Spectroscopic and mutational analysis of the blue-light photoreceptor AppA: a novel photocycle involving flavin stacking with an aromatic amino acid.
The flavoprotein AppA is a blue-light photoreceptor that functions as an antirepressor of photosynthesis gene expression in the purple bacterium Rhodobacter sphaeroides. Heterologous expression studies show that FAD binds to a 156 amino acid N-terminal domain of AppA and that this domain is itself photoactive. A pulse of white light causes FAD absorption to be red shifted in a biphasic process with a fast phase occurring in <1 micros and a slow phase occurring at approximately 5 ms. The absorbance shift was spontaneously restored over a 30 min period, also in a biphasic process as assayed by fluorescence quenching and electronic absorption analyses. Site-directed replacement of Tyr21 with Leu or Phe abolished the photochemical reaction implicating involvement of Tyr21 in the photocycle. Nuclear magnetic resonance analysis of wild-type and mutant proteins also indicates that Tyr21 forms pi-pi stacking interactions with the isoalloxazine ring of FAD. We propose that photochemical excitation of the flavin results in strengthening of a hydrogen bond between the flavin and Tyr 21 leading to a stable local conformational change in AppA.
Kraft
Brian J
BJ
Department of Biology, Indiana University, Bloomington, Indiana 47405, USA.
Masuda
Shinji
S
Kikuchi
Jun
J
Dragnea
Vladimira
V
Tollin
Gordon
G
Zaleski
Jeffrey M
JM
Bauer
Carl E
CE
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002942:Circular Dichroism; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry* / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry* / Q000235:genetics*; D005453:Fluorescence; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry; D010782:Photolysis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics*
C116785:AppA protein, Rhodobacter sphaeroides; D001426:Bacterial Proteins; D005420:Flavoproteins; D005182:Flavin-Adenine Dinucleotide; D014443:Tyrosine