Ligand binding to heme proteins: a comparison of cytochrome c variants with globins.
Ligand binding to heme proteins: a comparison of cytochrome c variants with globins.
复制标题
配体与血红素蛋白的结合：细胞色素 c 变体与球蛋白的比较
DOI：
10.1021/jp306775n
复制
发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Nienhaus
中科院分区：
文献类型：
--
作者：
Nienhaus;Nienhaus
我们研究了Cytcha突变体中一氧化碳（CO）的结合，其80位的蛋氨酸被丙氨酸、天冬氨酸和精氨酸取代，因此第六配位可用于配体结合。我们采用傅里叶变换红外 (FTIR) 光解差异光谱来检查突变产生的小血红素袋中血红素结合和光解的 CO（以及一氧化氮，NO）之间的相互作用。通过使用 FTIR 温度导数光谱 (TDS) 和纳秒闪光光解，确定了 CO 再结合的焓垒分布。在闪光光解实验中，大多数配体在皮秒时间尺度上与血红素铁重新结合，因此在纳秒尺度上仅可见分布的高势垒尾部。然而，通过在 TDS 表征屏障之前进行连续波激发，每个 Cytcm 分子都会被光激发多次，并且可以实现完全光解离，这可能是由于血红素袋内 CO 的旋转使氧面向血红素铁而产生的。显然，重新绑定之前的重新定向对重新绑定障碍构成了额外且重要的贡献。我们的实验表明，Cytco 的紧凑、刚性结构没有为蛋白质基质中的光解配体提供替代的结合位点。这些细胞突变体和血红蛋白中配体结合的比较强调了内部配体对接位点和配体迁移路线对于将配体结合功能传递给血红素蛋白的重要性。
We have studied the binding of carbon monoxide (CO) in mutants of Cytchaving its methionine at position 80 replaced by alanine, aspartate, and arginine, so that the sixth coordination is available for ligand binding. We have employed Fourier transform infrared (FTIR) photolysis difference spectroscopy to examine interactions of the heme-bound and photolyzed CO (and also nitric oxide, NO) in the small heme pocket created by the mutations. By using FTIR temperature derivative spectroscopy (TDS) and nanosecond flash photolysis, the enthalpy barrier distributions for CO rebinding were determined. In flash photolysis experiments, the majority of ligands rebind to the heme iron on picosecond time scales so that only the high-barrier tail of the distributions is visible on the nanosecond scale. By continuous wave excitation prior to TDS characterization of the barriers, however, each Cytcmolecule is photoexcited multiple times and complete photodissociation can be achieved, which likely arises from a rotation of the CO within the heme pocket so that the oxygen faces the heme iron. Apparently, reorientation prior to rebinding constitutes an additional and significant contribution to the rebinding barrier. Our experiments reveal that the compact, rigid structure of Cytcoffers no alternative binding sites for photodissociated ligands in the protein matrix. A comparison of ligand binding in these Cytcmutants and hemoglobins underscores the importance of internal ligand docking sites and ligand migration routes for conveying a ligand binding function to heme proteins.