Which copper is paramagnetic in the type 2/type 3 cluster of laccase?

Understanding the structure and function of the three copper atoms in the dioxygen reduction site of the blue oxidases such as laccase has been a long standing challenge. In the case of a widely studied derivative, known as type 2-depleted laccase, the removal of one copper from the cluster abolishes the EPR signal of the so-called type 2 copper. However, the present studies of isotopically enriched protein from Polyporus versicolor show that the readily replaceable copper is not active in the low-temperature EPR spectrum of fungal laccase or its difluoride adduct.

The type 2 copper of ascorbate oxidase.

Ascorbate oxidase, dissolved in Hepes or sodium phosphate buffers, was analyzed by EPR and activity measurements before and after storage at -30 degrees C and 77 K. The specific activity was somewhat higher in the phosphate buffer, about 3500-3700 Dawson units compared to about 3100 units of the enzyme dissolved in Hepes buffer. After storage at -30 degrees C the activity fell to 1400-2000 units in the phosphate buffer but only to 2600-2800 units in the Hepes buffer.

Effect of pH and ligand binding on the structure of the Cu site of the Met121Glu mutant of azurin from Pseudomonas aeruginosa.

A pH-dependent X-ray absorption fine structure (XAFS) study has been undertaken to provide a structural interpretation of the spectroscopic properties of the Met121 Glu mutant of azurin from Pseudomonas aeruginosa (Azp). Ligand binding studies have been carried out to investigate the effect of the cavity formed at the Cu site as a result of the mutation. The optical spectrum at pH 4 exhibits an intense band at approximately 600 nm and a weaker band at approximately 450 nm, typical for the blue copper proteins.

Development of a gas-phase oxygen biosensor using a blue copper-containing oxidase.

A gas-phase oxygen biosensor based on blue copper-containing oxidases was developed. Blue-oxidase enzymes, including laccase and ascorbate oxidase, have a blue chromophore prosthetic group, type 1 Cu+2, which can be reduced and decolorized with reducing substrates. When the enzyme is reoxidized with molecular oxygen, there is a concomitant return of the blue color.

Structural and spectroscopic studies of the copper site of stellacyanin.

The structure of the copper site in oxidized and reduced Rhus vernicifera stellacyanin has been studied by X-ray absorption (XAFS) spectroscopy at different pH values. Data for the oxidized protein are consistent with the fourth ligand being an O- or N-donating ligand rather than a cysteine from the disulfide bridge. The fourth ligand is not present in the inner coordination sphere, but makes a more distant interaction 2.

Crystallization and preliminary crystallographic data for the azurin mutant End-121 from Pseudomonas aeruginosa.

Pseudomonas aeruginosa azurin has been crystallized from a mutant where residues from Met 121 to Lys128 have been deleted from the protein. The crystals form pale-blue well formed prisms in the orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 60.79 (5), b = 123.

Structural characterization of azurin from Pseudomonas aeruginosa and some of its methionine-121 mutants.

Azurin from Pseudomonas aeruginosa and two mutants where the methionine ligand has been mutated have been studied in order to directly investigate the functional and structural significance of this ligand in the blue copper proteins. Reduction potentials, X-ray absorption fine structure (XAFS), electron paramagnetic resonance (EPR), and optical spectra are obtained in an attempt to provide a direct correlation between the spectrochemical properties and the immediate structure of this redox center.

Comparison of lignin peroxidase, horseradish peroxidase and laccase in the oxidation of methoxybenzenes.

Lignin peroxidase oxidizes non-phenolic substrates by one electron to give aryl-cation-radical intermediates, which react further to give a variety of products. The present study investigated the possibility that other peroxidative and oxidative enzymes known to catalyse one-electron oxidations may also oxidize non-phenolics to cation-radical intermediates and that this ability is related to the redox potential of the substrate. Lignin peroxidase from the fungus Phanerochaete chrysosporium, horseradish peroxidase (HRP) and laccase from the fungus Trametes versicolor were chosen for investigation with methoxybenzenes as a homologous series of substrates.

Proton-metal distance determination in cobalt(II) stellacyanin by 1H nuclear magnetic resonance relaxation measurements including Curie-spin effects: a proposed structure of the metal-binding region.

1H nuclear magnetic resonance (1H NMR) experiments on Co(II)-substituted stellacyanin have been performed. Large paramagnetic hyperfine shifts are observed, the whole spectrum covering a range of 190 ppm. Experiments were mainly performed at 270 MHz from which temperature and pH* dependencies of the out-shifted resonances were reported, as well as determinations of the longitudinal (T1) and transverse (T2) relaxation times.

The metal site of stellacyanin: EXAFS studies of the Cu(II), Cu(I), Ni(II) and Co(II) derivatives.

Extended X-ray absorption fine structure (EXAFS) studies of Cu(II) (oxidized), Cu(I) (reduced), Ni(II) and Co(II) stellacyanin from Rhus vernicifera are reported. For Cu(II) stellacyanin, the coordination by three close ligands, viz. 2 N and 1 S, with the presence of smaller shells pointing to imidazole coordination, indicates similarities with the coordination in other so-called type 1 or 'blue'-copper proteins.

Type 2-depleted fungal laccase.

Although copper is quantitatively removed from fungal laccase (Polyporus versicolor) by extended dialysis against high concentrations of cyanide, we have been unable to reconstitute the protein by addition of Cu(I) ions. However, two new methods for reversibly removing the type 2 Cu centre have been developed. The visible absorption at 610 nm, which is attributable to type 1 Cu, is unaffected by the procedure, but the absorbance of the type 3 Cu at 330 nm is decreased by 60 +/- 10%.

EPR of azurins from Pseudomonas aeruginosa and Alcaligenes denitrificans demonstrates pH-dependence of the copper-site geometry in Pseudomonas aeruginosa protein.

The X- and Q-band EPR spectra of Pseudomonas aeruginosa (63Cu)azurin and Alcaligenes denitrificans azurin have been measured at pH = 5.2 and 9.2, in the presence and absence of 40% glycerol.

63/65Cu and 1/2H2O isotope shifts in the low-temperature resonance Raman spectrum of fungal laccase.

Resonance Raman spectra are reported for the type 1 Cu site of fungal laccase at 295 and 77 K. The low-temperature spectra show enhanced resolution and reveal several weak bands not previously observed, as well as overtone and combination bands associated with the strong approximately equal to 400 cm-1 fundamentals. A novel low-temperature Raman difference technique has been used to obtain 63/65Cu and 1/2H2O isotope shifts.

Direct measurement of the self-exchange rate of stellacyanin by a novel e.p.r. method.

A method for reconstituting the blue copper protein stellacyanin with the stable copper isotopes 63Cu and 65Cu is reported. Small differences in the e.p.

Coordination environment for the type 3 copper center of tree laccase and CuB of cytochrome c oxidase as determined by electron nuclear double resonance.

A new rhombic EPR signal was recently discovered in the partially reduced type 2 copper-depleted Rhus vernicifera laccase (Reinhammar, B. (1983) J. Inorg.

A new copper(II) electron paramagnetic resonance signal in two laccases and in cytochrome c oxidase.

A new EPR signal from Cu2+ has been discovered in reductive experiments with type 2 copper-depleted laccase from Polyporus versicolor. A novel EPR signal has also been found in native laccase from Rhus vernicifera on oxidation of the reduced protein with H2O2. In reoxidation experiments with cytochrome c oxidase from beef heart, a new Cu2+ signal has been observed.

Spectroscopic and catalytic properties of Rhus vernicifera laccase depleted in type 2 copper.

1. The type 2 copper in Rhus vernicifera laccase was completely removed without loss of other types of copper. The properties of this protein derivative and the role of type 2 copper in the catalytic action of laccase was investigated.

The mechanism of electron transfer in laccase-catalysed reactions.

1. The reaction of the electron acceptors in Rhus vernicifera laccase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.

The reaction of mercaptans with tyrosinases and hemocyanins.

1. Titration of Neurospora tyrosinase with 2-mercaptoethanol shows that the increase of absorbance at 700 nm is directly correlated to the loss of enzymatic activity. Approximately 2 mol of 2-mercaptoethanol per mole of protein are needed for full development of the green, enzymatically inactive complex.

The state of copper in Neurospora laccase.

1. Neurospora crassa laccase has been prepared from the growth medium and studied by optical absorption, circular dichroism and electron paramagnetic resonance (EPR) spectroscopy. The molecular weight, the copper content and the amino acid composition have also been determined.

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.

Kinetic studies of Rhus vernicifera laccase. Evidence for multi-electron transfer and an oxygen intermediate in the reoxidation reaction.

1. The reoxidation of reduced Rhus vernicifera laccase (monophenol,dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.