A novel heme peroxidase from Raphanus sativus intrinsically resistant to hydrogen peroxide

All known heme Peroxidases are susceptible to self-inflicted oxidative damage. Substrate oxidation in peroxidases is a naturally imperfect process and damage emerges from the utilization of protein elements as electron sources through the catalytic intermediate Compound III. Given their wide application potential, the search for more stable heme peroxidases has been actively pursued in the past, albeit with limited success. Here, the identification and biochemical characterization of a novel H2O2-resistant isoenzyme is reported which was isolated from daikon radish (Raphanus sativus L. cv. daikon) and named Zo peroxidase (ZoPrx). Partial protein sequencing demonstrated that ZoPrx is more related to anionic isoenzyme 53 from Arabidopsis thaliana and to anionic isoenzyme 2 from Armoracia rusticana (HRPA2) than to any other protein known to date. The stability of ZoPrx was demonstrated by rigorous evaluation of resistance to H2O2 in the absence or presence of exogenous reductants which was consistent with porphyrin integrity. ZoPrx systematically outperformed the reference HRPA2 isoenzyme, a heme peroxidase known for its relative oxidative stability. The catalytic cycle of ZoPrx is conventional in the sense that the usual intermediate Compounds I and II were identified although reaction rates are significantly different compared to HRPA2. The distinctive characteristic of ZoPrx is that when Compound III is formed in the presence of excess H2O2, it is particularly more stable and, instead of engaging into the regular deactivation decay pathways, it commits to the ground state restoration decay pathway. ZoPrx is the only known case of a naturally occurring stable heme peroxidase based on intrinsic properties. Deeper study of the structural determinants underlying this property may eventually allow us to graft it into other peroxidases with established commercial potential.