Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines.

Steady-state and single-turnover kinetics for the oxidation of the N-substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4-10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k(cat)/K(m)) varied from 1 x10(7)M(-1)s(-1) to 2.6 x 108 M(-1)s(-1) at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pKa values of 4.9-5.7 and 7.7-9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8x 10(5) M(-1) s(-1) to 2.0 x 10(7) M(-1) s(-1) at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6 x 10(8)M(-1)s(-1) and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0 x 10(7) M(-1) s(-1) and 0.29 eV.