Time-resolved spectroscopic studies of the AppA blue-light receptor BLUF domain from Rhodobacter sphaeroides.
AppA is a blue-light and redox-responding regulator of photosynthesis gene expression in Rhodobacter sphaeroides. Detailed time-resolved fluorescence spectroscopy and subpicosecond transient absorption spectroscopy study of the BLUF domain is presented for wild-type AppA (AppAwt) and a photoinactive Y21F mutant of AppA. The main findings discussed here are that (1) time-resolved laser excitation studies on dark-adapted protein show that AppAwt and Y21F mutant protein exhibits a fluorescence decay with a lifetime of 0.6 ns. Dark-adapted AppAwt but not Y21F also exhibits slower fluorescence decay with a lifetime of 1.7 ns. Analysis of AppAwt that was light-excited to a stable light-adapted form prior to data collection shows monoexponential fluorescence decay with a lifetime of 1.0 ns. This component disappeared after 1 min of data collection after which the original "dark-adapted" values were recovered, demonstrating the presence of a approximately 1 min lifetime intermediate during the return of AppA from light- to dark-adapted form. (2) Transient absorption spectral analysis reveals a very fast rising of transient absorption (<1 ps) for AppAwt. This fast component is missing in the Y21F mutant, which lacks Tyr21, giving rise to a slower transient absorption at 4-6 ps. In the AppAwt transient spectra, most ground states recover within approximately 30 ps, compared to approximately 90-130 ps in the mutant Y21F. We propose that a temporary electron transfer occurs from Tyr21 to the N5 of flavin in AppAwt and is a triggering event for subsequent hydrogen-bond rearrangements. Dynamics of the AppA photocycle is discussed in view of the currently solved crystallographic structure of AppA.
Dragnea
Vladimira
V
Department of Biology, Indiana University, Bloomington, Indiana 47405, USA. dragnea@indiana.edu
Waegele
Matthias
M
Balascuta
Septimiu
S
Bauer
Carl
C
Dragnea
Bogdan
B
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D005420:Flavoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008027:Light; D015394:Molecular Structure; D010084:Oxidation-Reduction; D020283:Photoreceptors, Microbial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013997:Time Factors
C116785:AppA protein, Rhodobacter sphaeroides; D001426:Bacterial Proteins; D005420:Flavoproteins; D020283:Photoreceptors, Microbial