NMR study of the molecular and electronic structure of the heme cavity of Aplysia metmyoglobin. Resonance assignments based on isotope labeling and proton nuclear Overhauser effect measurements.
NMR study of the molecular and electronic structure of the heme cavity of Aplysia metmyoglobin. Resonance assignments based on isotope labeling and proton nuclear Overhauser effect measurements.
复制标题
海兔高铁肌红蛋白血红素腔的分子和电子结构的核磁共振研究。
DOI：
10.1021/bi00367a044
复制
发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Thanabal,V
中科院分区：
文献类型：
--
作者：
Pande,U;LaMar,GN;Lecomte,JT;Ascoli,F;Brunori,M;Smith,KM;Pandey,RK;Parish,DW;Thanabal,V
摘要：本文研究了软体动物Aaplasia limacina高自旋高铁肌红蛋白的~ 1H NMR特征，并与抹香鲸肌红蛋白（Mb）的~ 1H NMR特征进行了比较。海兔metMb表现出正常的酸碱转变，pK~ 7.8。在酸性形式中，血红素甲基和内消旋质子共振已通过NMR使用用选择性氘代氯化血红素重构的样品分配，并且在后一种情况下也通过2 H NMR分配。的甲基峰强度和位移模式的基础上，血红素旋转障碍，可以建立在AaplasiaMb;~ 20%的蛋白质表现出相反的血红素取向相比，在单晶中发现的。在-16和-35 ppm之间的高场区域中检测到三个中质子共振，表明这些质子的化学位移可以作为血红素蛋白中五配位活性位点的诊断探针，如先前所示的模型化合物中的情况。介观质子化学位移的温度依赖性与T~ xCurie行为有很大的偏离，反映了具有明显δ = 3/2特征的热可及Kramers双峰的存在。核Overhauser效应，NOE，对Aaplasia metMb的测量提供了单个血红素-丙酸酯共振的分配，并用于推断血红素侧链之间的空间邻近性。结合指定共振的超精细位移值、NOE连接性和NOE幅度，以定性地描述Aaprossia metMb相对于抹香鲸MbH 20的乙烯基和丙酸酯侧链的旋转迁移率和取向。因此，发现血红素侧链在前一种蛋白质中的空间钳位较小。从海兔的颊肌中提取的肌红蛋白Mb，1具有几个有趣的性质，
Revised Manuscript Received May 1, 1986 abstract: The* H NMR characteristics of the high-spin metmyoglobin from the mollusc Aplysia limacina have been investigated and compared with those of the myoglobin (Mb) from sperm whale. Aplysia metMb exhibits a normal acid alkaline transition with pK~ 7.8. In the acidic form, the heme methyl and meso proton resonances have been assigned by NMR using samples reconstituted with selectively deuterated hemins and in the latter case by 2H NMR as well. On the basis of the methyl peak intensities and shift pattern, heme rotational disorder could be established in AplysiaMb;~ 20% of the protein exhibits a reversed heme orientation compared to that found in single crystals. Three meso proton resonances have been detected in the upfield region between-16 and-35 ppm, showing that the chemical shift of such protons can serve as a diagnostic probe for a pentacoordinated active site in hemoproteins, as previously shownto be the case in model compounds. The temperature dependence of the chemical shift of the meso proton signals deviates strongly from the T~ x Curie behavior, reflecting the presence of a thermally accessible Kramers doublet with significant 5= 3/2 character. Nuclear Overhauser effect, NOE, measurements on Aplysia metMb have provided the assignment of individual heme-propionate resonances and were used to infer spatial proximity among heme side chains. The hyperfineshift values for assigned resonances, the NOE con-nectivities, and the NOE magnitudes were combined to reach a qualitative picture of the rotationalmobility and the orientation of the vinyl and propionate side chains of Aplysia metMb relative to sperm whale MbH20. Thus, it was found that the heme side chains are sterically less clamped in the former protein. e myoglobin, Mb, 1 from the buccal muscle of the sea hare Aplysia limacina possesses several interesting properties that