title: Functional Characterization of a LOV-Histidine Kinase Photoreceptor from Xanthomonas citri subsp. citri.
abstract: The blue-light (BL) absorbing protein Xcc-LOV from Xanthomonas citri subsp. citri is composed of a LOV-domain, a histidine kinase (HK) and a response regulator. Spectroscopic characterization of Xcc-LOV identified intermediates and kinetics of the protein's photocycle. Measurements of steady state and time-resolved fluorescence allowed determination of quantum yields for triplet (ΦT  = 0.68 ± 0.03) and photoproduct formation (Φ390  = 0.46 ± 0.05). The lifetime for triplet decay was determined as τT  = 2.4-2.8 μs. Fluorescence of tryptophan and tyrosine residues was unchanged upon light-to-dark conversion, emphasizing the absence of significant conformational changes. Photochemistry was blocked upon cysteine C76 (C76S) mutation, causing a seven-fold longer lifetime of the triplet state (τT  = 16-18.5 μs). Optoacoustic spectroscopy yielded the energy content of the triplet state. Interestingly, Xcc-LOV did not undergo the volume contraction reported for other LOV domains within the observation time window, although the back-conversion into the dark state was accompanied by a volume expansion. A radioactivity-based enzyme function assay revealed a larger HK activity in the lit than in the dark state. The C76S mutant showed a still lower enzyme function, indicating the dark state activity being corrupted by a remaining portion of the long-lived lit state.
authors.affiliation: Facultad de Ciencias Bioquímicas y Farmacéuticas, Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET, Universidad Nacional de Rosario, Rosario, Argentina.
authors.forename: Ivana
authors.initials: I
authors.lastname: Kraiselburd
authors.affiliation: Max-Planck-Institute for Chemical Energy Conversion, Mülheim, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Gutt
authors.affiliation: Department of Physics and Earth Sciences, University of Parma, Parma, Italy.
authors.forename: Aba
authors.initials: A
authors.lastname: Losi
authors.affiliation: Max-Planck-Institute for Chemical Energy Conversion, Mülheim, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Gärtner
authors.affiliation: Facultad de Ciencias Bioquímicas y Farmacéuticas, Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET, Universidad Nacional de Rosario, Rosario, Argentina.
authors.forename: Elena G
authors.initials: EG
authors.lastname: Orellano
mesh_terms: D000071677:Histidine Kinase; D008027:Light; D010100:Oxygen / Q000737:chemistry; D020283:Photoreceptors, Microbial / Q000378:metabolism*; D011494:Protein Kinases / Q000502:physiology*; D014974:Xanthomonas / Q000502:physiology*
chemical_list: D020283:Photoreceptors, Microbial; D011494:Protein Kinases; D000071677:Histidine Kinase; D010100:Oxygen