A retrospective on 30 years of using peroxidases to study fundamental components of heme enzyme structure–function relationships

Abstract

This review traces roughly 30 years of peroxidase engineering, built on half a century of prior enzymology and structural biology, as a case study in translational biomimicry. Beginning with the earlier work that defined Compound I and Compound II, we follow how structural biology established the peroxidase fold, how mechanistic work clarified the push–pull logic of heme reactivity, and how protein engineering has since tested and extended those principles. The central argument is that peroxidases are useful scaffolds because they combine a robust fold, native support for high-valent heme chemistry, and a modular active site that tolerates extensive redesign while remaining mechanistically interpretable. Using examples from cytochrome c peroxidase and ascorbate peroxidase, we show how biomimetic designs can probe proximal ligation, distal proton delivery, water organization, porphyrin-radical localization, and cofactor coupling in multimetal and heterocofactor systems. Collectively, this strategy of reconfiguring peroxidases to model more complex heme enzymes clarifies how the protein environment, rather than the porphyrin alone, determines heme enzyme function.