Purification and characterization of flavoproteins and cytochromes from the yellow bioluminescence marine bacterium Vibrio fischeri strain Y1.

Abstract

Several flavoproteins and cytochromes that occur as major components in extracts of the yellow bioluminescence Y1 strain of the marine bacterium Vibrio fischeri have been purified and characterized with respect to their mass (SDS/PAGE and matrix-assisted laser-desorption/ionization MS), chromatographic properties, N-terminal sequence, and spectroscopy (absorption, fluorescence emission and anisotropy decay). The investigated proteins were as follows: yellow fluorescence protein (YFP) with bound riboflavin, FMN or 6,7-dimethyl-8-ribityllumazine; a blue fluorescence protein (BFP) with bound 6,7-dimethyl-8-ribityllumazine, riboflavin, or 6-methyl-7-oxo-8-ribityllumazine; thioredoxin reductase with FAD as ligand; and two c-type diheme cytochromes, c551 and c554. We present evidence that the riboflavin-bound YFP has an N-terminal sequence corresponding to that published for the dimeric YFP. We show that an equilibrium replacement of the riboflavin can be made with excess lumazine derivative and that lumazine-bound YFP has different bioluminescence properties to those of the lumazine protein from Photobacterium leiognathi. BFP is a different protein again, and in the bacterial lysate it occurs in multiple forms, ligated to either riboflavin, lumazine, or the 7-oxolumazine derivative. The N-terminal sequence for BFP shows similarities to those of the YFP proteins and to lumazine protein 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 similar to those of thioredoxin reductases from Haemophilus influenzae and Escherichia coli. Cytochrome contaminations in previous preparations of YFP have been removed and are identified as the two c-type cytochromes c551 and c554. Both inhibit the NADH-induced bioluminescence in the 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 and c551 have absorption spectra similar to those of the respective cytochromes from the gram-negative bacteria Pseudomonas and Azotobacter.