pH and microwave power effects on the electron spin resonance spectra of Rhus vernicifera laccase and Cucumis sativus ascorbate oxidase

金沢大学理工研究域物質化学系The present study shows that the electron spin resonance (ESR) spectral features of Rhus laccase depend considerably on the pH value of the enzyme solution and the irradiated microwave power. Because of the local protein structure change, the type 1 copper is appreciably autoreduced at alkaline pH as monitored both by the ESR and absorption spectroscopies. In addition, the ESR signal of the type 2 copper, especially its g perpendicular to region, becomes prominent at alkaline pH. Protein dissociation from a water or an imidazole group coordinated to the type 2 copper is supposed to be responsible for this behavior. Besides above pH effects, the g perpendicular to component of the type 2 copper ESR signal is obscured with rising microwave power level. The power saturation behavior of native laccase and its derivatives reveals that the type 2 copper is more easily saturated than the type 1 copper. Cucumis ascorbate oxidase also exhibits similar behavior upon pH variation and microwave power saturation

Gene Synthesis, Expression, and Mutagenesis of Zucchini Mavicyanin: The Fourth Ligand of Blue Copper Center Is Gln.

The gene coding for the 109-amino-acid, non-glycosylated form of mavicyanin was synthesized and expressed inEscherichia coli.The recombinant protein refolded fromE. coliinclusion bodies was purified and characterized. Its spectroscopic properties are fully identical to those of mavicyanin isolated from zucchini, even in the absence of its carbohydrate moiety. The blue cooper center of mavicyanin strongly binds three ligands (2His and Cys) as well as many blue copper proteins. To disclose the fourth ligand of mavicyanin, Met was substituted for Gln95 by site-directed mutagenesis. The replacement changes from a rhombic EPR signal to an axial one and exhibits the quite similar absorption and CD spectra to those of plastocyanin. The midpoint potential of Gln95→Met mavicyanin shows the positive shift of 187 mV compared with the recombinant protein, being close to the values of plastocyanins. The differences of the spectroscopic and electrochemical properties between mavicyanin and its mutant demonstrate that the fourth ligand of mavicyanin is Gln95

Crystallization and preliminary X-ray diffraction analysis of a complex between the electron-transfer partners hexameric Cu-containing nitrite reductase and pseudoazurin

The hexameric Cu-containing nitrite reductase and its electron-donor protein pseudoazurin have been cocrystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 3.3 Å resolution using a synchrotron-radiation source

Reduction and oxidation processes of blue copper proteins, azurin, pseudoazurin, umecyanin, stellacyanin, plantacyanin, and plastocyanin approached by cyclic and potential step voltammetries

Direct electrochemistry of a series of blue copper proteins: azurin, pseudoazurin, umecyanin, stellacyanin, plantacyanin, and plastocyanin has been performed at a gold electrode modified with di-4-pyridyl disulfide and/or at a bare glassy carbon electrode. Well-resolved cyclic voltammograms with peak separation, ΔEp = 55—100 mV were obtained, allowing the facile determination of the redox potential of blue copper proteins. Protein molecules associate with the electrode surface through both electrostatic and hydrophobic interactions, of which the predominant one differs according to the combination of protein and electrode. Double-step voltammetry showed that redox processes of blue copper proteins depend profoundly on the translocation of the molecules (diffusion and/or change of orientation) at the electrode surface. The heterogeneous rate constants at pH 6.0 and 25 °C for both reduction and oxidation processes were independently determined to be the order of 10−3 to 10−4 cm s−1 by single-step voltammetry and were compared with those determined by cyclic voltammetry. Further, activation parameters for redox processes of some blue copper proteins have been determined from temperature dependence studies. The reaction pathway of blue copper proteins was discussed.出版者照会後に全文公

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the soluble domain of PPA0092, a putative nitrite reductase from Propionibacterium acnes

The soluble domain of a putative copper-containing nitrite reductase from P. acnes has been overexpressed, purified and crystallized. The crystal belonged to space group P213 and diffracted to 2.4 Å resolution