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.

Davide Doni,Javier Santos,Marco Bortolus,Paola Costantini,Donatella Carbonera,Marvin Schulz,Leonardo Passerini,Sylvain R A Marque,Gérard Audran

doi:10.3390/ijms21249619

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.

Highlights

Frataxin (FXN) is a small acidic protein that is highly conserved in most organisms, from bacterial to mammalian
Frataxin’s actual physiological function has been debated for a long time without reaching a general agreement; 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 participates in iron homeostasis
We use site-directed spin labeling coupled to electron paramagnetic resonance (SDSL Electron Paramagnetic Resonance (EPR)) to add new information on the effects of ferric and ferrous iron binding on the properties of human frataxin in vitro

Summary

Introduction

Frataxin (FXN) is a small acidic protein that is highly conserved in most organisms, from bacterial to mammalian. Human FXN is nuclear encoded, expressed in the cytoplasm as a precursor of 210 amino acids, and imported into the mitochondria, where it undergoes two-step maturation by the mitochondrial processing peptidase (MMP) [9,10,11]. MMP first cleaves a portion of the N-terminus, the mitochondrial import signal, originating an intermediate form (FXN 42–210), and with a further cleavage MMP produces the mature FXN 81–210, which is the most abundant form found both in normal individuals and in FRDA patients [9]. Sequence alignment studies have distinguished the N-terminal frataxin region, which is intrinsically unfolded and poorly conserved among the different species, from the C-terminus highly conserved block of about 100–120 amino acids, which is considered the most important part for protein function [12]. Despite harboring a potential iron binding site [15], it is often truncated (obtaining FXN 90–210) for in vitro studies

Results

Discussion

Conclusion