Differentiated, Promoter-specific Response of [4Fe-4S] NsrR DNA Binding to Reaction with Nitric Oxide.
Differentiated, Promoter-specific Response of [4Fe-4S] NsrR DNA Binding to Reaction with Nitric Oxide.
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DOI：
10.1074/jbc.m115.693192
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发表时间：
2016-04-15
期刊：
影响因子：
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通讯作者：
Le Brun NE
中科院分区：
文献类型：
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作者：
Crack JC;Svistunenko DA;Munnoch J;Thomson AJ;Hutchings MI;Le Brun NE
NsrR是一种铁-硫簇蛋白，调节许多细菌的一氧化氮(NO)应激反应。天蓝色链霉菌的NsrR调节自身和另外两个基因hmpA1和hmpA2的表达，这两个基因编码HmpA酶，预计可以解毒NO。NsrR仅在配位[4Fe-4S]簇时才与启动子DNA高亲和力结合。在这里，我们展示了[4Fe-4S]NsrR与NO的反应根据基因启动子的不同而不同地影响DNA结合。与hmpA2启动子的结合在每个簇的∼2 NO处被取消，尽管对于hMPA1和nsrR启动子，分别需要∼4和∼8 NO分子来取消结合。对NO反应的光谱和动力学研究揭示了一个快速的、多相的、非协调的过程，每个簇涉及多达8-10个NO分子，导致几个亚硝酸铁物种的形成。在每个簇的∼2 NO处观察到一个明显的中间体，在∼4和∼6 NO处观察到另外两个中间体。DNA结合对NsrR亚硝化反应无明显影响。这些结果表明，NsrR调节不同的启动子以响应不同浓度的NO。光谱证据表明，这是由不同的NO-FeS络合物实现的。
NsrR is an iron-sulfur cluster protein that regulates the nitric oxide (NO) stress response of many bacteria. NsrR from Streptomyces coelicolor regulates its own expression and that of only two other genes, hmpA1 and hmpA2, which encode HmpA enzymes predicted to detoxify NO. NsrR binds promoter DNA with high affinity only when coordinating a [4Fe-4S] cluster. Here we show that reaction of [4Fe-4S] NsrR with NO affects DNA binding differently depending on the gene promoter. Binding to the hmpA2 promoter was abolished at ∼2 NO per cluster, although for the hmpA1 and nsrR promoters, ∼4 and ∼8 NO molecules, respectively, were required to abolish DNA binding. Spectroscopic and kinetic studies of the NO reaction revealed a rapid, multi-phase, non-concerted process involving up to 8–10 NO molecules per cluster, leading to the formation of several iron-nitrosyl species. A distinct intermediate was observed at ∼2 NO per cluster, along with two further intermediates at ∼4 and ∼6 NO. The NsrR nitrosylation reaction was not significantly affected by DNA binding. These results show that NsrR regulates different promoters in response to different concentrations of NO. Spectroscopic evidence indicates that this is achieved by different NO-FeS complexes.