Kinetic, Spectral, and Structural Characterization of a Heme-Containing Peroxidase From the Skin of Cucurbita maxima

A heme-containing peroxidase was isolated and characterized from the skin of Cucurbita maxima (PKS). Large-scale purification of the enzyme was performed, yielding a stable and active preparation suitable for detailed biochemical analysis. The PKS's properties were investigated, including pH sensitivity, temperature stability, and the influence of ionic strength on its activity and its spectroscopic properties. Kinetic parameters were determined using fluorophenol substrates and compared to those of the extensively studied horseradish peroxidase (HRP), highlighting similarities and unique features. The primary structure determination of the purified PKS was conducted using protease digestion and subsequent MS fragment analysis. Coupled with genomic data from PKS, a corresponding protein sequence was determined, which was then used to generate a 3-dimensional structure for the PKS enzyme using computational approaches. In addition, a spectrophotometric analysis of the purified PKS was conducted, enabling the determination of molar extinction coefficients for the holoenzyme in its presumed 5-coordinate high-spin ferric state (as isolated) and its 6-coordinate high-spin ferric state upon complexation with fluoride ions. The dissociation constant for fluoride binding exhibited significant pH sensitivity, consistent with observations reported for HRP. Additionally, the PKS enzyme showed distinct spectral binding features when complexed with trifluoroacetic acid, whereas no such binding was observed with HRP. These findings provide insights into the biochemical and structural characteristics of this low-cost plant peroxidase. Furthermore, our results provide relevant information for advancing the potential uses of these peroxidases in biotechnological applications such as environmental remediation of perfluorinated carboxylic acids.