Cationic ascorbate peroxidase isoenzyme II from tea: structural insights into the heme pocket of a unique hybrid peroxidase.

Abstract

The novel class III ascorbate peroxidase isoenzyme II from tea leaves (TcAPXII), with an unusually high specific ascorbate peroxidase activity associated with stress response, has been characterized by resonance Raman (RR), electronic absorption, and Fourier transform infrared (FT-IR) spectroscopies. Ferric and ferrous forms and the complexes with fluoride, cyanide, and CO have been studied at various pH values. The overall blue shift of the electronic absorption spectrum, the high RR frequencies of the core size marker bands, similar to those of 6-coordinate low-spin heme, and the complex RR spectrum in the low-frequency region of ferric TcAPXII indicate that this protein contains an unusual 5-coordinate quantum mechanically mixed-spin heme. The spectra of both the fluoride and the CO adducts suggest that these exogenous ligands are strongly hydrogen-bonded with a residue that appears to be unique to this peroxidase. Electronic absorption spectra also emphasize structural differences between the benzhydroxamic acid binding sites of TcAPXII and horseradish peroxidases (HRPC). It is concluded that TcAPXII is a paradigm peroxidase since it is the first example of a hybrid enzyme that combines spectroscopic signatures, structural elements, and substrate specificities previously reported only for distinct class I and class III peroxidases.