Unno Masashi, Kumauchi Masato, Sasaki Jun, Tokunaga Fumio, Yamauchi Seigo
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai 980-8577, Japan.
Biochemistry. 2002 Apr 30;41(17):5668-74. doi: 10.1021/bi025508o.
Photoactive yellow protein (PYP) is a bacterial photoreceptor containing a 4-hydroxycinnamyl chromophore. Photoexcitation of PYP triggers a photocycle that involves at least two intermediate states: an early red-shifted PYP(L) intermediate and a long-lived blue-shifted PYP(M) intermediate. In this study, we have explored the active site structures of these intermediates by resonance Raman spectroscopy. Quantum chemical calculations based on a density functional theory are also performed to simulate the observed spectra. The obtained structure of the chromophore in PYP(L) has cis configuration and no hydrogen bond at the carbonyl oxygen. In PYP(M), the cis chromophore is protonated at the phenolic oxygen and forms the hydrogen bond at the carbonyl group. These results allow us to propose structural changes of the chromophore during the photocycle of PYP. The chromophore photoisomerizes from trans to cis configuration by flipping the carbonyl group to form PYP(L) with minimal perturbation of the tightly packed protein interior. Subsequent conversion to PYP(M) involves protonation on the phenolic oxygen, followed by rotation of the chromophore as a whole. This large motion of the chromophore is potentially correlated with the succeeding global conformational changes in the protein, which ultimately leads to transduction of a biological signal.
光活性黄色蛋白（PYP）是一种含有4-羟基肉桂发色团的细菌光感受器。PYP的光激发引发一个光循环，该循环涉及至少两个中间状态：一个早期红移的PYP(L)中间体和一个长寿命的蓝移PYP(M)中间体。在本研究中，我们通过共振拉曼光谱探索了这些中间体的活性位点结构。还进行了基于密度泛函理论的量子化学计算以模拟观察到的光谱。在PYP(L)中获得的发色团结构具有顺式构型，羰基氧处没有氢键。在PYP(M)中，顺式发色团在酚氧处质子化，并在羰基处形成氢键。这些结果使我们能够提出PYP光循环过程中发色团的结构变化。发色团通过翻转羰基从反式异构化为顺式构型，形成PYP(L)，对紧密堆积的蛋白质内部的扰动最小。随后转化为PYP(M)涉及酚氧上的质子化，随后发色团整体旋转。发色团的这种大的运动可能与蛋白质中随后的整体构象变化相关，这最终导致生物信号的转导。
