A circular dichroism study of the reactions of Rhus laccase with dioxygen.

Abstract

The interaction of Rhus laccase with O2 and its reduction intermediates leads to characteristic changes in the circular dichroism (CD) spectrum. Those occurring in the 300–400‐nm range can be resolved in terms of three distinct parameters: the intensity and exact position of a negative band at 310–330 nm and the intensity of the positive 370‐nm band. The former CD transition is absent in oxidized laccase (native as well as reoxidized), but appears in all cases which are assumed to involve enzyme‐bound O2 reduction intermediates. Peroxy‐laccase, prepared from the native‐oxidized enzyme and H2O2, displays the most intense band (at 330 nm), with the same anisotropy factor as oxy‐tyrosinases and oxy‐hemocyanins. A similar, though somewhat blue‐shifted, negative band is obtained from the presumed peroxy derivative produced by reacting partially reduced laccase with O2. Aerobic reduction of laccase with a limited amount of ascorbate leads to a transient spectrum which, characterized by a negative band at 330 nm and a strong enhancement of the (+) 370‐nm band intensity, is possibly related to a bound oxygen intermediate. After the first‐order decay of this transient (k= 5.1 × 10−4, s−1, 25°C, pH 7.0) a stable spectrum is established which clearly differs from that of native laccase and is attributed to a different, metastable form of the oxidized enzyme (C state). The CD spectrum of laccase formed during turnover at low ascorbate concentrations suggests that under these conditions peroxy intermediates and enzyme molecules in the C state are the major species present in the steady‐state phase. We propose a catalytic scheme for laccase which allows for the reactions of O2 with laccase molecules reduced to different extents and which suggests cooperation between the type 2 and type 3 sites in the bond‐breaking reduction of the peroxide intermediate.