Cu(II) nonspecifically binding chromate reductase NfoR promotes Cr(VI) reduction

Abstract

Cu(II)-enhanced microbial Cr(VI) reduction is common in the environment, yet its mechanism is unknown. The specific activity of chromate reductase, NfoR, from Staphylococcus aureus sp. LZ-01 was augmented 1.5-fold by Cu(II). Isothermal titration calorimetry and spectral data show that Cu(II) binds to NfoR nonspecifically. Further, Cu(II) stimulates the nitrobenzene reduction of NfoR, indicating that Cu(II) promotes electron transfer. The crystal structure of NfoR in complex with CuSO₄ (1.46 Å) was determined. The overall structure of NfoR-Cu(II) complex is a dimer that covalently binds with FMN and Cu(II)-binding pocket is located at the interface of the NfoR dimer. Structural superposition revealed that NfoR resembles the structure of class II chromate reductase. Site-directed mutagenesis revealed that Leu⁴⁶ and Phe¹²³ were involved in NADH binding, whereas Trp⁷⁰ and Ser⁴⁵ were the key residues for nitrobenzene binding. Furthermore, His¹⁰⁰ and Asp¹⁷¹ were preferential affinity sites for Cu(II) and that Cys¹⁶³ is an active site for FMN binding. Attenuation reductase activity in C163S can be partially restored to 54% wild type by increasing Cu(II) concentration. Partial restoration indicates dual-channel electron transfer of NfoR via Cu(II) and FMN. We propose a catalytic mechanism for Cu(II)-enhanced NfoR activity in which Cu(I) is formed transiently. Together, the current results provide an insight on Cu (II)-induced enhancement and benefit of Cr(VI) bioremediation.