Laccase anaerobic reduction

Laccase, 36 318, 329, 40 122 see also Blue copper oxidases amino-acid sequences, 40 141 anaerobic reduction, 40 158-160 biological function, 40 124 electrochemistry, 36 360 fungal, 40 145-152 evolution, 40 153-154 inhibition, 40 162 kinetic properties, 40 157-162 molecular and spectroscopic properties, 40 125-126... 

The anaerobic reduction of the trinuclear copper species monitored by the bleaching of the 330-nm band, the reappearance of the 600-nm band, and the disappearance of the type-2 EPR signal appear to be multiphasic processes in most cases. For fungal laccase, the process is monophasic with a unimolecular rate constant of 1.0 sec (225). Tree laccase with hydroquinone as a substrate (224) displays a pH depen-... 

In studies on the anaerobic reduction of tree laccase by hydroquinone and ascorbate (49), the existence of a plateau phase at low substrate concentration was reported for the reaction of the type 1 copper. This observation was explained in terms of an intramolecular reoxidation by the type 3 copper pair. A similar plateau phase is a dominant feature of the reduction of both chromophores of ascorbate oxidase by reductate (Figure 7). However, the plateau phase is only observed in the presence of "contaminating dioxygen rigorous removal of these dioxygen traces removes the plateau phase at all wavelengths. The reaction of reduced ascorbate oxidase with dioxygen is very rapid, k = 5 X 10 at pH... 

The kinetics if the anaerobic reduction of stellacyanin, plastocyanin, azurin, and laccase by [Fe(edta)] have been reported. Simple second-order behaviour was observed and the following rate constants, with their associated and values, were measured (at 25 °C and pH 7) 4.3x10 , 8.2x10 , 1.3x10 , and 2.6X 10 1 mol" s 3, 2, 2, and 13 kcal mol" and -21, -29, -37, and -5 cal K mol", respectively. The authors favour an outer-sphere mechanism for azurin, plastocyanin, and stellacyanin but conclude that laccase employs a pathway which requires specific protein activation (of ca. lOkcalmol" in A/f ) to accept the reductant. The kinetics of the reduction of laccase by [Fe(CN)6] are complicated, as they are for the autoxidation of reduced laccase. The results - for electron transfer between azurin and cytochrome c have already been mentioned. 

The stopped-flow technique has been used to study the anaerobic reduction of fungal laccase B by hydroquinone, ascorbate, and ferrocyanide, for which a model is formulated, and the reduction of caeruloplasmin by the hydrated electron has been followed by pulse radiolysis. The latter technique has also been used to study the inhibition of another copper-containing enzyme, superoxide dismutase, by cyanide ions. Comparison of the rates and activation parameters for the reduction by chromous ion of blue copper in laccase, stellacyanin, and spinach and French-bean plasto-cyanins indicates that reduction of the Cu(614) site in laccase may occur by intramolecular electron transfer from one of the Cu(330) sites. The value of 17.4 kcal mol for the activation enthalpy associated with the reduction of cytochrome c by the same species is consistent with a mechanism in which major conformational changes in the protein accompany electron transfer. 

Laccase contains one type 1 Cu and one type 2 Cu in addition to the type 3 pair. The copper can be reversibly removed from the type 2 site (to give T2D-laccase). Reconstitution may be accomplished by adding CuS04 or Cu1 under anaerobic conditions.958 Loss of type 2 copper has little effect on the redox potentials of the type 1 and type 3 copper, or on the electron-transfer reactivity of the type 1 copper. It appears that type 2 Cu is a substrate-binding site in the reduction pathway for the blue copper.959... 

The reaction of nitric oxide with laccase (76) and ascorbate oxidase (147) has been studied as well. Nitric oxide fully reduces fungal and tree laccase when it is added to the oxidized enzyme under anaerobic conditions. In addition the binding of one NO molecule to laccase can be detected. This is characterized by a new EPR signal and has been described as coordinated with the type-2 copper (76). Only the reduction of the type-1 copper has been observed when NO has been added to ascorbate oxidase under anaerobic conditions. 

Rhus vernicifera laccase is known to incorporate four tightly bound copper atoms distributed in three distinct sites. Type 1 copper is responsible for the intense blue colouration, and a second form of metal ion, type 2, is not associated with any specific spectroscopic absorption bands but has been inferred from e.s.r. studies. This centre is known to function as a strong anion-binding site. Type 3 copper is non-detectable by e.s.r. and is characterized by a u.v. absorption band (Amax ca. 330 nm). Anaerobic stopped-flow studies with hydroquinone (HgQ) have been made to investigate the mode of reduction at these centres. The type 1 and type 3 copper ions are reduced in parallel at comparable rates over a wide range of substrate concentrations and pH. The rate data are consistent with the mechanism... 

DET is fundamentally the simplest method of enzyme association and can be achieved by one of the three methods (a) physical adsorption, (b) electrostatic interaction, and (c) covalent bonding [23,24]. In 2006, Shleev et al. immobilized laccase on a functionalized carbon electrode, for example, via glutaraldehyde, to form a stable laccase film of one monolayer thickness [25]. The resulting enzyme association revealed bioelectrocatalytic activity for the reduction of dioxygen, and two distinct redox peaks were observed under anaerobic conditions. The redox peaks, at 99 and 530 mV versus Ag/AgCl, were attributed to the formal potentials of the type... 

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