Fourier transform infrared difference spectroscopy of photosystem II tyrosine D using site-directed mutagenesis and specific isotope labeling
Fourier transform infrared difference spectroscopy of photosystem II tyrosine D using site-directed mutagenesis and specific isotope labeling
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DOI：
10.1021/bi971521a
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发表时间：
1997-12-02
期刊：
影响因子：
2.9
通讯作者：
Berthomieu, C
中科院分区：
文献类型：
--
作者：
Hienerwadel, R;Boussac, A;Berthomieu, C
酪氨酸 D (Tyr(D)) 是光系统 II (PS II) 的侧路电子载体，已通过光诱导傅里叶变换红外 (FTIR) 差异光谱在集胞藻 PS II 护理复合体中进行了研究。 PCC 6803 使用先前优化的实验条件，在富含 PS II 的菠菜膜中生成纯 TyrD(.)/Tyr(D) FTIR 差异光谱 [Hienerwadel, R.、Boussac, A.、Breton, J. 和 Berthomieu, C. (1996) Biochemistry 35, 115447-115460]。 Tyr(D) 和 Tyr(D)(.) 的 IR 模式已通过酪氨酸侧链的特定 H-2- 或 C-13- 标记进行了鉴定。 Tyro 的 v(8a)(CC) 和 v(19)(CC) IR 模式分别在 1615 和 1513-1510 cm(-1) 处被识别，这些频率表明 Tyro 被质子化。将原位同位素敏感信号与溶解在不同溶剂中的模型化合物对甲基苯酚的信号进行比较，得出 Tyro 的 v(7'a)(CO) 和 delta(COH) 模式分别在 1275 和 1250 cm(-1) 处。结果表明，这些模式，特别是 δ(COH) IR 模式对于与酰胺 C=O 或与咪唑氮原子形成氢键配合物非常敏感。原位观察到的频率表明 Tyr(D) 与中性组氨酸的咪唑环形成氢键。对于自由基 Tyr(D)(.)，同位素敏感的 IX 模式在 1532 和 1503 cm(-1) 处被识别。 1503 cm(-1) 处的信号被分配给 Tyr(D)(.) 的 v(CO) 模式，因为它对参与 C4-O 键的环碳上的 C-13 标记敏感。 Gin 定点取代 D2-His 189 后对 Tyr(D) 和 Tyr(D)(.) IR 模式的扰动证实了 Tyr(D) 和 Tyr(D)(.) 与 D2-His189 之间存在氢键。在D20-His189Gln突变体中，Tyro在1267 cm(-1)处的v(7'a)(CO)模式和在大约1228 cm(-1)处的δ(COH)模式表明Tyr(D)和酰胺羰基(可能是D2-Gln189侧链的酰胺羰基)之间形成氢键。电子核双共振 (ENDOR) 测量表明 Tyr(D)(.) 在野生型中是氢键键合，但在突变体中则不然 [Tang, X.-S., Chrisholm, D, A., Dismukes, G. C., Brudwig, G. W., and Diner, B. A. (1993) Biochemistry 32, 13742-13748]。 Tyr(D)(.)在1497 cm(-1)处的v(CO)模式与WT PS]I相比下移了6 cm(-1)，表明氢键导致Tyr(.)的v(CO) IR模式频率上移，来自Gin侧链v(C=O)模式的IR信号被认为在Tyr(D)和1692 cm(-1)处有贡献Tyr(D)(.) 分别表示，这些频率与突变体中 Tyr(D)(.) 形成时氢键的断裂一致。 WT PS II 在 1503 cm(-1) 处观察到的 Tyr(D)(.) v(CO) 模式频率介于 D2-Wis189Gln 突变体中在 1497 cm(-1) 处观察到的频率和在贝拉特缓冲液中通过 UV 照射形成的 Tyr(.) 在 1513 cm(-1) 处观察到的频率之间，表明 PS II 中 Tyr(D)(.) 的氢键比溶液中的氢键更弱或更少。还研究了 D2-His189 在 TyrD(.) 形成时质子摄取中的作用。
Tyrosine D (Tyr(D)), a side path electron carrier of photosystem II (PS II), has been studied by light-induced Fourier transform infrared (FTIR) difference spectroscopy in PS II care complexes of Synechocystis sp. PCC 6803 using the experimental conditions previously optimized to generate the pure TyrD(.)/Tyr(D) FTIR difference spectrum in PS II-enriched membranes of spinach [Hienerwadel, R., Boussac, A., Breton, J., and Berthomieu, C. (1996) Biochemistry 35, 115447-115460]. IR modes of Tyr(D) and Tyr(D)(.) have been identified by specific H-2- or C-13-Iabeling of the tyrosine side chains. The v(8a)(CC) and v(19)(CC) IR modes of Tyro are identified at 1615 and 1513-1510 cm(-1), respectively, These frequencies show that Tyro is protonated. Comparison of isotope-sensitive signals in situ with those of the model compound p-methylphenol dissolved in different solvents leads to the assignment of the v(7'a)(CO) and delta(COH) modes of Tyro at 1275 and 1250 cm(-1), respectively. It is shown that these modes and in particular the delta(COH) IR mode are very sensitive to the formation of hydrogen-bonded complexes with amide C=O or with imidazole nitrogen atoms. The frequencies observed in situ show that Tyr(D) is hydrogen-bonded to the imidazole ring of a neutral histidine. For the radical Tyr(D)(.), isotope-sensitive IX modes are identified at 1532 and 1503 cm(-1). The signal at 1503 cm(-1) is assigned to the v(CO) mode of Tyr(D)(.) since it is sensitive to C-13-labeling at the ring carbon involved in the C4-O bond. The perturbation of Tyr(D) and Tyr(D)(.) IR modes upon site-directed replacement of D2-His 189 by Gin confirms that a hydrogen bond exists between both Tyr(D) and Tyr(D)(.) and D2-His189. In the D20-His189Gln mutant, the v(7'a)(CO) mode of Tyro at 1267 cm(-1) and the delta(COH) mode at approximate to 1228 cm(-1) show that a hydrogen bond is formed between Tyr(D) and an amide carbonyl, probably that of the D2-Gln189 side chain. Electron nuclear double resonance (ENDOR) measurements have shown that Tyr(D)(.) is hydrogen-bonded in the wild type but not in the mutant [Tang, X.-S., Chrisholm, D, A., Dismukes, G. C., Brudwig, G. W., and Diner, B. A. (1993) Biochemistry 32, 13742-13748]. The v(CO) mode of Tyr(D)(.) at 1497 cm(-1) is downshifted by 6 cm(-1) compared To WT PS]I, indicating that hydrogen bonding induces a frequency upshift of the v(CO) IR mode of Tyr(.), IR signals from the Gin side chain v(C=O) mode are proposed to contribute at 1659 and 1692 cm(-1) in the Tyr(D) and Tyr(D)(.) states, respectively, These frequencies are consistent with the rupture of a hydrogen bond upon Tyr(D)(.) formation in the mutant. The frequency of the v(CO) mode of Tyr(D)(.), observed al 1503 cm(-1) for WT PS II, is intermediate between that observed at 1497 cm(-1) in the D2-Wis189Gln mutant and at 1513 cm(-1) for Tyr(.) formed by UV irradiation in berate buffer, suggesting weaker or fewer hydrogen bonds for Tyr(D)(.) in PS II than in solution. The role of D2-His189 in proton uptake upon TyrD(.) formation is also investigated.