Structural studies of the trinuclear cluster of Rhus vernicifera laccase

Catherine Miller, Purdue University

Abstract

Presented here is an investigation of the trinuclear copper center in the blue copper protein laccase. This study utilizes two different derivatives of laccase. In Chapter 2, tree laccase, a multi-copper oxidase, is studied by using anion binding. We have refined a useful incubation method for preparing adducts of tree laccase with inhibitor anions; however, the technique should be generally applicable. The procedure involves incubating a previously frozen aqueous solution at subzero temperatures. Factors influencing the amount of adduct that forms include the nature of the buffer, pH changes that occur at subzero temperatures and the presence of glycerol. The results reveal several important factors to consider in designing or interpreting low-temperature spectroscopic investigations. Chapter 3 characterizes a mixed isotope derivative of laccase. Multifrequency EPR studies of the mixed isotope derivative (MID) show evidence that suggests that different copper ions of the type 2/type 3 cluster are EPR active at low temperature and at room temperature. This previously unsuspected structural reorganization is of considerable interest because it is indicative of the flexibility which is required of a unit that must bind the various partially reduced forms of oxygen. This chapter also describes and demonstrates that using the complexing ligand, acetylacetonate, and taking advantage of the difference in magnetic moments between $\sp{63}$Cu and $\sp{65}$Cu, the isotopic composition of laccase derivatives can be determined and hence, the viability of the various modifications supported. Cu(acac)$\sb2$ analysis of several incubation experiments as well as the mixed isotope derivative reveal that there is an exchange on the average of one copper. The most easily replaced or facile copper is the type 2 copper. Insignificant exchange is seen in the type 1 site and the type 2 site, and therefore exchange does not compromise the mixed-isotope derivative. Having established the viability of the mixed isotope derivative, the binding of fluoride and azide to the trinuclear cluster was investigated. Results suggest that fluoride binds to a Cu-63 which would be one of the type 3 coppers. The azide studies are not as clear and the possibility of bridging between two coppers, the type 2 ($\sp{65}$Cu) and the type 3 ($\sp{63}$Cu) is discussed in chapter 4.

Degree

Ph.D.

Advisors

McMillin, Purdue University.

Subject Area

Chemistry

COinS