Exploring the process–structure–function relationship of horseradish peroxidase through investigation of pH- and heat induced conformational changes

• Conformational changes of peroxidase were correlated with inactivation.
• At optimum pH value, the Trp117 is exposed at surface of molecule.
• A flexible conformation at lower pH and at higher temperature was outlined.
• The secondary structure appeared preserved against temperature.
• The tertiary structure around heme was more affected.

Given the importance of peroxidase as an indicator for the preservation of vegetables by heat treatment, the present study is focused on enzyme behavior under different pH and temperature conditions, in terms of process–structure–function relationships. Thus, the process–structure–function relationship of peroxidase was investigated by combining fluorescence spectroscopy, in silico prediction methods and inactivation kinetic studies. The fluorescence spectra indicated that at optimum pH value, the Trp117 residue is not located in the hydrophobic core of the protein. Significant blue- and red-shifts were obtained at different pH values, whereas the heat-treatment did not cause significant changes in Trp and Tyr environment. The ANS and quenching experiments demonstrated a more flexible conformation at lower pH and respectively at higher temperature. On the other hand molecular dynamics simulations at different temperatures highlighted that the secondary structure appeared better preserved against temperature, whereas the tertiary structure around the heme was more affected. Temperature dependent changes in the hydrogen bonding and ion paring involving amino acids from the heme-binding region (His170 and Asp247) might trigger miss-coordination of the heme iron atom by His170 residue and further enzyme activity loss.

Transpared surf representation of peroxidase (Armoracia rusticana) structure. The cationic aminoacids are marked in blue. The figure was drawn using VMD software.Figure optionsDownload as PowerPoint slide