Frataxin's actual physiological function has been debated for a long time without reaching a general agreement; however, it is commonly accepted that the protein is involved in the biosynthetic iron-sulfur cluster (ISC) machinery, and several authors have pointed out that it also participates in iron homeostasis. In this work, site-directed spin labeling coupled to electron paramagnetic resonance (SDSL EPR) is used to add new information on the effects of ferric and ferrous iron binding on the properties of human frataxin in vitro. Using SDSL EPR and relating the results to fluorescence experiments commonly performed to study iron binding to FXN, the authors produced evidence that ferric iron causes reversible aggregation without preferred interfaces in a concentration-dependent fashion, starting at relatively low concentrations (micromolar range), whereas ferrous iron binds without inducing aggregation. Moreover, our experiments show that the ferrous binding does not lead to changes of protein conformation. The data reported in this study reveal that the currently reported binding stoichiometries should be taken with caution. The use of a spin label resistant to reduction, as well as the comparison of the binding effect of Fe2+ in wild type and in the pathological D122Y variant of frataxin, allowed the characterization of the Fe2+ binding properties of different protein sites and highlight the effect of the D122Y substitution on the surrounding residues. The authors suggest that both Fe2+ and Fe3+ might play a relevant role in the context of the proposed FXN physiological functions.

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