Functional Characterization of a LOV‐Histidine Kinase Photoreceptor from Xanthomonas citri subsp. citri |
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| Authors: | Ivana Kraiselburd Alexander Gutt Aba Losi Wolfgang Gärtner Elena G. Orellano |
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| Affiliation: | 1. Facultad de Ciencias Bioquímicas y Farmacéuticas, Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET, Universidad Nacional de Rosario, Rosario, Argentina;2. Max‐Planck‐Institute for Chemical Energy Conversion, Mülheim, Germany;3. Department of Physics and Earth Sciences, University of Parma, Parma, Italy |
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| 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. |
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