Crystal structural investigations of heme protein derivatives resulting from reactions of aryl- and alkylhydroxylamines with human hemoglobin
Crystal structural investigations of heme protein derivatives resulting from reactions of aryl- and alkylhydroxylamines with human hemoglobin
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DOI：
10.1016/j.jinorgbio.2023.112304
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发表时间：
2023-07-04
影响因子：
3.9
通讯作者：
Richter-Addo，George B.
中科院分区：
文献类型：
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作者：
Powell，Samantha M.;Wang，Bing;Richter-Addo，George B.
苯羟胺（PhNHOH）和亚硝基苯（PhNO）与人血红蛋白四聚体（Hb）相互作用形成亚硝基苯加合物Hb（PhNO）。这些相互作用也经常导致红细胞中高铁血红蛋白的形成。我们利用紫外-可见光谱和X-射线晶体学，以确定形成的初级和次级产品时，PhNHOH和相关的烷基羟胺（RNHOH; R = Me，t-Bu）与人铁血红蛋白反应。我们发现，用MeNHOH，主要产物是Hb[α-FeIII（H2O）][β-FeII（MeNO）]，其中亚硝基甲烷与β亚基结合，而不是与α亚基结合。分离亚硝基氯霉素（CAMNO）加合物的尝试导致我们分离出Hb[α-FeII][β-FeII-cySOx]{CAMNO}产物（cySOx =氧化半胱氨酸），其中CAMNO位于蛋白质外部同一四聚体的β2和α2亚基之间的溶剂区域中。我们还观察到β cys 93残基已被氧化。在t-BuNHOH的情况下，我们证明分离的产物是β-半色素Hb[α-FeIII（H2O）][β-FeIII（His）2]{t-BuNHOH}，其中β血红素已向溶剂外部滑动4.4 Å以适应双His血红素配位。当使用PhNHOH时，得到类似的β-半色素Hb[α-FeIII（H2O）][β-FeIII（His）2-cySOx]{PhNHOH}。我们的结果首次揭示了人血红蛋白衍生物中β-半色素的X射线结构测定。我们的UV-vis和X-射线晶体结构结果表明，虽然Hb（PhNO）和Hb（RNO）复合物可以形成为初级产物，但试图通过结晶分离这些产物可能导致其次级产物的结构确定，这些次级产物可能含有进一步蛋白质降解的β-半色素。
Phenylhydroxylamine (PhNHOH) and nitrosobenzene (PhNO) interact with human tetrameric hemoglobin (Hb) to form the nitrosobenzene adduct Hb(PhNO). These interactions also frequently lead to methemoglobin formation in red blood cells. We utilize UV–vis spectroscopy and X-ray crystallography to identify the primary and secondary products that form when PhNHOH and related alkylhydroxylamines (RNHOH; R = Me,t-Bu) react with human ferric Hb. We show that with MeNHOH, the primary product is Hb[α-FeIII(H2O)][β-FeII(MeNO)], in which nitrosomethane is bound to the β subunit but not the α subunit. Attempts to isolate a nitrosochloramphenicol (CAMNO) adduct resulted in our isolation of a Hb[α-FeII][β-FeII-cySOx]{CAMNO}product (cySOx = oxidized cysteine) in which CAMNO was located outside of the protein in the solvent region between the β2 and α2 subunits of the same tetramer. We also observed that the βcys93 residue had been oxidized. In the case oft-BuNHOH, we demonstrate that the isolated product is the β-hemichrome Hb[α-FeIII(H2O)][β-FeIII(His)2]{t-BuNHOH}, in which the β heme has slipped ∼4.4 Å towards the solvent exterior to accommodate the bis-His heme coordination. When PhNHOH is used, a similar β-hemichrome Hb[α-FeIII(H2O)][β-FeIII(His)2-cySOx]{PhNHOH}was obtained. Our results reveal, for the first time, the X-ray structural determination of a β-hemichrome in a human Hb derivative. Our UV–vis and X-ray crystal structural result reveal that although Hb(PhNO) and Hb(RNO) complexes may form as primary products, attempted isolation of these products by crystallization may result in the structural determination of their secondary products which may contain β-hemichromes en route to further protein degradation.