The Reversible Removal of one Specific Copper(II) from Fungal Laccase

Abstract

One of the four copper atoms of fungal laccase forms a Cu1+‐chelate with bathocuproine disulfonate at pH 4.0 in 1 M guanidine hydrochloride and in the presence of excess ascorbate. Electron paramagnetic resonance (EPR) analysis of the native and copper‐depleted proteins has shown that one of the two Cu2+ in the protein has been removed under these conditions. The Cu2+ removed has been identified as the Type 2 or “non‐blue” Cu2+. The spectral characteristics of the other Cu2+ present in the molecule (the Type 1 Cu2+) appear unchanged after removal of the second Cu2+. The copper‐depleted protein is almost completely devoid of enzymic activity after removal of the Type 2 Cu2+ but both the activity and original copper content can be restored after incubation of the copper‐depleted protein with Cu2+ and ascorbate under anaerobic conditions. No activity is restored by the addition of Cu2+ alone. The rate of removal of the Type 2 Cu2+ closely parallels the rate of decrease of enzymic activity of the protein during the reaction with the chelating agent. These results show that the Type 2 Cu2+ is an integral part of the laccase molecule and has a functional role in the catalytic reaction.

A Study of the Reduction and Oxidation of Human Ceruloplasmin

“…This proposal is not unique because the data can also be explained by a model in which both chromophores receive electrons directly from the reducing substrate. Also, the data considered here are incomplete because the investigation does not deal with the function of Type 2 copper which undoubtedly is essential for the oxidase activity [6,12].…”

“…Therefore, this copper has been studied only by low-temperature electron paramagnetic resonance spectroscopy. However, there is evidence that it is essential for oxidase activity [6,12].…”

X‐ray crystallographic characterisation of type‐2‐depleted ascorbate oxidase from zucchini

“…Several methods have been described for laccase from the fungus Polyporus versicolor [13,141, and from the Japanese lacquer tree Rhus vernicifera [15 -181, and for ascorbate oxidase from zucchini . All procedures except one work under reducing conditions with metal-chelating reagents, such as EDTA, dimethyl glyoxime, bathocuproine disulfonate or nitrilotriacetate.…”

Laccase: enzyme revisited and function redefined

“…They are a diverse group of multi-copper proteins with broad substrate specifi city, originally discovered in the exudates of Rhus vernicifera, the Japanese lacquer tree [10], and subsequently were demonstrated as a fungal enzyme as well [11][12]. Laccase has been extensively examined since the mid seventies and a number of reviews have appeared on the subject [13][14][15][16][17][18][19][20]. Broad families of organisms share laccases with diverse biological functions including breakdown of cellulose to provide nutrients to fungi, lignifi cation of plant cell walls and production of melanin in the insect midgut as a primitive immune defense against parasites [1].…”