Substrate-Engaged Formation of Reactive Oxygen Intermediates in Metalloenzymes: Mechanisms and Implications for Biocatalysis

Abstract Reactive oxygen intermediates in metalloenzymes are generally viewed as products of metal-centered O2 or H2O2 activation that are tuned by the surrounding protein environment. Increasing mechanistic evidence, however, shows that substrates can actively participate in the generation and evolution of these oxidants. In this Perspective, we discuss a substrate-engaged framework for reactive oxygen intermediate formation in metalloenzymes. Two major modes of substrate participation are emphasized. In H2O2-dependent heme enzymes, substrates can functionally replace or complement distal acid–base residues by providing transferable protons, organizing hydrogen-bond networks, and/or enabling proton-coupled electron transfer, thereby promoting productive O–O bond cleavage and ferryl/Compound I formation. In O2-activating heme and non-heme iron enzymes, substrates or substrate-derived intermediates can contribute electron density or reducing equivalents to metal–O2 adducts, generate radical-coupled intermediates, and guide metal–oxygen species into catalytically competent geometries. These examples indicate that substrate recognition, oxygen activation, and substrate oxidation are often coupled within a single catalytic manifold. This substrate-engaged view complements the traditional metal-centered paradigm and provides mechanistic principles for discovering and designing oxidative biocatalytic reactions in which substrates help generate, control, and productively consume reactive oxygen intermediates.