U32 enzymes are a new class of bacterial O2-independent hydroxylases.1 Members of this family perform key reactions in quinone biosynthesis and RNA modification.2-5 Their functions are hypothesized to serve roles in bacterial adaptation to hypoxic and anoxic conditions.6-7 Thus, these enzymes could be unique therapeutic targets for pathogens that form biofilms or infect human body sites with low oxygen availability. However, the catalytic mechanism and structure of U32 oxidases are unknown. Here, we characterize the structure and redox properties of iron-sulfur clusters in U32 enzymes. Our studies reveal common features across U32 oxidases, including the presence of labile [Fe4S4] clusters and the requirement for two clusters per protein complex. We observe substrate-induced perturbation of the iron-sulfur clusters, suggesting direct interaction and a potential role in substrate activation. Based on these studies, we propose mechanistic hypotheses for O2-independent hydroxylation catalyzed by U32 oxidases. Lastly, we analyze the distribution of U32 enzymes across bacterial phyla and explore the potential for novel oxidases within this family. This work expands our understanding of the structure and function of iron-sulfur cluster-dependent enzymes and redox chemistry in anaerobic bacteria.