Time-resolved crystallography is a powerful technique to elucidate molecular mechanisms at both spatial (angstroms) and temporal (picoseconds to seconds) resolutions. We recently discovered an unusually slow reaction at room temperature that occurs on the order of days: the in crystalline reverse oxidative decay of the chemically labile (6S)-5,6,7,8-tetrahydrofolate in complex with its producing enzyme Escherichia coli dihydrofolate reductase. Here, we report the critical analysis of a representative dataset at an intermediate reaction time point. A quinonoid-like intermediate state lying between tetrahydrofolate and dihydrofolate features a near coplanar geometry of the bicyclic pterin moiety, and a tetrahedral sp 3 C6 geometry is proposed based on the apparent mFo-DFc omit electron densities of the ligand. The presence of this intermediate is strongly supported by Bayesian difference refinement. Isomorphous Fo-Fo difference map and multi-state refinement analyses suggest the presence of end-state ligand populations as well, although the putative intermediate state is likely the most populated. A similar quinonoid intermediate previously proposed to transiently exist during the oxidation of tetrahydrofolate was confirmed by polarography and UV-vis spectroscopy to be relatively stable in the oxidation of its close analog tetrahydropterin. We postulate that the constraints on the ligand imposed by the interactions with the protein environment might be the origin of the slow reaction observed by time-resolved crystallography.

中文翻译：

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时间分辨的X射线晶体学捕获缓慢反应的四氢叶酸中间体。

时间分辨晶体学是一种阐明空间（埃）和时间（皮秒至秒）分辨率的分子机制的强大技术。我们最近发现，在室温下发生的反应异常缓慢，发生的时间大约为几天：化学不稳定的（6S）-5,6,7,8-四氢叶酸与其产生的酶二氢叶酸大肠埃希菌在晶体中发生逆向氧化降解。还原酶。在这里，我们报告了在中间反应时间点对代表性数据集的关键分析。位于四氢叶酸和二氢叶酸之间的类醌型中间态具有双环蝶呤部分的近共面几何形状，并且基于表观的mFo-DFc省略了配体的电子密度，提出了四面体sp 3 C6几何形状。贝叶斯差异细化强烈支持该中间体的存在。同构的Fo-Fo差异图和多态优化分析表明，也存在末端态配体，尽管假定的中间态可能是人口最多的。通过极谱法和UV-可见光谱法证实了先前提出的在四氢叶酸的氧化过程中瞬时存在的类似醌类中间体在其紧密类似的四氢蝶呤的氧化中相对稳定。我们假设与蛋白质环境相互作用对配体施加的限制可能是时间分辨晶体学观察到的缓慢反应的起源。同构的Fo-Fo差异图和多态优化分析表明，也存在末端态配体，尽管假定的中间态可能是人口最多的。通过极谱法和UV-可见光谱法证实了先前提出的在四氢叶酸的氧化过程中瞬时存在的类似醌类中间体在其紧密类似的四氢蝶呤的氧化中相对稳定。我们假设与蛋白质环境相互作用对配体施加的限制可能是时间分辨晶体学观察到的缓慢反应的起源。同构的Fo-Fo差异图和多态优化分析表明，也存在末端态配体，尽管假定的中间态可能是人口最多的。通过极谱法和UV-可见光谱法证实了先前提出的在四氢叶酸的氧化过程中瞬时存在的类似醌类中间体在其紧密类似的四氢蝶呤的氧化中相对稳定。我们假设与蛋白质环境相互作用对配体施加的限制可能是时间分辨晶体学观察到的缓慢反应的起源。极谱法和紫外可见光谱法证实了先前提出的在四氢叶酸氧化过程中短暂存在的类似醌类中间体，在其紧密类似的四氢蝶呤的氧化过程中相对稳定。我们假设与蛋白质环境相互作用对配体施加的限制可能是时间分辨晶体学观察到的缓慢反应的起源。通过极谱法和UV-可见光谱法证实了先前提出的在四氢叶酸的氧化过程中瞬时存在的类似醌类中间体在其紧密类似的四氢蝶呤的氧化中相对稳定。我们假设与蛋白质环境相互作用对配体施加的限制可能是时间分辨晶体学观察到的缓慢反应的起源。