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

Davide Doni,Sylvain R A Marque,Marco Bortolus,Donatella Carbonera,Javier Santos,Paola Costantini,Marta Meggiolaro,Gérard Audran

doi:10.3390/biomedicines9121763

Abstract

Frataxin (FXN) is a highly conserved mitochondrial protein whose deficiency causes Friedreich’s ataxia, a neurodegenerative disease. The precise physiological function of FXN is still unclear; however, there is experimental evidence that the protein is involved in biosynthetic iron–sulfur cluster machinery, redox imbalance, and iron homeostasis. FXN is synthesized in the cytosol and imported into the mitochondria, where it is proteolytically cleaved to the mature form. Its involvement in the redox imbalance suggests that FXN could interact with mitochondrial superoxide dismutase (SOD2), a key enzyme in antioxidant cellular defense. In this work, we use site-directed spin labelling coupled to electron paramagnetic resonance spectroscopy (SDSL-EPR) and fluorescence quenching experiments to investigate the interaction between human FXN and SOD2 in vitro. Spectroscopic data are combined with rigid body protein–protein docking to assess the potential structure of the FXN-SOD2 complex, which leaves the metal binding region of FXN accessible to the solvent. We provide evidence that human FXN interacts with human SOD2 in vitro and that the complex is in fast exchange. This interaction could be relevant during the assembly of iron-sulfur (FeS) clusters and/or their incorporation in proteins when FeS clusters are potentially susceptible to attacks by reactive oxygen species.

Highlights

We previously produced a library of FXN site-directed mutants across the protein where a native amino acid was mutated to cysteine and labeled with a spin probe to study the effect of Fe2+/3+ binding [32]
This study stemmed from previous work by Han et al [29], which reported on the interaction between yeast FXN and superoxide dismutases (SODs)
SOD2, since Han and coworkers estimated that at physiological concentrations a complex between FXN and SOD1 could not be formed within the mitochondria [29]

Summary

Introduction

Frataxin (FXN) is a small, soluble protein that is highly conserved in most organisms, from bacteria to mammalian. The neurodegenerative disease Friedreich’s ataxia (FRDA; OMIM 229300) [1] results from low FXN expression, primarily caused by an abnormal GAA triplet repeat expansion in the first intron of the frataxin gene. In addition to low expression levels of FXN, several point mutations of FXN, including nonsense, missense, insertions, and deletions, have been associated with compound heterozygous FRDA patients [1,2,3,4]. The principal effect of FRDA is a large depletion of proteins containing iron–sulfur clusters (ISC) as cofactors, such as respiratory chain complexes (complex I-III) or other enzymes, Biomedicines 2021, 9, 1763.

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Results

Conclusion