PURIFICATION AND CHARACTERIZATION OF FLAVOPROTEINS AND CYTOCHROMES FROM THE YELLOW BIOLUMINESCENCE MARINE BACTERIUM VIBRIO-FISCHERI STRAIN Y1

Several flavoproteins and cytochromes that occur as major components i n extracts of the yellow bioluminescence Y1 strain of the marine bacte rium Vibrio fischeri have been purified and characterized with respect to their mass (SDS/PAGE and matrix-assisted laser-desorption/ionizati on MS), chromatographic properties, N-terminal sequence, and spectrosc opy (absorption, fluorescence emission and anisotropy decay). The inve stigated proteins were as follows: yellow fluorescence protein (YFP) w ith bound riboflavin, FMN or 6,7-dimethyl-8-ribityllumazine; a blue fl uorescence protein (BFP) with bound 6,7-dimethyl-8-ribityllumazine, ri boflavin, or 6-methyl-7-oxo-8-ribityllumazine; thioredoxin reductase w ith FAD as ligand; and two c-type diheme cytochromes, c551 and c554. W e present evidence that the riboflavin-bound YFP has an N-terminal seq uence corresponding to that published for the dimeric YFP. We show tha t an equilibrium replacement of the riboflavin can be made with excess lumazine derivative and that lumazine-bound YFP has different biolumi nescence properties to those of the lumazine protein from Photobacteri um leiognathi. BFP is a different protein again, and in the bacterial lysate it occurs in multiple forms, ligated to either riboflavin, luma zine, or the 7-oxolumazine derivative. The N-terminal sequence for BFP shows similarities to those of the YFP proteins and to lumazine prote in and riboflavin synthase from Photobacterium. BFP in any form has no bioluminescence or riboflavin-synthase activity. A 70-kDa fluorescent flavoprotein with FAD as ligand has an N-terminal sequence highly sim ilar to those of thioredoxin reductases from Haemophilus influenzae an d Escherichia coli. Cytochrome contaminations in previous preparations of YFP have been removed and are identified as the two c-type cytochr omes c551 and c554. Both inhibit the NADH-induced bioluminescence in t he reductase/luciferase system with the luciferases from P. leiognathi and V. fischeri. The N-terminal amino acid sequence of the cytochrome (c551) corresponds to a diheme cytochrome c4. The spectral properties of c554 are similar to those of other c5 cytochromes, and both c554 a nd c551 have absorption spectra similar to those of the respective cyt ochromes from the gram-negative bacteria Pseudomonas and Azotobacter.