Rapid Oxidation Following Photoreduction in the Avian Cryptochrome4 Photocycle
Rapid Oxidation Following Photoreduction in the Avian Cryptochrome4 Photocycle
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鸟类隐花色素 4 光循环中光还原后的快速氧化
DOI：
10.1021/acs.biochem.0c00495
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发表时间：
2020
期刊：
影响因子：
2.9
通讯作者：
Okano Toshiyuki
中科院分区：
文献类型：
--
作者：
Otsuka Hiroaki;Mitsui Hiromasa;Miura Kota;Okano Keiko;Imamoto Yasushi;Okano Toshiyuki
鸟类的磁感受被认为发生在视网膜上。虽然其分子机制尚不清楚，但磁场依赖的形成和含自由基光中间体的稳定性被认为在称为自由基对机制的假设中起关键作用。鸡隐色素4 （cCRY4）已被确定为候选磁接受分子，因为它在视网膜中表达，并且在蓝光吸收时能够形成稳定的黄素中性自由基（FADH●）。在这里，我们使用毫秒闪光光解来研究cCRY4在存在和不存在二硫苏糖醇（DTT）的情况下的光循环；在两种条件下检测FAD的阴离子自由基形式（FAD●-）。利用在闪光光解和紫外-可见光谱学中获得的光谱数据，我们估计了光中间体的绝对吸光度光谱，从而使我们能够将每个光谱分解为其单独的组分。值得注意的是，在没有DTT的情况下，大约37%和63%的FAD●-分别被氧化成fadox和质子化形成FADH●。奇异值分解分析表明存在两种FAD●-分子种，每一种都注定被氧化成FADOXor质子化成FADH●。还检测到酪氨酸中性自由基；然而，它可能伴随着FAD●-的氧化而衰变。基于这些结果，我们考虑了在FAD -生成之前或在FAD -氧化过程中分叉的发生，并讨论了酪氨酸自由基在自由基对机制中所起的潜在作用。
Avian magnetoreception is assumed to occur in the retina. Although its molecular mechanism is unclear, magnetic field-dependent formation and the stability of radical-containing photointermediate(s) are suggested to play key roles in a hypothesis called the radical pair mechanism. Chicken cryptochrome4 (cCRY4) has been identified as a candidate magnetoreceptive molecule due to its expression in the retina and its ability to form stable flavin neutral radicals (FADH●) upon blue light absorption. Herein, we used millisecond flash photolysis to investigate the cCRY4 photocycle, in both the presence and absence of dithiothreitol (DTT); detecting the anion radical form of FAD (FAD●–) under both conditions. Using spectral data obtained during flash photolysis and UV–visible photospectroscopy, we estimated the absolute absorbance spectra of the photointermediates, thus allowing us to decompose each spectrum into its individual components. Notably, in the absence of DTT, approximately 37% and 63% of FAD●–was oxidized to FADOXand protonated to form FADH●, respectively. Singular value decomposition analysis suggested the presence of two FAD●–molecular species, each of which was destined to be oxidized to FADOXor protonated to FADH●. A tyrosine neutral radical was also detected; however, it likely decayed concomitantly with the oxidation of FAD●–. On the basis of these results, we considered the occurrence of bifurcation prior to FAD●–generation, or during FAD●–oxidization, and discussed the potential role played by the tyrosine radical in the radical pair mechanism.