| Abstract
| - Phototropin is a blue-light receptor involved in the phototropic response of higher plants. Thephotoreceptor comprises a protein kinase domain and two structurally similar flavin-mononucleotide (FMN)binding domains designated LOV1 and LOV2. Blue-light irradiation of recombinant LOV2 domains inducesthe formation of a covalent adduct of the thiol group of a functional cysteine in the cofactor-binding pocketto C(4a) of the FMN. Cysteine-to-alanine mutants of LOV domains are unable to form that adduct butgenerate an FMN radical upon illumination. The recombinant C450A mutant of the LOV2 domain of Avenasativa phototropin was reconstituted with universally and site-selectively 13C-labeled FMN and the 13C NMRsignals were unequivocally assigned. 13C NMR spectra were acquired in darkness and under blue-lightirradiation. The chemical shifts and the coupling patterns of the signals were not affected by irradiation.However, under blue-light exposure, exceptionally strong nuclear-spin polarization was developed in theresonances belonging to certain carbons of the FMN's isoalloxazine moiety. An enhancement of the NMRabsorption was observed for the signals of C(5a), C(7), and C(9). NMR lines in emission were detected forthe signals belonging to C(2), C(4), C(4a), C(6), C(8), and C(9a). The signal of C(10a) remained in absorptionbut was slightly attenuated. In contrast, the intensities of the NMR signals belonging to the carbons of theribityl side chain of FMN were not affected by light. The observation of spin-polarized 13C-nuclei in theNMR spectra of the mutant LOV2 domain is clear evidence for radical-pair intermediates in the reactionsteps following optical sample excitation.
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