Antagonistic effects of typical veterinary antibiotic and herbicide on Cu/Zn superoxide dismutase activity: Competitive binding at the dimer interface

Veterinary antibiotics and herbicides could be simultaneously ingested by livestock (especially cattle) due to their co-occurrence in feed, disrupting redox homeostasis. However, limited information is available on the combined molecular-level effects of these agrochemicals on antioxidant enzymes. In this study, sulfadiazine (SD) and metamitron (MET) were respectively selected as the representatives of veterinary antibiotics and herbicides to explore their combined toxic effects on SOD and the underlying molecular mechanisms, using multi-spectroscopic technologies, X-ray photoelectron spectroscopy (XPS), and molecular docking. Binding and conformation characterizations revealed that, at specific compound-to-enzyme ratios, SOD inactivation predominantly originated from direct binding of test chemicals at key sites rather than compound-induced conformational changes. Furthermore, fluorescence quenching analysis demonstrated 1:1 binding stoichiometry, indicating the only one preferential binding site for SD and MET in SOD. An integrated 'interaction-microenvironment-conformation' evaluation framework was established to judge the joint toxic effects between SD and MET, suggesting that these chemicals competitively interacted with the same binding site to antagonistically inhibit SOD activity. Considering the proximal amino acid residues (such as glutamate, arginine, and tyrosine residues) confirmed by activity test, synchronous fluorescence, and XPS, a key binding pocket in the SOD inter-subunit cavity was identified as the interaction site for both SD and MET. Computational simulations further validated and visualized the binding characteristics of test chemicals within this key SOD dimer interface. This study elucidates the interaction characteristics between SOD and typical agrochemicals, providing a new insight and methodology for assessing the molecular-level health risks of mixed agrochemicals.