15N-NMR characterization of His residues in and around the active site of FeSOD.
15N-NMR characterization of His residues in and around the active site of FeSOD.
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FeSOD 活性位点及其周围的 His 残基的 15N-NMR 表征。
DOI：
10.1016/j.bbapap.2009.11.009
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
Vathyam,Surekha
中科院分区：
文献类型：
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作者：
Miller,Anne-Frances;Yikilmaz,Emine;Vathyam,Surekha
我们利用15N-核磁共振技术观察了含铁超氧化物歧化酶(FeSOD)活性部位及其周围的组氨酸(His)侧链。在氧化状态下，我们观察到所有的非配体His侧链，而在还原状态下，我们可以根据非配体His‘、来自两个主链的顺磁位移信号和谷氨酰胺69(Gln69)的侧链来解释咪唑光谱区域的所有信号。我们也观察到来自结扎FII的His‘的信号。这些都证实了Q69H和Q69E突变都没有强烈地影响FeII电子结构，尽管EmThey产生的电子结构分别增加了250 mV和>；660 mV。在Q69H突变体中，我们观察到两个新的信号，可归因于取代Gln69的活性部位引入的组氨酸。一个对应于质子化的N，另一个对应于强烈的顺磁位移，达到500ppm。强烈的顺磁效应支持His69与FeII配位的溶剂分子之间存在氢键，这是基于结晶学的假设。根据先前His69是中性的信息，我们推断移位的N不是质子化的。因此，我们认为这个N代表一个从配位溶剂中接受氢键的位置，代表着这个氢键的极性与WT(野生型)FeSOD蛋白中的极性相反。我们还提供了证据表明，底物类似物在FEII配位范围外与FeIISOD结合，影响Gln69，但不直接参与His30。
We have exploited15N-NMR to observe histidine (His) side chains in and around the active site of Fe-containing superoxide dismutase (FeSOD). In the oxidized state, we observe all the non-ligand His side chains and in the reduced state we can account for all the signals in the imidazole spectral region in terms of the non-ligand His′, paramagnetically displaced signals from two backbone amides, and the side chain of glutamine 69 (Gln69). We also observe signals from the His′ that ligate FeII. These confirm that neither the Q69H nor the Q69E mutation strongly affects the FeIIelectronic structure, despite the 250 mV and >660 mV increases in Emthey produce, respectively. In the Q69H mutant, we observe two new signals attributable to the His introduced into the active site in place of Gln69. One corresponds to a protonated N and the other is strongly paramagnetically shifted, to 500 ppm. The strong paramagnetic effects support the existence of an H-bond between His69 and the solvent molecule coordinated to FeII, as proposed based on crystallography. Based on previous information that His69 is neutral, we infer that the shifted N is not protonated. Therefore, we propose that this N represents a site of H-bond acceptance from coordinated solvent, representing a reversal of the polarity of this H-bond from that in WT (wild-type) FeSOD protein. We also present evidence that substrate analogs bind to FeIISOD outside the FeIIcoordination sphere, affecting Gln69 but without direct involvement of His30.