title: On the role of aromatic side chains in the photoactivation of BLUF domains.
abstract: BLUF (blue-light sensing using FAD) domain proteins are a novel group of blue-light sensing receptors found in many microorganisms. The role of the aromatic side chains Y21 and W104, which are in close vicinity to the FAD cofactor in the AppA BLUF domain from Rhodobacter sphaeroides, is investigated through the introduction of several amino acid substitutions at these positions. NMR spectroscopy indicated that in the W104F mutant, the local structure of the FAD binding pocket was not significantly perturbed as compared to that of the wild type. Time-resolved fluorescence and absorption spectroscopy was applied to explore the role of Y21 and W104 in AppA BLUF photochemistry. In the Y21 mutants, FADH*-W* radical pairs are transiently formed on a ps time scale and recombine to the ground state on a ns time scale. The W104F mutant shows a spectral evolution similar to that of wild type AppA but with an increased yield of signaling state formation. In the Y21F/W104F double mutant, all light-driven electron-transfer processes are abolished, and the FAD singlet excited-state evolves by intersystem crossing to the triplet state. Our results indicate that two competing light-driven electron-transfer pathways are available in BLUF domains: one productive pathway that involves electron transfer from the tyrosine, which leads to signaling state formation, and one nonproductive electron-transfer pathway from the tryptophan, which leads to deactivation and the effective lowering of the quantum yield of the signaling state formation. Our results are consistent with a photoactivation mechanism for BLUF domains where signaling state formation proceeds via light-driven electron and proton transfer from the conserved tyrosine to FAD, followed by a hydrogen-bond rearrangement and radical-pair recombination.
authors.affiliation: Biophysics Group, Department of Physics and Astronomy, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1081, 1081HV Amsterdam, The Netherlands.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Gauden
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Grinstead
authors.forename: Wouter
authors.initials: W
authors.lastname: Laan
authors.forename: Ivo H M
authors.initials: IH
authors.lastname: van Stokkum
authors.forename: Marcela
authors.initials: M
authors.lastname: Avila-Perez
authors.forename: K C
authors.initials: KC
authors.lastname: Toh
authors.forename: Rolf
authors.initials: R
authors.lastname: Boelens
authors.forename: Robert
authors.initials: R
authors.lastname: Kaptein
authors.forename: Rienk
authors.initials: R
authors.lastname: van Grondelle
authors.forename: Klaas J
authors.initials: KJ
authors.lastname: Hellingwerf
authors.forename: John T M
authors.initials: JT
authors.lastname: Kennis
mesh_terms: D019943:Amino Acid Substitution; D024322:Amino Acids, Aromatic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004583:Electrons; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism; D008027:Light; D008954:Models, Biological; D008956:Models, Chemical; D019906:Nuclear Magnetic Resonance, Biomolecular; D010777:Photochemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011522:Protons
chemical_list: D024322:Amino Acids, Aromatic; D011522:Protons; D005182:Flavin-Adenine Dinucleotide