Class I heme peroxidases: Characterization of soybean ascorbate peroxidase

An efficient expression system [D. A. Dalton ct al. Arch. Biochem. Biophys. 328, 1-8, 1996) for soybean nodule ascorbate peroxidase (APX) has, for the first time, been used to generate enzyme in large enough quantities for de tailed biophysical analysis. The recombinant APX has been characterized by electronic absorption, EPR, NMR and circular dichroism spectroscopies, and by electrochemistry, Electronic, EPR, and NMR spectra are consistent with a high-spin ferric resting state for the enzyme at 298 R. Low-temperature EP R (7 K) and electronic absorption (77 K) experiments indicate formation of a low-spin heme derivative at these temperatures. The midpoint reduction po tential for the Ee(III)/Fe(II) redox couple, determined by spectroelectroch emistry, is -159 +/- 2 mV vs SHE (pH 7.0, 25.0 degrees C, mu =0.10 M). Circ ular dichroism spectra of pea and soybean APXs are very similar, indicating common structural features for the two enzymes. The melting temperature of soybean APX, as monitored by circular dichroism spectroscopy, is 49 degree s C, These results represent the first detailed spectroscopic and electroch emical analysis of soybean ascorbate peroxidase and are discussed in the br oader context of other class I peroxidases. (C) 1998 Academic Press.