Structural and chemical studies of the type 2 sites in copper oxidases
Abstract
Using a derivative of laccase in which the Type 1 copper has been selectively replaced by the EPR-silent Hg(II) ion, a room temperature EPR spectrum of the type 2 copper in laccase has been obtained. This spectrum is markedly better resolved than those reported earlier in the literature. EPR spectra of the derivative were also recorded at several temperatures between 123 and 299 K to explore the temperature-dependent conformational change about the type 2 copper first reported by Morpurgo (Morpurgo, L., Calabrese, L., Desideri, A., & Rotilio, G. (1981) Biochem. J., 193, 639-642). At the temperature extremes (123 K, 299 K), single well-defined species are present, but at intermediate temperatures (between 213 and 253 K), the presence of multiple structures is indicated. Azide and fluoride binding have also been studied as a function of temperature. The results suggest that each anion interacts preferentially with the type 3 coppers in fluid solution and that these adducts can be trapped by rapidly cooling the sample to 123 K. Annealing the adducts at 253 K permits rearrangement and binding at an equatorial position of the type 2 copper. This pathway to anation at the type 2 site contrasts sharply with previous studies which required a large excess of anions, and it reveals important insight into the flexibility of the type 2/type 3 cluster in laccase. Remetalation studies of ceruloplasmin have been performed toward the preparation of a mixed-metal derivative. A procedure for the reconstitution of the enzyme has been developed which results in a product which has spectroscopic properties indistinguishable from those of the native enzyme. It has been shown that Hg(II) binds to the apo-protein in a normal metal binding site. The presence of Hg(II) inhibits the uptake of Cu(I) by the apo-enzyme. A derivative which possesses no type 1 copper spectral properties has been prepared, but at present it is only a minor product. Attempts to increase the yield of this apparent mixed-metal derivative have been unsuccessful.
Degree
Ph.D.
Advisors
McMillin, Purdue University.
Subject Area
Chemistry|Chemistry
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