红外翻译

Detection of protein-cofactor interactions by means protein-cof inter of Fourier transfor m infrared spectroscopy our ansf infr
spectroscop
Takumi Noguchi
Biophysical Chemistry Laboratory, RIKEN
Abstract Fourier transform infrared(FTIR)spectroscopy was used to study protein-cofactor interactions in pho- tosystem II. The FTIR spectrum of each cofactor was measured as a dif frence spectrum upon its light-induced reaction. The spectrum included the e bands of the protein moiety coupled to the cofactor, which were identified using selective isotopic labeling of amino acids. Analysis of these FTIR bands provided insight into the protein-cofactor interaction as well as enzymatic reactions.
Introduction A number of proteins possess organic compounds and metal ions as cofactors. The chemical and physical properties of these cofactors are tuned in the binding pockets of the proteins to express their functions. Thus, it is of importance to study protein-cofactor interactions to clarify the mechanism of enzymatic reactions. A powerful method for studying protein-cofactor interactions is Fourier transform infrared (FTIR) spectroscopy, which can directly detect chemical bonds and bonds. In FTIR, infrared absorption of molecular interactions such as hydrogen molecules is detected using a Michelson interferometer, which gives very precise wavenumbers of spectra. Hence, FTIR is capable of detecting subtle spectral changes and For changes (A <104 ), making it possible to study detailed structural reactions in the active site of a large protein complex of as much as 1000 kDa. such FTIR measurements, membrane samples can be used and a relatively small amount of protein (e.g., 200g) is needed. This article introduces the light-induced FTIR difference technique to study protein-cofactor interactions with examples from our recent results on photosystem II (PSII).
Cofactors in photosystem II PS II is a protein complex (Mr ≈ 500kDa) that performs light-induced electron -1- transfer and oxygen evolution in the early process of photosynthesis in plants and cyanobacteria. Various pigments and metal ions are bound to the protein to achieve the PS II function (Fig. 1). The primary donor chlorophyll, P680, is first excited by excitation energy transfer from antenna chlorophylls, and ejects an electron to the pheophytin molecule. The electron is subsequently transferred to the primary (QA ) and then the secondary (QB ) quinone acceptors. On the electron donor side, the hole moves from P680+ to the tyrosine YZ and then to the Mn-cluster, where water is oxidized to evolve molecular oxygen (Fig. 1). Fig.1. Structural model of photosystem II.
FTIR detection of protein-cofactor interactions in photosystem II The FTIR spectrum of a targeted cofactor in the PSII complex can be obtained by measuring a difference spectrum upon the
light-induced reaction of the cofactor. For instance, the spectrum of the QA electron acceptor was measured as a lightminus-dark dif frence in the presence of an inhibitor to block further electron e transfer to QB. The obtained QA/QA dif frence spectrum includes structural e information not only of the quinone molecule itself but also of the protein moiety coupled to QA . For example, several bands of the imidazole group of a His residue were identified in the QA /QA spectrum using the PS II complex in which nitrogen atoms of His side chains were selectively labeled with 15 N (Fig. 2).1) Analyzing these His bands as well as the C=O stretching band of QA at 1478 cm1 -2- showed the presence of a strong hydrogen bond between the C=O group of QA and the imidazole N-H (Fig. 3).1, 2) The structures of amino-acid ligands of the oxygen-evolving Mn-cluster were also studied using FTIR. The dif frence spectrum upon the first intermediate e transition (S1→S2), induced by single flash illumination, showed several COO stretching bands of carboxylate groups at 1600-1500cm1 (asymmetric stretch) and 1450–1350 cm1 (symmetric stretch), indicating that several Asp or Glu residues serve as the ligands of the Mn-cluster.3) Also, the N-H (2900–2500cm1 ) and CN in the S2 /S1 (1113 cm1 ) stretching vibrations of a His side chain were identified spectrum by selective 15 N-His labeling.4) From these His bands, it was proposed that the His ligand of the Mn-cluster has a structure in which Nτ is coordinated to the Mn ion and the Nπ-H is strongly hydrogen bonded.4) Furthermore, measurement of the S2 /S1 spectrum with the 13C-Tyr-labeled complex identified the COH (1254 cm1 ) and C=C (1521 cm1 ) vibra- tions of a Tyr side chain, probably the redox active YZ , cou- pled to the Mn-cluster through a hydrogen-bond network.5) This hydrogen-bond network between the Mn-cluster and YZ was suggested to be a pathway of proton release from the substrate water coordinated to the Mn ion.5) Recently, we have detected the O-H stretching vibration of substrate water at 3618/3585cm1 in the S2/S1 spectrum and discussed the changes in the hydrogen-bonding interaction of the water molecule upon S2 formation.6) Furthermore, we have succeeded in measuring all the flash-induced transitions of intermediates (S2→S3 , S3→ S0 , S0→S1 ) by repeating cycles of successive flash illumination (1 s interval), spectral measurement between the flashes and the following dark adaptation (Fig. 4). It was seen that the structural changes in the protein conformations and carboxylate groups in the S1→ S2→ S3 transitions are reversed in the S3 → S0 → S1 transitions.7) -3- Fig.2.FTIR dif frence spectrum of QA-/QA(upper trace) and His e bands abstracted by 15 N-labeling of His side chains (lower trace). Fig.3. Predicted interactions of QA with the protein. -4- Fig.4.Flash-induced FTIR difference spectra of the S-state transitions of the oxygen-evolving Mn-cluster. As seen in the above examples, FTIR is a suitable method for the investigation of enzymatic reactions as well as protein-cofactor interactions. Such detection of the proteincofactor interactions in PS II will be essential in understanding the role of protein in controlling electron-transfer reactions. Also, monitoring the reactions of substrate water, amino-acid ligands and protein conformations in S-state transitions by FTIR is expected to be a most crucial method for elucidating the molecular mechanism of photosynthetic oxygen evolution. References
1) T. Noguchi, Y. Inoue, and X.-S. Tang: Biochemistry 38, 399 (1999). 2) T. Noguchi, J. Kurreck, Y. Inoue, and G. Renger: Biochem-istry 38, 4846(1999). 3) T. Noguchi, T. Ono, and Y. Inoue: Biochim. Biophys. Acta1228, 189 (1995). 4) T. Noguchi, Y. Inoue, and X.-S. Tang: Biochemistry 38,10187 (1999). 5) T. Noguchi, Y. Inoue, and X.-S. Tang: Biochemistry 36,14705 (1997). 6) T. Noguchi and M. Sugiura: Biochemistry 39, 10943 (2000). -5- 7) T. Noguchi and M. Sugiura: Biochemistry 40, 1497 (2001).
利用傅立叶变换红外光谱检测蛋白质辅助
因子的相互作用
Takumi Noguchi
生物物理化学实验室，理化学研究所
摘要:红外光谱仪用来研究蛋白质辅助因子在光合作用 2 中的相互作用。

每一辅酶红外光谱测定时其作为光诱导反应差光谱。

光谱包括采用选择性同位素标记的氨基酸蛋白质耦合因子的谱带。

傅里叶变换红外光谱的分析提供了认识蛋白质辅助因子的相互作用以及酶反应。

引言大量的蛋白质控制着有机物和金属离子作为辅助因子。

这些辅助因子的化学物理性质受到蛋白质结合口袋的调节来表现他们的职能。

因此，研究蛋白质辅助因子的相互作用阐明酶反应的作用机制是很重要的。

一种研究蛋白质相互作用的强有力的方法是傅立叶变换红外光谱法（FTIR），它可以直接检测化学键与分子的相互作用如氢键。

在傅里叶红外光谱（FTIR）中，分子的红外吸收使用迈克尔逊干涉仪检测，它能给出非常精确地光谱波数。

因此，傅里叶红外光谱法能够检测到光谱的微妙变化（A <104），从而使在活性部分研究高达100kDa 的大量的蛋白质复合体精细结构的变化以及反应成为可能。

对于这样的傅里叶变换红外光谱（FTIR）测量，只需要相对少量的蛋白质（例如，200 微克）制作成样品膜。

本论文介绍了光致傅里叶变换红外光谱（FTIR）不同技术从我们在光合系统II （PSII）中最新研究成果的例子去研究蛋白质辅助因子相互作用。

在光合系统II(PSII)中的辅助因子-6- 光合系统II 是在植物和蓝藻的光合作用早期复合蛋白(Mr≈500kDa)质执行光致电子转移和氧的演化。

各种色素和金属离子受到蛋白质的约束控制完成光合系统II 的功能（图1）。

主要供体叶绿素，P680，首先受到从天线叶绿素以及放出一个电子的脱镁叶绿素的激励能激发。

随后电子转移到首要的（QA），然后到次级的（QB）醌受体。

在电子供体方面，从P680+移动到酪氨酸YZ 然后到锰复合物，其中水分解演变出氧分子（图1）。

图1 光合作用II结构模型FTIR 检测光合系统II 中蛋白质辅助因子的相互作用一个在光合系统II 中的复合蛋白质目标辅助因子在光致反应上通过测量可以获得不同的频谱的傅里叶变换红外光谱。

例如，测量出的QA 电子受体的光谱存在light-minus-dark 与抑制剂阻止电子进一步转移到QB 的不一样。

获得的QA/QA 不同光谱所包含的结构信息不仅仅是醌分子本身也包含蛋白质的一部分基团与QA 的耦合。

例如，在光合系统II 中的蛋白质复合物选择性地利用侧链上的氮原子（15N）示踪，残留的咪唑组的一些谱带在QA-/QA 光谱中被鉴定（图2）[1]。

分析这些谱带在C=O 伸缩振动带中的1478cm-1 显示出在C=O 基团和咪唑N-H 之间有强氢键的存在（图3）[1,2]。

-7- 图2 QA-/QA（上游）和它的吸收带以及利用15N 对它的侧链示踪（下游）的不同FTIR 光谱图3 QA 与蛋白质的相互作用推断同样对氨基酸的配位体锰复合物的氧演变的结构进行了傅里叶变换红外光谱研究。

光谱不同的是第一个中介过度1→S2）（S 包括了单脉冲照射，1600 -1500 在cm-1 处呈现出一些羧根的COO-伸缩振动带（非对称伸缩）和
1450-1350 cm- 1（对称伸缩）表明一些天门冬氨酸和谷氨酸的残基作为锰复合物的配位体[3]。

此外，，S2/S1 光谱中一个侧链N-H（2900-2500cm-1）和CN（1113cm-1）伸缩振动被被鉴别，15N 已被选择性标记[4]。

从这些谱带中，有人提议是锰复合物结构中的配位基，τ对于锰离子是协调的以及Nπ-H 存在一个强烈的氢键[4]。

N 此外，测量的S2/S1 光谱，利用13C 标记的酪氨酸鉴定COH（1254cm-1）和C=C（1521cm-1）为酪氨酸一个侧链上的振动，很可能是通过氢键网络耦合到一起的酪氨酸YZ 和锰复合物的氧化还原反应[5]。

这个在锰复合物和YZ 之间的氢键网络被提出为质子释放-8- 通道与水基质中的锰离子相协调[5]。

最近，我们已经发现在S2/S1 光谱中O-H 在水基质中的伸缩振动在3618/3585cm-1 处，以及讨论在氢键合水分子的相互作用下的S2 的结构[6]。

此外，我们已经成功地通过重复连续光照（1 秒的间隔）测量了所有的脉冲引起的中介物（S2→S3,S3→S0,S0→S1）的跃迁，光谱测量之间的闪烁和暗适应后（图4）。

有人看到，在蛋白质的结构中的构象变化和羧酸根在S1 →S2→S3 转换中与S3→S0→S1 转换中是相反的[7]。

图 4 脉冲引起的S 态跃迁以及锰复合物的氧演变导致傅里叶变换红外光谱的差异在上述例子中，傅里叶变换红外光谱法是一种合适的研究酶反应以及蛋白质辅助因子之间的相互作用的方法。

蛋白质辅助因子间的相互作用的发现在了解蛋白质控制电子转移作用所扮演的角色是必不可少的。

此外，检测水基质中的氨基酸配体以及蛋白质结构在S 态的跃迁，FTIR 应该为一种最重要的阐明光合作用的氧演变的分子机制的方法。

参考文献[1] T. Noguchi, Y. Inoue, and X.-S. Tang:Biochemistry 38, 399 (1999). [2] T. Noguchi, J. Kurreck, Y. Inoue, and G. Renger: Biochem-istry 38, 4846 (1999). -9- [3] T. Noguchi, T. Ono, and Y. Inoue: Biochim. Biophys. Acta1228, 189 (1995). [4] T. Noguchi, Y. Inoue, and X.-S. Tang: Biochemistry 38,10187 (1999). [5] T. Noguchi, Y. Inoue, and X.-S. Tang: Biochemistry 36,14705 (1997). [6] T. Noguchi and M.Sugiura: Biochemistry 39, 10943 (2000). [7] T. Noguchi and M.Sugiura: Biochemistry 40, 1497 (2001).。