Aromatic Residue F443 Modulates the Dimer Interface and Activity of Pseudomonas mandelii Glutathione Reductase

Abstract

Glutathione reductase (GR) is a homodimeric flavoenzyme that regenerates reduced glutathione, essential for cellular redox balance, with an active site cleft at the upper dimer interface. However, the role of intramolecular interactions within each monomer in maintaining the dimer interface and influencing enzyme activity remains unclear. This study investigates how hydrophobic interactions within each monomer near the dimer interface, particularly involving L416 (α12 helix) and F443 (α14 helix), affect the stability and activity of GR from the psychrotrophic bacterium Pseudomonas mandelii JR-1 (PmGR). Mutations at L416 (L416A, L416E, L416Q) preserved the dimeric form during native polyacrylamide gel electrophoresis at 4 and 50 °C but reduced catalytic function, with activity progressively declining at 50 and 60 °C. In contrast, F443A remained dimeric only at 4 °C, while F443E and L416A/F443A disrupted hydrophobic interactions, causing dimer dissociation at 4 °C and severely impairing enzyme activity. F443L, however, retained dimerization under all tested conditions. Despite these disruptions, all mutants except F443A retained their secondary structure. These findings demonstrate that L416 in the longer α12 helix modulates enzyme kinetics without destabilizing the dimer interface, whereas hydrophobic interactions involving F443 in the shorter α14 helix are critical for dimer integrity and activity. This study highlights the essential role of hydrophobic interactions within each monomer, particularly those involving F443, alongside dimer interface interactions, in maintaining GR stability and activity, offering valuable insights for GR engineering in oxidative stress defense.