Initial characterization of the primary photochemistry of AppA, a blue-light-using flavin adenine dinucleotide-domain containing transcriptional antirepressor protein from Rhodobacter sphaeroides: a key role for reversible intramolecular proton transfer from the flavin adenine dinucleotide chromophore to a conserved tyrosine?
The flavin adenine dinucleotide (FAD)-containing photoreceptor protein AppA (in which the FAD is bound to a novel so-called BLUF domain) from the purple nonsulfur bacterium Rhodobacter sphaeroides was previously shown to be photoactive by the formation of a slightly redshifted long-lived intermediate that is thought to be the signaling state. In this study, we provide further characterization of the primary photochemistry of this photoreceptor protein using UV-Vis and Fourier-transform infrared spectroscopy, pH measurements and site-directed mutagenesis. Available evidence indicates that the FAD chromophore of AppA may be protonated in the receptor state, and that it becomes exposed to solvent in the signaling state. Furthermore, experimental data lead to the suggestion that intramolecular proton transfer (that may involve [anionic] Tyr-17) forms the basis for the stabilization of the signaling state.
Laan
Wouter
W
Laboratory for Microbiology, Swammerdam Institute for Life Sciences, BioCentrum Amsterdam, University of Amsterdam, The Netherlands.
van der Horst
Michael A
MA
van Stokkum
Ivo H
IH
Hellingwerf
Klaas J
KJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D015226:Dinucleoside Phosphates / Q000737:chemistry* / Q000235:genetics; D008027:Light; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry; D011522:Protons; D012242:Rhodobacter sphaeroides / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared; D015534:Trans-Activators / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*
D017931:DNA Primers; D015226:Dinucleoside Phosphates; D011522:Protons; D015534:Trans-Activators; C012276:diadenosine pyrophosphate; D014443:Tyrosine