Richter Gerald, Weber Stefan, Römisch Werner, Bacher Adelbert, Fischer Markus, Eisenreich Wolfgang
Technische Universität München, Lehrstuhl für Organische Chemie und Biochemie, Lichtenbergstrasse 4, 85747 Garching, Germany.
J Am Chem Soc. 2005 Dec 14;127(49):17245-52. doi: 10.1021/ja053785n.
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 (13)C-labeled FMN and the (13)C NMR signals were unequivocally assigned. (13)C 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 (13)C-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.
向光素是一种参与高等植物向光性反应的蓝光受体。该光受体由一个蛋白激酶结构域和两个结构相似的黄素单核苷酸（FMN）结合结构域组成，分别称为LOV1和LOV2。重组LOV2结构域的蓝光照射会诱导辅因子结合口袋中一个功能性半胱氨酸的巯基与FMN的C(4a)形成共价加合物。LOV结构域的半胱氨酸到丙氨酸突变体无法形成该加合物，但在光照下会产生FMN自由基。用普遍和位点选择性（13）C标记的FMN重建了燕麦向光素LOV2结构域的重组C450A突变体，并明确归属了（13）C NMR信号。在黑暗和蓝光照射下采集了（13）C NMR光谱。信号的化学位移和耦合模式不受照射影响。然而，在蓝光照射下，FMN异咯嗪部分某些碳的共振中出现了异常强烈的核自旋极化。观察到C(5a)、C(7)和C(9)信号的NMR吸收增强。检测到属于C(2)、C(4)、C(4a)、C(6)、C(8)和C(9a)信号的发射NMR谱线。C(10a)的信号仍处于吸收状态，但略有衰减。相比之下，FMN核糖醇侧链碳的NMR信号强度不受光的影响。在突变体LOV2结构域的NMR光谱中观察到自旋极化的（13）C核，这清楚地证明了光学样品激发后反应步骤中的自由基对中间体。
