Light-induced structural changes of apoprotein and chromophore in the sensor of blue light using FAD (BLUF) domain of AppA for a signaling state

Abstract

AppA is a new class blue-light receptor controlling photosynthesis gene expression in the purple bacterium Rhodobacter sphaeroides and retains a characteristic flavin adenine dinucleotide (FAD)-binding domain named the "sensor of blue light using FAD" (BLUF). AppA functions as an antirepressor controlling transcription of photosynthesis genes through the direct association with a transcriptional repressor PpsR in a blue-light-dependent manner [Masuda and Bauer (2002) Cell 110, 613-623]. Illumination of AppA induces a red shift in the UV-visible absorption of FAD, which results in a signaling state of AppA. Light-induced Fourier transform infrared (FTIR) difference spectrum of the AppA BLUF domain showed relatively simple features, which were mainly composed of two sets of derivative-shaped sharp bands at 1709(-)/1695(+) and 1632(+)/1619(-) cm(-)(1). We have developed an in vitro reconstitution method, by which a fully functional BLUF domain was reconstituted from free FAD and an apoprotein for the BLUF domain of AppA. An AppA BLUF domain that consisted of an apoprotein isotopically labeled with (13)C and unlabeled FAD was constituted using this method, and hydrated and deuterated samples were applied to FTIR spectroscopic analyses. When the spectra for the reconstituted domain were compared with those for uniformly (15)N- and (13)C-labeled or deuterated domains as well as for the unlabeled domain, the IR bands responsible for the light-induced changes in the FAD chromophore and apoprotein were identified. Unexpectedly, the light-induced spectrum of the unlabeled BLUF domain of AppA was predominantly composed of multiple apoprotein bands, while a C(4)=O stretching of an isoalloxazine ring was the only band exclusively assigned to FAD. The results showed that relatively large structural changes occur in the protein backbone of the BLUF domain of AppA upon illumination. These changes were discussed in relation to the mechanistic role of the BLUF domain in the process of blue-light perception by AppA.