Effects of Fe<sup>2+</sup>/Fe<sup>3+</sup> Binding to Human Frataxin and Its D122Y Variant, as Revealed by Site-Directed Spin Labeling (SDSL) EPR Complemented by Fluorescence and Circular Dichroism Spectroscopies
Frataxin is a highly conserved protein whose deficiency results in the neurodegenerative disease Friederich’s ataxia. 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 b...
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MDPI AG
2020-12-01
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| Series: | International Journal of Molecular Sciences |
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| author | Davide Doni Leonardo Passerini Gérard Audran Sylvain R. A. Marque Marvin Schulz Javier Santos Paola Costantini Marco Bortolus Donatella Carbonera |
| author_facet | Davide Doni Leonardo Passerini Gérard Audran Sylvain R. A. Marque Marvin Schulz Javier Santos Paola Costantini Marco Bortolus Donatella Carbonera |
| author_sort | Davide Doni |
| collection | DOAJ |
| description | Frataxin is a highly conserved protein whose deficiency results in the neurodegenerative disease Friederich’s ataxia. 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-sulphur cluster (ISC) machinery, and several authors have pointed out that it also participates in iron homeostasis. In this work, we use site-directed spin labeling coupled to electron paramagnetic resonance (SDSL EPR) to add new information on the effects of ferric and ferrous iron binding on the properties of human frataxin <i>in vitro</i>. Using SDSL EPR and relating the results to fluorescence experiments commonly performed to study iron binding to FXN, we 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 Fe<sup>2+</sup> in wild type and in the pathological D122Y variant of frataxin, allowed us to characterize the Fe<sup>2+</sup> binding properties of different protein sites and highlight the effect of the D122Y substitution on the surrounding residues. We suggest that both Fe<sup>2+</sup> and Fe<sup>3+</sup> might play a relevant role in the context of the proposed FXN physiological functions. |
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| language | English |
| last_indexed | 2024-03-10T14:00:02Z |
| publishDate | 2020-12-01 |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-6110282b0cf246ddbebb39ebc1e2ef752023-11-21T01:15:09ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672020-12-012124961910.3390/ijms21249619Effects of Fe<sup>2+</sup>/Fe<sup>3+</sup> Binding to Human Frataxin and Its D122Y Variant, as Revealed by Site-Directed Spin Labeling (SDSL) EPR Complemented by Fluorescence and Circular Dichroism SpectroscopiesDavide Doni0Leonardo Passerini1Gérard Audran2Sylvain R. A. Marque3Marvin Schulz4Javier Santos5Paola Costantini6Marco Bortolus7Donatella Carbonera8Department of Biology, University of Padova, Viale G. Colombo 3, 35131 Padova, ItalyDepartment of Chemical Sciences, University of Padova, Via F. Marzolo 1, 35131 Padova, ItalyInstitut de Chimie Radicalaire, Aix Marseille Universitè, CNRS, ICR, UMR 7273, Case 551, Ave Escadrille Normandie Niemen, CEDEX 20, 13397 Marseille, FranceInstitut de Chimie Radicalaire, Aix Marseille Universitè, CNRS, ICR, UMR 7273, Case 551, Ave Escadrille Normandie Niemen, CEDEX 20, 13397 Marseille, FranceInstitut de Chimie Radicalaire, Aix Marseille Universitè, CNRS, ICR, UMR 7273, Case 551, Ave Escadrille Normandie Niemen, CEDEX 20, 13397 Marseille, FranceDepartamento de Química Biológica, Instituto de Biociencias, Biotecnología y Biomedicina (iB3-UBA), Facultad de Ciencia Exactas y Naturales, Universidad de Buenos Aires, Intendente Güiraldes 2160—Ciudad Universitaria, 1428EGA CONICET, Godoy Cruz 2290, Buenos Aires C1425FQB, ArgentinaDepartment of Biology, University of Padova, Viale G. Colombo 3, 35131 Padova, ItalyDepartment of Chemical Sciences, University of Padova, Via F. Marzolo 1, 35131 Padova, ItalyDepartment of Chemical Sciences, University of Padova, Via F. Marzolo 1, 35131 Padova, ItalyFrataxin is a highly conserved protein whose deficiency results in the neurodegenerative disease Friederich’s ataxia. 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-sulphur cluster (ISC) machinery, and several authors have pointed out that it also participates in iron homeostasis. In this work, we use site-directed spin labeling coupled to electron paramagnetic resonance (SDSL EPR) to add new information on the effects of ferric and ferrous iron binding on the properties of human frataxin <i>in vitro</i>. Using SDSL EPR and relating the results to fluorescence experiments commonly performed to study iron binding to FXN, we 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 Fe<sup>2+</sup> in wild type and in the pathological D122Y variant of frataxin, allowed us to characterize the Fe<sup>2+</sup> binding properties of different protein sites and highlight the effect of the D122Y substitution on the surrounding residues. We suggest that both Fe<sup>2+</sup> and Fe<sup>3+</sup> might play a relevant role in the context of the proposed FXN physiological functions.https://www.mdpi.com/1422-0067/21/24/9619frataxinironEPRfluorescenceCDFe-S cluster assembly machinery |
| spellingShingle | Davide Doni Leonardo Passerini Gérard Audran Sylvain R. A. Marque Marvin Schulz Javier Santos Paola Costantini Marco Bortolus Donatella Carbonera Effects of Fe<sup>2+</sup>/Fe<sup>3+</sup> Binding to Human Frataxin and Its D122Y Variant, as Revealed by Site-Directed Spin Labeling (SDSL) EPR Complemented by Fluorescence and Circular Dichroism Spectroscopies International Journal of Molecular Sciences frataxin iron EPR fluorescence CD Fe-S cluster assembly machinery |
| title | Effects of Fe<sup>2+</sup>/Fe<sup>3+</sup> Binding to Human Frataxin and Its D122Y Variant, as Revealed by Site-Directed Spin Labeling (SDSL) EPR Complemented by Fluorescence and Circular Dichroism Spectroscopies |
| title_full | Effects of Fe<sup>2+</sup>/Fe<sup>3+</sup> Binding to Human Frataxin and Its D122Y Variant, as Revealed by Site-Directed Spin Labeling (SDSL) EPR Complemented by Fluorescence and Circular Dichroism Spectroscopies |
| title_fullStr | Effects of Fe<sup>2+</sup>/Fe<sup>3+</sup> Binding to Human Frataxin and Its D122Y Variant, as Revealed by Site-Directed Spin Labeling (SDSL) EPR Complemented by Fluorescence and Circular Dichroism Spectroscopies |
| title_full_unstemmed | Effects of Fe<sup>2+</sup>/Fe<sup>3+</sup> Binding to Human Frataxin and Its D122Y Variant, as Revealed by Site-Directed Spin Labeling (SDSL) EPR Complemented by Fluorescence and Circular Dichroism Spectroscopies |
| title_short | Effects of Fe<sup>2+</sup>/Fe<sup>3+</sup> Binding to Human Frataxin and Its D122Y Variant, as Revealed by Site-Directed Spin Labeling (SDSL) EPR Complemented by Fluorescence and Circular Dichroism Spectroscopies |
| title_sort | effects of fe sup 2 sup fe sup 3 sup binding to human frataxin and its d122y variant as revealed by site directed spin labeling sdsl epr complemented by fluorescence and circular dichroism spectroscopies |
| topic | frataxin iron EPR fluorescence CD Fe-S cluster assembly machinery |
| url | https://www.mdpi.com/1422-0067/21/24/9619 |
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