Absorption and emission spectroscopic characterization of BLUF protein Slr1694 from Synechocystis sp. PCC6803 with roseoflavin cofactor.
The wild-type BLUF protein Slr1694 from Synechocystis sp. PCC6803 (BLUF=blue-light sensor using FAD) has flavin adenosine dinucleotide (FAD) as natural cofactor. This light sensor causes positive phototaxis of the marine cyanobacterium. In this study the FAD cofactor of the wild-type Slr1694 was replaced by roseoflavin (RoF) and the roseoflavin derivatives RoFMN and RoFAD during heterologous expression in a riboflavin auxotrophic E. coli strain. An absorption and emission spectroscopic characterization of the cofactor-exchanged-Slr1694 (RoSlr) was carried out both under dark conditions and under illuminated conditions. The behaviour of RoF embedded in RoSlr in aqueous solution at pH 8 is compared with the behaviour of RoF in aqueous solution. The fluorescence of RoF and RoSlr is quenched by photo-induced twisted intra-molecular charge transfer at room temperature with stronger effect for RoF. The fluorescence quenching is diminished at liquid nitrogen temperature. Light exposure of RoSlr causes irreversible conversion of the protein embedded roseoflavins to 8-methylamino-flavins, 8-dimethylamino-lumichrome and 8-methylamino-lumichrome.
Zirak
P
P
Institut II - Experimentelle und Angewandte Physik, Universität Regensburg, Universitätstrasse 31, D-93053 Regensburg, Germany.
Penzkofer
A
A
Mathes
T
T
Hegemann
P
P
D000042:Absorption; D001426:Bacterial Proteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008027:Light; D010782:Photolysis; D012256:Riboflavin / Q000031:analogs & derivatives / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D046939:Synechocystis / Q000737:chemistry*
D001426:Bacterial Proteins; C009191:roseoflavin; D012256:Riboflavin