Department of Electrical Engineering and Bioscience, Graduate School of Sciences and Engineering, Waseda University, TWIns, Wakamatsucho 2-2, Shinjuku-Ku, Tokyo 162-8480, Japan.
Department of Biophysics, Division of Biological Sciences, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-Ku, Kyoto 606-8502, Japan.
Biochemistry. 2020 Sep 29;59(38):3615-3625. doi: 10.1021/acs.biochem.0c00495. Epub 2020 Sep 17.
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 FAD and 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 FAD or 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.
鸟类的磁受体被认为存在于视网膜中。尽管其分子机制尚不清楚，但磁场依赖性的形成和含有自由基的光中间产物的稳定性被认为在称为自由基对机制的假设中起着关键作用。鸡隐花色素 4（cCRY4）由于其在视网膜中的表达及其在吸收蓝光后能够形成稳定的黄素中性自由基（FADH），因此被认为是一种候选的磁受体分子。在此，我们使用毫秒级闪光光解来研究 cCRY4 的光循环，分别在存在和不存在二硫苏糖醇（DTT）的情况下进行检测；在两种情况下均检测 FAD 的阴离子自由基形式（FAD）。利用闪光光解和紫外可见光光谱学获得的光谱数据，我们估计了光中间产物的绝对吸收光谱，从而使我们能够将每个光谱分解为其各个组成部分。值得注意的是，在没有 DTT 的情况下，FAD 分别有约 37%和 63%被氧化为 FAD 和质子化为 FADH。奇异值分解分析表明存在两种 FAD 分子物种，每种分子物种都注定要被氧化为 FAD 或质子化为 FADH。还检测到酪氨酸中性自由基；然而，它可能与 FAD 的氧化同时衰减。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可能发挥的作用。
