164168 20251127172930.0 doi 10.1016/j.ijbiomac.2025.148860 sideral 146338 ART-2025-146338 eng Minjarez-Saenz, Martha (orcid)0000-0001-7947-8813 A π-stacking gate for redox control in flavin ferredoxin-thioredoxin reductases 2025 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 Access copy available to the general public Unrestricted info:eu-repo/grantAgreement/ES/AEI/PID2023-150714OB-I00 info:eu-repo/grantAgreement/ES/DGA/E35-23R info:eu-repo/grantAgreement/ES/MICINN-AEI/PID2019-110900GB-I00 info:eu-repo/grantAgreement/ES/MICINN/PID2022-136369NB-I00 info:eu-repo/semantics/openAccess by-nc https://creativecommons.org/licenses/by-nc/4.0/deed.es info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion Correa-Pérez, Víctor Universidad de Zaragoza Rivero, Maria Isabel Universidad de Zaragoza Sadeghi, Sheila J. Calvo, Javier Velázquez-Campoy, Adrián Universidad de Zaragoza (orcid)0000-0001-5702-4538 Gago, Federico Buey, Rubén M. Martínez-Júlvez, Marta Medina, Milagros Universidad de Zaragoza (orcid)0000-0001-8743-0182 Balsera, Monica 1002 060 Universidad de Zaragoza Dpto. Bioq.Biolog.Mol. Celular Área Bioquímica y Biolog.Mole. 333 (2025), 148860 [11 pp.] Int. j. biol. macromol. International journal of biological macromolecules 0141-8130 2112644 http://zaguan.unizar.es/record/164168/files/texto_completo.pdf Versión publicada 2462990 http://zaguan.unizar.es/record/164168/files/texto_completo.jpg?subformat=icon icon Versión publicada oai:zaguan.unizar.es:164168 articulos driver 2025-11-27-15:16:21 ARTICLE