Photodynamics of the small BLUF protein BlrB from Rhodobacter sphaeroides.
The BLUF protein BlrB from the non-sulphur anoxyphototrophic purple bacterium Rhodobacter sphaeroides is characterized by absorption and emission spectroscopy. BlrB expressed from E. coli binding FAD, FMN, and riboflavin (called BrlB(I)) and recombinant BlrB containing only FAD (called BlrB(II)) are investigated. The dark-adapted proteins exist in two different receptor conformations (receptor states) with different sub-nanosecond fluorescence lifetimes (BLUF(r,f) and BLUF(r,sl)). Some of the flavin-cofactor (ca. 8%) is unbound in thermodynamic equilibrium with the bound cofactor. The two receptor conformations are transformed to putative signalling states (BLUF(s,f) and BLUF(s,sl)) of decreased fluorescence efficiency and shortened fluorescence lifetime by blue-light excitation. In the dark at room temperature both signalling states recover back to the initial receptor states with a time constant of about 2s. Quantum yields of signalling state formation of about 90% for BlrB(II) and about 40% for BlrB(I) were determined by intensity dependent transmission measurements. Extended blue-light excitation causes unbound flavin degradation (formation of lumichrome and lumiflavin-derivatives) and bound cofactor conversion to the semiquinone form. The flavin-semiquinone further reduces and the reduced flavin re-oxidizes back in the dark. A photo-dynamics scheme is presented and relevant quantum efficiencies and time constants are determined.
Institut II -- Experimentelle und Angewandte Physik, Universität Regensburg, Universitätstrasse 31, D-93053 Regensburg, Germany.
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Zirak
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Penzkofer
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Schiereis
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Hegemann
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Jung
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Schlichting
D001665:Binding Sites; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000378:metabolism; D010777:Photochemistry; D010786:Photoreceptor Cells / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D012242:Rhodobacter sphaeroides / Q000737:chemistry*; D013053:Spectrophotometry; D013997:Time Factors
D005420:Flavoproteins; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D010727:Phosphoric Diester Hydrolases