The AxqNOR-BRIL and Val495Ala-BRIL expression plasmid was made by GenScript (Hong Kong), with the AxqNOR gene (NorZ) truncated (Δ747 to Δ763) to accommodate the apocytochrome b562 [BRIL562, Protein Data Bank (PDB) accession code: 1M6T] fusion partner at the C terminus of AxqNOR. The chimeric gene (qNOR-BRIL) was then ligated between the Nde I and Xho I sites of a pET-26b (+) plasmid, allowing a hexa-histidine tag to be attached at the C terminus of BRIL562. Site-directed mutants of qNOR were generated using the QuikChange II Kit (Agilent) using the wild-type AxqNOR plasmid as a template vector. Mutations were confirmed by DNA sequencing before use.

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