Friedreich's ataxia (FRDA) is linked to a deficiency of frataxin (FXN), a mitochondrial protein involved in iron-sulfur cluster synthesis. FXN is a small protein with an a/b fold followed by the C-terminal region (CTR) with a nonperiodic structure that packs against the protein core. In the present study, we explored the impact of the alteration of the CTR on the stability and dynamics of FXN. We analyzed several pathological and rationally designed CTR mutants using complementary spectroscopic and biophysical approaches. The pathological mutation L198R yields a global destabilization of the structure correlating with a significant and highly localized alteration of dynamics, mainly involving residues that are in contact with L198 in wild-type FXN. , which is closely related to the FRDA-associated mutant FXN 81-193, conserves a globular shape with a native-like structure. However, the truncation of the CTR results in an extreme alteration of global stability and protein dynamics over a vast range of timescales and encompassing regions far from the CTR, as shown by proton-water exchange rates and 15 N-relaxation measurements. Increased sensitivity to proteolysis, observed in vitro for both mutants, suggests a faster degradation rate in vivo, whereas the enhanced tendency to aggregate exhibited by the truncated variant may account for the loss of functional FXN, with both phenomena providing an explanation as to why the alteration of the CTR causes FRDA. These results contribute to understanding how stability and activity are linked to protein motions and they might be useful for the design of target-specific ligands to control local protein motions for stability enhancement.

2‐Cys peroxiredoxins are peroxidases devoid of prosthetic groups that mediate in the defence against oxidative stress and the peroxide activation of signaling pathways. This dual capacity relies on the high reactivity of the conserved peroxidatic and resolving cysteines, whose modification embraces not only the usual thiol–disulfide exchange but also higher oxidation states of the sulfur atom. These changes are part of a complex system wherein the cooperation with other post‐translational modifications – phosphorylation, acetylation – may function as major regulatory mechanisms of the quaternary structure. More importantly, modern proteomic approaches have identified the oxyacids at cysteine residues as novel protein targets for unsuspected post‐translational modifications, such as phosphorylation that yields the unusual sulfi(o)nic–phosphoryl anhydride. In this article, we review the biochemical attributes of 2‐Cys peroxiredoxins that, in combination with complementary studies of forward and reverse genetics, have generated stimulating molecular models to explain how this enzyme integrates into cell signaling in vivo.

2-Cys peroxiredoxins (2-Cys Prx) are ubiquitous thiol-containing peroxidases that have been implicated in antioxidant defense and signal transduction. Although their biochemical features have been extensively studied, little is known about the mechanisms that link the redox activity and non-redox processes. Here we report that the concerted action of a nucleoside triphosphate and Mg(2+) on rapeseed 2-Cys Prx reversibly impairs the peroxidase activity and promotes the formation of high molecular mass species. Using protein intrinsic fluorescence in the analysis of site-directed mutants, we demonstrate that ATP quenches the emission intensity of Trp179, a residue close to the conserved Cys175. More importantly, we found that ATP facilitates the autophosphorylation of 2-Cys Prx when the protein is successively reduced with thiol-bearing compounds and oxidized with hydroperoxides or quinones. MS analyses reveal that 2-Cys Prx incorporates the phosphoryl group into the Cys175 residue yielding the sulfinic-phosphoryl [Prx-(Cys175)-SO(2)PO(3)(2-)] and the sulfonic-phosphoryl [Prx-(Cys175)-SO(3)PO(3)(2-)] anhydrides. Hence, the functional coupling between ATP and 2-Cys Prx gives novel insights into not only the removal of reactive oxygen species, but also mechanisms that link the energy status of the cell and the oxidation of cysteine residues.