Copper (II) binding of NAD(P)H- flavin oxidoreductase (NfoR) enhances its Cr (VI)-reducing ability

Microbes can reduce hexavalent chromium Cr (VI) to the less toxic and soluble trivalent Cr (III). Copper stimulates microbial reduction of Cr (VI) by the Bacillus, Ochrobactrum, and Gluconobacter species; however, the mechanism remains unclear. In our study, the rate of Cr (VI) reduction by Staphylococcus aureus LZ-01 was increased by 210 % when supplemented with 60 μM Cu (II). A putative NAD(P)H-flavin oxidoreductase gene (nfoR) was upregulated under Cr (VI) stress. NfoR-knockout mutant displayed impaired reduction of Cr (VI) and Cu (II)-enhanced Cr (VI) reduction by nfoR isogenic mutant was attenuated in the presence of Cu (II). In vitro tests showed an increased V _max value of 25.22 μM min^-1 mg^-1 NfoR in the presence of Cu (II). Together, these results indicate that NfoR is responsible for Cu (II) enhancement. Isothermal titration calorimetry (ITC) assays confirmed the interaction of NfoR with Cu (II) at the dissociation constant of 85.5 μM. Site-directed mutagenesis indicates that His100, His128, and Met165 residues may be important for Cu (II) binding, while Cys163 is necessary for the FMN binding of NfoR. These findings show that Cu (II)-enhanced NfoR belongs to a new branch of Cr (VI) reductases and profoundly influences Cr (VI) reduction.