Characteristic structure and environment in FAD cofactor of (6-4) photolyase along function revealed by resonance Raman spectroscopy
Characteristic structure and environment in FAD cofactor of (6-4) photolyase along function revealed by resonance Raman spectroscopy
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DOI：
10.1021/jp062998b
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发表时间：
2006-08-24
影响因子：
3.3
通讯作者：
Kitagawa, Teizo
中科院分区：
文献类型：
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作者：
Li, Jiang;Uchida, Takeshi;Kitagawa, Teizo
嘧啶-嘧啶酮(6-4)光产物和环丁烷嘧啶二聚体(CPD)是紫外线照射引起的主要DNA损伤，而(6-4)光裂合酶是一种以黄素腺嘌呤二核苷酸(FAD)作为辅因子的酶，通过光照特异性修复前者。我们研究了来自拟南芥的 (6-4) 光裂合酶的共振拉曼光谱，该光裂合酶具有中性半醌型和氧化形式的 FAD，分别通过 568.2 和 488.0 nm 的激发选择性增强强度。首次对中性半醌进行了DFT计算。 H2O中1606 cm(-1)处中性半醌的标记带在各种黄酮酶中频率最低，在D2O中明显分裂为1594和1608 cm(-1)两个可比较的带，同样，H2O中1522 cm(-1)处的标记带也分裂为1456、1508和1536 cm(-1)三个带。 D2O。这种 D2O 效应只有在被氧化一次并再次光还原形成半醌后才能被识别，但不能通过溶剂的简单 H/D 交换来识别。在明显低频（1621、1576 cm(-1)）和带分裂（1508/1493、1346/1320 cm(-1)）处观察到一些氧化形式的拉曼带。这些拉曼光谱特征表明强氢键相互作用（在 N5-H、N1）、相当疏水的环境以及 FAD 辅因子苯环中的缺电子特征，这似乎专门控制 (6-4) 光裂合酶的反应性。
A pyrimidine-pyrimidone (6-4) photoproduct and a cyclobutane pyrimidine dimer (CPD) are major DNA lesions induced by ultraviolet irradiation, and (6-4) photolyase, an enzyme with flavin adenine dinucleotide (FAD) as a cofactor, repairs the former specifically by light illumination. We investigated resonance Raman spectra of (6-4) photolyase from Arabidopsis thaliana having neutral semiquinoid and oxidized forms of FAD, which were selectively intensity enhanced by excitations at 568.2 and 488.0 nm, respectively. DFT calculations were carried out for the first time on the neutral semiquinone. The marker band of a neutral semiquinone at 1606 cm(-1) in H2O, whose frequency is the lowest among various flavoenzymes, apparently splits into two comparable bands at 1594 and 1608 cm(-1) in D2O, and similarly, that at 1522 cm(-1) in H2O does into three bands at 1456, 1508, and 1536 cm(-1) in D2O. This D2O effect was recognized only after being oxidized once and photoreduced to form a semiquinone again, but not by simple H/D exchange of solvent. Some Raman bands of the oxidized form were observed at significantly low frequencies (1621, 1576 cm(-1)) and with band splittings (1508/1493, 1346/1320 cm(-1)). These Raman spectral characteristics indicate strong H-bonding interactions (at N5-H, N1), a fairly hydrophobic environment, and an electron-lacking feature in benzene ring of the FAD cofactor, which seems to specifically control the reactivity of (6-4) photolyase.