FUNCTIONAL AND STRUCTURAL STUDIES OF RHUS LACCASE (TYPE-2 COPPER, TRANSFER, MERCURY DERIVATIVE)
Abstract
Laccase was found to donate copper to copper-deficient proteins that contain only one copper site, which may be similar to ones found in vivo. The donation of copper depends upon protein heterogeneity. The site of heterogeneity appears to be the type-3 site and may involve the same fraction of protein which has previously been observed to contain a reduced type-3 site. It is possible that in the natural environment, laccase exists in two different forms, each with a specific function. The reconstitution of apolaccase was examined, and a preparative procedure devised such that all of the spectral characteristics of the remetalated protein were identical to those of native laccase. The quality of reconstitution was dependent of the method used to remove the copper from the native enzyme. The preparation and characterization of a mixed-metal derivative of laccase containing mercury(II) in the type-1 binding site are presented. Metal analyses of this derivative indicate one equivalent of mercury and three equivalents of copper are bound to the protein. Spectral studies indicated no optical absorbance features due to the type-1 copper are present, but an absorbance band due to the type-3 copper is present. The EPR spectrum of the protein contains features due specifically to the type-2 copper. Low-temperature multifrequency EPR techniques were used to assign an N(,3)O donor set to the type-2, and revealed a conformational change in the type-2 structure upon fluoride-binding at 77 K. The mercury derivative has a hydroquinone activity that is markedly different than the native enzyme.
Degree
Ph.D.
Subject Area
Biochemistry
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