemical studies

Density functional methods have been applied to investigate the properties of the active site of copper-containing nitrite reductases and possible reaction mechanisms for the enzyme catalysis. The results for a model of the active site indicate that a hydroxyl intermediate is not formed during the catalytic cycle, but rather a state with a protonated nitrite bound to the reduced copper. Electron affinity calculations indicate that reduction of the T2 copper site does not occur immediately after nitrite binding. Proton affinity calculations are indicative of substantial pK(a) differences between different states of the T2 site. The calculations further suggest that the reaction does not proceed until uptake of a second proton from the bulk solution. They also indicate that Asp-92 may play both a key role as a proton donor to the substrate, and a structural role in promoting catalysis. In the D92N mutant another base, presumably a nearby histidine (His-249) may take the role as the proton donor. On the basis of these model calculations and available experimental evidence, an ordered reaction mechanism for the reduction of nitrite is suggested. An investigation of the binding modes of the nitric oxide product and the nitrite substrate to the model site has also been made, indicating that nitric oxide prefers to bind in an end-on fashion to the reduced T2 site.     

An assessment of the relative contributions of redox and steric issues to laccase specificity towards putative substrates

Laccases catalyze the one-electron oxidation of a broad range of substrates coupled to the 4 electron reduction of O2 to H2O. Phenols are typical substrates, because their redox potentials (ranging from 0.5 to 1.0 V vs. NHE) are low enough to allow electron abstraction by the T1 Cu(II) that, although a relatively modest oxidant (in the 0.4-0.8 V range), is the electron-acceptor in laccases. The present study comparatively investigated the oxidation performances of Trametes villosa and Myceliophthora thermophila laccases, two enzymes markedly differing in redox potential (0.79 and 0.46 V). The oxidation efficiency and kinetic constants of laccase-catalyzed conversion of putative substrates were determined. Hammett plots related to the oxidation of substituted phenols by the two laccases, in combination with the kinetic isotope effect determination, confirmed a rate-determining electron transfer from the substrate to the enzyme. The efficiency of oxidation was found to increase with the decrease in redox potential of the substrates, and the Marcus reorganisation energy for electron transfer to the T1 copper site was determined. Steric hindrance to substrate docking was inferred because some of the phenols and anilines investigated, despite possessing a redox potential compatible with one-electron abstraction, were scarcely oxidised. A threshold value of steric hindrance of the substrate, allowed for fitting into the active site of T. villosa laccase, was extrapolated from structural information provided by X-ray analysis of T. versicolor lac3B, sharing an identity of 99% at the protein level, thus enabling us to assess the relative contribution of steric and redox properties of a substrate in determining its susceptibility to laccase oxidation. The inferred structural threshold is compatible with the distance between two phenylalanine residues that mark the entrance to the active site. Interaction of the substrate with other residues of the active site is commented on.     

Morphology‐controlled Electrodeposition of Copper Nanospheres onto FTO for Enhanced Photocatalytic Hydrogen Production

Using CuSO₄ as the copper source, nanostructured copper with four different morphologies was obtained by electrodeposition method on FTO substrates. The as‐synthesized Cu/FTO samples were characterized by X‐ray diffraction (XRD), diffuse reflectance spectroscopy (DRS), Raman, X‐ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), energy‐dispersive X‐ray spectroscopy (EDS), and transmission electron microscopy (TEM). The effects of electrodeposition potential and electrodeposition time on the Cu/FTO samples and the photocatalytic performance were investigated systematically. The results showed that the Cu/FTO samples were well‐crystallized and the morphologies could be changed from nanoslices to nanodendrites structure with the negative shift of the depositing potential. The electrodeposition potential and time have a significant effect on the amount of H₂ evolution. The obtained Cu nanospheres which were prepared at the potential of −0.65 V for 600 s showed the best photocatalytic behavior. The mechanisms for the photocatalytic activities were also discussed.