Hydrogen Bond Switching among Flavin and Amino Acid Side Chains in the BLUF Photoreceptor Observed by Ultrafast Infrared Spectroscopy
Hydrogen Bond Switching among Flavin and Amino Acid Side Chains in the BLUF Photoreceptor Observed by Ultrafast Infrared Spectroscopy
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DOI：
10.1529/biophysj.108.139246
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发表时间：
2008-11-15
影响因子：
3.4
通讯作者：
Kennis, John T. M.
中科院分区：
文献类型：
--
作者：
Bonetti, Cosimo;Mathes, Tilo;Kennis, John T. M.
BLUF结构域构成了最近发现的一类在细菌和真核藻类中发现的光感受器蛋白。BLUF结构域通过FAD辅因子对蓝光敏感，该辅因子参与与附近氨基酸侧链（包括高度保守的酪氨酸和谷氨酰胺）的广泛氢键网络。通过超快红外光谱法评估了特定氨基酸侧链在与FAD的超快氢键转换反应中的参与，FAD是BLUF结构域光活化的基础。FAD的蓝光吸收导致FAD（中心点-）的形成和酪氨酸环振动模式在皮秒时间尺度上的漂白，这表明从酪氨酸到FAD的电子转移构成了主要的光化学。这种解释是支持的动力学同位素效应的H/D交换的荧光衰减的情况下。FAD（中心点-）随后质子化，在皮秒时间尺度上产生FADH（中心点），这可以通过FAD N5质子化位点出现N-H弯曲模式和FADH（中心点）C = N伸缩标记模式来证明，其中酪氨酸作为可能的质子供体。FADH（中心点）在67 ps（D2 O中为180 ps）内被再氧化，从而在FAD周围形成长寿命的氢键转换网络。这种氢键开关显示来自酪氨酸的C-OH伸缩和FAD C4=O和C=N伸缩的红外特征，这表明在所有这些位点的氢键强度增加。研究结果支持了先前假设的谷氨酰胺通过光驱动的自由基对机制旋转180度作为氢键转换的决定因素。
BLUF domains constitute a recently discovered class of photoreceptor proteins found in bacteria and eukaryotic algae. BLUF domains are blue-light sensitive through a FAD cofactor that is involved in an extensive hydrogen-bond network with nearby amino acid side chains, including a highly conserved tyrosine and glutamine. The participation of particular amino acid side chains in the ultrafast hydrogen-bond switching reaction with FAD that underlies photoactivation of BLUF domains is assessed by means of ultrafast infrared spectroscopy. Blue-light absorption by FAD results in formation of FAD(center dot-) and a bleach of the tyrosine ring vibrational mode on a picosecond timescale, showing that electron transfer from tyrosine to FAD constitutes the primary photochemistry. This interpretation is supported by the absence of a kinetic isotope effect on the fluorescence decay on H/D exchange. Subsequent protonation of FAD(center dot-) to result in FADH(center dot) on a picosecond timescale is evidenced by the appearance of a N-H bending mode at the FAD N5 protonation site and of a FADH(center dot) C = N stretch marker mode, with tyrosine as the likely proton donor. FADH(center dot) is reoxidized in 67 ps (180 ps in D2O) to result in a long-lived hydrogen-bond switched network around FAD. This hydrogen-bond switch shows infrared signatures from the C-OH stretch of tyrosine and the FAD C4=O and C=N stretches, which indicate increased hydrogen-bond strength at all these sites. The results support a previously hypothesized rotation of glutamine by similar to 180 degrees through a light- driven radicalpair mechanism as the determinant of the hydrogen- bond switch.