Dinitrosyliron complexes and the mechanism(s) of cellular protein nitrosothiol formation from nitric oxide
Dinitrosyliron complexes and the mechanism(s) of cellular protein nitrosothiol formation from nitric oxide
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DOI：
10.1073/pnas.0710416106
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发表时间：
2009-03-24
影响因子：
11.1
通讯作者：
Lancaster, Jack R., Jr.
中科院分区：
文献类型：
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作者：
Bosworth, Charles A.;Toledo, Jose C., Jr.;Lancaster, Jack R., Jr.
亚硝基硫醇（RSNO），由硫醇和一氧化氮（中心点NO）的代谢产物形成，涉及各种生理和病理生理过程，尽管它们在生物学上形成的确切机制尚不清楚。几个候选亚硝化途径涉及中心点NO与O-2，活性氧（ROS）和过渡金属的反应。我们开发了一种使用细胞外亚铁氰化物的策略，以确定在我们的条件下，细胞内蛋白RSNO的形成发生从细胞内的中心点NO的反应，而不是从细胞外室的亚硝化反应物的细胞进入。使用这种方法，我们发现，在RAW 264.7细胞RSNO的形成只发生在非常低的（< 8 μ M）O-2浓度和显示零级中心点NO浓度的依赖性。事实上，即使在O-2水平< 1 μ M时，RSNO的形成也不受抑制。此外，细胞内可螯合铁库（CIP）的螯合使RSNO形成减少> 50%。一种可能的金属依赖性、O-2非依赖性的亚硝化途径是硫醇与二亚硝基铁络合物（DNIC）的反应，DNIC由中心点NO与CIP反应在细胞中形成。在我们的条件下，DNIC的形成，如RSNO的形成，被抑制约50%后螯合不稳定的铁。DNIC和RSNO在氧化还原循环剂5，8-二甲氧基-1，4-萘醌过量产生ROS期间也增加。总之，这些数据强烈表明，细胞RSNO是由来自CIP的DNIC通过转亚硝化作用从自由中心点NO形成的。我们详细研究了细胞内RSNO形成的动力学和机制。
Nitrosothiols (RSNO), formed from thiols and metabolites of nitric oxide (center dot NO), have been implicated in a diverse set of physiological and pathophysiological processes, although the exact mechanisms by which they are formed biologically are unknown. Several candidate nitrosative pathways involve the reaction of center dot NO with O-2, reactive oxygen species (ROS), and transition metals. We developed a strategy using extracellular ferrocyanide to determine that under our conditions intracellular protein RSNO formation occurs from reaction of center dot NO inside the cell, as opposed to cellular entry of nitrosative reactants from the extracellular compartment. Using this method we found that in RAW 264.7 cells RSNO formation occurs only at very low (< 8 mu M) O-2 concentrations and exhibits zero-order dependence on center dot NO concentration. Indeed, RSNO formation is not inhibited even at O-2 levels < 1 mu M. Additionally, chelation of intracellular chelatable iron pool (CIP) reduces RSNO formation by > 50%. One possible metal-dependent, O-2-independent nitrosative pathway is the reaction of thiols with dinitrosyliron complexes (DNIC), which are formed in cells from the reaction of center dot NO with the CIP. Under our conditions, DNIC formation, like RSNO formation, is inhibited by approximate to 50% after chelation of labile iron. Both DNIC and RSNO are also increased during overproduction of ROS by the redox cycler 5,8-dimethoxy-1,4-naphthoquinone. Taken together, these data strongly suggest that cellular RSNO are formed from free center dot NO via transnitrosation from DNIC derived from the CIP. We have examined in detail the kinetics and mechanism of RSNO formation inside cells.