Structural and chemical studies of Rhus vernicifera laccase
Abstract
The electronic spectra of Co(II) in the type 1 and type 3 sites have been obtained and isolated by the aid of the mercury derivative of laccase in which the type 1 site has been blocked out by the spectroscopically silent Hg(II) ion. The spectral data provide insights into the structure of these metal sites. Comparisons with the Co(II) derivatives of the simple proteins which contain either only the type 1 site or only the type 3 site show a great similarity in the spectral features. The results also resolve the previous controversy regarding the Co(II)-substituted laccase (Larrabee & Spiro (1979) Biochem. Biophys. Res. Comm., 88, 753-760; Sakurai et al. (1988) Inorg. Chim. Acta, 152, 139-143). The type 2 depleted form of isotopically enriched laccase has been prepared and subsequently reconstituted with a different copper isotope. The room temperature S-band EPR spectrum shows that the type 2 copper site contains the second isotope, but the low temperature X-band spectrum indicates that the type 2 signal originates from a different copper isotope. The results provide direct evidence of a temperature-dependent structural reorganization and a change in the nature of the antiferromagnetic coupling that occurs within the type 2/type 3 cluster of laccase. The reaction of type 2 depletion has also been systematically studied. Several factors are crucial for the process; they are: the redox potential, the presence of a Cu(II) chelating agent, and highly charged anions. The reaction can be conducted in a broad range of redox potentials and does not require specific reagents. Moreover, Cu(I) is released from the reduced protein when an extremely low potential and a Cu(I) chelator are employed. Thus a mechanism is proposed in which that copper is dissociated as copper(I) and subsequently oxidized to copper(II) suppressing recombination with the T2D derivative. Silver derivatives have been prepared by introducing silver ion into either a Hg derivative or T2D laccase. The products have been characterized as HgCuCuAg or AgCuCuAg. Both derivatives give rise to about 0.7 type 2 like EPR features but no type 1 signal in the best preparations. Therefore, the two derivatives possess very similar structural features except that the type 1 site is occupied by Hg and Ag ions, respectively. In the trinuclear site, about half of the copper is in the oxidized form responsible for the EPR signal.
Degree
Ph.D.
Advisors
McMillin, Purdue University.
Subject Area
Biochemistry|Chemistry
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