Orientation of a key glutamine residue in the BLUF domain from AppA revealed by mutagenesis, spectroscopy, and quantum chemical calculations.

The flavin-adenine-dinucleotide-binding BLUF domain constitutes a new class of blue-light receptors, and the N-terminal domain of AppA is a representative of this family. A crystal structure of the BLUF domain from AppA suggested that a conserved Gln63 forms a hydrogen bond with the flavin N5 atom. Upon light excitation, this residue is proposed to undergo a approximately 180 degrees rotation that leads to a rearrangement of a hydrogen bonding network. However, crystallographic studies on the other BLUF proteins claimed an opposite orientation for the glutamine residue. In this communication, we have revealed the presence of a Gln63-to-N5 hydrogen bond in the dark state of AppA by a combined approach of mutagenesis, spectroscopy, and quantum chemical calculations. The present finding supports the view that the reorientation of the Gln63 side chain is a key event in the signaling state formation of BLUF proteins.