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

Steady-state and single-turnover kinetics for the oxidation of the N-substi tuted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Copri nus 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 x 10(7) M-1 s(-1) to 2.6 x 10(8) 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 pK(a) val ues of 4.9-5.7 and 7.7-9.7 that significantly affected the activity of pero xidase. Single-turnover experiments showed that the limiting step of PT oxi dation was reduction of compound II and second-order rate constants were ob tained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecul ar rate constants varied from 1.8 x 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 co mpound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere ele ctron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as P O, 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.