A histidine-rich metal binding domain at the N terminus of Cu,Zn-superoxide dismutases from pathogenic bacteria
A histidine-rich metal binding domain at the N terminus of Cu,Zn-superoxide dismutases from pathogenic bacteria
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DOI：
10.1074/jbc.m010527200
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发表时间：
2001-08-10
影响因子：
4.8
通讯作者：
Rotilio, G
中科院分区：
文献类型：
--
作者：
Battistoni, A;Pacello, F;Rotilio, G
来自致病菌的一组Cu， zn超氧化物歧化酶的特征是具有高度暴露和移动构象的富含组氨酸的n端延伸。这一特点使得这些蛋白质可以通过固定化金属亲和层析在一个步骤中很容易地纯化。杜氏嗜血杆菌和副流感嗜血杆菌的铜、锌超氧化物歧化酶在可见区显示异常的吸收光谱，这是由于铜在n端区结合。无铜酶的重构实验表明，在铜可用性有限的条件下，这种金属离子最初结合在n端区域，随后转移到活性位点。为铜从酶亚基的n端结构域转移到位于不同二聚体分子上的活性位点的分子间途径提供了证据。用EDTA孵育能迅速去除N端结合的铜，但对活性位点的铜离子结合效果较差。这表明n端组氨酸的金属结合在动力学上更有利，但催化位点结合铜的亲和力更高。我们认为，在体内金属饥饿条件下，富含组氨酸的n端区域构成了一个参与金属摄取的金属结合域。该结构域的特殊生物学重要性是通过观察到它的存在增强了质周Cu， zn -超氧化物歧化酶对吞噬细胞杀伤的保护作用来推断的。
A group of Cu,Zn-superoxide dismutases from pathogenic bacteria is characterized by histidine-rich N-terminal extensions that are in a highly exposed and mobile conformation. This feature allows these proteins to be readily purified in a single step by immobilized metal affinity chromatography. The Cu,Zn-superoxide dismutases from both Haemophilus ducreyi and Haemophilus parainfluenzae display anomalous absorption spectra in the visible region due to copper binding at the N-terminal region. Reconstitution experiments of copper-free enzymes demonstrate that, under conditions of limited copper availability, this metal ion is initially bound at the N-terminal region and subsequently transferred to an active site. Evidence is provided for intermolecular pathways of copper transfer from the N-terminal domain of an enzyme subunit to an active site located on a distinct dimeric molecule. Incubation with EDTA rapidly removes copper bound at the N terminus but is much less effective on the copper ion bound at the active site. This indicates that metal binding by the N-terminal histidines is kinetically favored, but the catalytic site binds copper with higher affinity. We suggest that the histidine-rich N-terminal region constitutes a metal binding domain involved in metal uptake under conditions of metal starvation in vivo. Particular biological importance for this domain is inferred by the observation that its presence enhances the protection offered by periplasmic Cu,Zn-superoxide dismutase toward phagocytic killing.