Laccase azide binding

Figure 21. Comparison of the spectroscopically effective models for azide binding at the blnuclear copper active site in hemocyanln and the trlnuclear copper cluster site in laccase.

It is noteworthy that the proximity of the copper sites in ceruloplasmin, and, indeed, the involvement of most of the correct ligand histidines, were predicted some time ago by Ryden (1982, 1984) strictly on the basis of sequence homologies to plastocyanin. A similar prediction was made for laccase based on sequence similarities around the cysteine regions (Briving et al, 1980). Proximity of the type II site to the type III site (e.g., a trinuclear site) was also predicted by Solomon and co-workers (Allen-dorf et al., 1985 Spira-Solomon et al, 1986) on the basis of spectroscopic analysis of azide binding to laccase. What could not have been foreseen... 

Figure 18. Comparison of half-met hemocyanin with the half-met type 3 (in T2D) laccase copper sites. A EPR spectra and binding constants of exogenous azide binding. B Spectroscopically effective structural models for exogenous ligand binding to the half-met derivatives and their relation to differences in dioxygen reactivity.

Figure 20. Azide binding to native laccase. A Difference electronic absorption spectra at 298 K (see reference 89). B Difference MCD spectra at 4.9 K (5 T). C Changes in EPRy LTMCDy and absorption intensities plotted as a function of increasing azide concentration. D EPR of laccase titrated with azide. Arrow indicates the new signal present at g = 1.86 and 8 K.

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. 

The binding of azide to the T3 and T2 sites in native laccase has been probed via absorption and low temperature MCD spectroscopic studies of the —> Cu(II) charge transfer transitions in the... 

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 binding of azide to laccase as well as to ascorbate oxidase has been studied extensively by Solomon and co-workers (79, 80, 97, 171)... 

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