The Na+-translocating NADH-quinone reductase (NQR) from Vibrio alginolyticus is composed of six subunits (NqrA to NqrF). We previously demonstrated that both NqrB and NqrC subunits contain a flavin cofactor covalently attached to a threonine residue. Fluorescent peptide fragments derived from the NqrB and NqrC subunits mere applied to a matrix-assisted laser desorption ionization time-of-flight mass spectrometer, and covalently attached flavin was identified as FMN in both subunits, From post-source decay fragmentation analysis, it was concluded that FMN is attached by a phosphate group to Thr-235 in the NqrB subunit and to Thr-223 in the NqrC subunit, The phosphoester binding of FMN to a threonine residue reported here is a new type of flavin attachment to a polypeptide. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.