A π-stacking gate for redox control in flavin ferredoxin-thioredoxin reductases

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.