Dual activation of azoreductase SaCinD by Copper(II) via upregulated expression and activity enhancement

Copper can accelerate microbial detoxification of dyes, yet the precise impact of Cu(II) on azoreductase expression and function remains unclear. Here, a Cu(II)-dependent expression of gene SaCinD, encoding azoreductase, was identified from azo-reducing Staphylococcus aureus LZ-01. Flow cytometry and fluorescence microscopy analysis revealed that the copper regulator SaCopR manipulated the gene expression of SaCinD. Moreover, the K_m and V_max values of SaCinD towards Acid Red were 30.1 μM and 1.2 μM/min/mg, with the presence of Cu(II) leading to a 1.5-fold increase in V_max. ITC analysis confirmed Cu(II) binds to SaCinD with a dissociation constant of 69.23 μM. Acute toxicity assessments of degradation products demonstrated reduced toxicity levels compared to the original dyes. Site-directed mutagenesis confirmed that key residues Lys94 and Leu156 were potential Cu(II) binding sites of SaCinD. Mutants F98A and Y24A showed a 1.3-fold increase in the Acid Red activity, restoring the ability to metabolize Methyl Orange, albeit with a trade-off of reduced T_m values by 5°C. Simulated wastewater scenarios illustrated the exceptional decoloration capabilities of SaCinD. Our study proposes a novel Cu(II)-mediated dual activation mechanism for microbial detoxification, involving Cu(II)-induced SaCinD expression and Cu(II)-enhanced SaCinD activity, offering a promising approach for bioremediation of co-contaminated wastewater.