1H NMR study of dynamics and thermodynamics of heme rotational disorder in native and reconstituted hemoglobin A.
1H NMR study of dynamics and thermodynamics of heme rotational disorder in native and reconstituted hemoglobin A.
复制标题
天然和重构血红蛋白 A 中血红素旋转紊乱的动力学和热力学的 1H NMR 研究。
DOI：
10.1021/bi00366a045
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
LaMar,GN
中科院分区：
文献类型：
--
作者：
Yamamoto,Y;LaMar,GN
加州大学戴维斯分校化学系1986年3月25日收到，95616摘要：用核磁共振波谱详细研究了血红素和载脂蛋白的反应，以阐明血红蛋白(Hb)重组产生天然蛋白的条件。最初形成的全蛋白以具有两个亚基的异构体的混合物的形式存在，这些亚基具有两个不同的亚铁血红素取向，绕中轴旋转180圈[La Mar，G.N.，Yamamoto，Y.，Jue，T.，Smith，K.M.，&Pandey，R.K.(1985)BioChemical 24,3826-3831]，我们在这里详细描述了血红素重定向的速率和机制，并表明Met-Aquo和Met-azido衍生物的速率显著不同，并且在两个亚基中都高度依赖于pH，从而允许在任一亚单位中选择性平衡。这种异构体的非平衡混合物可以以甲基叠氮化合物的形式被动力学捕获，并以这种亚稳态形式储存数月。对于运动控制的血红素定向无序Hb，对甲基叠氮衍生物的各个亚基进行了明确的~1H核磁共振指认，其α和0亚基中分别有约2%和10%的平衡血红素无序。比较各种血红素旋转无序的Hb衍生物的核磁共振谱表明，这种无序在所研究的所有形式中都可观察到，但最容易被识别为血红素无序，最容易在Met-azido络合物中被监测到。血红素紊乱的结构序列似乎在外围比在血红素的轴向相互作用中表现得更强烈。初步研究表明，氧合-Hb的自氧化速率和Met-Aquo-Hb的叠氮化亲和力都取决于血红素的取向。脱脂蛋白和血红素之间的反应不会像最初认为的那样在几毫秒内产生纯的天然全蛋白(Gibson&Antonini，1960；Rose&Olson，1983)，但最初提供了~1：1的全蛋白与血红素的混合物，它们相对于-中轴旋转无序(图1)(La Mar等人，1985)。这种亚稳定的亚铁血红素取向的平衡，以产生基本上由单晶X射线衍射定义的结构(Perutz，1970；Fermi，1975；Baldwin&Chothia，1979)需要几个小时到许多天。最重要的是，中间体不会随着时间的推移而完全消失，而在天然HB1A的制备中，对该中间体的核磁共振谱特征的鉴定表明，至少在一个亚基中存在很大程度的平衡血红素取向无序(La Mar等人，1985)。F这项研究得到了美国国立卫生研究院(HL-16087)和美国国家科学基金会(CHE-84-
Department of Chemistry, University of California, Davis, California 95616 Received March 25, 1986 abstract: The reaction of heme and apoprotein has been studied in detail by NMR spectroscopy in order to elucidate the conditions for reconstitution of hemoglobin (Hb) to yield the native protein. The initially formed holoprotein exists as a mixture of isomers with individual subunits possessing the two heme orientations differing by a 180 rotation about the,-meso axis [La Mar, G. N., Yamamoto, Y., Jue, T., Smith, K. M., & Pandey, R. K.(1985) Biochemistry 24, 3826-3831], We characterize in detail herein the rates and mechanism of heme reorientation andshow that the rates differ dramatically for met-aquo and met-azido derivatives and are highly pH dependent in both subunits in a fashion that allows selective equilibration in either subunit. Nonequilibrium mixtures of such isomers can be kinetically trapped inthe met-azido form and stored in this metastable form for many months. With kineticallycontrolled heme orientationally disordered Hb, unambiguousassignment of'H NMR resonances to individual subunits has been made for the met-azido derivative, which demonstrates~ 2% and 10% equilibrium heme disorder in the a-and 0-subunits, respectively. Comparison of the NMR spectra of various heme rotationally disordered Hb derivatives indicates that this disorder is observable in all forms studied, but is most easily recognized as heme disorder and most convenientlymonitored in the met-azido complex. Structural con-sequences of heme disorder appear to manifest themselves much more strongly in peripheral than axial interactions at the heme. Preliminary studies reveal that both therate of autoxidation of oxy-Hb and the azide affinity of met-aquo-Hb depend on the orientation of the heme. e reaction between apohemoglobin and heme does not yield the pure native holoprotein within a few milliseconds, as originally thought (Gibson & Antonini, 1960; Rose & Olson, 1983), but affords initially a~ 1: 1 mixture of the holoprotein with the heme rotationally disordered with respect to the,-meso axis (Figure 1)(La Mar et al., 1985). The equilibration of this metastableheme orientation to yield the structure essentially as defined by single crystal X-ray diffraction (Perutz, 1970; Fermi, 1975; Baldwin & Chothia, 1979) takes several hours to many days. Most importantly, the intermediate does not completely disappear with time, and the identification of NMR spectral characteristics of this intermediate in preparations of native Hb1 A indicate that a significant degree of equilibrium heme orientation disorder exists within at least one of the subunits (La Mar et al., 1985). f This research was supported by grants from the National Institutes of Health (HL-16087) and the National Science Foundation (CHE-84-