Laccase spectra

Characterization of the Type 2 Depleted Derivative of Laccase. The model for the coupled blnuclear copper site in hemocyanln and tyrosinase (Figure 7) may now be compared to the parallel site in laccase which contains a blue copper (denoted Type 1 or Tl), a normal copper (Type 2, T2), and a coupled binuclear copper (Type 3, T3) center. As shown in Figures 8a and b, native laccase has contributions from both the Tl and T2 copper centers in the EPR spectrum (the T3 cupric ions are coupled and hence EPR nondetectable as in hemocyanln), and an intense absorption band at associated with the Tl center (a thlolate —> Cu(II) CT transition).(14) The only feature in the native laccase spectra believed to be associated with the T3 center was the absorption band at 330 nm (e 3200 M cm ) which reduced with two electrons, independent of the EPR signals.(15) Initial studies have focussed on the simplified Type 2 depleted (T2D) derlvatlve(16) in which the T2 center has been reversibly removed. From Figure 8 the T2 contribution is clearly eliminated from the EPR spectrum of T2D and the Tl contribution to both the EPR and absorption spectrum remains. 

The conjugation of catechin on poly(allylamine) using ML as catalyst was examined under air. During the conjugation, the reaction mixture turned brown and a new peak at 430 nm was observed in the UV-vis spectrum. At pH 7, the reaction rate was the highest. The conjugation hardly occurred in the absence of laccase, indicating that the reaction proceeded via enzyme catalysis. 

Many multiple copper containing proteins (e.g., laccase, ascorbate oxidase, hemo-cyanin, tyrosinase) contain so-called type III copper centers, which is a historical name (cf. Section 5.8 for type I and type II copper) for strongly exchange-coupled Cu(II) dimers. In sharp contrast to the ease with which 5=1 spectra from copper acetate are obtained, half a century of EPR studies on biological type III copper has not produced a single triplet spectrum. Why all type III centers have thus far remained EPR silent is not understood. 

Turning to the X-ray absorption edge spectrum of the T3 site In T2D laccase (Figure 10a), a peak Is observed below 898A eV. This energy Is characteristic of Cu(I) while the shape of the peak suggests that It is due to three coordinate copper. The amount of reduced copper present can then be quantitated from the normalized edge intensities of copper model complexes with the appropriate... 

Type III copper(II), found for example in Rhus laccase, is ESR inactive. Although copper(II) is present no ESR spectrum can be obtained. Recent magnetic susceptibility measurements on Rhus laccase indicate an antiferromagnetically coupled cop-per(II) dimer. 

N) Native laccase, 7.0 X 10 s M, pH 7.0. (Rj Fully reduced laccase after adding 1.54 X 10 M ascorbic acid. (l)-(7) Spectra obtained 20 min after each successive addition of 2.8 X 10 5 M HtOt. Addition of another 2.8 X 10 5 M HtOt increased the absorbance at 330 nm by 0.02 units (not shown). Further addition of HtOt did not change the spectrum. Insert the difference spectrum of peroxide-treated laccase-native laccase in the near-uv. 

For the type 3 center, the antiferromagnetic coupling leads to an S = 0 ground state that cannot split in a magnetic field. Thus, this site does not exhibit C-term intensity and the low-temperature MCD spectrum of native laccase will be dominated by the intense C-terms associated with paramagnetic copper centers (89, 90). 

Upon oxidation of T2D, spectral changes104 are also observed for the type 1 copper site (Fig. 41), indicating intersite interaction. The type 1 parallel hyperfine increases to 42.9 x 10-4 cm-1, intensity of the Blue band decreases (Ac614 —300 M-1 cm-1) and the resonance Raman spectrum of the Blue site shows a significant increase in intensity of a 382 cm-1 vibration. These changes demonstrate that the geometry of the type 1 site is affected by oxidation of the type 3 copper in T2D laccase. 

The reoxidation studies on laccase and ascorbate oxidase are listed in Table IX. The reoxidation of the type-1 copper and of the trinuclear copper site occurs at a rate of 5 x 10 M" sec" both for tree laccase 134) and for ascorbate oxidase 135). During reoxidation with H2O2, an 02 " intermediate is formed in several minutes, which is documented for tree laccase by changes in the CD spectrum 136) and for ascorbate oxidase in the formation of an absorption band at 350 nm... 

In the case of Polyporus laccase, Malkin et al. (95) have differentially removed the non-blue Cu(II) from the protein. This inactivates the enzyme but leaves the intense blue color intact. The activity and original copper content can be restored by adding Cu(II) and ascorbate (95). Anions such as F" and CN" appear to inhibit by reacting with the non-blue copper (66). Fluoride, for example, appears to react exclusively with the non-blue Cu(II) since the super hyperfine lines from the fluoride nucleus appear exclusively on the non-blue Cu(II) hyperfine lines in the ESR spectrum, and the blue Cu(II) hyperfine lines remain unaltered (Figure 6) (96). Figure 6 is an ESR spectrum taken at a... 

Type I Cu(II) is the blue copper responsible for the unusually intense absorption at 614 nm in laccase. Its EPR spectrum is unusual with low g values and very small hyperfine splittings. Type II Cu(II) shows a more normal EPR spectrum and is thus responsible for the so called low field line in the EPR spectrum of laccase (in spite of considerable overlap with the type I spectrum). There are no optical absorptions which strongly depend on the oxidation state of the type II copper in laccase and it is thus considered transparent. It is well established89 that anions such as F- and N3 strongly bind to type II Cu(II) in laccase. In the case of F- binding91, the low field EPR line was split by the 19F nucleus, the enzyme was inactivated, and a very high binding constant measured. 

Aasa, Branden, Deimin, Malmstrom, Reinhammer, Vanngard and Andreasson examined the product of 02 oxidation of reduced laccase 30 ms after mixing by a rapid freeze technique101,102). They found a stable intermediate (at 77 k) which contained fully oxidized type I Cu(II) even at 30 ms but still reduced type II Cu(I) as evidenced by the EPR spectra. Optical stop flow102) measurements at 614 and 330 nm also showed the oxidation of type I and type III copper as early as 25 ms. The intermediate optical spectrum was more intense than the product at 340 nm and the decay of the intermediate at 25 °C was slow (t1/2 20 s) and similar to the rate of... 

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