title: Unusual spectral properties of bacteriophytochrome Agp2 result from a deprotonation of the chromophore in the red-absorbing form Pr.
abstract: Phytochromes are widely distributed photoreceptors with a bilin chromophore that undergo a typical reversible photoconversion between the two spectrally different forms, Pr and Pfr. The phytochrome Agp2 from Agrobacterium tumefaciens belongs to the group of bathy phytochromes that have a Pfr ground state as a result of the Pr to Pfr dark conversion. Agp2 has untypical spectral properties in the Pr form reminiscent of a deprotonated chromophore as confirmed by resonance Raman spectroscopy. UV/visible absorption spectroscopy showed that the pKa is >11 in the Pfr form and ∼7.6 in the Pr form. Unlike other phytochromes, photoconversion thus results in a pKa shift of more than 3 units. The Pr/Pfr ratio after saturating irradiation with monochromatic light is strongly pH-dependent. This is partially due to a back-reaction of the deprotonated Pr chromophore at pH 9 after photoexcitation as found by flash photolysis. The chromophore protonation and dark conversion were affected by domain swapping and site-directed mutagenesis. A replacement of the PAS or GAF domain by the respective domain of the prototypical phytochrome Agp1 resulted in a protonated Pr chromophore; the GAF domain replacement afforded an inversion of the dark conversion. A reversion was also obtained with the triple mutant N12S/Q190L/H248Q, whereas each single point mutant is characterized by decelerated Pr to Pfr dark conversion.
authors.affiliation: From the Botanical Institute, Karlsruhe Institute of Technology, Kaiserstrasse 2, D-76131 Karlsruhe, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Zienicke
authors.forename: Isabel
authors.initials: I
authors.lastname: Molina
authors.forename: René
authors.initials: R
authors.lastname: Glenz
authors.forename: Patrick
authors.initials: P
authors.lastname: Singer
authors.forename: Dorothee
authors.initials: D
authors.lastname: Ehmer
authors.forename: Francisco Velazquez
authors.initials: FV
authors.lastname: Escobar
authors.forename: Peter
authors.initials: P
authors.lastname: Hildebrandt
authors.forename: Rolf
authors.initials: R
authors.lastname: Diller
authors.forename: Tilman
authors.initials: T
authors.lastname: Lamparter
mesh_terms: D016960:Agrobacterium tumefaciens / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010834:Phytochrome / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D010834:Phytochrome
keywords: Bacterial Protein Kinases; Bacterial Signal Transduction; Bilin; Biliprotein; Domain Swap Mutants; Flash Photolysis; Photobiology; Photoreceptors; Raman Spectroscopy; UV Spectroscopy
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