Laura Morpurgo

Selective removal of type 2 copper from Rhus vernicifera laccase

Rhus vernicifera lactase contains, like fungal lactase, one Type 1, one Type 2 and two Type 3 copper ions [I] , which can all be removed by cyanide at pH 8.0 in a reversible process [2]. In the case of fungal lactase a method is described [3] which allows reversible removal of only the Type 2 copper. In view of the many analogies presented by these two proteins we have investigated the possibility of obtaining, also in the case of Rhus vemicifera lactase, selective removal of Type 2 copper. A method for the preparation of a protein depleted of Type 2 copper is reported in this communication.

Reconstitution of stellacyanin as a case of direct Cu(I) transfer between yeast copper thionein and ‘blue’ copper apoprotein

It was of interest to examine whether yeast Cu‐thionein could be used to transfer the thiolate bound copper directly into the copper binding site of ‘blue’ apoproteins which contain free thiol groups. In particular apo‐stellacyanin was used in the present study and it was found to be able to accept Cu(I) from yeast Cu‐thionein, without any detectable unspecific Cu(II) intermediate, both aerobically and anaerobically.

Anion complexes of Cu(II) bovine carbonic anhydrase.

Abstract 1. The stability constants of some anion complexes of bovine Cu(II) carbonic anhydrase have been determined. They are found to be three-four orders of magnitude higher than those of the corresponding complexes of bovine superoxide dismutase, aqueous copper, and copper compounds of low molecular weight. Stabilization can be achieved either through the additional formation of a hydrogen bond between the anion and the protein in some cases or through donor-acceptor or charge-transfer type interactions in halide complexes. 2. Room temperature experiments show that the EPR spectrum of the cyanide complex is unaffected by pH, so that the change observed by Taylor and Coleman (1) must be produced by freezing. 3. The properties-of halide complexes of Cu(II), Co(II), and Zn(II) carbonic anhydrases are discussed and it is proposed that they give rise to pentacoordinate metal species. This may explain previous NMR data (2) without excluding water as a metal ligand at low pH. 4. The p K a = 8.0 and the HCO 3 − binding constant ( K HCO 3 = 3.5 × 10 3 ) for Cu(II)BCA are discussed in relation to the lack of activity of Cu(II) carbonic anhydrases.

Reaction of N,N-diethyldithiocarbamate and other bidentate ligands with Zn, Co and Cu bovine carbonic anhydrases: Inhibition of the enzyme activity and evidence for stable ternary enzyme-metal-ligand complexes

The reactions with N,N-diethyldithiocarbamate (DDC) of zinc, cobalt and copper carbonic anhydrase from bovine erythrocytes were investigated. The native zinc enzyme was inhibited by DDC, but no removal of zinc could be detected even at a very high [ligand]/[protein] ratio. At identical pH values a larger inhibitory effect was found for the cobalt enzyme. The metal was removed by DDC from the protein at pH less than 7.0. No cobalt removal occurred at pH 10, where a stable ternary complex with the enzyme-bound Co(II) was detected. Its optical and EPR spectra are indicative of five-coordinate Co(II). The reaction of the Cu(II) enzyme with stoichiometric chelating agent was marked by the appearance of an electronic transition at 390 nm (epsilon = 4300 M-1 X cm-1). Metal removal from the copper enzyme readily occurred as the ligand was in excess over the metal, with parallel appearance of a band at 440 nm, which was attributed to the free Cu(II)-DDC complex. Also, in the case of the copper enzyme an alkaline pH was found to stabilize the ternary adduct with the diagnostic 390 nm band. EPR spectra showed that the ternary adduct is a mixture of two species, both characterized by the presence in the EPR spectrum of a superhyperfine structure from two protein nitrogens and by a low g parallel value, indicative of coordination to sulfur ligands. It is suggested that the two species contain the metal as penta- and hexacoordinated, respectively. Measurements of the longitudinal relaxation time, T1, of the water protons suggested that water coordination is retained in the latter case. Hexacoordination with retention of water is also proposed for the Cu(II) derivatives with the bidentate oxalate and bicarbonate anions, unlike the corresponding Co(II) derivatives, which are pentacoordinated. Different coordination of Co(II) and Cu(II) adducts may be relevant to the difference of activity of the two substituted enzymes.

LIGAND BINDING TO THE BLUE COPPER CENTER OF HORSE LIVER ALCOHOL DEHYDROGENASE

The catalytic zinc ion of horse liver alcohol dehy- drogenase (LADH) is liganded by 2 thiol groups (Cys 46 and 174) and one imidazole nitrogen (His 67) its fourth ligand being water [ 11. Incorporation of Cu*+ into the catalytic zinc binding site of horse liver alcohol dehydrogenase (EC 1 .l .l .l) produces a metastable cupric protein with optical and EPR spectra reminiscent of the so-called ‘blue’ copper proteins [2]. The blue Cu*+ center in cupric alcohol dehydrogenase differs how- ever from the known blue protein metal centers since at least one co-ordination site is occupied by a water molecule. This water molecule is in rapid exchange with solvent water [3]. Small ligand molecules such as pyrazole, an ethanol-competitive inhibitor of the zinc enzyme, are also able to coordinate to the copper ion [2]. This makes possible the use of the copper LADH system as a model not only for the study of structural features of blue cupric centers but also of its chemical behaviour, e.g., the redox reaction mechanisms. Here, we describe results of EPR and optical absorption spectroscopy studies of several complexes of Cu-LADH with small anionic ligand molecules, such as cyanide, azide, imidazole and 2-mercaptoethanol. These com- plexes were found to be metastable and their EPR spectra were indicative of a geometry more tetragonal than in the unliganded cupric enzyme. Binding of NAD+ and NADH to the cupric enzyme gave rise to EPR spectra similar to those observed in ‘blue’ copper centers. 2.

Anion binding to Rhus vernicifera laccase

Abstract 1. 1. At least two sites of Rhus vernicifera laccase are involved in the reaction with N 3 − , that is the Type 2 Cu(II), at the higher anion concentrations, and possibly the EPR non-detectable copper, near to stoichiometric concentrations. 2. 2. The spectral features of Type I Cu(II) do not change in the presence of N 3 − , but its redox potential increases. Conversely in the presence of a reducing species, such as ferrocyanide, the N 3 − affinity of Type 2 Cu(II) increases considerably indicating cooperativity of the two copper sites. 3. 3. Inhibition of the ferrocyanide-oxidase activity of laccase by N 3 − can be related to binding to Type 2 Cu(II) as both are strongly pH dependent. The values of N 3 − binding constants calculated from inhibition experiments, which are higher than those obtained in the titration experiments, can be explained by the reduction of the protein during catalytic turnover. 4. 4. Data on CNS − and CNO − binding are included.

Full, reversible copper removal from ascorbate oxidase

Anaerobic treatment with cyanide of reduced ascorbate oxidase causes total depletion of copper. No significant amount of the metal is reincorporated when the apo-enzyme is incubated with cupric ions, but it is upon incubation with a stoichiometric amount (eight mol per mol of native enzyme) of a Cu(I) complex stable in air [Cu(I)(thiourea)3]Cl. The yield in reconstituted protein is higher under anaerobic conditions (85-90%) than in air (70-75%). By treatment with less than stoichiometric amounts of [Cu(I)(thiourea)3]Cl the apo-protein binds copper preferentially at the blue copper site. As a consequence the recovery of enzymatic activity is percentually lower than copper reincorporation.

Is the catalytic mechanism of bacteria, plant, and mammal copper-TPQ amine oxidases identical?

This short review is mostly concerned with the work carried out in Rome on the copper amine oxidase from bovine serum (BSAO). The first target was the copper oxidation state and its relationship with the organic cofactor. It was found that copper is not reduced on reaction with amines under anaerobic conditions or along the catalytic cycle and that it is not within bonding distance of the quinone cofactor. The copper stability in the oxidised state was supported by BSAO ability to oxidise benzylhydrazine, a slow substrate, in the presence of N,N-diethyldithiocarbamate (DDC) and by the substantial catalytic activity of Co(2+)-substituted BSAO. Parallel work established that only one subunit of the dimeric enzyme readily binds reagents of the carbonyl group. Flexible hydrazides with a long aromatic tail were found to be highly specific inhibitors, suggesting the presence of an extended hydrophobic region at the catalytic site. A study by stopped-flow transient spectroscopy and steady state kinetics led to the formulation of a simplified, yet complete and consistent, catalytic mechanism for BSAO that was compared with that available for lentil seedling amine oxidase (LSAO). As in other copper amine oxidases, BSAO is inactivated by H(2)O(2) produced in the catalytic reaction, while the cofactor is stabilised in its reduced state. A conserved tyrosine hydrogen-bonded to the cofactor might be oxidised.

The role of copper in bovine serum amine oxidase.

SummaryThe role of copper in bovine serum amine oxidase was investigated by studying the effect of copper-binding inhibitors on the reactions of the pyrroloquinoline quinone carbonyl and on the reaction with oxygen. Hydrazines and hydrazides were used as carbonyl reagents and one of the hydrazines, benzylhydrazine, which was found to behave as a pseudo-substrate, was used to probe the reaction with oxygen. The presence ofN,N-diethyldithiocarbamate, a chelator that binds copper irreversibly, did not prevent the reactions at the carbonyl, but slowed down their rate and modified the conformation of the adducts. The same happened to the reaction with oxygen, which was slowed down but not abolished. Copper, which was never seen in the reduced state, thus appears to control all reactions without being directly involved in the binding of either hydrazines or oxygen. The enzyme functionality was in fact preserved upon substitution of copper with cobalt. The specific activity of the cobalt-substituted enzyme was only reduced to about 40% the native amine oxidase value. This is the first case so far in which the role of copper can be performed by a different metal ion.

A ferrocyanide charge-transfer complex of bovine superoxide dismutase. Relevance of the zinc imidazolate bond to the redox properties of the enzyme.

Abstract Ferrocyanide does not reduce the bovine superoxide dismutase copper at pH 3.0 as it does at higher pH (1,2) but binds at the copper site giving a pink-violet charge-transfer complex. Similar reactions occur between ferrocyanide and Cu(II) bovine carbonic anhydrase or Cu(II) diethylenetriamine near neutral pH. The non-reducibility of superoxide dismutase Cu(II) at low pH suggests that its redox potential depends on the conformation of the site and on the presence of the zinc-imidazolate bond.