Studies of Rhus vernicifera laccase using chemical and physical methods

Angela Schmidt Klemens, Purdue University

Abstract

Extended x-ray absorption fine structure (EXAFS) and x-ray absorption near edge structure (XANES) data are reported for mercury substituted derivatives of the blue copper proteins plastocyanin, azurin, laccase and stellacyanin. The EXAFS data confirm that the mercury is selectively substituted at the type 1 (blue) site in laccase, and provide the first direct structural information for this metal-binding site. The first coordination spheres of the four proteins are identical within experimental uncertainty, consisting of two nitrogens at ca. 2.24 Aand one sulfur a ca. 2.35 A. The EXAFS results suggest that the Hg(II) centers in Hg(II)-stellacyanin and Hg(II)-azurin contain additional scattering atoms at ca. 3.0 A. By analogy to the crystal structure of Cu(II)-azurin, these scatterers are most likely peptide oxygens. Additional scatterers at this distance are less evident for the Hg(II) sites in laccase and plastocyanin. In contrast to the overall similarity in the EXAFS spectra, the Hg(II) XANES spectra provide clear evidence for a structural difference in stellacyanin compared with the other three proteins. The significance of these observations in relation to the native proteins is discussed. A new procedure for the selective removal of the type 2 copper from tree laccase is presented. The type 2 copper is removed by dialysis against a redox buffer containing ferri- and ferrocyanide, as well as EDTA. More than 25% of the total copper is removed from laccase during the procedure, but a pure t2d sample is readily obtained by means of a cation exchange column. In the resulting purified t2d (type 2 depleted) protein, the type 1 copper is fully oxidized, while the type 3 site is reduced to the extent of ca. 85%. The type 3 coppers can be reoxidized by treatment with excess hydrogen peroxide. The derivative has only about 5% of the activity of the native enzyme. Reconstitution of the t2d protein is achieved by incubation with Cu(I), and the remetalated protein exhibits the complete activity and the spectral properties of the native enzyme. The new reaction system used to type 2 deplete laccase has resulted in new insights into the type 2 depletion process. These insights are discussed, as are some type 2 depletion mechanisms which were proposed as a result of these studies. Unsuccessful attempts to prepare HgCu2 laccase, the type 2 depleted form of the mercury derivative of laccase, are discussed. Several approaches were taken, including attempts to type 2 deplete HgCu$\sb3$ laccase, to add Hg(II) to t2d laccase, and to add stoichiometric amounts of Hg(II) and Cu(I) to apolaccase. Insights from these results are discussed.

Degree

Ph.D.

Advisors

McMillin, Purdue University.

Subject Area

Chemistry

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