Structural determinants of hydrogen peroxide tolerance in plant peroxidases: Distinct H₂O₂ accommodation and...

Hydrogen peroxide is both the oxidizing substrate of plant peroxidases and a major cause of their irreversible inactivation. Understanding how structurally related peroxidases accommodate H₂O₂ is therefore relevant to enzyme robustness and catalytic performance. Here, three class III plant peroxidases, horseradish peroxidase (HRP), peanut peroxidase (PNP), and royal palm tree peroxidase (RPTP) were compared using a reproducible structure-based workflow that evaluated heme geometry, distal-pocket accessibility, geometry-constrained H₂O₂ sampling, protein-derived electrostatic potentials, and residue-level peroxide contacts. Relative heme exposure differed substantially among the enzymes, with HRP showing the lowest exposure and RPTP the highest, indicating that cofactor burial alone does not account for their distinct peroxide responses. Geometry-constrained sampling revealed a markedly smaller population of sterically allowed and productive-like H₂O₂ configurations in HRP than in PNP and RPTP. Across independent sampling seeds, productive-like configurations represented 23.1% of allowed poses in HRP, 66.8% in PNP, and 78.1% in RPTP. Electrostatic analysis further distinguished RPTP by a predominantly positive distal-to-proximal potential difference across productive-like configurations, whereas HRP and PNP showed predominantly negative values. Residue-contact analysis confirmed conservation of the catalytic His42-Arg38 framework and indicated stronger local steric restriction around Phe41 in HRP. Taken together, these results show that the three enzymes differ in how their distal heme environments accommodate H₂O₂ and suggest that peroxide tolerance reflects a combination of accessibility, local geometry, and electrostatic organization rather than heme burial alone. These structural trends are qualitatively consistent with published biochemical evidence on peroxide processing and inactivation.