Schmidt Marius, Nienhaus Karin, Pahl Reinhard, Krasselt Angela, Anderson Spencer, Parak Fritz, Nienhaus G Ulrich, Srajer Vukica
Physikdepartment E17, Technische Universität München, James Franck Strasse, 85747 Garching, Germany.
Proc Natl Acad Sci U S A. 2005 Aug 16;102(33):11704-9. doi: 10.1073/pnas.0504932102. Epub 2005 Aug 5.
By using time-resolved x-ray crystallography at room temperature, structural relaxations and ligand migration were examined in myoglobin (Mb) mutant L29W from nanoseconds to seconds after photodissociation of carbon monoxide (CO) from the heme iron by nanosecond laser pulses. The data were analyzed in terms of transient kinetics by fitting trial functions to integrated difference electron density values obtained from select structural moieties, thus allowing a quantitative description of the processes involved. The observed relaxations are linked to other investigations on protein dynamics. At the earliest times, the heme has already completely relaxed into its domed deoxy structure, and there is no photo-dissociated CO visible at the primary docking site. Initial relaxations of larger globin moieties are completed within several hundred nanoseconds. They influence the concomitant migration of photo-dissociated CO to the Xe1 site, where it appears at approximately 300 ns and leaves again at approximately 1.5 ms. The extremely long residence time in Xe1 as compared with wild-type MbCO implies that, in the latter protein, the CO exits the protein from Xe1 predominantly via the distal pocket. A well-defined deligated state is populated between approximately 2 micros and approximately 1 ms; its structure is very similar to the equilibrium deoxy structure. Between 1.5 and 20 ms, no CO is visible in the protein interior; it is either distributed among many sites within the protein or has escaped to the solvent. Finally, recombination at the heme iron occurs after >20 ms.
通过在室温下使用时间分辨X射线晶体学，在纳秒激光脉冲使一氧化碳（CO）从血红素铁光解离后的纳秒到秒的时间范围内，研究了肌红蛋白（Mb）突变体L29W中的结构弛豫和配体迁移。通过将试验函数拟合到从选定结构部分获得的积分差分电子密度值，根据瞬态动力学对数据进行分析，从而对所涉及的过程进行定量描述。观察到的弛豫与蛋白质动力学的其他研究相关。在最早的时间，血红素已经完全弛豫到其圆顶状脱氧结构，并且在初级对接位点没有可见的光解离CO。较大球蛋白部分的初始弛豫在几百纳秒内完成。它们影响光解离CO向Xe1位点的伴随迁移，CO在大约300 ns出现在该位点，并在大约1.5 ms再次离开。与野生型MbCO相比，在Xe1中极长的停留时间意味着，在后者蛋白质中，CO主要通过远端口袋从Xe1离开蛋白质。在大约2微秒到大约1毫秒之间形成了一个明确的去配位状态；其结构与平衡脱氧结构非常相似。在1.5到20毫秒之间，在蛋白质内部没有可见的CO；它要么分布在蛋白质内的许多位点，要么已经逃逸到溶剂中。最后，在>20毫秒后血红素铁发生重组。
