Cuprous State Research Articles

Detoxification of copper and zinc from anaerobic digestate effluent by indigenous bacteria: Mechanisms, pathways and metagenomic analysis

The presence of organic-complexed copper and zinc in anaerobic digestate effluent (ADE) poses persistent ecological toxicity. This study investigated the detoxification performance and biotic responses of indigenous bacteria against ethylene diamine tetraacetic acid (EDTA)-complexed Cu(II) and Zn(II). Heavy metals (HMs) stress induced reactive oxygen species (ROS) generation and enhanced extracellular polymeric substances (EPS) secretion. At a Cu(II) influent concentration of 20.0 mg·L−1, indigenous bacteria removed 88.2% of Cu(II) within nine days. The majority of copper and zinc sequestered by bacteria were stored in the cell envelope, with over 50% of copper and 60% of zinc being immobilized. Transmission electron microscopy mapping (TEM-mapping) revealed significant mineralization of copper and zinc on the cell wall. Proteins abundant in EPS, alongside humic acid-like substances, effectively adsorbed HMs. Indigenous bacteria exhibited the capacity to reduce cupric to the cuprous state and cupric is preferentially reduced to cuprous before reaching reducing capacity saturation. Sulfur precipitation emerges as a crucial pathway for Zn(II) removal. Metagenomic analysis indicated that indigenous bacteria upregulated genes related to HMs homeostasis, efflux, and DNA repair, enhancing its resistance to high concentrations HMs. This study provided theoretical guidance for employing bacterial consortia to eliminate HMs in complex aquatic environments.

A comparative study of the thermal and hydrothermal aging effect on Cu-SSZ-13 for the selective catalytic reduction of NO with NH3

In this work, the characterizations of Cu-SSZ-13 after hydrothermal aging (HTA) and thermal aging (TA) at different temperatures (750, 800, and 850 °C) are compared, and the differences between those two serious aged samples are analyzed. With this data, the effect of steam on catalysts deactivation during hydrothermal aging is analyzed. The TA at 750 and 800 °C causes the dealumination and the agglomeration of Cu 2+ ions to CuO, resulting in the activity loss of Cu-SSZ-13. The presence of steam upon HTA at 750 and 800 °C aggravates the catalyst deactivation by increasing the Al detachment and the Cu 2+ agglomeration. The structure and cupric state are almost the same in the Cu-SSZ-13 after TA and HTA at 850 °C, respectively, indicating that the steam has little influence on the deactivation. The formation of CuAl 2 O 4 spinel is the primary reason for the deactivation after both HTA and TA at 850 °C, probably attributed to the strong interaction between Cu 2+ ions and framework Al sites at high temperatures.

Inflammation accelerates copper-mediated cytotoxicity through induction of six-transmembrane epithelial antigens of prostate 4 expression.

Copper is an essential trace metal, but imbalance in copper homeostasis can induce oxidative damage. Inflammation is a fundamental element of various pulmonary diseases. Although a positive relationship between copper and chronic pulmonary diseases has been reported, the underlying reasons are still not clear. The copper level in the sputum of patients with various pulmonary diseases was measured. An inflammatory model was established to evaluate the impact of inflammation on copper uptake in the lung. We found that the level of sputum copper was increased in patients with various pulmonary diseases, especially chronic obstructive pulmonary disease and asthma. Then, we confirmed that mice with pulmonary inflammation were susceptible to copper-mediated oxidative damage because of copper overload in lung tissue. Further investigation demonstrated that interleukin (IL)-17 and tumor necrosis factor (TNF)-α exerted synergistic effects in airway epithelial cells by upregulating the expression of six-transmembrane epithelial antigens of prostate 4 (STEAP4), a metalloreductase that reduces extracellular copper ions from the cupric state to the cuprous state and facilitates copper uptake. Inhibition of STEAP4 decreased the copper uptake of cells and inhibited copper-mediated oxidative damage. Moreover, we demonstrated that the upregulation of STEAP4 by IL-17 and TNF-α was largely dependent on TNF receptor-associated factor 4 (TRAF4). Traf4-/- mice were resistant to copper-mediated oxidative damage. Our data suggest a novel IL-17/TNF-α-TRAF4-STEAP4 axis that regulates copper homeostasis.

Coupled transport of electrons and protons in a bacterial cytochrome c oxidase-DFT calculated properties compared to structures and spectroscopies.

After a general introduction to the features and mechanisms of cytochrome c oxidases (CcOs) in mitochondria and aerobic bacteria, we present DFT calculated physical and spectroscopic properties for the catalytic reaction cycle compared with experimental observations in bacterial ba3 type CcO, also with comparisons/contrasts to aa3 type CcOs. The Dinuclear Complex (DNC) is the active catalytic reaction center, containing a heme a3 Fe center and a near lying Cu center (called CuB) where by successive reduction and protonation, molecular O2 is transformed to two H2O molecules, and protons are pumped from an inner region across the membrane to an outer region by transit through the CcO integral membrane protein. Structures, energies and vibrational frequencies for Fe-O and O-O modes are calculated by DFT over the catalytic cycle. The calculated DFT frequencies in the DNC of CcO are compared with measured frequencies from Resonance Raman spectroscopy to clarify the composition, geometry, and electronic structures of different intermediates through the reaction cycle, and to trace reaction pathways. X-ray structures of the resting oxidized state are analyzed with reference to the known experimental reaction chemistry and using DFT calculated structures in fitting observed electron density maps. Our calculations lead to a new proposed reaction pathway for coupling the PR → F → OH (ferryl-oxo → ferric-hydroxo) pathway to proton pumping by a water shift mechanism. Through this arc of the catalytic cycle, major shifts in pKa's of the special tyrosine and a histidine near the upper water pool activate proton transfer. Additional mechanisms for proton pumping are explored, and the role of the CuB+ (cuprous state) in controlling access to the dinuclear reaction site is proposed.

Revisiting dithiadiaza macrocyclic chelators for copper-64 PET imaging.

Synthesis and characterisation of a dithiadiaza chelator NSNS2A, as well as copper complexes thereof are reported in this paper. Solution structures of copper(i/ii) complexes were calculated using density functional theory (DFT) and validated by both NMR and EPR spectroscopy. DFT calculations revealed a switch in the orientation of tetragonal distortion upon protonation, which might be responsible for poor stability of the Cu(II)NSNS2A complex in aqueous media, whilst the same switch in tetragonal distortion was experimentally observed by changing the solvent. The chelator was radiolabeled with 64Cu and evaluated using PET/MRI in rats. Despite a favorable redox potential to stabilize the cuprous state in vivo, the 64Cu(II)NSNS2A complex showed suboptimal stability compared to its tetraazamacrocyclic analogue, 64Cu(TE2A), with a significant 64Cu uptake in the liver.

Stable cuprous active sites in Cu+-graphitic carbon nitride: Structure analysis and performance in Fenton-like reactions.

Cu+-based catalysts have great potential in Fenton reactions under neutral pH conditions. However, cuprous (Cu+) materials are instable in the aqueous environment. Herein, using the cheap precursors, a Cu+-graphitic carbon nitride complex with an efficient Fenton-like activity as well as relative stability was prepared. 99.2% removal of Rhodamine B with an initial concentration of 50 mg/L could be attained in 1 h. Several experimental techniques are employed to study the structure of this catalyst. Results show that after the addition of Cu, the graphitic carbon nitride (g-C3N4) network is partially destroyed and the reduced Cu is therefore firmly embedded in the fragmentary g-C3N4 sheet. The X-ray adsorption fine spectra illustrates the chemical state and the local structure of the bonded Cu. Due to the strong orbital hybridization, Cu+ could be stabilized through the coordination with pyridinic N. A two-coordinate structure with a bond length of 1.90 Å is confirmed and this structure is not changed even after the Fenton-like reaction. Singlet oxygen (1O2) and hydroxyl radicals (HO•) are produced by the rapid interaction of bonded Cu+ with H2O2 and the resulting Cu2+ can be easily reduced to its cuprous state due to its structure stability, leading to its high activity in the Fenton-like reaction.

An investigation into the leaching behaviour of copper oxide minerals in aqueous alkaline glycine solutions

Copper oxide minerals are normally extracted by acidic leaching followed by copper recovery with solvent extraction and electrowinning. However, copper oxide deposits often containing large amounts of acid consumable gangue leads to a very high acid consumption. In addition, if the oxide deposit also contains precious metals and iron bearing minerals, significant (lime) neutralisation costs are incurred to establish the conditions for subsequent cyanidation, with concomitant production of gypsum, potential formation of silver-locking jarosites and potential gel formation when acids interact with layer silicate minerals. In this study, an alternative aqueous alkaline glycine system has been employed to evaluate the batch leaching behaviour of copper oxide mineral specimens of the minerals azurite, chrysocolla, cuprite and malachite. The effects of glycine concentration and pH were investigated at ambient temperature and atmospheric pressure. Glycine concentration and pH both had a major effect on the copper extraction. Complete extraction of copper from azurite was achieved in <6h when glycine to copper ratio was 8:1. However, further investigation established the optimum leaching conditions as being pH11 and glycine to copper ratio of 4:1. Under such conditions 95.0%, 91.0%, 83. 8% and 17.4% of copper was extracted after 24h from the azurite, malachite, cuprite, and chrysocolla mineral specimens respectively. While the dissolution rates of the copper oxide minerals are markedly slower than acid leaching, the selective dissolution of copper over acid-consuming gangue minerals shows much potential. It was shown through UV–Vis spectroscopy that dissolved copper is in the cupric state and forms a neutral copper-glycinate complex under alkaline conditions. The study has shown that copper extraction from malachite, azurite and cuprite in aqueous alkaline glycine solutions is fast, whereas Cu extraction from chrysocolla was found to be poor and slow.

PH and Solvent H/D Isotope Effects on the Thermodynamics and Kinetics of Electron Transfer for Electrode-Immobilized Native and Urea-Unfolded Stellacyanin

The thermodynamics of Cu(II) to Cu(I) reduction and the kinetics of the electron transfer (ET) process for Rhus vernicifera stellacyanin (STC) immobilized on a decane-1-thiol coated gold electrode have been measured through cyclic voltammetry at varying pH and temperature, in the presence of urea and in D(2)O. Immobilized STC undergoes a limited conformational change that mainly results in an enhanced exposure of one or both copper binding histidines to solvent which slightly stabilizes the cupric state and increases histidine basicity. The large immobilization-induced increase in the pK(a) for the acid transition (from 4.5 to 6.3) makes this electrode-SAM-protein construct an attractive candidate as a biomolecular ET switch operating near neutral pH in molecular electronics. Such a potential interest is increased by the robustness of this interface against chemical unfolding as it undergoes only moderate changes in the reduction thermodynamics and in the ET rate in the presence of up to 8 M urea. The sensitivity of these parameters to solvent H/D isotope effects testifies to the role of protein solvation as effector of the thermodynamics and kinetics of ET.

Copper Catalyst Activation Driven by Photoinduced Electron Transfer: A Prototype Photolatent Click Catalyst

PET cat. While the copper(II) tren ketoprofenate precatalyst 1 (see picture) is inactive at room temperature in methanol, it is quantitatively and rapidly reduced to its cuprous state upon light irradiation to provide a highly reactive click catalyst. By simply introducing air into the reaction medium the catalysis can be switched off and then switched on again by bubbling argon followed by irradiation.

Theoretical study of role of H 2O molecule on initial stage of reduction of O 2 molecule in active site of cytochrome c oxidase

Mechanism of formation of [Fe–O–O–H, Cu B] from [Fe–O–O, Cu B] in active site of cytochrome c oxidase was theoretically investigated. It was found that the H 2O molecule is hydrogen-bonded to both Tyr244 and His290, and is a key molecule to produce Fe–O–O–H. This H 2O molecule plays a role as a carrier of a proton toward Fe–O–O to yield [Fe–O–O–H, Cu B]. Cu B atom is formally changed from cuprous state to cupric state through transportation of a proton by the H 2O molecule. A new reaction mechanism, which we call ‘water-proton transport’ (WPT) mechanism, is proposed for the initial stage of reduction of the O 2 molecule.

Crystal structure of nitrous oxide reductase from Paracoccus denitrificans at 1.6 A resolution.

N2O is generated by denitrifying bacteria as a product of NO reduction. In denitrification, N2O is metabolized further by the enzyme N2O reductase (N2OR), a multicopper protein which converts N2O into dinitrogen and water. The structure of N2OR remained unknown until the recent elucidation of the structure of the enzyme isolated from Pseudomonas nautica. In the present paper, we report the crystal structure of a blue form of the enzyme that was purified under aerobic conditions from Paracoccus denitrificans. N2OR is a head-to-tail homodimer stabilized by a multitude of interactions including two calcium sites located at the intermonomeric surface. Each monomer is composed of two domains: a C-terminal cupredoxin domain that carries the dinuclear electron entry site known as Cu(A), and an N-terminal seven-bladed beta-propeller domain which hosts the active-site centre Cu(Z). The electrons are transferred from Cu(A) to Cu(Z) across the subunit interface. Cu(Z) is a tetranuclear copper cluster in which the four copper ions (Cu1 to Cu4) are ligated by seven histidine imidazoles, a hydroxyl or water oxygen and a bridging inorganic sulphide. A bound chloride ion near the Cu(Z) active site shares one of the ligand imidazoles of Cu1. This arrangement probably influences the redox potential of Cu1 so that this copper is stabilized in the cupric state. The treatment of N2OR with H2O2 or cyanide causes the disappearance of the optical band at 640 nm, attributed to the Cu(Z) centre. The crystal structure of the enzyme soaked with H2O2 or cyanide suggests that an average of one copper of the Cu(Z) cluster has been lost. The lowest occupancy is observed for Cu3 and Cu4. A docking experiment suggests that N(2)O binds between Cu1 and Cu4 so that the oxygen of N2O replaces the oxygen ligand of Cu4. Certain ligand imidazoles of Cu1 and Cu2, as well as of Cu4, are located at the dimer interface. Particularly those of Cu2 and Cu4 are parts of a bonding network which couples these coppers to the Cu(A) centre in the neighbouring monomer. This structure may provide an efficient electron transfer path for reduction of the bound N2O.

Paramagnetic species in the plasma of dogs with lymphoma prior to and after treatment with doxorubicin: An ESR study

Doxorubicin is a potent cytostatic drug which is applied for the treatment of various kinds of malignant diseases. In spite of the routine use of this drug its major adverse effect, the dose-dependent cardiotoxicity, cannot be prevented yet. However, several clinical trials indicated that iron chelators are able to moderate the noxious effect more efficiently than radical scavenging antioxidants. This in turn supports the idea that doxorubicin–iron complexes are involved in triggering the cardiotoxicity of this drug by catalyzing the formation of oxygen radicals. However, both the mode of generation of doxorubicin–iron complexes and the consequences in vivo are not understood so far. In order to figure out whether or not doxorubicin can utilize iron from the transport protein transferrin for complex formation and prooxidative activities we studied the redox state of iron and its regulatory control by ceruloplasmin and ascorbate in the plasma of dogs suffering from malignant lymphoma by electron spin resonance spectroscopy. The respective electron spin resonance intensities prior to and after treatment with doxorubicin were compared with those from healthy controls. Our results revealed that dogs with lymphoma exhibit lower levels of paramagnetic copper in ceruloplasmin (−22%) and iron in transferrin (−33%) than healthy animals. Likewise the concentration of ascorbate radicals was lower in patients with lymphoma than in healthy subjects. The decreased cupric state of ceruloplasmin is equivalent to a diminished ferroxidase activity in plasma and therefore indicates indirectly an impaired antioxidant activity in these patients. Administration of doxorubicin in vivo further reduced the concentration of paramagnetic copper (−18%) and iron (−13%) while the concentration of ascorbate radicals remained unchanged. This decrease was also seen during the in vitro incubation of plasma with doxorubicin suggesting a direct interaction of the drug with the paramagnetic metal species. Model experiments revealed that the effect is based on a doxorubicin-induced release of iron from transferrin which is enhanced by ascorbate and the subsequent formation of doxorubicin–iron complexes. This mechanism was shown to trigger the formation of hydroxyl radicals from H 2O 2 and to cause an oxidation of the antioxidant ceruloplasmin. Our data demonstrate that cardiotoxic doxorubicin–iron complexes are not only formed in cardiomyocytes itself as generally assumed, but are also present in the circulation. Therefore, these findings provide an additional rationale for potential benefit of iron chelators during doxorubicin chemotherapy.

Superoxide-Superoxide Oxidoreductase Activity of the Captopril-Copper Complex

Background The purpose of this study was to determine whether the interaction of captopril, an angiotensin-converting enzyme inhibitor, with copper could modify the superoxide dismutase activity of this metal. Results may help to explain the interaction of captopril with reactive oxygen species in the stunned myocardium where substantial mobilization of copper and iron in the coronary flow following ischemia has been reported. Methods An assay that generates superoxide anion radicals without the intervention of metal ions was utilized. In addition, direct EPR analysis was applied to assess the redox state of copper during reactions. Results Captopril-copper complex inhibited the superoxide-mediated reduction of nitroblue tetrazolium. In addition, captopril-copper complex was able to suppress formazan production by potassium superoxide. Direct EPR analysis showed that copper was reduced to the cuprous state by captopril and remained in this state in the course of the reaction. Captopril was also stable during the dismutation reaction. Conclusions We conclude that cuprous-captopril complex is a catalytic species with properties different from those of Cu 2+ alone. A model in which sulfur acts as electron acceptor/donor in place of the metal is proposed and a mechanism of action for this complex is discussed.

Copper Effects on Ion Transport across Lamprey Erythrocyte Membrane: Cl−/OH− Exchange Induced by Cuprous Ions

We studied the effects of prelytic copper concentrations on cell volume, intracellular pH, and ion transport in lamprey erythrocytes. Ion fluxes and pH were measured by radioactive tracer technique, patch clamp, and flame photometry. Prelytic CuSO4 concentration of 100 μM caused anion-dependent intracellular acidification and increase in Cl− influx after 2 min lag-phase. In the presence of ascorbate copper effect was amplified and lag-phase was skipped. Pretreatment of the cells with N-phenyl maleimide abolished copper-induced changes completely. Copper treatment caused an increase in Na+ fluxes in both directions and a net Na+ uptake. Copper-induced Na+ transport was partially amiloride(MIA)-sensitive representing Na+/H+ exchange. The nature of the amiloride-insensitive fraction of copper-activated Na+ influx remains unknown. Cell swelling after 15 min of copper exposure induced regulatory volume decrease response involving KCl extrusion via K+ and Cl− volume-sensitive channels. We suggest that the effects of copper on ion transport fit the following sequence of events: (i) cupric ions are reduced to cuprous state on the membrane surface, (ii) electroneutral pairs CuCl and CuOH mediate chloride/hydroxyl exchange, as shown before for trialkyltin, dissipating transmembrane pH gradient, and (iii) changes in intracellular pH result in the activation of the Na+/H+ exchange and consecutive volume changes cause the RVD response.

Preparation of Cu&lt;sup&gt;+&lt;/sup&gt;-Containing Phosphate Glasses

Cu+-containing calcium phosphate glasses have been prepared by remelting Cu-containing glass (a) under a controlled atmosphere of low oxygen pressure, and (b) with silicon powder as a reducing agent. About 96% of copper became cuprous ion by remelting under an oxygen partial pressure of 10-10Pa at 1400°C for 8h. However, further reduction of oxygen pressure was not effective but led to the vaporization of phosphor, resulting in an increase in the devitrification tendency of the melt. Remelting with silicon powder was much more effective in reducing Cu2+ to Cu+ state. More than 99% of copper could be reduced to cuprous state by the remelting at 1250°C for 0.5h when the ratio of silicon to copper, Si/Cu, was equal to 2.5 or larger. The emission intensity from the Cu+-containing glass became maximum when Si/Cu=2.5.

Utilization of Copper as a Paramagnetic Probe for the Binuclear Metal Center of Phosphotriesterase

Bacterial phosphotriesterase catalyzes the hydrolysis of organophosphate triesters. To be active, the enzyme requires that two divalent cations are bound. These metal ions are bound in close proximity to one another as a binuclear center. To characterize the structure and function of the binuclear metal binding sites, we have prepared the copper-substituted enzyme. The kinetic data indicate that this enzyme is essentially inactive toward the hydrolysis of phosphotriesters. The EPR signal arising from the copper-substituted enzyme is nearly axial, with g|| = 2.24 and g⊥ = 2.05 and shows at least seven superhyperfine transitions in the g⊥ region with A⊥ = 1.45 × 10−3 cm−1. These splittings are consistent with the direct ligation of more than one nitrogen to the metal center. The average spin quantitation of copper-substituted enzymes are 0.6 spin/Cu, approximately half of that observed for noninteracting Cu2+ ions. The spin intensity increases to ca. 1 spin/Cu when samples are denatured with acid. The binding of metal ions to the designated α and β sites is highly synergistic (i.e., the metal ions bind in pairs). Mixed metal complexes of the type Cu/X and X/Cu were prepared. When X is a diamagnetic ion (Zn2+ or Cd2+), the spin quantitation increases, but when X is the paramagnetic Co2+ ion, the spin quantitation decreases. This behavior indicates that the low spin intensities observed for copper-substituted phosphotriesterase arise from spin-coupling of the two adjacent Cu2+ ions. The addition of dithiothreitol, ascorbate, or dithionite to the copper-substituted phosphotriesterase results in nearly the complete loss of spin intensity. This indicates that the bound coppers can be reduced to the cuprous state.

In situ e.s.r. monitoring of the coordination and oxidation states of copper in Cu-ZSM-5 up to 500°C in flowing gas mixtures: 2. Interaction with CH 4 and CO

A flow cell was used for the monitoring of the state of copper ions in Cu-ZSM-5 in situ at high temperatures by e.s.r. Reduction of the most reactive square-planar coordinated Cu 2+ ions to Cu + occurs at 500°C in methane flow. The reoxidation of cuprous to cupric ions at 500°C by NO or O 2 is very fast; and, at 500°C, in mixtures of CH 4 with excess oxygen, most of the copper in CuH-ZSM-5 retains the cupric state. The formation of weak adsorption complexes between isolated Cu 2+ cations and CO molecules at 200°C leads to a measurable change in coordination. An activated formation of strong adsorption complexes, with charge transfer, takes place on treatment of the CuH-ZSM-5 by CO at 100–300°C. Subsequent heating in He flow removes all ligands at T > 400°C. At 500°C, in CO flow a slow reduction of Cu 2+ takes place. Cooling of the CuH-ZSM-5 in a mixture [CO + NO] is accompanied by a change in Cu 2+ coordination which is indicative of a mixed adsorption complex.

Electrochemical preparation of cuprous oxide powder: Part I. Basic electrochemistry

The preferred process for the production of cuprous oxide powder is via the anodic dissolution of copper in alkaline solution of sodium chloride. The principal reactions are as follows: $$\begin{gathered} Cu + nCl^ - = CuCl_n^{1 - n} (n = 2, 3) \hfill \\ 2H_2 O + 2e = H_2 \uparrow + 2OH^ - \hfill \\ 2CuCl_n^{1 - n} + 2OH^ - = Cu_2 O \downarrow + 2nCl^ - + H_2 O \hfill \\ \end{gathered} $$ In the present investigation the basic electrode processes were studied systematically under a broad range of conditions using linear sweep voltammetry. Variables studied include the concentration of sodium chloride and sodium hydroxide (i.e., alkalinity), temperature of the solution, two categories of additives (an inhibitor for preventing the deposition of spongy metallic copper powder on the cathodes, and a chemical reducing agent for reducing the cupric ions to the cuprous state), and the effect of carbonate ions (resulting from the spontaneous absorption of carbon dioxide from the air by sodium hydroxide). Useful guidelines concerning the electrolysis conditions, additives, and the concentration limit of carbonate ions have been established. The proper operating conditions can be considered to be as follows: 80–85°C, NaCl 240–260 gl−1, NaOH below 1 gl−1. Conditions pertaining to the use of additives are the following: calcium gluconate 0–5 gl−1, Na2CrO4 below 0.5 gl−1, Na2Cr2O7 below 0.25 gl−1, NH2OH·HCl below 2.5 gl−1, N2H4·H2O below 2.5 gl−1, sucrose 0–5 gl−1. Special attention must be given to eliminate or reduce the presence of carbonate ions in the electrolyte below 0.25 gl−1 Na2CO3.

Chemical Reactivity of Metal Particles Produced by Laser Ablation into Liquids

ABSTRACTSpectroscopic evidence of the chemical differences between metal particles produced by laser ablation of metals into reactive fluid solvents and metal particles produced by less energetic means is presented. Specifically. 1) ablation of copper into fluid methanol produces metal particles in which some fraction of the metal is present In the cuprous oxidation state and 2) ablation of nickel into tetrahydrofuran results In metal particles less reactive to the solvent than those produced by ultrasound promoted chemical reduction.

Characterization of copper atoms in bilirubin oxidase by spectroscopic analyses.

Bilirubin oxidase [EC 1.3.3.5], purified from the culture medium of Myrothecium verrucaria, was found to contain two blue copper atoms per protein molecule with a molecular weight of ca. 52 kDa. The two copper atoms were estimated to be in the all cupric state by the cuproine colorimetric method and also atomic absorption analysis. We could remove the reduce cuprous ions from the holo enzyme by adding ascorbate, followed by a KCN solution, yielding an apo-enzyme with no activity. The apo-enzyme can be reconstituted with Cu or other divalent cations such as Co, Fe, and Cd, with accompanying recovery of the enzyme activity. The activity recovery depended upon the species of cation employed; Cu being most effective, an almost 100% recovery, and Cd the least, only a 25% recovery. We could obtain information on the copper ions and their coordination structure by spectroscopic analyses of the apo- and reconstituted enzymes, obtaining such as absorption, CD, MCD, and XPS spectra. The bilirubin oxidase catalyzed-reaction was a second order reaction with respect to copper bound with protein. The donor set was of the CuSS*N2 (S = Cys, S* = Met, N = His) type, i.e., the same as in the case of blue copper proteins. On studying the Co-substituted enzyme, it was revealed that the copper site of the enzyme had a 4-coordinated structure.