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首页> 外文期刊>Biochemistry >~19F NMR of Trifluoroacetyl-Labeled Cysteine Mutants of Myoglobin: Structural Probes of Nitric Oxide Bound to the H93G Cavity Mutant
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~19F NMR of Trifluoroacetyl-Labeled Cysteine Mutants of Myoglobin: Structural Probes of Nitric Oxide Bound to the H93G Cavity Mutant

机译：三氟乙酰基标记的肌红蛋白半胱氨酸突变体的〜19F NMR：一氧化氮与H93G空腔突变体结合的结构探针

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摘要

Nitric oxide (NO) binds to the myoglobin (Mb) cavity mutant, H93G, forming either a 5- or 6-coordinate Fe-NO heme complex. The H93G mutation replaces the proximal histidine of Mb with glycine, allowing exogenous ligands to occupy the proximal binding site. In the absence of the covalently attached proximal ligand, NO could bind to H93G from the proximal side of the heme rather than the typical diatomic binding pocket on the distal side when the 5-coordinate complex forms. The question of whether NO binds on the distal or proximal side was addressed by 19F NMR. Site-directed mutagenesis was used to introduce unique cysteine residues at the protein surface on either the distal (S58C) or proximal (L149C) side, approximately equidistant from and perpendicular to the heme plane of both wild-type and H93G Mb. The cysteine thiols were alkylated with 3-bromo-l,1,1-trifluoroacetone to attach a trifluoroacetyl group at the mutation site. 19F NMR spectra of 5-coordinate, NO bound S58C/H93G and L149C/H93G double mutants depict peaks with line widths of 100 and 23 Hz, respectively. As fluorine peaks broaden with increasing proximity to paramagnetic centers, such as 5-coordinate Fe- NO, the 19F NMR data are consistent with NO binding in the distal heme pocket of H93G, even in the absence of a sixth axial ligand. Additionally, 19F NMR spectra are reported for deoxy, oxy, CO, met CN, and met H2O forms of the labeled cysteine mutants. These results demonstrate that the fluorine probes are sensitive to subtle conformational changes in the protein structure due to ligation and oxidation state changes of the heme iron in Mb.
机译：一氧化氮（NO）与肌红蛋白（Mb）腔突变体H93G结合，形成5或6坐标的Fe-NO血红素复合物。 H93G突变用甘氨酸替代了Mb的近端组氨酸，使外源配体占据了近端结合位点。在没有共价连接的近端配体的情况下，当形成5坐标复合物时，NO可以从血红素的近端结合到H93G，而不是远端的典型双原子结合口袋。 19 F NMR解决了NO是否结合在远端或近端的问题。定点诱变用于在远端（S58C）或近端（L149C）侧的蛋白质表面上引入独特的半胱氨酸残基，与野生型和H93G Mb的血红素平面大约等距并垂直。用3-溴-1,1,1-三氟丙酮将半胱氨酸硫醇烷基化以在突变位点连接三氟乙酰基。 5坐标，NO结合的S58C / H93G和L149C / H93G双突变体的19F NMR光谱分别描绘了线宽为100和23 Hz的峰。由于氟峰随着与顺磁性中心（例如5坐标的Fe-NO）的接近程度增加而加宽，因此即使没有第六个轴向配体，19F NMR数据也与H93G远端血红素腔中的NO结合一致。另外，报道了标记的半胱氨酸突变体的脱氧，氧，CO，CN和H2O形式的19F NMR光谱。这些结果表明，由于Mb中血红素铁的连接和氧化态变化，氟探针对蛋白质结构的细微构象变化敏感。

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