Abstract
This study investigates the impact of aromatic cluster side-chain interactions in Grx3 (SpGrx3) from the psychrophilic Arctic bacterium Sphingomonas sp. Grx3 is a class I oxidoreductase with a unique parallel arrangement of aromatic residues in its aromatic cluster, unlike the tetrahedral geometry observed in Trxs. Hydrophilic-to-hydrophobic substitutions were made in the aromatic cluster, in β1 (E5V and Y7F), adjacent β2 (Y32F and Y32L), both β1 and β2 (E5V/Y32L), and short α2 (R47F). The hydrophobic substitutions, particularly those at or near Tyr7 (E5V, Y7F, Y32F, and R47F), increased melting temperatures and conformational stability, whereas disrupting β1-β2 interactions (Y32L and E5V/Y32L) led to structural instability of SpGrx3. However, excessive hydrophobic interactions (Y7F and E5V/Y32L) caused protein aggregation at elevated temperatures. All mutations resulted in a reduction in α-helical content and an increase in β-strand content. The R47F mutant, which formed dimers and exhibited the highest β-strand content, showed increased conformational flexibility and a significant decrease in catalytic rate due to the disturbance of β1-α2 interactions. In summary, the configuration of the aromatic cluster, especially Tyr7 in the buried β1 and Arg47 in the short α2, played crucial roles in maintaining the active conformation of SpGrx3 and preventing its protein aggregation. These modifications, reducing hydrophobicity in the central β-sheet, distinguish Grx3 from other Trx-fold proteins, highlighting evolutionary divergence within the Trx-fold superfamily and its functional versatility.