Redox sensitive human mitochondrial aconitase and its interaction with frataxin: In vitro and in silico studies confirm that it takes two to tango

• Published in: Free radical biology & medicine 2023-03, Vol.197, p.71-84
• Main Authors: Mansilla, Santiago, Tórtora, Verónica, Pignataro, Florencia, Sastre, Santiago, Castro, Ignacio, Chiribao, Ma. Laura, Robello, Carlos, Zeida, Ari, Santos, Javier, Castro, Laura
• Format: Article
• Language: English
• Subjects: Aconitate Hydratase - metabolism; Electron Spin Resonance Spectroscopy; Frataxin; Humans; Iron-Binding Proteins - genetics; Iron-Binding Proteins - metabolism; Iron-sulfur protein; Iron-Sulfur Proteins - genetics; Iron-Sulfur Proteins - metabolism; Mitochondria; Mitochondrial aconitase; Oxidation-Reduction; protein‐protein interaction; Superoxides - metabolism; Tricarboxylic acid cycle (TCA cycle) (Krebs cycle)
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2023.01.028

Mitochondrial aconitase (ACO2) has been postulated as a redox sensor in the tricarboxylic acid cycle. Its high sensitivity towards reactive oxygen and nitrogen species is due to its particularly labile [4Fe–4S]2+ prosthetic group which yields an inactive [3Fe–4S]+ cluster upon oxidation. Moreover, ACO2 was found as a main oxidant target during aging and in pathologies where mitochondrial dysfunction is implied. Herein, we report the expression and characterization of recombinant human ACO2 and its interaction with frataxin (FXN), a protein that participates in the de novo biosynthesis of Fe–S clusters. A high yield of pure ACO2 (≥99%, 22 ± 2 U/mg) was obtained and kinetic parameters for citrate, isocitrate, and cis-aconitate were determined. Superoxide, carbonate radical, peroxynitrite, and hydrogen peroxide reacted with ACO2 with second-order rate constants of 108, 108, 105, and 102 M−1 s−1, respectively. Temperature-induced unfolding assessed by tryptophan fluorescence of ACO2 resulted in apparent melting temperatures of 51.1 ± 0.5 and 43.6 ± 0.2 °C for [4Fe–4S]2+ and [3Fe–4S]+ states of ACO2, sustaining lower thermal stability upon cluster oxidation. Differences in protein dynamics produced by the Fe–S cluster redox state were addressed by molecular dynamics simulations. Reactivation of [3Fe–4S]+-ACO2 by FXN was verified by activation assays and direct iron-dependent interaction was confirmed by protein-protein interaction ELISA and fluorescence spectroscopic assays. Multimer modeling and protein-protein docking predicted an ACO2-FXN complex where the metal ion binding region of FXN approaches the [3Fe–4S]+ cluster, supporting that FXN is a partner for reactivation of ACO2 upon oxidative cluster inactivation. [Display omitted] •Structural and kinetic data regarding human ACO2 is presented herein•Oxidative inactivated human ACO2 is reactivated by iron-loaded frataxin (FXN)•Experimental and molecular docking evidence of ACO2-FXN complex is shown•FXN-ACO2 interactions stand for ACO2 as a mitochondrial redox regulator