Redox cycling nitroxide limits cellular iron availability and selectively inhibits iron-sulfur cluster metabolism.

Iron-sulfur clusters (ISCs) are redox active cofactors for essential proteins with diverse functions. We demonstrate that Tempol, a redox cycling nitroxide, limits iron bioavailability in a manner distinct from iron chelators, mainly via its effect on ascorbate and iron redox balance. This non-canonical iron limitation triggers upregulation of IRP2 and HIF1α, proteins whose degradation is ferrous iron-dependent, while disrupting ISC synthesis only in a subset of cell lines. Suppression of ISC synthesis inhibits ISC-dependent enzymes, destabilizes ISC proteins, and reduces cell viability, particularly in cells dependent on ISC protein ACO2. These effects are reversed by the reducing agent ascorbate, a cofactor required for multiple enzymes, such as the HIF1α prolyl hydroxylases. Tempol treatment also inhibits ferroptosis, an oxidative form of cell death catalyzed by reduced iron. These results demonstrate ascorbate and cellular iron redox state are essential in iron homeostasis, which is proposed to underlie pathological conditions from neurodegeneration to cancer.