Barry B A, el-Deeb M K, Sandusky P O, Babcock G T
Department of Chemistry, Michigan State University, East Lansing 48824.
J Biol Chem. 1990 Nov 25;265(33):20139-43.
Deuteration at selected positions on the phenol ring and at the beta-methylene carbon for the YD.tyrosine radical in Photosystem II in the cyanobacterium Synechocystis 6803 was achieved by growing the organism under conditions in which it is a functional aromatic amino acid auxotroph (Barry, B. A., and Babcock, G. T. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7099-7103). A series of model tyrosine radicals, also deuterated at specific sites on the aromatic ring and the methylene group, was generated by UV irradiation of frozen solutions. The EPR spectra of the specifically deuterated in vivo radicals confirm that YD.is a tyrosine; nevertheless its spectra differ from those of the tyrosine models. By comparing the EPR spectra of the specifically deuterated radicals with those of the fully protonated, the hyperfine couplings of the various protons of both YD.and the model compound radicals were determined. For both species, the unpaired electron spin density distribution is essentially identical and follows an odd-alternant pattern with high rho values at the carbons ortho and para to the tyrosine phenol oxygen; the meta positions have low spin densities. The differences in EPR spectral characteristics for the two radicals are rationalized as arising from variations in the conformation of the beta-methylene group with respect to the phenol head group. Considering these EPR results and those reported for other model and naturally occurring tyrosine radicals, we conclude that this situation is general; there is little deviation in this class of compounds from the odd-alternant spin density distribution; variations in EPR lineshapes arise primarily from variations in beta-methylene orientation. The conformation of the -CH2- group in biologically active tyrosine radicals deviates from that observed in the models and may be functionally significant. Because the EPR spectrum of YZ., the second redox active tyrosine radical in Photosystem II, is identical to that of YD., we conclude that the two radicals are in similar protein environments, a conclusion that is supported by the protein sequences in the vicinity of the two radicals.
通过在蓝藻集胞藻6803中使其成为功能性芳香族氨基酸营养缺陷型的条件下培养该生物体，实现了对光系统II中YD.酪氨酸自由基的酚环上选定位置以及β-亚甲基碳的氘代（巴里，B.A.，和巴布科克，G.T.（1987年）《美国国家科学院院刊》84，7099 - 7103）。通过对冷冻溶液进行紫外线照射，生成了一系列同样在芳香环和亚甲基的特定位置进行了氘代的模型酪氨酸自由基。体内特定氘代自由基的电子顺磁共振（EPR）光谱证实YD.是一种酪氨酸；然而其光谱与酪氨酸模型的光谱不同。通过将特定氘代自由基的EPR光谱与完全质子化的自由基的光谱进行比较，确定了YD.和模型化合物自由基中各个质子的超精细耦合。对于这两种物质，未成对电子自旋密度分布基本相同，并且遵循奇数交替模式，在酪氨酸酚氧的邻位和对位碳处具有高的ρ值；间位具有低自旋密度。两种自由基在EPR光谱特征上的差异被解释为是由于β-亚甲基基团相对于酚头基团的构象变化所致。考虑到这些EPR结果以及其他模型和天然存在的酪氨酸自由基的报道结果，我们得出结论，这种情况是普遍的；这类化合物在奇数交替自旋密度分布方面几乎没有偏差；EPR线形的变化主要源于β-亚甲基取向的变化。生物活性酪氨酸自由基中 -CH2- 基团的构象与模型中观察到的不同，并且可能具有功能意义。由于光系统II中第二个氧化还原活性酪氨酸自由基YZ.的EPR光谱与YD.的相同，我们得出结论，这两个自由基处于相似的蛋白质环境中，这一结论得到了两个自由基附近蛋白质序列的支持。
