Spectroscopic and binding studies of azide to type-2-copper-depleted ascorbate oxidase from zucchini

Summary

Binding of azide to type-2-copper-depleted (T2D) zucchini ascorbate oxidase, containing reduced type-3 Cu centers, and met-T2D ascorbate oxidase, containing oxidized type-3 Cu centers, has been studied spectroscopically. In both cases titration with azide in 0.1 M phosphate pH 6.8 produces a broad near-ultraviolet band with maximum at 455 nm (Δe ≈ 2500 M⁻¹ cm⁻¹, with respect to the met-T2D enzyme) and shoulder at 390 nm (Δe ≈ 1700 M⁻¹ cm⁻¹), that are assigned toπ(azide)→Cu(II) ligand-to-metal charge transfer (LMCT) transitions. This is accompanied by a reduction of absorbance at 330 nm in the met-T2D) enzyme adduct (Δe ≈ −1400 M⁻¹ cm⁻¹). A broad circular dichroic band of negative sign between 370–480 nm corresponds to the LMCT absorption band. Analysis of the titration data indicates that one azide ion binds independently to each of the binuclear T3 Cu couples with low affinity (K = 50 M⁻¹). The ESR signal of the T1 Cu observed in frozen solutions of the T2D enzyme is also perturbed by the addition of azide. The analogies in the azide-binding characteristics between ascorbate oxidase and laccase are discussed.