Cadmium-113 NMR of metallothionein: direct evidence for the existence of polynuclear metal binding sites
Cadmium-113 NMR of metallothionein: direct evidence for the existence of polynuclear metal binding sites
复制标题
金属硫蛋白的镉 113 NMR：多核金属结合位点存在的直接证据
DOI：
10.1021/ja00520a028
复制
发表时间：
1979
影响因子：
15
通讯作者：
I. Armitage
中科院分区：
文献类型：
--
作者：
J. Otvos;I. Armitage
金属硫蛋白具有显著的金属结合能力（~ 6 - 7 g-atoms/mol，通常为Zn ~（2+）和Cd ~（2+）），其结构基础引起了人们的极大兴趣，因为最近有人提出该蛋白参与金属解毒和/或代谢。Ia· 2金属硫蛋白（Metallothionein，Ia· 2 Metallothionein）是一种小分子蛋白质（molwt，~ 6800），广泛存在于包括人在内的多种动物的肾脏和肝脏中，Cd ~（2+）、Zn ~（2+）和其它重金属可诱导其合成。3蛋白质的氨基酸组成非常不寻常，因为完全不存在组氨酸和芳香族氨基酸残基，而蛋白质中总共60个氨基酸中约有20个是半胱氨酸残基。所有20个半胱氨酸都显示通过硫醇连接参与金属连接。13-4由于在所有金属硫蛋白中半胱氨酸/二价金属离子的比例接近3，因此推测蛋白质中的金属以分离的带负电荷的三硫醇络合物形式存在，即[Me 2+（Cys-）3]。鉴于已知的Cd 2+形成多核络合物的倾向，其来源于硫醇硫原子作为两个或三个Cd 2+离子之间的桥的能力，涉及低聚金属结合位点的6种替代结构同样可能存在于金属硫蛋白中。本文报道了在兔肝脏金属硫蛋白II的113 Cd核磁共振谱中观察到广泛的113 Cd-、13 Cd自旋耦合。这一结果提供了第一个直接的证据，在这种蛋白质中存在的多核金属簇排列。纯金属硫蛋白II（金属硫蛋白的主要同种蛋白之一）从反复注射96%富集的113 CdCl 2的兔的肝脏中分离。通过在Diaflo UM-2膜上超滤将蛋白质浓缩至约14 mM，用于NMR。通过原子吸收测定并标准化为总计7克金属原子/摩尔蛋白质的蛋白质金属含量为4.4克原子的113 Cd 2+和2.6克原子的Zn 2 +/摩尔。非完整金属含量反映了天然蛋白质的已知异质性，这是由于存在含有不同相对量的Zn 2+和Cd 2+的物质。图1A中金属硫蛋白II的113 Cd NMR谱显示出五个分辨的共振峰，其中心位于0.1M CdClOq低场的604、613、623、641和668 ppm处。这些化学位移值与以前的报告7一致，表明金属硫蛋白中的每个Cd 2+离子参与广泛的硫连接。从一系列Cd 2 +-烷基硫醇盐络合物中观察到的去屏蔽程度和硫连接程度之间的定性相关性来看，8金属硫蛋白中的每个Cd 2+离子可能与至少三个半胱氨酸硫配体配位。然而，根据现有的
The structural basis for the remarkable metal binding ca-pacity of metallothionein (—6—7 g-atoms/mol, usually Zn2+ and Cd2+) is of considerable interest owing to the recent pos-tulated involvement of this protein in metaldetoxification and/or metabolism. Ia· 2 Metallothionein is a small protein (mol wt,~ 6800) located in the kidney and liver of a wide varietyof animal species, including man, and its synthesis can be induced by the administrationof Cd2+, Zn2+, and other heavy metals. 3 The amino acid composition of the protein is very unusual in that there is a complete absence of histidine and aromatic amino acid residues while about 20 out of the total of 60 amino acids in the protein are cysteine residues. Ia, 2c All 20 cysteines have been shown to participate in metal ligation via mercaptide linkages. 13-4 Since the ratio of cysteine/divalent metal ion in all metallothioneins is close to 3, it has been postulated that the metals in the protein exist as isolated, negatively charged trimercaptide complexes, 5 ie,[Me2+(Cys-) 3]~. In view of the known propensity of Cd2+ to form polynuclear complexes deriving from the ability of mercaptide sulfur atoms to act as bridges between two or three Cd2+ ions, 6 alternate structures involving oligomeric metal binding sites are equally likely to exist in metallothionein. In this paper, we report the observa-tion of extensive ll3Cd-, l3Cd spin coupling in the 1 l3Cd NMR spectrum of metallothionein II from rabbit liver. This result provides the first direct evidence for the existence of a polynuclear metal cluster arrangement in this protein. Pure metallothionein II (one of the major isoproteins of metallothionein) was isolated'3 from the livers of rabbits subjected to repeated injections of 96% enriched 1 l3CdCl2. Protein was concentrated for NMR to~ 14 mM by ultrafil-tration on a Diaflo UM-2 membrane. The metal content of the protein determined by atomic absorption and normalized to a total of 7 g-atoms of metal/mol of protein was 4.4 g-atoms of ll3Cd2+ and 2.6 g-atoms of Zn2+/mol. The nonintegral metal content reflects the known heterogeneity of the native protein which is due to the presence of species containing dif-ferent relative amounts of Zn2+ and Cd2+. 3 113Cd spectra at 19.96 MHz were obtained on an extensively modified Bruker HFX-90 spectrometer and at 44.37 MHz on a Bruker CXP-200 spectrometer.The 1 l3Cd NMR spectrum of metallothionein II in Figure 1A exhibits five resolved resonances centered at 604, 613, 623, 641, and 668 ppm downfieldfrom 0.1 M CdClOq. These chemical shift values are consistent with previous reports7 and indicate that each Cd2+ ion in metallothionein is involved in extensive sulfur ligation. From the qualitative correlation be-tween degree of deshielding and extent of sulfur ligation ob-served in a series of Cd2+-alkylthiolate complexes, 8 it is likely that every Cd2+ ion in metallothionein is coordinated to at least three cysteine sulfur ligands. However, on the basis of available