BLUE COPPER SPECTRA AND THE METALATION OF LACCASE
Abstract
The charge transfer of azurin and plastocyanin are analyzed in detail. The number and relative energies of the low-lying charge transfer bands attributable to cysteine and methionine sulfur are considered with reference to the energy levels associated with the sulfur donors and to experimental work on relevant complexes. Two cysteine-based charge transfer transitions are assigned to bands that occur near 620 nm and 770 nm. Energy considerations suggest that the band in the vicinity of 550 nm is due to a methionine (--->) Cu(II) transition. The presence of a disulfide bond in stellacyanin is determined by thiol titrations. It is possible that this disulfide is involved in copper coordination. If so, the band in the vicinity of 550 nm in the visible spectrum of stellacyanin can be assigned to a cystine Cu(II) transition. The removal and reincorporation of copper from chinese laccase is discussed. The greatest success is achieved when Cu(I) added slowly to the apoprotein, allowed to incubate and then purified using cation-exchange chromatography at pH 7.0. The remetalated protein has EPR and fluorescence spectra identical to those of native laccase. Significant restoration of optical absorbances is observed upon remetalation of apolaccase. The remetalated protein also displays EPR and fluorescence spectra identical to those of native laccase. The preparation of a mixed-metal hybrid of laccase is presented. The hybrids discussed contain a non-native metal located specifically in the Type 1 site and copper in the Type 2 and Type 3 sites. Attempts are made to prepare Cd(II)-Cu(,3) and Hg(II)-Cu(,3) hybrids. Using the discussed procedure, only the preparation of a Hg(II)-Cu(,3) hybrid is possible. The Hg-derivative contains no optical absorbance due to Type 1 copper but does exhibit an absorbance due to Type 3 copper. The mercury and copper analyses indicates one and three site equivalents, respectively, are present in the Hg(II)-Cu(,3) hybrid. The EPR spectrum contains features due solely to Type 2 copper.
Degree
Ph.D.
Subject Area
Chemistry
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