Covalently bound flavin in the NqrB and NqrC subunits of Na+-translocatingNADH-quinone reductase from Vibrio alginolyticus

Na+-translocating NADH-quinone reductase (NQR) from the marine bacterium Vi brio alginolyticus is composed of six subunits (NqrA to NqrF). On SDS-PAGE of the purified complex, NqrB and NqrC subunits mere found to give yellow-g reen fluorescent bands under UV illumination. Both the NqrB and NqrC, elect roeluted from the gel, had an absorption maximum at 448 nm, and the fluores cence excitation maxima at 365 and 448 nm and the emission maximum at 514 n m, The electroeluted NqrB and NqrC, respectively, were identified from thei r N-terminal amino acid sequences. These results clearly indicated that the NqrB and NqrC subunits have covalently bound flavins. The two subunits wer e digested by protease and then the fluorescent peptide fragments mere sepa rated by a reversed-phase high performance liquid chromatography. N-Termina l amino acid sequence analyses of the fluorescent peptides revealed that th e flavia is linked to Thr-235 in the NqrB and Thr-223 in the NqrC subunits, This is the first example that the flavin is linked to a threonine residue . The amino acid sequence around the flavin-linked threonine was well conse rved between NqrB and NqrC. Identification of the flavin group is in progre ss. (C) 2000 Federation of European Biochemical Societies.