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Photochemically induced dynamic nuclear polarization in a C450A mutant of the LOV2 domain of the Avena sativa blue-light receptor phototropin

Richter, Gerald, Weber, Stefan, Römisch, Werner, Bacher, Adelbert, Fischer, Markus and Eisenreich, Wolfgang 2005. Photochemically induced dynamic nuclear polarization in a C450A mutant of the LOV2 domain of the Avena sativa blue-light receptor phototropin. Journal of the American Chemical Society 127 (49) , pp. 17245-17252. 10.1021/ja053785n

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Phototropin is a blue-light receptor involved in the phototropic response of higher plants. The photoreceptor 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 induces the formation of a covalent adduct of the thiol group of a functional cysteine in the cofactor-binding pocket to C(4a) of the FMN. Cysteine-to-alanine mutants of LOV domains are unable to form that adduct but generate an FMN radical upon illumination. The recombinant C450A mutant of the LOV2 domain of Avena sativa phototropin was reconstituted with universally and site-selectively 13C-labeled FMN and the 13C NMR signals were unequivocally assigned. 13C NMR spectra were acquired in darkness and under blue-light irradiation. 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 the resonances belonging to certain carbons of the FMN's isoalloxazine moiety. An enhancement of the NMR absorption was observed for the signals of C(5a), C(7), and C(9). NMR lines in emission were detected for the signals belonging to C(2), C(4), C(4a), C(6), C(8), and C(9a). The signal of C(10a) remained in absorption but was slightly attenuated. In contrast, the intensities of the NMR signals belonging to the carbons of the ribityl side chain of FMN were not affected by light. The observation of spin-polarized 13C-nuclei in the NMR spectra of the mutant LOV2 domain is clear evidence for radical-pair intermediates in the reaction steps following optical sample excitation.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Chemistry
Publisher: American Chemical Society
ISSN: 00027863
Last Modified: 29 Apr 2016 01:33

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