Molecular mechanism investigation on the interactions of copper (II) ions with glutathione peroxidase 6 from Arabidopsis thaliana

Accumulation of copper (II) ions in plant leads to the excessive reactive oxygen species (ROS) which attributes to the depletion of the antioxidants in the cell and destruction to antioxidant enzymes. The antioxidant enzyme glutathione peroxidase has been used as biomarkers to reflect metal-induced oxidative stress. However, the underlying toxic mechanisms of the copper ions(II)-induced oxidative damage to plants remain unknown. In the work, a detailed molecular interaction of copper (II) ions with Arabidopsis thaliana glutathione peroxidase 6 (AtGPX6) in relation with poisonous effects of exposure to heavy metal was investigated by multiple spectroscopic techniques. The intrinsic fluorescence of AtGPX6 was quenched upon the addition of copper (II) ions by the combination of static and dynamic quenching mechanisms accompanied by complex formation and conformational changes. A single binding site was revealed for AtGPX6 towards copper ions. The binding process was hydrophobic effect accompanied by positive entropy change and enthalpy change. The secondary structure of AtGPX6 was changed by the addition of copper ions investigated by synchronous fluorescence spectroscopy, ultraviolet-visible absorption spectroscopy and circular dichroism spectroscopy, resulting in loosened and deconstructed protein skeleton and increased hydrophobicity around the Trp residues. This study helps to illuminate the detailed interactions between copper ions and plant glutathione peroxidase and elucidate the destructive mechanism to antioxidative defense system caused by heavy metal exposure at molecular level.