Effects of Fe2+/Fe3+ Binding to Human Frataxin and Its D122Y Variant, as Revealed by Site-Directed Spin Labeling (SDSL) EPR Complemented by Fluorescence and Circular Dichroism Spectroscopies

Abstract

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 in vitro. 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 Fe2+ in wild type and in the pathological D122Y variant of frataxin, allowed us to characterize the Fe2+ binding properties of different protein sites and highlight the effect of the D122Y substitution on the surrounding residues. We suggest that both Fe2+ and Fe3+ might play a relevant role in the context of the proposed FXN physiological functions.

A Combined Spectroscopic and In Silico Approach to Evaluate the Interaction of Human Frataxin with Mitochondrial Superoxide Dismutase

“…The experiments with Fe 3+ were performed on the same solution as above with the addition of 1 μL of Fe 3+ stock solution. As previously verified [ 32 ], this volume of acidic iron solution does not change the pH of the solution.…”

“…Human mitochondrial SOD (SOD2) was purchased as a His-tag recombinant protein from Technical Novusbio; the SOD2 buffer was Tris HCl buffer (20 mM, pH 8) containing 20% glycerol. The M-TETPO label was synthesized as previously described [ 32 ].…”

“…Heterologous expression and purification of human wild-type FXN and mutants A plasmid containing the coding sequence of human wild-type mature FXN, pET-9b/FXN(90–210), was previously obtained in our laboratory [ 33 ]. FXN mutants were obtained as previously described [ 32 ]. The two additional frataxin mutants used in this work, H177C and S202C, were obtained using the pET-9b/FXN(90–210) plasmid as a template and the couples of primers listed in the SI.…”

“…We make use of the site-directed spin labelling technique (SDSL) coupled to electron paramagnetic resonance spectroscopy (EPR). This is a powerful tool for detecting changes in structure, dynamics, and oligomerization in proteins that was previously used to detect the effects of iron binding to FXN [ 32 ]. The EPR investigation is completed by endogenous tryptophan fluorescence experiments and Molecular Dynamics (MD) simulations.…”

Molecular Details of the Frataxin–Scaffold Interaction during Mitochondrial Fe–S Cluster Assembly

“…Iron binding is also not exclusively localized to the α-helix 1 region; frataxin contains a pool of additional acidic ligands capable of participating in binding iron on the β-strands 1 and 2 [55]. Specifically, FXN D122Y mutation lowers the frataxin Fe(II)-binding affinity but does not impact NFS1 binding, so the potential role of this and additional acidic residues within the β-sheet region related to interacting with metal may be important during assembly [56,57]. Assuming frataxin iron binding is in part driven by availability of Fe(II) from the mitochondrial matrix free iron pool, the flexibility of these iron-binding sites provide for a dynamic interaction between protein binding partners in an iron-dependent manner.…”