Abstract
Rieske oxygenases (ROs) are a diverse family of nonheme iron enzymes that catalyze a wide array of oxidative transformations in both catabolic and biosynthetic pathways. Their catalytic repertoire spans dioxygenation, monooxygenation, oxidative N- and O-dealkylation, desaturation, sulfoxidation, C-C bond formation, N-oxygenation, and C-N bond cleavage─reactions that are often challenging to achieve selectively through synthetic methods. These diverse functions highlight the increasing importance of ROs in natural product biosynthesis and establish them as promising candidates for biocatalytic applications. Despite extensive study, our understanding of how ROs orchestrate these diverse reactions at the molecular level remains incomplete. In particular, the transient, dynamic nature of electron transfer events and the limited structural characterization of oxygen-bound intermediates hinder our understanding of how structural features govern electron transfer efficiency, O(2) activation, and the origins of their catalytic diversity. Recent findings challenge traditional views of the RO catalytic cycle and underscore the importance of integrating static structural data with dynamic studies of redox interactions. In this Perspective, we explore emerging insights into the structural and mechanistic basis of RO function. We focus on how the architecture of the oxygenase component shapes reactivity, electron transfer, and redox partner interactions. Finally, we discuss current limitations and future opportunities in harnessing ROs for biocatalysis, emphasizing the potential of engineering approaches─particularly the optimization of redox partner compatibility─to expand their functional utility.