Studies on the copper sites in Polyporus versicolor laccase

Phillip Maurice Hanna, Purdue University

Abstract

A simple colorimetric test for the Cu(I) content in blue copper proteins is described. The procedure is based on the formation of a complex between Cu(I) and 2,2$\sp\prime$-biquinoline in an acetic acid. Laccase samples contain an endogenous reductant not attributed to the type 1 cysteine residue which can reduce 0.14 $\pm$ 0.04 moles of Cu(II) per mole of protein; however, the addition of EDTA eliminates the interference. Two new methods for removing type 2 copper in Polyporus versicolor laccase is described. The visible absorption at 610 nm, attributed to type 1 copper, remains unaffected. Absorbance at 330 nm is decreased due to partial reduction of the binuclear type 3 copper center, although a change in the molar absorptivity of the oxidized chromophore is possible. Removal of the type 2 copper causes an absorbance increase around 435 nm, and is assigned to the semi-reduced form of the type 3 pair. In the e.p.r. spectrum of the type 2-depleted enzyme the type 1 copper signal exhibits well resolved ligand hyperfine splitting which is simulated on the basis of contributions from two nitrogen and two hydrogen nuclei (A$\sp{\rm H}$ $\approx$ A$\sp{\rm N}$ $\approx$ 25 MHz). The hydrogens are assumed to be attached to the $\beta$ carbon of the covalently bonded cysteine ligand. Enzyme activity, lacking in this derivative, returns fully when the type 2 copper is restored. Although copper is quantitatively removed from Polyporus versicolor laccase by extended dialysis against various reagents, the resulting apoprotein does not reconstitute with copper(I) ions. The type 1 Cu(II) bleaches at high pH. Thus, e.p.r. studies on the type 2 and a type 3 centers are possible when the native and type 2-depleted enzymes are dialyzed to pH 8.8, respectively. Low frequency e.p.r. suggests the ligation about the type 2 copper in Rhus vernicifera and Polyporus versicolor laccases are very similar. Evidence for copper migration into the type 2 site in the type 2-depleted enzyme at high pH may be prevented by a new mixed-metal derivative, as Cd(II) has been selectively bound in the vacant type 2 site in the type 2-depleted derivative of Rhus laccase.

Degree

Ph.D.

Advisors

McMillin, Purdue University.

Subject Area

Chemistry

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