Highlights•First detailed characterization of multi-FMN-containing proteins.•Two bacterial flavoproteins, containing 3 and 4 covalent flavin cofactors, respectively, could be produced and purified as recombinant proteins.•The fluorescence and redox properties have been determined.•The crystal structures of two single FMN-containing domain have been elucidated.•Mutant proteins were prepared with altered fluorescence properties.•Reduced FMN-containing protein can consume molecular oxygen.