Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and iron(III)-superoxide dismutase from Escherichia coli.
Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and iron(III)-superoxide dismutase from Escherichia coli.
复制标题
大肠杆菌铁 (II) 和铁 (III) 超氧化物歧化酶的氨基酸特异性同位素标记和活性位点 NMR 研究。
DOI：
10.1023/a:1008344210662
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发表时间：
2000
影响因子：
2.7
通讯作者：
Miller,AF
中科院分区：
文献类型：
--
作者：
Sorkin,DL;Miller,AF
我们已经开发并使用了多种氨基酸特异的同位素标记方案来获得来自大肠杆菌的铁(II)和铁(III)-超氧化物歧化酶(Fe(II)和Fe(III)-超氧化物歧化酶)的活性部位的1H核磁共振波谱的确定归属。尽管高自旋Fe(III)具有严重的驰豫特性，但我们已经能够在Fe(III)超氧化物歧化酶中指定配体His的δ1质子接近100ppm，β和α质子集体在20到50ppm之间。在还原状态下，除了7个配基质子外，我们已经分配了所有的配基质子，在大多数情况下是残基专一性的。一对先前未报道的25.9ppm和22.1ppm的广谱已被确凿地指定为Asp156的β质子，取代了先前的指定(ming等人。(1994)Inorg.化学，33，83-87)。我们利用较高的温度来解析以前未观察到的邻位配体His的质子共振，并利用特定的同位素标记来区分δ2和ε1质子的可能性。这些是最接近Fe(II)的蛋白质质子，因此它们具有最宽的(∼4000赫兹)和最难检测到的共振。我们的工作允许用氢键网络和Fe(II)电子态来解释化学位移、pH依赖和H/D交换率的温度依赖关系。有趣的是，Fe(II)-SOD的轴向His配体化学位移与沼泽红假单胞菌细胞色素c‘的轴向His配体化学位移相似(Bertini等人)。(1988)Inorg.这表明Fe(II)超氧化物歧化酶的赤道His2Asp−连接能够重现血红素与Fe~(2+)配位的一些电子性质，从而可能是化学性质。
We have developed and employed multiple amino acid-specific isotopic labeling schemes to obtain definitive assignments for active site1H NMR resonances of iron(II)- and iron(III)-superoxide dismutase (Fe(II)SOD and Fe(III)SOD) fromEscherichia coli.Despite the severe relaxivity of high-spin Fe(III), we have been able to assign resonances to ligand His′ δ1 protons near 100 ppm, and β and α protons collectively between 20 and 50 ppm, in Fe(III)SOD. In the reduced state, we have assigned all but 7 ligand protons, in most cases residue-specifically. A pair of previously unreported broad resonances at 25.9 and 22.1 ppm has been conclusively assigned to the β protons of Asp 156, superseding earlier assignments (Ming et al. (1994)Inorg. Chem.,33, 83–87). We have exploited higher temperatures to resolve previously unobserved ortho-like ligand His proton resonances, and specific isotopic labeling to distinguish between the possibilities of δ2 and ε1 protons. These are the closest protein protons to Fe(II) and therefore they have the broadest (∼4000 Hz) and most difficult to detect resonances. Our assignments permit interpretation of temperature dependences of chemical shifts, pH dependences and H/D exchange rates in terms of a hydrogen bond network and the Fe(II) electronic state. Interestingly, Fe(II)SOD's axial His ligand chemical shifts are similar to those of the axial His ligand ofRhodopseudomonas palustriscytochrome c′ (Bertini et al. (1988)Inorg. Chem.,37, 4814–4821) suggesting that Fe(II)SOD's equatorial His2Asp−ligation is able to reproduce some of the electronic, and thus possibly chemical, properties of heme coordination for Fe2+.