A π-stacking gate for redox control in flavin ferredoxin-thioredoxin reductases
Minjarez-Saenz, Martha ; Correa-Pérez, Víctor (Universidad de Zaragoza) ; Rivero, Maria Isabel (Universidad de Zaragoza) ; Sadeghi, Sheila J. ; Calvo, Javier ; Velázquez-Campoy, Adrián (Universidad de Zaragoza) ; Gago, Federico ; Buey, Rubén M. ; Martínez-Júlvez, Marta ; Medina, Milagros (Universidad de Zaragoza) ; Balsera, Monica
Resumen: Flavin ferredoxin-thioredoxin reductases (FFTRs) constitute a unique class of enzymes that transfer electrons from low-potential ferredoxins (Fdxs) to thioredoxins (Trxs) through a flavin cofactor. They are widely distributed across bacterial lineages, including cyanobacteria and anaerobes such as Clostridium, where they function either as the sole Trx reductase or in parallel with canonical NADPH-dependent or iron–sulfur systems. A distinguishing feature of cyanobacterial FFTRs is a C-terminal tail containing a conserved tryptophan (Trp) that engages in a π-stacking interaction with the flavin—a motif absent in clostridial orthologs. In this study, we use the cyanobacterial FFTR from Gloeobacter violaceus as a model to demonstrate that the C-terminal tail and its conserved aromatic residue modulate the FAD electronic environment, electron transfer efficiency, and Fdx donor interactions. Mutants lacking the tail or Trp exhibited increased flavin solvent exposure, a redox potential shift of over 200 mV toward less negative values, altered reduction kinetics, impaired electron flow to the redox-active disulfide, and reduced specificity for Fdx binding. These results reveal a dual role for the C-terminal tail: it establishes a productive donor-binding interface and shapes the FAD environment to meet the thermodynamic and kinetic requirements for efficient intramolecular electron transfer to the redox-active disulfide. Collectively, these findings provide mechanistic insight into how peripheral structural features, such as FAD-aromatic π-stacking interactions, govern flavin reactivity, donor specificity, and redox behavior in cyanobacterial FFTRs, underscoring their relevance and offering a framework for engineering redox-active biocatalysts for synthetic biology and metabolic applications
Idioma: Inglés
DOI: 10.1016/j.ijbiomac.2025.148860
Año: 2025
Publicado en: International journal of biological macromolecules 333 (2025), 148860 [11 pp.]
ISSN: 0141-8130
Financiación: info:eu-repo/grantAgreement/ES/AEI/PID2023-150714OB-I00
Financiación: info:eu-repo/grantAgreement/ES/DGA/E35-23R
Financiación: info:eu-repo/grantAgreement/ES/MICINN-AEI/PID2019-110900GB-I00
Financiación: info:eu-repo/grantAgreement/ES/MICINN/PID2022-136369NB-I00
Tipo y forma: Article (Published version)
Área (Departamento): Área Bioquímica y Biolog.Mole. (Dpto. Bioq.Biolog.Mol. Celular)
You must give appropriate credit, provide a link to the license, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use. You may not use the material for commercial purposes.
Exportado de SIDERAL (2025-11-27-15:16:21)
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Idioma: Inglés
DOI: 10.1016/j.ijbiomac.2025.148860
Año: 2025
Publicado en: International journal of biological macromolecules 333 (2025), 148860 [11 pp.]
ISSN: 0141-8130
Financiación: info:eu-repo/grantAgreement/ES/AEI/PID2023-150714OB-I00
Financiación: info:eu-repo/grantAgreement/ES/DGA/E35-23R
Financiación: info:eu-repo/grantAgreement/ES/MICINN-AEI/PID2019-110900GB-I00
Financiación: info:eu-repo/grantAgreement/ES/MICINN/PID2022-136369NB-I00
Tipo y forma: Article (Published version)
Área (Departamento): Área Bioquímica y Biolog.Mole. (Dpto. Bioq.Biolog.Mol. Celular)
You must give appropriate credit, provide a link to the license, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use. You may not use the material for commercial purposes. Exportado de SIDERAL (2025-11-27-15:16:21)
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Record created 2025-11-27, last modified 2025-11-27