Molecular manipulation of oxygen binding proteins and new development of precise structure-function studies on them

从本项目中获得了以下成果。1.用人工突变方法(IMAI)控制血红蛋白(Hb)的氧亲和力；2.微观氧平衡常数的测定及镁离子和铬离子的测定；-卟啉作为脱氧和氧合血红素的模型使用金属取代杂化HBS(Morimoto)，3.通过模块交换和分析具有分子内二硫键的肌红蛋白(Mb)的配体结合动力学(Morishima)来构建新的珠蛋白，4.鉴定根瘤杆菌(Iizuka)的氧声波蛋白FixL的第五轴向配体，5.利用紫外共振拉曼光谱(Kitagawa)检测HbNO(+IHP)T态和脱氧Hb T态之间在α1-β2接触处的结构差异，6.使用突变体和化学修饰的HBs(Nagai)鉴定作为T-态标记的在287 nm处负CD带的氨基酸残基，7.n…的比较通过对Mb晶体(GO)的X射线衍射数据进行精密简正模分析，获得了更多的中微子衍射数据和随温度变化的涨落数据，8.用不同光学探针(Kihara)的停流法研究了Mb折叠过程中血红素基团的作用，9.血红素-珠蛋白接触的变化对Mb功能的影响很小，而在卟啉的介观位置引入氮原子对Mb功能(Neya)有很大的影响，10.环节动物Hb的巨大尺寸是通过连接链的凝集素样糖粘合和确定连接链在Hb分子中的位置(Gotoh)来维持的。11头巾壳中的Mb与吲哚胺2，3-双加氧酶高度同源，这表明前者是由后者(Suzuki)进化而来的，12 Cerithdea Mb的自氧化伴随着二聚体到单体的转化(Shikama)和13。人胎儿Hb的氧平衡曲线被优化以在低氧压环境下获得高效的氧气传输，而人类成人Hb的氧平衡曲线被优化以最大限度地利用Bohr效应(Kobayashi)。较少

From the present project the following results were obtained. 1.Control of oxygen affinity of hemoglobin (Hb) with cooperativity preserved by means of artificial mutations (Imai), 2.determination of microscopic oxygen equilibrium constants and test of Mg^<2+> and Cr^<3+>-porphyrins as the model for deoxy-and oxyhemes using metal-substituted hybrid Hbs (Morimoto), 3.constitution of novel globins by module exchanges and analysis of ligand binding kinetics of myoglobin (Mb) with intramolecular disulfide bond (Morishima), 4.identification of the fifth axial ligand for the oxygen sonsor protein FixL of root nodule bacterium (Iizuka), 5.detection of structural differences at the alpha1-beta2 contacts between HbNO (+IHP) T-state and deoxyHb T-state by means of ultraviolet resonance Raman spectroscopy (Kitagawa), 6.identification of the amino acid residues contributing to the negative CD band at 287 nm as a T-state marker using mutant and chemically modified Hbs (Nagai), 7.comparison between n … More eutron diffraction data for Mb crystal and temperature-dependence data of fluctuation obtained by applying the precision normal mode analysis to X-ray diffraction data for Mb crystal (Go), 8.the role of heme group in the folding process of Mb studied by stopped-flow method using various optical probes (Kihara), 9.changes in heme-globin contacts cause only small effects on Mb functions whereas introduction of nitrogen atoms into the meso position of porphyrin causes drastic effects on Mb functions (Neya), 10.the giant size of the annelid Hb is maintained by lectin-like sugar adhesion via linker chains and determination of the location of the linker chains in the Hb molecule (Gotoh), 11.Mb from turban shell shows high homologous with indoleamine 2,3-dioxygenase, suggesting that the former evolved from the latter (Suzuki), 12.the autooxidation of Cerithidea Mb is accompanied with dimer-to-monomer conversion (Shikama) and 13.the oxygen equilibrium curve for human fetal Hb is optimized to attain highly efficient oxygen transport under low oxygen pressure environments while that for human adult Hb is optimized to utilize the Bohr effect at maximum (Kobayashi). Less