Removal of type 2 Cu from ascorbate oxidase and laccase by reaction with n,n-diethyldithiocarbamate

The type 2 Cu of ascorbate oxidase from zucchini peelings can be rapidly removed by reaction with a tenfold excess N,N-diethyldithiocarbamate (DDC) in air, while other chelating agents, such as EDTA, require anaerobic reducing conditions. The type 2 Cu of laccase from Rhus vernicifera is never removed under aerobic conditions. In anaerobiosis and in the presence of a reducing agent, EDTA is also unable to remove the copper unless a smaller lipophilic molecule (DDC or dimethylglyoxime) is present, acting as a mediator. Type 1 Cu is not involved in the reaction of ascorbate oxidase with DDC, but reduction of type 3 Cu is probably required for type 2 Cu depletion, suggesting interdependence of type 2 and type 3 copper. Type 2 Cu is less exposed in laccase, possibly because of the large carbohydrate content of this protein.     

Copper toxicity: evidence for the conversion of cupric to cuprous copper in vivo under anaerobic conditions.

We have determined the toxicity to cells of Escherichia coli B of cupric copper applied under aerobic and anaerobic conditions in two ways. The growth of cells in liquid medium incorporating cupric copper shows differential inhibition, comparing aerobic and anaerobic conditions--toxicity being greater under anoxia. The growth of colonies upon agar plates incorporating cupric copper does not show such a differential effect. We conclude that colonies on plates are largely anoxic even when incubated aerobically. EPR spectra of cells obtained at various times after application of cupric copper under anoxic conditions indicate the conversion of a considerable proportion of the Cu(II) to a non-paramagnetic species, probably Cu(I). We demonstrate that Cu(I) is more toxic than Cu(II) to cells when applied under anoxic conditions and conclude that the difference in toxicity of Cu(II) applied to cells under aerobic and anaerobic conditions results from the greater extent of reduction of Cu(II) to Cu(I) under anaerobic conditions.     

Cu(I) analysis of blue copper proteins.

A simple colorimetric test for the Cu(I) content in blue copper proteins is described. The procedure is based on the formation of a complex between Cu(I) and 2,2'-biquinoline in an acetic acid medium. Analyses of spinach plastocyanin, Pseudomonas aeruginosa azurin and Rhus vernicifera stellacyanin show that the cysteine residue in the type 1 site does not induce Cu(II) reduction under our conditions. There is evidence in laccase samples for the presence of an endogenous reductant that can reduce 0.14 +/- 0.04 mol of Cu(II)/mol of protein; however, the addition of EDTA eliminates the interference. The analysis shows that 25 +/- 2% of the type 3 copper ions are in the reduced form in the resting enzyme and that 80 +/- 15% of the type 3 copper ions are reduced in preparations of type-2-depleted laccase. There is growing interest in the development of chemically modified forms of laccase, and our method should be very useful for establishing the valence state of the metal centres in the various derivatives.     

Copper(I)-bleomycin: structurally unique complex that mediates oxidative DNA strand scission

Copper(I)-bleomycin [Cu(I) X BLM] was characterized in detail by 13C and 1H NMR. Unequivocal chemical shift assignments for Cu(I) X BLM and Cu(I) X BLM X CO were made by two-dimensional 1H-13C correlated spectroscopy and by utilizing the observation that Cu(I) X BLM was in rapid equilibrium with Cu(I) and metal-free bleomycin, such that individual resonances in the spectra of BLM and Cu(I) X BLM could be correlated. The binding of Cu(I) by bleomycin involves the beta-aminoalaninamide and pyrimidinyl moieties, and possibly the imidazole, but not N alpha of beta-hydroxyhistidine. Although no DNA strand scission by Cu(II) X BLM could be demonstrated in the absence of dithiothreitol, in the presence of this reducing agent substantial degradation of [3H]DNA was observed, as was strand scission of cccDNA. DNA degradation by Cu(I) X BLM was shown not to depend on contaminating Fe(II) and not to result in the formation of thymine propenal; the probable reason(s) for the lack of observed DNA degradation in earlier studies employing Cu(II) X BLM and dithiothreitol was (were) also identified. DNA strand scission was also noted under anaerobic conditions when Cu(II) X BLM and iodosobenzene were employed. If it is assumed that the mechanism of DNA degradation in this case is the same as that under aerobic conditions (i.e., with Cu(I) X BLM + O2 in the presence of dithiothreitol), then Cu X BLM must be capable of functioning as a monooxygenase in its degradation of DNA.     

Kinetic studies of Rhus vernicifera laccase. Role of the metal centers in electron transfer.

The reactions of Rhus vernicifera (monophenol,dihydroxyphenylalanine: oxygen oxidoreductase, EC 1.14.18.1) with the reducing substrates hydroquinone and ascorbic acid have been investigated with the stopped-flow technique. Rhus laccase appears to be present in two molecular forms with a pH-sensitive equilibrium constant regulating the relative concentrations of each species. A model for the reaction of Rhus laccase with reducing substrates has been formulated. The model is similar to one formulated earlier for the anaerobic reduction of laccase from Polyporus versicolor (Andréasson, L.-E., Malstr?m, B.G., Str?mberg, C. and V?nng?rd, T. (1973) Eur. J. Biochem. 34, 434-439) and accounts for the reduction also of this enzyme. The essentials of the model are as follows: Electrons are taken up from reductants one at a time. The type 1 Cu2+ has a central role in mediating the transfer of at least one of the electrons needed for the reduction of the co-operative two-electron acceptor. Intramolecular reactions determine the concentrations of two molecular forms of the enzyme and influence the rate of reduction of the two-electron acceptor. The model, which has been used for successful simulations of the anaerobic reduction of Rhus laccase, is capable of explaining the reduction of laccases also in the presence of the inhibitor F-. In addition, the model gives an explanation of the behaviour of the laccases when reducing substrates and O2 are simultaneously present and is consistent with earlier observations of the post-steady-state reduction of the type 1 Cu2+ and the two-electron accetor (Holwerda, R.A. and Gray, H.B. (1974) J. Am. Chem. Soc. 96, 6008-6022).     

Enhanced biodegradation of cyclotetramethylenetetranitramine (HMX) under mixed electron-acceptor condition

The biodegradation of cyclotetramethylenetetranitramine, commonly known as 'high melting explosive' (HMX), under various electron-acceptor conditions was investigated using enrichment cultures developed from the anaerobic digester sludge of Thibodaux sewage treatment plant. The results indicated that the HMX was biodegraded under sulfate reducing, nitrate reducing, fermenting, methanogenic, and mixed electron accepting conditions. However, the rates of degradation varied among the various conditions studied. The fastest removal of HMX (from 22 ppm on day 0 to < 0.05 ppm on day 11) was observed under mixed electron-acceptor conditions, followed in order by sulfate reducing, fermenting, methanogenic, and nitrate reducing conditions. Under aerobic conditions, HMX was not biodegraded, which indicated that HMX degradation takes place under anaerobic conditions via reduction. HMX was converted to methanol and chloroform under mixed electron-acceptor conditions. This study showed evidence for HMX degradation under anaerobic conditions in a mixed microbial population system similar to any contaminated field sites, where a heterogeneous population exists.     

Comparative Characterization of Methods for Removal of Cu(II) from the Active Sites of Fungal Laccases

Copper-containing sites of laccases isolated from the Basidiomycetes Coriolus hirsutus and Coriolus zonatus were characterized by optical methods and EPR spectroscopy. Methods for preparation of fungal laccase derivatives free from type 2 copper ions were compared. The data of EPR spectroscopy and spectrophotometric titration of copper sites showed that only a modified method based on the use of bathocuproine as a chelator for type 2 copper yielded laccase derivatives completely free from type 2 copper. The original enzymes can be reconstituted from the derivatives by dialysis under anaerobic conditions, resulting in complete recovery of native conformation of the protein molecule and the structure of the copper-containing site.     

Investigation of Cu(I)-dependent 2,4,5-trihydroxyphenylalanine quinone biogenesis in Hansenula polymorpha amine oxidase

Copper, a mediator of redox chemistries in biology, is often found in enzymes that bind and reduce dioxygen. Among these, the copper amine oxidases catalyze the oxidative deamination of primary amines utilizing a type(II) copper center and 2,4,5-trihydroxyphenylalanine quinone (TPQ), a covalent cofactor derived from the post-translational modification of an active site tyrosine. Previous studies established the dependence of TPQ biogenesis on Cu(II); however, the dependence of cofactor formation on the biologically relevant Cu(I) ion has remained untested. In this study, we demonstrate that the apoform of the Hansenula polymorpha amine oxidase readily binds Cu(I) under anaerobic conditions and produces the quinone cofactor at a rate of 0.28 h(-1) upon subsequent aeration to yield a mature enzyme with kinetic properties identical to the protein product of the Cu(II)-dependent reaction. Because of the change in magnetic properties associated with the oxidation of copper, electron paramagnetic resonance spectroscopy was employed to investigate the nature of the rate-limiting step of Cu(I)-dependent cofactor biogenesis. Upon aeration of the unprocessed enzyme prebound with Cu(I), an axial Cu(II) electron paramagnetic resonance signal was found to appear at a rate equivalent to that for the cofactor. These data provide strong evidence for a rate-limiting release of superoxide from a Cu(II)(O(2)(.)) complex as a prerequisite for the activation of the precursor tyrosine and its transformation for TPQ. As copper is trafficked to intracellular protein targets in the reduced, Cu(I) state, these studies offer possible clues as to the physiological significance of the acquisition of Cu(I) by nascent H. polymorpha amine oxidase.     

A novel mixed valence form of Rhus vernicifera laccase and its reaction with dioxygen to give a peroxide intermediate bound to the trinuclear center

Rhus vernicifera laccase, in a novel mixed valence state [T1oxT23red: type 1 Cu as Cu(II), and type 2 and 3 Cus as Cu(I)], was formed by reacting Cu(I) on the type 2 Cu-depleted laccase [T1oxT3red: type 1 Cu as Cu(II) and type 3 Cus as Cu(I)] under argon. Contrary to T1oxT3red, T1oxT23red was highly reactive with dioxygen, and gave the three transient bands at 340, 475, and 680 nm due to the two-electron reduced form of dioxygen [charge transfer bands from peroxide to Cu(II)]. The first order decays were highly dependent on pH, which led to the successful detection of the intermediate for ca. 2 h at pH 7.5. Another mixed valence derivative, T12oxT3red [type 1 and type 2 Cus as Cu(II), and type 3 Cus as Cu(I)] prepared through the action of Cu(II) on T1oxT3red was not reactive with dioxygen, but showed high enzyme activity as to the oxidation of N,N-dimethyl-p-phenylenediamine. The whole reaction mechanism of the reduction of dioxygen by laccase was proposed based on the present results together with data for the former detection and characterization of the three-electron reduced form of dioxygen [Huang, H. et al. (1999) J. Biol. Chem. 274, 46, 32718-32724].     

Thiol-copper(I) and disulfide-dicopper(I) complex O2-reactivity leading to sulfonate-copper(II) complex or the formation of a cross-linked thioether-phenol product with phenol addition

In order to better understand copper mediated oxidative chemistry via ligand-Cu(I)/O(2) reactivity employing S-donor ligands for copper, O(2)-reactivity studies of the copper(I) complexes (1 and 2, Chart 2) have been carried out with a tridentate N(2)S thiol ligand (1-(N-methyl-N-(2-(pyridin-2-yl)ethyl)amino)propane-2-thiol; L(SH)) or its oxidized disulfide form (L(SS)). Reactions of [L(SH)Cu(I)](+) (1) and [L(SS)(Cu(I))(2)(X)(2)](2+) (2) with O(2) give approximately 90% and approximately 70% yields of [L(SO3)Cu(II)(MeOH)(2)](+) (3), respectively, where L(SO3) is S-oxygenated sulfonate; 3 was characterized by electrospray ionization (ESI) mass spectrometry and X-ray crystallography. Mimicking TyrCys galactose oxidase cofactor biogenesis, a new C-S bond is formed (within new thioether moiety L(SPhOH)) from cuprous complex (both 1 and 2) dioxygen reactivity in the presence of 2,4-tBu(2)-phenolate. In addition, the disulfide ligand (L(SS)) reacts with 2equiv. cupric ion salts and the phenolate to efficiently give the cross-linked product L(SPhOH) in high yield (>90%) under anaerobic conditions. Separately, complex [L(SPhO)Cu(II)(ClO(4))] (4), possessing the cross-linked L(SPhOH), was characterized by ESI mass spectrometry and X-ray crystallography.     

Cu(0) as the reaction additive in purge-free ATRP-assisted DNA detection

We report here the use of Cu(0) as a reducing reagent to eliminate dissolved oxygen-induced ATRP catalyst oxidation and radical chain termination. It eliminates the need for an inert environment as in a conventional ATRP reaction and allows DNA detection to be conducted in a purge-free fashion. 2-Hydroxyethylmethacrylate (HEMA) is the model monomer in our study, and Cu(I)/2,2'-dipyridyl-based complexes are used as the reaction catalyst. The amount of polymer chains grafted on the substrate upon reaction termination is quantified by measuring the final film thicknesses using ellipsometry. Synthesis of PHEMA films atop both small molecules and ssDNA in the presence of a limited amount of air at room temperature is achieved by adding Cu(0) as the reducing reagent. No compromised DNA detection sensitivity is noticed, though a 10:1 ratio of Cu(0):Cu(I) is required to achieve optimal polymer growth. Successful employment of Cu(0) as the reducing agent eliminates the cumbersome purging process in ATRP and renders the ATRP-assisted DNA sensing method more portable, simpler, and more time-efficient.     

1H NMR studies of the reactions of copper(I) and copper(II) with D-penicillamine and glutathione.

Reactions of copper ions with D-penicillamine (PSH) have been monitored by 1H NMR spectroscopy in the presence and absence of glutathione (GSH) under aerobic as well as anaerobic conditions. In D2O solution at pD = 7.4, PSH coordinates to Cu+ to form PS(-)-Cu+ under argon atmosphere as revealed from the broadening of each signal. In the presence of dioxygen, the complex was converted to the well-characterized purple cluster species consisting of Cu+, Cu2+, and PS2-. Addition of GSH into this solution quickly decomposed the cluster by the reduction of Cu2+ to Cu+. The cluster species was, however, reproduced after several hours because of the oxidation of Cu+ back to Cu2+. The solution containing both PSH and GSH formed three possible disulfides, PSSP, GSSG, and PSSG, under aerobic conditions. Addition of Cu+ to this solution again produced the purple cluster through several redox reactions. On the basis of these results, it was concluded that the co-existence of PSH and/or PSSP with Cu+ and/or Cu2+ leads to the formation of the stable cluster species regardless of the presence or absence of the other thiols such as GSH. This must be one of the reasons why PSH works in living cells as an effective drug for the Wilson disease.     

Copper- and Arene-Catalyzed Cleavage of DNA

DNA was found to be cleaved by arenes and copper(II) salts in neutral solutions. The efficiency of this reaction is comparable with the DNA cleavage by such systems as Cu(II)–phenanthroline and Cu(II)–ascorbic acid in efficiency, but, unlike them, it does not require the presence of an exogenous reducing agent or hydrogen peroxide. The Cu²⁺–arene system does not cleave DNA under anaerobic conditions. Catalase, sodium azide as well as bathocuproine, a specific chelator of Cu(I), completely inhibit the reaction. Our results suggest that Cu(I) ions, superoxide radical and singlet oxygen participate in this reaction. It was shown by EPR and spin traps that the reaction proceeds with the formation of alkoxyl radicals capable of inducing breaks in DNA molecules. An efficient cleavage of DNA in the Cu(II)–o-bromobenzoic acid system requires the generation of radicals under the conditions of formation of a specific copper–DNA–o-bromobenzoic acid complex, in which copper ions are likely to be coordinated with oxygen atoms of the DNA phosphate groups.     

Combined effect of copper and initial pH of the culture on production of laccase and manganese peroxidase by Stereum hirsutum (Willd) Pers

Stereum hirsutum is a white-rot fungus which produces laccase and manganese peroxidase as part of its ligninolytic system. Maximal ligninases production (under the conditions studied) was obtained in the media containing 250 microM Cu++ along with a pH value of 5.5. The fitting of ligninase production to a linear equation showed negative interaction between increasing values of pH and concentration of copper.     

Copper-catalyzed cleavage of DNA by arenes

DNA was found to be cleaved in neutral solutions containing arenes and copper (II) salts. The reaction is comparable in efficiency with the DNA cleavage by such systems as Cu(II)-phenanthroline and Cu(II)-ascorbic acid, but, in contrast to the latter, the system Cu(2+)-arene does not require the presence of an exogenous reducing agent or hydrogen peroxide. The system Cu(2+)-arene does not cleave DNA under anaerobic conditions. Catalase, sodium azide, and bathocuproine, which is a specific chelator of Cu(I), completely inhibit the reaction. The data obtained allow one to suppose that Cu(I) ions, superoxide radical, and singlet oxygen participate in the reaction. It has been shown by the EPR method using spin traps that the reaction proceeds with formation of alkoxyl radicals, which can insert breaks in the DNA molecule. For effective cleavage of DNA in the Cu(II)-o-bromobenzoic acid system, the radicals have to be generated by a specific copper-DNA-o-bromobenzoic acid complex, in which copper ions are most probably coordinated with oxygen atoms of the DNA phosphate groups. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.     

Quantitative Cu(I) determination using X-ray absorption edge spectroscopy: oxidation of the reduced binuclear copper site in type 2 depleted Rhus laccase

We report a procedure, through difference comparison of X-ray absorption edge spectra, for the quantitative determination of Cu(I) content in copper complexes of mixed oxidation state composition. This technique is tested on copper model systems and then used to quantitatively determine that untreated T2D Rhus laccase contains 70 +/- 15% Cu(I). Whereas excess ferricyanide is demonstrated not to alter the Cu(I) content of the untreated T2D, aqueous peroxide and nitrite at pH 6.0 are shown to oxidize the cuprous type 3 site and generate met T2D protein forms.     

Characterization and immobilization of the laccase from Pleurotus ostreatus and its use for the continuous elimination of phenolic pollutants

A laccase, the only ligninolytic enzyme produced by the basidiomycete Pleurotus ostreatus strain RK 36 was purified to homogeneity and characterized. The enzyme is a monomeric protein with a molecular weight of 67 000 Da and an isoelectric point of 3.6. Type I and type III Cu(2+) centers were identified by spectrophotometry. With syringaldazine as substrate laccase showed the highest oxidation rates at pH 5.8, 50 degrees C, and in 40 mM phosphate buffer. Among the tested stabilization parameters laccase retained most of its activity in high ionic buffer, pH 10, -20 degrees C, in the presence of 10 mM benzoic acid and with 35% ethylene glycol respectively. Crude laccase was covalently immobilized to Eupergit((R))C. Benzoate was found to stabilize the enzyme during the immobilization process. The activity loss of laccase during 10 days at 25 degrees C storage was 2% on average. Continuous elimination of 2,6-dimethoxyphenol by immobilized laccase was carried out in a packed bed reactor followed by filtration of the formed precipitate. The solubility of the polymerisates of oxidized syringaldazine, o-dianisidine, and 2,6-dimethoxyphenol with respect to temperature, pH-value and organic solvents were examined. The precipitates were found to be insoluble under non-extreme environmental conditions.     

Effect of temperature on ppp(A2''p)nA binding protein activities in rabbit reticulocyte lysates and other mammalian extracts

New epr features consistent with a novel type of Cu(II) are observed in partially reduced Type 2 copper depleted laccase molecules. Cu(II) hyperfine lines appear near 2590 G and 2770 G, and a rhombic g1 feature is also observed. These reflect a Cu(II) emergent on reductive disruption of the binuclear Type 3 site in T2D laccase. Additionally, much of the new, magnetically isolated Cu(II) is retained on full reoxidation of partly reduced Type 2 copper depleted laccase. The proportion of disrupted Type 3 Cu(II) sites remaining after reoxidation appears to depend on the prior distribution of electrons within T2D laccase.     

Cu2+ suppresses GABA(A) receptor-mediated responses in rat sacral dorsal commissural neurons

The effect of copper ions (Cu(2+)) on gamma-aminobutyric acid (GABA)-induced responses in acutely dissociated neurons from the rat sacral dorsal commissural nucleus (SDCN) was investigated using a nystatin-perforated patch recording configuration under voltage clamp conditions. The application of Cu(2+) to SDCN neurons reversibly suppressed the GABA (10 microM)-activated Cl(-) current (I(GABA)) in a concentration-dependent manner (1-1000 microM; IC(50) = 24.5 microM). In the presence of Cu(2+) (30 microM), the concentration-response curve of GABA was shifted rightward without reducing I(GABA) recorded under the maximally effective concentration of GABA, thus indicating a dependence of Cu(2+) action on GABA concentration. Inhibition of GABA (10 microM) responses by 30 microM Cu(2+) was essentially voltage independent and was not accompanied by a shift in the reversal potential of the currents. Cu(2+) antagonized the suppressive effect of Zn(2+) in a concentration-dependent manner, suggesting competition between Cu(2+) and Zn(2+) for similar binding sites. These data demonstrate that Cu(2+) is a potent inhibitor of GABA(A) receptor-mediated responses, implying a possible modulatory effect of Cu(2+) on GABAergic synaptic transmission in the mammalian SDCN.     

Effect of nutrient nitrogen and manganese on manganese peroxidase and laccase production by Lentinula (Lentinus) edodes

Abstract Lentinula (Lentinus) edodes , strain LS4, produces manganese-dependent peroxidase (MnP) and laccase, but not lignin peroxidase, when grown on a defined medium with glucose as sole carbon source. MnP production is suppressed by nitrogen whereas highest levels of laccase were observed when the fungus was grown under high nitrogen (26 mM) conditions. Both the titre and time of appearance of MnP were affected by the concentration of Mn in the culture medium with highest enzyme levels recorded in cultures supplemented with 1.1 ppm Mn. Purified MnP from L. edodes LS4 has an apparent M _r of 59000 and a p I of 5.6, and differs in several respects from a MnP isolated from L. edodes grown on a commercial wood substrate.