Kinetics of N-substituted phenothiazines and N-substituted phenoxazines oxidation catalyzed by fungal laccases

Abstract

Laccase-catalyzed oxidation of N-substituted phenothiazines and N-substituted phenoxazines was investigated at pH 5.5 and 25A degrees C. The recombinant laccase from Polyporus pinsitus (rPpL) and the laccase from Myceliophthora thermophila (rMtL) were used. The dependence of initial reaction rate on substrate concentration was analyzed by applying the laccase action scheme in which the laccase native intermediate (NI) reacts with a substrate forming reduced enzyme. The reduced laccase produces peroxide intermediate (PI) which in turn decays to the NI. The calculated constant (k(ox)) values of the PI formation are (6.1 +/- 3.1)x10(5) M(-1)s(-1) for rPpL and (2.5 +/- 0.9)x10(4) M(-1)s(-1) for rMtL. The bimolecular constants of the reaction of the native intermediate with electron donor (kred) vary in the interval from 2.2x10(5) to 2.1x10(7) M(-1)s(-1) for rPpL and from 1.3x10(2) to 1.8x10(5) M(-1)s(-1) for rMtL. The larger reactivity of rPpL in comparison to rMtL is associated with the higher redox potential of type I Cu of rPpL. The variation of k(red) values for both laccases correlates with the change of the redox potential of substrates. Following outer sphere (Marcus) electron transfer mechanism the calculated activationless electron transfer rate and the apparent reorganization energy are 5.0x107 M(-1)s(-1) and 0.29 eV, respectively.