Photocycle of the flavin-binding photoreceptor AppA, a bacterial transcriptional antirepressor of photosynthesis genes.
The flavoprotein AppA from Rhodobacter sphaeroides contains an N-terminal domain belonging to a new class of photoreceptors designated BLUF domains. AppA was shown to control photosynthesis gene expression in response to blue light and oxygen tension. We have investigated the photocycle of the AppA BLUF domain by ultrafast fluorescence, femtosecond transient absorption, and nanosecond flash-photolysis spectroscopy. Time-resolved fluorescence experiments revealed four components of flavin adenine dinucleotide (FAD) excited-state decay, with lifetimes of 25 ps, 150 ps, 670 ps, and 3.8 ns. Ultrafast transient absorption spectroscopy revealed rapid internal conversion and vibrational cooling processes on excited FAD with time constants of 250 fs and 1.2 ps, and a multiexponential decay with effective time constants of 90 ps, 590 ps, and 2.7 ns. Concomitant with the decay of excited FAD, the rise of a species with a narrow absorption difference band near 495 nm was detected which spectrally resembles the long-living signaling state of AppA. Consistent with these results, the nanosecond flash-photolysis measurements indicated that formation of the signaling state was complete within the time resolution of 10 ns. No further changes were detected up to 15 micros. The quantum yield of the signaling-state formation was determined to be 24%. Thus, the signaling state of the AppA BLUF domain is formed on the ultrafast time scale directly from the FAD singlet excited state, without any apparent intermediate, and remains stable over 12 decades of time. In parallel with the signaling state, the FAD triplet state is formed from the FAD singlet excited state at 9% efficiency as a side reaction of the AppA photocycle.
Gauden
Magdalena
M
Department of Biophysics, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands.
Yeremenko
Sergey
S
Laan
Wouter
W
van Stokkum
Ivo H M
IH
Ihalainen
Janne A
JA
van Grondelle
Rienk
R
Hellingwerf
Klaas J
KJ
Kennis
John T M
JT
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D056931:Cryptochromes; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005415:Flavins / Q000737:chemistry / Q000378:metabolism*; D005420:Flavoproteins / Q000737:chemistry* / Q000378:metabolism*; D008956:Models, Chemical; D036103:Nanotechnology / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010782:Photolysis; D010788:Photosynthesis / Q000235:genetics; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000037:antagonists & inhibitors*; D012242:Rhodobacter sphaeroides / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence / Q000379:methods; D013053:Spectrophotometry / Q000379:methods; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*
C116785:AppA protein, Rhodobacter sphaeroides; D001426:Bacterial Proteins; D056931:Cryptochromes; D005415:Flavins; D005420:Flavoproteins; D010446:Peptide Fragments; D012097:Repressor Proteins; D014157:Transcription Factors; D005182:Flavin-Adenine Dinucleotide