Aromatic Residue Variations in the Central β‑Sheet Influence Stability and Activity of E. coli Glutaredoxin 3.

Bacterial glutaredoxin Grx3 is a Class I oxidoreductase with a canonical thioredoxin (Trx) fold but exhibits greater conformational flexibility than Trx due to the absence of one α-helix and one β-strand. In Escherichia coli Grx3 (EcGrx3), the adjacent β1 and β3 strands contain Tyr6 and Phe56, which interact with Arg46 in the α2 helix via hydrogen bonding and cation-π interactions, forming a stabilizing Tyr6-Arg46-Phe56 network. To investigate how aromatic residue variation at these sites affects EcGrx3 stability and activity, we introduced substitutions at Phe56 (F56A, F56S, F56I, F56Y, and F56W), Tyr6 (Y6F), and a double mutant (Y6F/F56Y). All mutants showed reduced melting temperatures and increased sensitivity to guanidinium chloride (GdmCl)-induced unfolding. Although F56Y and F56W can form cation-π interactions with Arg46, they exhibited the lowest thermal stability but distinct functional outcomes. F56Y retained wild-type-like activity and flexibility and was most resistant to chemical denaturation, while F56W, with higher α-helix content and rigidity, showed the highest catalytic efficiency but was highly GdmCl-sensitive. Aliphatic substitutions (F56A and F56I) caused moderate destabilization, while polar (F56S) and charged (F56E) mutants were more disruptive. Y6F significantly reduced both α-helix content and catalytic efficiency. These results demonstrate that both cation-π interactions and hydrophobic packing at position 56 are critical for EcGrx3 stability, with Phe56 providing optimal balance. Tyr6 stabilizes the β1-α2 interface via hydrogen bonding, and both residues are critical for α-helix formation. Together, these findings highlight how aromatic variation within the central β-sheet contributes to structural and functional adaptation in the Trx-fold superfamily.