Mechanism of binding of NO to soluble guanylyl cyclase: implication for the second NO binding to the heme proximal site.
Mechanism of binding of NO to soluble guanylyl cyclase: implication for the second NO binding to the heme proximal site.
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DOI：
10.1021/bi300105s
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发表时间：
2012-04-03
期刊：
影响因子：
2.9
通讯作者：
Tsai AL
中科院分区：
文献类型：
--
作者：
Martin E;Berka V;Sharina I;Tsai AL
可溶性鸟苷酸环化酶 (sGC) 是形成第二信使环 GMP (cGMP) 的关键酶，是一氧化氮 (NO) 的真实传感器。 NO 与 sGC 的结合导致酶活性的强烈激活。 NO与sGC结合的多个分子和步骤已被证实，但第二个NO的靶点和详细的结合机制仍存在争议。在本研究中，我们使用 15NO 和 14NO 以及厌氧顺序混合冷冻淬灭 EPR 来明确证实血红素 Fe 是第二个 NO 的目标。第二步 NO 结合的观察速率对 NO 浓度的线性依赖性高达 600 s−1，不仅表明第二个 NO 的结合位点与第一步中的结合位点（即血红素的近端位点）不同，而且支持了一种协调机制，其中 His105 近端配体的解离与第二个 NO 分子的结合同时发生。计算机建模成功预测了一组五配位 NO 复合物与远端或近端位点上的配体形成的动力学，并支持从瞬时双 NO sGC 复合物的远端选择性释放 NO。因此，正如细胞色素c'所证明的，在第二个NO结合后，形成含有近端NO的五配位NO-sGC。
Soluble guanylyl cyclase (sGC), the key enzyme for the formation of second messenger cyclic GMP (cGMP), is an authentic sensor for nitric oxide (NO). Binding of NO to sGC leads to strong activation of the enzyme activity. Multiple molecules and steps of NO binding to sGC have been implicated, but the target of the second NO and the detailed binding mechanism remain controversial. In this study, we used 15NO and 14NO and anaerobic sequential mixing-freeze quench EPR to unambiguously confirm that the heme Fe is the target of the second NO. Linear dependence on NO concentrations up to 600 s−1 for the observed rate of the second step of NO binding not only indicates that the binding site of the second NO is different from that in the first step, i.e. the proximal site of the heme, but also support a concerted mechanism in which the dissociation of the His105 proximal ligand occurs simultaneously with the binding of the second NO molecule. Computer modeling successfully predicts the kinetics of formation of a set of five-coordinate NO complexes with the ligand on either the distal or proximal site and supports a selective release of NO from the distal side of the transient bis-NO sGC complex. Thus, as has been demonstrated with cytochrome c', a five-coordinate NO-sGC containing a proximal NO is formed after the binding of the second NO.