The Type 3 copper site is intact but labile in Type 2-depleted laccase

We report results of experiments designed to characterize the Type 1 and Type 3 copper sites in Rhus laccase depleted of Type 2 copper (T2D). Use of the Lowry method for determining protein concentration yielded the value 5620 +/- 570 M-1 cm-1 for the extinction of the 615-nm absorption band of this protein. Anaerobic reductive titrations with Ru(NH)3)6(2)+ and Cr(II)aq ions established the presence of three electron-accepting centers, which are reduced in a complex manner. Treatment of T2D laccase with a 70-fold excess of H2O2 induced a new shoulder at 330 nm (delta epsilon = 660 M-1 cm-1), as well as intensity perturbations at 280 and 615 nm. Comparison of difference spectra show that this 330-nm band derives from a Type 3 copper-bound peroxide and not from a reoxidized Type 3 site. Dioxygen reoxidation of ascorbate-reduced T2D laccase produced new difference bands at 330 nm (delta epsilon = 770 M-1 cm-1) and 270 nm (delta epsilon = 13,000 M-1 cm-1), the former assigned to a bound peroxide which is a dioxygen reduction intermediate. In the corresponding epr spectrum of this material new Cu(II) g parallel features (A parallel approximately 130 G) indicative of an isolated copper ion and a triplet signal near 3,400 G were observed, originating from the Type 3 sites of separate T2D laccase molecules. Reoxidation by ferricyanide or by dioxygen as mediated by iron hexacyanide did not produce these changes. Thus the magnetism of the reoxidized Type 3 site in T2D laccase can be perturbed as a consequence of aerobic turnover. The suggestion is advanced that there are presently three forms of T2D laccase, possibly metastable conformational isotypes, accounting for the apparently contradictory reports on the properties of this protein.