Reactivity of Glass-Embedded Met Hemoglobin Derivatives toward External NO: Implications for Nitrite-Mediated Production of Bioactive NO
Reactivity of Glass-Embedded Met Hemoglobin Derivatives toward External NO: Implications for Nitrite-Mediated Production of Bioactive NO
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DOI：
10.1021/ja903364h
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发表时间：
2009-09-02
影响因子：
15
通讯作者：
Friedman, Joel M.
中科院分区：
文献类型：
--
作者：
Navati, Mahantesh S.;Friedman, Joel M.
在标准条件下，由于反应物和中间体的浓度低，许多蛋白质反应极难分析。一个恰当的例子是血红蛋白与亚硝酸盐和一氧化氮的几种反应。在目前的工作中，玻璃基质被用来动态控制外部引入的气态NO的进入速度，并与几种不同的血红蛋白衍生物（包括亚硝酸盐、硝酸盐和水晶体）发生反应。这种新颖而通用的方法揭示了亚硝酸盐和其他配体（包括硝酸盐、水和内咪唑）之间的明显区别。对于硝酸盐、水和内部远端咪唑，观察到的光谱变化表明，进入远端血红素口袋的NO可以有效地将这些配体从铁血红素铁中取代。相反，当配体是亚硝酸盐时，产生的初始光谱表明形成了具有明显的类铁性质的中间体。光谱和DAF荧光对中间体的反应与最近提出的亚硝酸盐酸酐酶反应一致。这个提议的中间体特别重要，因为它代表了一个依赖亚硝酸盐的催化过程的途径，在这个过程中，Hb产生相对长寿的生物活性形式的NO，如s -亚硝基谷胱甘肽。在低pH值或当玻璃被广泛干燥时，不能形成这种中间体，这与远端血红素口袋内的反应物和残留物侧链的特定构象的要求是一致的。
Many protein reactions are exceedingly difficult to dissect under standard conditions due to low concentrations of reactants and intermediates. A case in point are several proposed reactions of hemoglobin with both nitrite and nitric oxide. In the present work, glassy matrices are used to dynamically control the rate at which externally introduced gaseous NO accesses and reacts with several different met Hb derivatives including the nitrite, nitrate, and aquomet forms. This novel yet general approach reveals a clear difference between nitrite and other ligands including nitrate, water, and an internal imidazole. For nitrate, water, and the internal distal imidazole, the observed spectral changes indicate that NO entering the distal heme pocket is effective in displacing these ligands from the ferric heme iron. In contrast, when the ligand is nitrite, the resulting initial spectra indicate the formation of an intermediate that has distinctly ferrous-like properties. The spectrum and the response of DAF fluorescence to the presence of the intermediate are consistent with a recently proposed nitrite anhydrase reaction. This proposed intermediate is especially significant in that it represents a pathway for a nitrite-de pendent catalytic process whereby Hb generates relatively long-lived bioactive forms of NO such as S-nitrosoglutathione. The failure to form this intermediate either at low pH or when the glass is extensively dried is consistent with the requirement for a specific conformation of reactants and residue side chains within the distal heme pocket.