Redox sensitive human mitochondrial aconitase and its interaction with frataxin: In vitro and in silico studies confirm that it takes two to tango
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, A...
| Autores: | , , , , , , , , , |
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| Tipo de recurso: | artículo |
| Estado: | Versión publicada |
| Fecha de publicación: | 2023 |
| País: | Argentina |
| Institución: | Consejo Nacional de Investigaciones Científicas y Técnicas |
| Repositorio: | CONICET Digital (CONICET) |
| Idioma: | inglés |
| OAI Identifier: | oai:ri.conicet.gov.ar:11336/225425 |
| Acceso en línea: | http://hdl.handle.net/11336/225425 |
| Access Level: | acceso abierto |
| Palabra clave: | ACONITASE FRATAXIN MITOCHONDRIA INTERACTION https://purl.org/becyt/ford/1.6 https://purl.org/becyt/ford/1 |
| Sumario: | 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. |
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