The reversible depletion and reconstitution of a copper ion in Coprinus cinereus laccase followed by spectroscopic techniques

The specific activities of crude and purified Coprinus cinereus laccase preparations could be enhanced by a factor of 10–12 by activation with copper ions. The copper to protein contents of purified non-activated laccase were 2.3 ± 0.1 compared to 3.3 ± 0.1 in purified activated laccase indicating that only a fraction of the laccase can be activated. Purified laccase not activated with copper ions shows in isoelectric focusing four bands in order of decreasing pI in a ratio 1/5/3/1 where only bands I and II had laccase activity. Purified activated laccase showed only three bands (I, II and III) in the ratio 5/4/1 all with some laccase activity. The pH profile of the activity for activated and non-activated laccase showed identical behavior indicating that the active forms were the same. The change in UV–Vis around 330 nm following the depletion and reconstitution of the enzyme combined with activity measurements supports the reversibility of the selective removal and insertion of copper ions at the type 2 site. The circular dichroism spectrum of activated purified laccase has characteristic changes around 350 nm relative to non-activated laccase indicative of changes at the type 2/type 3 sites. The difference between the electron paramagnetic resonance spectra of non-activated and activated C. cinereus laccase indicates that a fraction of the non-activated purified laccase contained a copper(II) signal with a coupling constant between a type 1 and a type 2 copper(II). This electron paramagnetic resonance signal could be explained by an induced asymmetry in the type 3 site due to a missing type 2 copper ion.

The reversible and selective removal and insertion of the type 2 copper ions in Coprinus cinereus laccase is investigated. The influence of the type 2 copper ions on the spectroscopic and catalytic properties is probed by laccase activity and various spectroscopic methods.Figure optionsDownload as PowerPoint slide