Laccase spectroscopy

Low-temperature MCD spectroscopy was used to probe the effects of binding the exogenous ligand azide to native laccase (89, 90). Titration of the native enzyme with azide produces two N3 - Cu(II) charge-transfer transitions one at 500 nm and a second more intense band at... 

The low-temperature MCD and absorption titration studies (Figure 10) have determined that azide binds to both the type 2 and type 3 centers with similar binding constants. A series of chemical perturbations and stoichiometry studies have shown that these effects are associated with the same azide. This demonstrates that one N3 bridges between the type 2 and type 3 centers in laccase. These and other results from MCD spectroscopy first defined the presence of a trinuclear copper cluster active site in biology (89). At higher azide concentration, a second azide binds to the trinuclear site in laccase. Messerschmidt et al. have determined from X-ray crystallography that a trinuclear copper cluster site is also present in ascorbate oxidase (87, 92) and have obtained a crystal structure for a two-azide-bound derivative (87). It appears that some differences exist between the two-azide-bound laccase and ascorbate oxidase derivatives, and it will be important to spectroscopically correlate between these sites. 

Cole et al. (97) studied the electronic structure of the laccase trinuclear copper active site by the use of absorption, circular dichroism, and low-temperature magnetic circular dichroism spectroscopies. The assigned ligand field transition energies indicated that all three coppers have tetragonal geometries and that the two type-3 coppers are inequivalent. 

This paper summarizes briefly the physicochemistry and enzymology of plant copper oxidases with particular emphasis on polyphenol oxidase and laccase. A brief comparative discussion of other naturally occurring copper proteins and artificial copper proteins is appropriate when discussing the physicochemistry of the copper site itself. In the case of the copper proteins listed in Table I, we know a great deal more about the copper site than about the physicochemistry of the rest of the protein molecule. This is primarily a result of the availability of sophisticated spectroscopic techniques such as optical spectroscopy (both absorption and circular dichroism) and electron spin resonance which are applicable to the electronic transitions of the copper ion. On the other hand, protein chemistry has progressed more slowly. Many of the proteins are large and complex multisubunit enzymes, difficult to purify, and often unstable. There are several excellent reviews on this group of proteins (59, 60, 61, 62). 

Laccase contains four copper atoms and catalyzes the four-electron reduction of dioxygen to water. X-Ray absorption edge spectroscopy has been used to determine the oxidation states of copper in Rhus vernicifera laccase, following the reaction of the reduced enzyme with dioxygen (202). This study included the incorporation of mercury(II) in the Type 1 copper site (see Section IV,B). The results demonstrate that the Type 2/Type 3 trinuclear copper site, as found in ascorbate oxidase (103), represents the minimal active site required for the multielectron reduction of dioxygen. 

Faced with the problem of elucidating the individual roles of the diflFerent copper centers in the blue oxidases, the researcher has naturally focused in recent years on the laccases (9). Being easier to isolate, better characterized, and containing fewer copper atoms than cemloplasmin or ascorbate oxidase, the laccases from the Japanese lacquer tree Rhus vernicifera and the fungus Polyporus versicolor have been the subject of several transient kinetic studies in the millisecond range, that is, studies using stopped-flow spectrophotometry and rapid-freeze EPR spectroscopy (9,49,50). 

General Procedure for the Biotransformation of BP with Laccase/Linear-dendritic complexes. Fine powder of BP (0.010 g) was added to preformed laccase/[G-2]-PEG5000-[G-2] complexes and the mixture was stirred for additional 12 h. The mediator was added last and the reaction was followed by UV-Vis spectroscopy over extended time period. Control mixtures of laccase and BP with linear copolymers (Brij-35 and Igepal CO-990) and the same mediator were prepared and analyzed spectroscopically for biocatalytic activity under identical conditions. 

In extension to the above experiments the usefulness of the laccase/[G2]-PEG5k-[G2] complex for biotransformations of highly hydrophobic substrates is evaluated with BP and HBT. The reaction is followed by UV-Vis spectroscopy. Figure 7. It is seen that, despite the notable difference in the intensities, the spectral characteristics of reaction mixtures with BP (Figure 7A) are rather similar to those of mixtures without BP (Figure 7B). Obviously HBT is not spectroscopically suitable as mediator for the analysis of laccase mediated transformations of BP, since previous studies have shown that it can also be oxidized by laccase, and the reaction produces) possess absorption maxima in the same spectral region as the BP quinones (19, 20). 

Microemulsions with ILs acted as catalytic activity enhancer for oxidases. Zhou et al. [95] reported a water-m-[bmim][PFJ microemulsion system stabilized by TX-lOO that enhances the catalytic activity of hgnin peroxidase (LiP) and laccase. Optimum molar ratios of water to TX-lOO were 8 and >20 for UP and laccase, respectively. Compared to pure or water-saturated [bmim][PFJ, the derived microemulsion evidenced enhanced catalytic activity. Use O/W for oU-in-water ivation effect of [bmim][PFJ on LiP and laccase. Xue et al. [96] reported timable enzyme (laccase) activity in a microemulsion system, water/AOT+TX-100/[bmim][PFJ. The solution of IL [bmim][Cl] and polar organic solvent formamide (FA) were used to form a nonaqueous microemulsion as [bmim][Cl]-FA/TX-100/cyclohexane (Rg. 10.8) at 25 0.1°C, reported by Wei et al. [97]. By means of electrical conductivity, dynamic hght scattering (DLS) and UV-Vis spectroscopy measurements, microstructures, internal phases, and size regime were explored of the aforesaid microemulsion system. UV-Vis studies using CoCl indicated metal salt dissolution by microemulsion. 

AOx, CP, and tree and fungal laccases (Figure 8.5) [69]. Comparisons among the ground-state distribution of the Cu—S(cys) stretching vibration in the T1 copper site in each of these proteins show that they differ in strength. The Cu—S(cys) bond is one of the three essential coordination complexes of copper that defines the T1 site in MCOs and other copper proteins and is the major factor in electron transfer from the substrate to the trinuclear center [40,85]. Hence, RR spectroscopy can provide valuable information on how subatomic structural variations at this coordination can drive the functional properties of electron transfer at the T1 site. 

IN SITU X-RAY SPECTROSCOPY OF ENZYMATIC CATALYSIS LACCASE-CATALYZED OXYGEN REDUCTION... 

Arruda TA, Chakraborty L, Lawton JS, Calabrese-Barton S, Atanassov P, Mukeijee S. Direct observation of oxygen binding and reduction in laccase T. versicolor) by in situ X-ray absorption spectroscopy. ChemElectroChem 2014 under review. 

A recent suggestion that e.s.r. spectroscopy may be unable to detect certain copper(n) centres in such enzymes as laccase and caeruloplasmin because they exist in the form of spin-paired cupric dimeric units has led... 

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