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.

A polyacrylamide gel containing an engineered hexameric hemoprotein as a cross-linking unit toward redox-responsive materials

“…S5†). 66 Upon addition of dithionite, this CD signal shifts to 438 nm, indicating that the Fe( ii ) states of heme molecules are also bound in the protein matrix of HTHP (Fig. S6†).…”

“…The axial ligand of the heme cofactor in each monomer of HTHP is a tyrosine residue located at position 45 of the protein. The Fe( iii ) state is highly stabilized in tyrosine-coordinated hemoproteins, 66 inspiring significantly redox-dependent heme-binding behavior. Furthermore, the remarkably high thermal stability ( T m > 130 °C) and the ability to replace heme with synthetic cofactors are useful features for development of a cross-linking unit.…”

A Supramolecular Assembly of Hemoproteins Formed in a Star-Shaped Structure via Heme–Heme Pocket Interactions

“…66.5 • C [24] and we suspect that denaturation of Cyt b 562 does not occur in our system because the reaction temperature is well below both reported temperatures. However, previous studies have indicated that T m has an effect on the binding affinity of heme for Cyt b 562 [26]. On the other hand, the thermostability of HTHP may also contribute to the heme-heme pocket interaction resulting in conjugation of the artificial heme to its binding site.…”

“…The spectra of Cyt b 562 are derived from a low spin hexa-coordinated heme species with His102 and Met7 axial residues (Figure 2A), while the HTHP spectra are generated by a penta-coordinated high spin heme species ligated by a Tyr45 axial residue (Figure 2B). The UV-vis spectrum of the ferric state of Cyt b 562 has characteristic absorption peaks at 417 nm, 532 nm and 562 nm [24], while absorption peaks of a ferric state of HTHP are typically observed at 402 nm, 500 nm, 534.5 nm and 623 nm [25,26] (Figure 3A). Thus, ferric UV-Vis spectra can conveniently distinguish the presence of the individual proteins when their apo-forms are mixed with sub-equivalent concentrations of heme molecules.…”

“…Hydrodynamic diameters of the fractionated components are obviously larger than the apoHTHP hydrodynamic diameter of 6 nm and reported Cyt b 562 diameter of ca. 2.5 nm [26] ) n and apoHTHP are prepared according to our reported methods [18]. All other reagents were commercially available and used as received or otherwise specified.…”