Iron regulatory proteins play a principal role in maintaining cellular iron homeostasis by post-transcriptionally regulating factors responsible for iron uptake, utilization, and storage. An E3 ubiquitin ligase complex containing FBXL5 targets IRP2 for proteasomal degradation under iron- and oxygen-replete conditions, whereas FBXL5 itself is degraded when iron and oxygen availability decreases. FBXL5 contains a hemerythrin-like (Hr) domain at its N terminus that mediates its own differential stability. Here, we investigated the iron- and oxygen-dependent conformational changes within FBXL5-Hr that underlie its role as a cellular sensor. As predicted, FBXL5-Hr undergoes substantive structural changes when iron becomes limiting, accounting for its switch-like behavior. However, these same changes are not observed in response to oxygen depletion, indicating that this domain accommodates two distinct sensing mechanisms. Moreover, FBXL5-Hr does not behave as a dynamic sensor that continuously samples the cellular environment, assuming conformations in equilibrium with ever-changing cellular iron levels. Instead, the isolated domain appears competent to incorporate iron only at or near the time of its own synthesis. These observations have important implications for mechanisms by which these metabolites are sensed within mammalian cells.