Characterizationof the Iron–Sulfur Clusterin the NCOA4 Fragment (383–522) and Its Interaction with Ferritin

Ferritin degradation pathways, particularly NCOA4-mediated ferritinophagy, are crucial for maintaining iron homeostasis. Here, we demonstrate the coexistence of two NCOA4 isoforms, one iron–sulfur cluster-free and one iron–sulfur cluster-bound, in oxygenated cell cultures. Using a combination of spectroscopic and analytical techniques, in vitro characterization of the NCOA4 fragment (383–522), denoted NCOA4-D, revealed a predominance of monomeric species with a relatively stable [2Fe-2S] cluster under normoxic conditions. The results demonstrate distinct interactions between NCOA4-D isoforms and ferritin, underscoring the influence of cellular oxygen and iron concentrations on NCOA4’s regulatory functions, pathways, and ferritin’s fate. Our findings suggest that different NCOA4-initiated degradation pathways may concurrently occur in cells and highlight the necessity of further exploring the role of the Fe–S cluster in NCOA4 as an iron-sensing mechanism for maintaining cellular iron homeostasis.