The single NqrB and NqrC subunits in the Na+-translocating NADH: Quinone oxidoreductase (Na+-NQR) from Vibrio cholerae each carry one covalently attached FMN

► The Na+ -translocating NADH:quinone oxidoreductase is composed of six subunits. ► Each NqrB and NqrC subunit carries one riboflavin phosphate bound to threonine. ► Phosphodiester-linked flavins are released by β-elimination with LiOH. ► The threonine residues in the flavin binding sites are identified by mass spectroscopy.