Characterization of a plant paraperoxidase from Curcurbita pepo using magnetic circular dichroism: direct evidence for cyanide ligation in the ferric resting state

Abstract

A series of ferrous, ferric, and oxoferryl derivatives of two plant peroxidase isozymes isolated from zucchini peels has been examined using electronic absorption and magnetic circular dichroism (MCD) spectroscopy. Isozyme A 1 has spectroscopic properties that are very similar to those of a typical peroxidase enzyme such as horseradish peroxidase. Isozyme C 1, on the other hand, has abnormal spectroscopic properties in the ferric resting state and has been termed a paraperoxidase. In general, the ligand adducts of both isozymes in all oxidation states have spectral properties that compare favorably with the corresponding ligand derivatives of horseradish peroxidase (HRP). Thus, the central heme iron of both isozymes is likely coordinated to a proximal histidine ligand. The unusual spectral properties of the native low-spin ferric state of isozyme C 1 appears to be due to the presence of cyanide as an exogenous sixth ligand. The cyanide can be formed by the hydrogen peroxidase dependent oxidation of thiocyanate. Oxidation of thiocyanate results in formation of a product, hypothiocyanite (OSCN −), which can be further oxidized by excess H 2O 2 to form cyanide. Therefore we propose the most likely cause of the unusual spectral properties of isozyme C 1, the paraperoxidase, is the presence of cyanide as an exogenous ligand in the ferric resting state.