Blue light perception in plants -: Detection and characterization of a light-induced neutral flavin radical in a C450A mutant of phototropin
Blue light perception in plants -: Detection and characterization of a light-induced neutral flavin radical in a C450A mutant of phototropin
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DOI：
10.1074/jbc.m205509200
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发表时间：
2003-03-28
影响因子：
4.8
通讯作者：
Richter, G
中科院分区：
文献类型：
--
作者：
Kay, CWM;Schleicher, E;Richter, G
将燕麦趋光蛋白及其C450 A突变体的LOV 2结构域表达为重组融合蛋白，并通过光学光谱、电子顺磁共振和电子-核双共振进行了检测。在照射（420-480 nm）后，LOV 2 C450 A突变蛋白即使在不存在外源电子供体的情况下也给出黄素自由基的光学吸收光谱特征，从而证明处于其光生三重态的黄素单核苷酸（FMN）辅因子是LOV 2结构域内氧化还原活性氨基酸残基的有效氧化剂。LOV 2 C450 A突变体中的FMN自由基是N（5）-质子化的，这表明接近FMN的局部pH足够酸性，使得野生型蛋白中的半胱氨酸残基也可能被质子化。的光生FMN自由基的电子顺磁共振分析给出了信息的几何和电子结构和环境的FMN辅因子。实验确定的FMN自由基的超精细耦合点的高度限制的非成对电子自旋在isoalloxazine部分的离域。根据这些结果，讨论了在LOV结构域中形成FMN-C（4a）-半胱氨酰加合物的可能的自由基对机制。
The LOV2 domain of Avena sativa phototropin and its C450A mutant were expressed as recombinant fusion proteins and were examined by optical spectroscopy, electron paramagnetic resonance, and electron-nuclear double resonance. Upon irradiation (420-480 nm), the LOV2 C450A mutant protein gave an optical absorption spectrum characteristic of a flavin radical even in the absence of exogenous electron donors, thus demonstrating that the flavin mononucleotide (FMN) cofactor in its photogenerated triplet state is a potent oxidant for redox-active amino acid residues within the LOV2 domain. The FMN radical in the LOV2 C450A mutant is N(5)-protonated, suggesting that the local pH close to the FMN is acidic enough so that the cysteine residue in the wild-type protein is likely to be also protonated. An electron paramagnetic resonance analysis of the photogenerated FMN radical gave information on the geometrical and electronic structure and the environment of the FMN cofactor. The experimentally determined hyperfine couplings of the FMN radical point to a highly restricted delocalization of the unpaired electron spin in the isoalloxazine moiety. In the light of these results a possible radical-pair mechanism for the formation of the FMN-C(4a)-cysteinyl adduct in LOV domains is discussed.