Dipartimento di Fisica, National Enterprise for nanoScience and nanoTechnology CNR-INFM, Università degli Studi di Parma, 43100 Parma, Italy.
Proc Natl Acad Sci U S A. 2009 Nov 10;106(45):18984-9. doi: 10.1073/pnas.0905433106. Epub 2009 Oct 22.
Neuroglobin (Ngb), a member of the globin superfamily, was found in the brain of vertebrates and is suggested to play a neuroprotective function under hypoxic conditions by scavenging nitrogen monoxide (NO) through a dioxygenase activity. In order for such a reaction to efficiently take place and to minimize the release of reactive intermediates in the cytosol, the cosubstrates O(2) and NO and other unstable reaction intermediates should bind sequentially to docking sites in the protein matrix. We have characterized the accessibility of these sites by analyzing the geminate CO rebinding kinetics to the heme moiety observed upon nanosecond flash photolysis of the Ngb-CO complex encapsulated in silica gels. The geminate rebinding phase showed a remarkable complexity, revealing the presence of a system of secondary docking sites where ligands are stored for hundreds of microseconds. Most kinetics steps display little temperature dependence, demonstrating that ligands can easily migrate through the cavities, except for the slowest reaction intermediate, possibly reflecting a structural conformational change reshaping the system of cavities. This conformational change is unrelated with distal His E7 binding to the heme, as it persists for the HE7L mutant. Overall, data are consistent with the presence of a discrete system of docking sites, possibly acting as reservoirs for the putative cosubstrates and for other reactive species involved in the physiologically relevant reaction.
神经球蛋白（Ngb）是球蛋白超家族的成员，在脊椎动物的大脑中被发现，据推测，它通过一种双加氧酶活性清除一氧化氮（NO），从而在缺氧条件下发挥神经保护功能。为了使这种反应有效地发生，并最大限度地减少细胞质中反应中间体的释放，应将共底物 O(2)和 NO 以及其他不稳定的反应中间体顺序结合到蛋白质基质中的对接位点上。我们通过分析在硅胶中封装的 Ngb-CO 复合物的纳秒闪光光解后观察到的血红素部分的成对 CO 再结合动力学，来表征这些位点的可及性。成对再结合相表现出显著的复杂性，揭示了存在一个二级对接位点系统，其中配体可以储存数百微秒。大多数动力学步骤几乎没有温度依赖性，表明配体可以很容易地通过腔迁移，除了最慢的反应中间体，可能反映了结构构象变化重塑了腔系统。这种构象变化与远端 His E7 与血红素的结合无关，因为它在 HE7L 突变体中仍然存在。总体而言，数据与离散的对接位点系统的存在一致，该系统可能充当假定共底物和参与生理相关反应的其他反应性物质的储库。
