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Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) is a redox-driven sodium pump that generates a transmembrane electrochemical Na+ potential and operates in the respiratory chain of various bacteria. This enzyme contains two FMN residues as redox-active prosthetic groups attached by a phosphoester bond to threonine residues in subunits NqrB and NqrC. Covalently bound flavin is a common redox-active prosthetic group that can be attached to proteins by different bond types, yet the mechanisms of the posttranslational modification remain largely unknown. We have shown that covalent binding of an FMN residue to proteins via a phosphoester bond is not an autocatalytic process and instead requires a specific flavin transferase, ApbE. This enzyme catalyses Mg2+-dependent transfer of the FMN moiety from FAD to a threonine residue of a flavoprotein. Also it was shown that flavinylation of the Na+-NQR subunits proceeds in bacterial periplasm. As post-translational modification usually succeeds protein integration into the membrane, our findings suggest a periplasmic orientation of the flavin-binding domains of NqrC and NqrB in mature Na+-NQR. These data let us to propose a new mechanism of Na+-NQR functioning, involving an electroneutral Na+/electron symport. The periplasmic location of the bound FMN residues in Na+-NQR also suggests that this covalent bonding may help to prevent cofactor loss to the external medium. apbE-like genes are found in the majority of known bacterial genomes. Although apbE-like genes are present in all nqr-containing bacterial genomes, many apbE-containing microorganisms contain no nqrC or nqrB-like genes in their genomes. This may mean that the corresponding organisms have as yet unrecognized ApbE-dependent proteins that also use a covalently bound FMN residue as a prosthetic group.