Received 3 July 2008; Accepted 13 October 2008 Central European Journal of Biology 1 Institute of Biochemistry, Department of Enzyme Chemistry, 08662 Vilnius, Lithuania 2 Vilnius Gediminas Technical University, Faculty of Fundamental Sciences, Department of Chemistry and Bioengineering, 10223 Vilnius, Lithuania Kinetics of N-substituted phenothiazines and N-substituted phenoxazines oxidation catalyzed by fungal laccases Lidija Tetianec 1* , Juozas Kulys 1,2 Abstract: Laccase-catalyzed oxidation of N-substituted phenothiazines and N-substituted phenoxazines was investigated at pH 5.5 and 25 o 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 lac- case 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)×10 5 M -1 s -1 for rPpL and (2.5±0.9)×10 4 M -1 s -1 for rMtL. The bimolecular constants of the reaction of the native intermediate with electron donor (k red ) vary in the interval from 2.2×10 5 to 2.1×10 7 M -1 s -1 for rPpL and from 1.3×10 2 to 1.8×10 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 cal- culated activationless electron transfer rate and the apparent reorganization energy are 5.0×10 7 M -1 s -1 and 0.29 eV, respectively. Keywords: Laccase • Kinetics • Phenoxazine • Phenothiazine • Oxygen • Bimolecular rate constant • Redox potential * E-mail: lidija@bchi.lt © Versita Warsaw and Springer-Verlag Berlin Heidelberg. Research Article 1. Introduction Multicopper oxidoreductase - laccase (EC 1.10.3.2) catalyzes the 4 e - reduction of O 2 to H 2 O by using copper centers of three different types. The electrons are taken up at the blue type 1 (T1) Cu site and transferred ~13 Å to the trinuclear Cu cluster, composed of a normal type 2 (T2) and a coupled-binuclear type 3 (T3) site, where the O 2 reduction occurs [1-3]. The T2 Cu site is held in the protein by two His residues and has a water- derived OH − ligand external to the cluster, whereas the OH − bridged T3 Cu site is held by three His at each Cu. The reaction of O 2 with the fully reduced enzyme (E red ) produces the peroxide intermediate (PI) of laccase following native intermediate (NI) generation [1-3]. The estimated rate of reduced laccase interaction with oxygen is ~2×10 6 M -1 s -1 and the peroxide intermediate decomposition is more than 350 s -1 [2]. In the absence of reducing substrate, the NI slowly decays to the resting enzyme (RE), in which the one remaining oxygen atom of the O 2 is terminally bound as OH − to the T2 site [1]. The slow rate of NI decay (~0.034 s -1 ) conlicts with the much higher turnover number, indicating that the resting enzyme is not involved in the catalytic cycle and that the NI is the only catalytically relevant fully oxidized form of the laccase. The application of laccase in biotechnology is associated with substrate oxidation using molecular oxygen [4]. Typically the laccases are used in concert with other oxidoreductases and in presence of mediators Cent. Eur. J. Biol. • 4(1) • 2009 • 62–67 DOI: 10.2478/s11535-008-0050-5 62 - 10.2478/s11535-008-0050-5 Downloaded from PubFactory at 08/18/2016 05:38:49AM via free access