Chromophore Exchange in the LOV2 Domain of the Plant Photoreceptor Phototropin1 from Oat

• Published in: Biochemistry (Easton) 2005-03, Vol.44 (8), p.3050-3055
• Main Authors: Dürr, Harald, Salomon, Michael, Rüdiger, Wolfhart
• Format: Article
• Language: English
• Subjects: Avena - chemistry; Avena - metabolism; Cryptochromes; Disulfides - analysis; Electron Spin Resonance Spectroscopy; Flavin Mononucleotide - metabolism; Flavin-Adenine Dinucleotide - metabolism; Flavins - metabolism; Flavoproteins - chemistry; Flavoproteins - metabolism; Kinetics; Light; Magnetic Resonance Spectroscopy; Models, Molecular; Photosynthetic Reaction Center Complex Proteins - chemistry; Photosynthetic Reaction Center Complex Proteins - metabolism; Plant Proteins - chemistry; Plant Proteins - metabolism; Protein Conformation; Riboflavin - metabolism; Spectrophotometry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0478897

Phototropins are a family of plant photoreceptors mediating blue light responses such as phototropism, leaf expansion, chloroplast relocation, and stomatal opening. Characteristic for phototropins are two LOV domains which, when expressed in heterologous systems, each carry a single flavin mononucleotide (FMN) chromophore. Here we describe removal of FMN from the LOV2 domain of Avena sativa using a hydrophobic matrix and successful incorporation of flavin adenine dinucleotide (FAD), riboflavin, and 5'-malonyl-riboflavin into the resulting apoprotein; 5-deaza-FMN was not incorporated under the applied conditions. The chromoproteins reconstituted with the various flavins showed absorption spectra and photocycle almost identical to those of the native LOV2 domain and that reconstituted with FMN except for the kinetics:  LOV2−riboflavin and LOV2−5'-malonyl-riboflavin showed more rapid regeneration in the dark. LOV2−FAD can be hydrolyzed to LOV2−FMN with phosphodiesterase, indicating that the adenosine part extrudes from the protein. Together with the data from the X-ray structure (Crosson, S., and Moffat, K. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 2995−3000), the results allow us to decide which of the chromophore−protein interactions are essential for the reconstitution process.