Several biological activities depend on iron–sulfur clusters ([Fe-S]). Even though they are well-known in several organisms their function and metabolic pathway were poorly understood in the majority of the organisms. We propose to use the amoeba Dictyostelium discoideum, as a biological model to study the biosynthesis of [Fe-S] at the molecular, cellular and organism levels. First, we have explored the D. discoideum genome looking for genes corresponding to the subunits that constitute the molecular machinery for Fe-S cluster assembly and, based on the structure of the mammalian supercomplex and amino acid conservation profiles, we inferred the full functionality of the amoeba machinery. After that, we expressed the recombinant mature form of D. discoideum frataxin protein (DdFXN), the kinetic activator of this pathway. We characterized the protein and its conformational stability. DdFXN is monomeric and compact. The analysis of the secondary structure content, calculated using the far-UV CD spectra, was compatible with the data expected for the FXN fold, and near-UV CD spectra were compatible with the data corresponding to a folded protein. In addition, Tryptophan fluorescence indicated that the emission occurs from an apolar environment. However, the conformation of DdFXN is significantly less stable than that of the human FXN, (4.0 vs. 9.0 kcal mol−1, respectively). Based on a sequence analysis and structural models of DdFXN, we investigated key residues involved in the interaction of DdFXN with the supercomplex and the effect of point mutations on the energetics of the DdFXN tertiary structure. More than 10 residues involved in Friedreich’s Ataxia are conserved between the human and DdFXN forms, and a good correlation between mutational effect on the energetics of both proteins were found, suggesting the existence of similar sequence/function/stability relationships. Finally, we integrated this information in an evolutionary context which highlights particular variation patterns between amoeba and humans that may reflect a functional importance of specific protein positions. Moreover, the complete pathway obtained forms a piece of evidence in favor of the hypothesis of a shared and highly conserved [Fe-S] assembly machinery between Human and D. discoideum.

The mitochondrial cysteine desulfurase NFS1 is an essential PLP-dependent enzyme involved in iron–sulfur cluster assembly. The enzyme catalyzes the desulfurization of the l-Cys substrate, producing a persulfide and l-Ala as products. In this study, we set the measurement of the product l-Ala by NMR in vitro by means of 1H NMR spectra acquisition. This methodology provided us with the possibility of monitoring the reaction in both fixed-time and real-time experiments, with high sensitivity and accuracy. By studying I452A, W454A, Q456A, and H457A NFS1 variants, we found that the C-terminal stretch (CTS) of the enzyme is critical for function. Specifically, mutation of the extremely conserved position W454 resulted in highly decreased activity. Additionally, we worked on two singular variants: “GGG” and C158A. In the former, the catalytic Cys-loop was altered by including two Gly residues to increase the flexibility of this loop. This variant had significantly impaired activity, indicating that the Cys-loop motions are fine-tuned in the wild-type enzyme. In turn, for C158A, we found an unanticipated increase in l-Cys desulfurase activity. Furthermore, we carried out molecular dynamics simulations of the supercomplex dedicated to iron–sulfur cluster biosynthesis, which includes NFS1, ACP, ISD11, ISCU2, and FXN subunits. We identified CTS as a key element that established interactions with ISCU2 and FXN concurrently; we found specific interactions that are established when FXN is present, reinforcing the idea that FXN not only forms part of the iron–sulfur cluster assembly site but also modulates the internal motions of ISCU2.

Here we present the development of a new model system for Friedreich's Ataxia (FA) using D. discoideum (Dd). FA is a rare disease caused by disfunction of frataxin (FXN), a protein involved in Fe-S cluster assembly machinery. We firstly investigated the conservation of function between human and D. discoideum. In this work we show that DdFXN can substitute the human version in the interaction and activation of Fe-S assembly supercomplex. DdFXN can in vitro displace HsFXN in competition assays and also it can activate cysteine desulfurase activity in the context of human Fe-S assembly supercomplex. We then manage to edit fxn locus and isolated clone 8, a defective mutant with undetectable levels of frataxin. Clone 8 presents landmarks of frataxin deficiency such as decrease in Fe-S cluster dependent enzymatic functions, growth rate reduction and increase sensitivity to oxidative stress. Besides these phenotypes, shared with other FA models, clone 8 presents defects in the multicellular developmental program induced by starvation in this protist. We then assessed the rescuing capacity of DdFXN G122V, a version that mimics a human variant presented in some FA patients. While expression of DdFXN G122V rescues growth and enzymatic activities defects as well as DdFXN does, multicellular development defects were only partially rescued. This work opens the door to develop drug or treatment screenings that would help to design, and/or evaluate therapeutical strategies. Besides this biological model offers a wide range of possibilities to easily explore diverse phenotypes in FA.