Substitution of an amino acid residue axially coordinating to the heme molecule in hexameric tyrosine-coordinated hemoprotein to enhance peroxidase activity

Abstract

To convert an originally tyrosine-coordinated heme to histidine-coordinated heme in hexameric tyrosine-coordinated hemoprotein, HTHP, Tyr45, a residue coordinating to the heme cofactor, and Arg25 located in the distal site are replaced with Phe45 and His25, respectively in each of the subunits of the protein. The obtained HTHP mutant (HTHP[Formula: see text] was characterized by SDS-PAGE, ESI-TOF MS, dynamic light scattering measurements and size exclusion chromatography. These analyses indicate that HTHP[Formula: see text] maintains its stable hexameric structure with the altered ligation of each of the heme cofactors. Comparison of UV-vis absorption spectra of the ferric-, ferrous-, CO- and CN-forms of HTHP[Formula: see text] with those of several well-known His-ligated hemoproteins indicates that heme is coordinated by the His25 residue. The reaction of HTHP[Formula: see text] with cumene hydroperoxide produces both cumyl alcohol and acetophenone in a 2.3:1 ratio, indicating that heterolytic O–O bond cleavage dominantly occurs to form the two-electron oxidized species known as compound I. Peroxidase activity of HTHP[Formula: see text] is found to follow Michaelis–Menten kinetics. The [Formula: see text] values of HTHP[Formula: see text] for H[Formula: see text]O[Formula: see text]-dependent oxidation of ABTS and guaiacol are 10- and 100-fold higher, respectively, than those of wild type HTHP (HTHP[Formula: see text]. The [Formula: see text]/[Formula: see text] values of HTHP[Formula: see text] for both substrates are increased 30-fold relative to that of HTHP[Formula: see text]. Moreover, HTHP[Formula: see text] is capable of promoting catalytic sulfoxidation of thioanisole with H[Formula: see text]O[Formula: see text] with a turnover number ca. 2-fold higher than that of HTHP[Formula: see text]. The present findings demonstrate that proximal His ligation to the heme is significantly effective to increase the peroxidase activity in the HTHP matrix.