Kim Jooyoung, Park Jaeheung, Lee Taegon, Lim Manho
Department of Chemistry and Chemistry Institute of Functional Materials, Pusan National University, Busan, 609-735 Korea.
J Phys Chem B. 2009 Jan 8;113(1):260-6. doi: 10.1021/jp804656t.
Rebinding dynamics of CO to carboxymethyl cytochrome c (Ccytc), a chemically modified cytochrome c to bind ligands in its ferrous form, in D(2)O solution at 283 K after photodeligation, was investigated using femtosecond vibrational spectroscopy. The stretching mode of CO bound to the protein shows four stretching bands near 1962 cm(-1). Time-resolved spectra of the bound CO revealed a slight band-position-dependent rebinding kinetics, suggesting that the geminate rebinding of CO depends on the conformation of the protein. The overall rebinding kinetics of CO to Ccytc was more than 1000 times faster than that to myoglobin (Mb), a ligand-binding protein, and is also faster than a model heme, microperoxidase-8 in viscous solvent. The efficient rebinding of CO to Ccytc was attributed to the longer retention of the dissociated CO near the active binding site by the organized protein matrix of Ccytc. The spectra of the dissociated CO reveal a fast-growing band in the picosecond time scale that is assigned to CO in D(2)O solvent. The ultrafast CO escape to bulk solution is consistent with its 3D structure showing a sizable opening in the active site. It appears that most of the dissociated CO rebinds within 1 ns, except for those that escape to the bulk solution through the opening. The CO rebinding in Ccytc indicates that the primary heme pocket in Mb, located near the active site and holding the dissociated ligand for longer than tens of nanoseconds, has a specific structure to suppress CO rebinding.
利用飞秒振动光谱研究了在283K的重水（D₂O）溶液中，光解络合后一氧化碳（CO）与羧甲基细胞色素c（Ccytc，一种经化学修饰的细胞色素c，能以亚铁形式结合配体）的再结合动力学。与蛋白质结合的CO的伸缩模式在1962 cm⁻¹附近显示出四个伸缩带。结合态CO的时间分辨光谱揭示了一种轻微的带位置依赖性再结合动力学，这表明CO的双分子再结合取决于蛋白质的构象。CO与Ccytc的整体再结合动力学比其与配体结合蛋白肌红蛋白（Mb）的再结合动力学快1000倍以上，也比粘性溶剂中的模型血红素微过氧化物酶-8快。CO与Ccytc的有效再结合归因于Ccytc有组织的蛋白质基质使解离的CO在活性结合位点附近保留更长时间。解离态CO的光谱显示在皮秒时间尺度上有一个快速增长的带，该带归属于重水（D₂O）溶剂中的CO。CO向本体溶液的超快逸出与其三维结构一致，该结构显示活性位点有一个相当大的开口。似乎除了那些通过开口逸出到本体溶液中的CO外，大多数解离的CO在1纳秒内重新结合。Ccytc中的CO再结合表明，位于活性位点附近且能将解离的配体保持数十纳秒以上的Mb中的主要血红素口袋具有抑制CO再结合的特定结构。
