Functional Characterization of a LOV-Histidine Kinase Photoreceptor from Xanthomonas citri subsp. citri

IVANA KRAISELBURD; ALEXANDER GUTT; ABA LOSI; WOLFGANG GÄRTNER; ELENA G. ORELLANO

Photochemistry and Photobiology

The American Society of Photobiology

Año: 2015 vol. 91 p. 1123 - 1132

1751-1097

The blue-light (BL) absorbing protein Xcc-LOV fromXanthomonas citri subsp. citri is composed of a LOV-domain,a histidine kinase (HK) and a response regulator. Spectroscopiccharacterization of Xcc-LOV identified intermediatesand kinetics of the protein?s photocycle. Measurements ofsteady state and time-resolved fluorescence allowed determinationof quantum yields for triplet (ΦT = 0.68 ± 0.03) andphotoproduct formation (Φ390 = 0.46 ± 0.05). The lifetimefor triplet decay was determined as sT = 2.4?2.8 ls. Fluorescenceof tryptophan and tyrosine residues was unchangedupon light-to-dark conversion, emphasizing the absence ofsignificant conformational changes. Photochemistry wasblocked upon cysteine C76 (C76S) mutation, causing aseven-fold longer lifetime of the triplet state (sT = 16?18.5 ls). Optoacoustic spectroscopy yielded the energy contentof the triplet state. Interestingly, Xcc-LOV did notundergo the volume contraction reported for other LOVdomains within the observation time window, although theback-conversion into the dark state was accompanied by avolume expansion. A radioactivity-based enzyme functionassay revealed a larger HK activity in the lit than in the darkstate. The C76S mutant showed a still lower enzyme function,indicating the dark state activity being corrupted by aremaining portion of the long-lived lit state.