A novel CXXC motif-mediated chromate reduction mechanism in the high-activity Cr(VI) reductase BCR005.

Chromate reductases are central to Cr(VI) bioremediation, yet their catalytic mechanisms remain largely uncharacterized. Here we report BCR005, a high-efficiency native chromate reductase with a kcat of 20.8 s⁻¹ , which is 5.6- and 9.9-fold higher than the two previously reported native chromate reductases with available kcat values (ChrR, 3.7 s⁻¹; NemA, 2.1 s⁻¹). rBCR005 (recombinant BCR005 fused with a C-terminal His-tag) and its four CXXC motif mutants (C10S, C13S, DS, and DA) were heterologously expressed and purified. Ellman's assay confirmed that the CXXC motif forms an intramolecular disulfide bond. The DS and DA double mutants showed complete loss of catalytic activity, whereas the single mutants retained partial activity (77% and 90% of rBCR005 Vmax, respectively), indicating functional redundancy between the two cysteine residues. Surface plasmon resonance (SPR) analysis revealed that the CXXC motif serves as the chromate-binding site rather than the FMN-binding site. FMN binding was found to be a prerequisite for chromate recognition, and the two cysteine residues exhibit clear functional asymmetry: Cys10 plays a dominant role in chromate recognition and conformational stability, whereas Cys13 acts as a gatekeeper of the FMN-binding pocket. The disulfide bond itself is not essential for catalysis but contributes to maintaining the spatial coupling of the FMN- and chromate-binding pockets to enable efficient electron transfer from NADPH to Cr(VI). To our knowledge, this is the first report of a functional CXXC motif in any characterized chromate reductase, revealing a previously unreported regulatory mechanism. These findings provide a well-defined molecular target for the rational engineering of high-efficiency biocatalysts for chromium bioremediation.