Ligand-dependent modulation of ROS response in soybean by Cu(II)-amino acid complexes

Copper speciation plays a central role in modulating redox reactivity and biological responses under oxidative stress conditions, for example in soybean (Glycine max (L.) Merr.) under salt stress. In this study, a series of Cu(II)-amino acid complexes (Cu(aa)₂) containing glycine, serine, histidine, tryptophan and tyrosine were synthesized and characterized by elemental analysis, infrared spectroscopy, mass spectrometry, and ultraviolet-visible spectroscopy. Their reactivity toward superoxide was evaluated using the riboflavin-nitro blue tetrazolium assay and potassium superoxide kinetic assays as chemical models of reactive oxygen species (ROS) interaction. The complexes displayed ligand-dependent superoxide reactivity, with Cu(His)₂ showing the highest kinetic response toward KO₂-derived superoxide species. Selected compounds were subsequently evaluated in soybean plants subjected to acute salt stress to investigate the relationship between the copper coordination environment and physiological responses. Histochemical analyses revealed marked differences among treatments: whereas CuCl₂ promoted increased ROS accumulation and phytotoxicity, Cu(aa)₂ complexes reduced superoxide and hydrogen peroxide levels while maintaining lower toxicity. Among the evaluated compounds, Cu(His)₂ exhibited the most consistent behavior, combining high chemical reactivity with effective ROS reduction in planta and moderate copper accumulation in leaf tissues. These findings demonstrate that ligand coordination strongly influences copper behavior, decoupling in vitro reactivity from biological outcome under stress conditions. Overall, the results establish a structure-reactivity-biological response relationship for Cu(II)-amino acid complexes and highlight the importance of copper speciation in the design of safer redox-active compounds for biological applications.