Stepwise isotope editing of [FeFe]-hydrogenases exposes cofactor dynamics
Stepwise isotope editing of [FeFe]-hydrogenases exposes cofactor dynamics
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[FeFe] - 氢化酶的逐步同位素编辑揭示辅因子动态变化
DOI：
10.1073/pnas.1606178113
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发表时间：
2016-07-26
影响因子：
11.1
通讯作者：
Stripp, Sven Timo
中科院分区：
文献类型：
--
作者：
Senger, Moritz;Mebs, Stefan;Stripp, Sven Timo
[FeFe]-氢化酶的六铁辅助因子（h簇）是自然界中最有效的h -2形成催化剂。它包括一个双铁活性位点，具有活性氧化态（H-ox）的三个一氧化碳（CO）和两个氰化物（CN-）配体和一个抑制态（H-ox-CO）的CO配体。双原子配体是辅助因子结构变化的敏感报告基团。利用衰减全反射傅立叶变换红外光谱实时监测了其振动动力学。（CO）-C-13气体暴露、蓝光或红光照射以及三种不同的[FeFe]-氢化酶蛋白的控制水化组合产生8种H-ox和16种H-ox-CO，具有所有可能的同位素交换模式。广泛的密度泛函理论计算揭示了羰基配体的振动模式耦合，并将每个红外光谱独特地分配到特定的标记模式。对于H-ox-CO，实验和计算的红外频率之间的一致性提高了一个数量级，对于辅助因子的远端铁离子的顶端CN-，而不是顶端CO。对于H-ox，提出了两个具有部分旋转配体的等可能异构体。这些结构之间的相互转换意味着配体在h簇上的动态重定向。我们的位点选择性(CO)-C-13同位素编辑与计算物种分配相结合的实验方案为催化循环中功能中间体的表征开辟了新的视角。
The six-iron cofactor of [FeFe]-hydrogenases (H-cluster) is the most efficient H-2-forming catalyst in nature. It comprises a diiron active site with three carbon monoxide (CO) and two cyanide (CN-) ligands in the active oxidized state (H-ox) and one additional CO ligand in the inhibited state (H-ox-CO). The diatomic ligands are sensitive reporter groups for structural changes of the cofactor. Their vibrational dynamics were monitored by real-time attenuated total reflection Fourier-transform infrared spectroscopy. Combination of (CO)-C-13 gas exposure, blue or red light irradiation, and controlled hydration of three different [FeFe]-hydrogenase proteins produced 8 H-ox and 16 H-ox-CO species with all possible isotopic exchange patterns. Extensive density functional theory calculations revealed the vibrational mode couplings of the carbonyl ligands and uniquely assigned each infrared spectrum to a specific labeling pattern. For H-ox-CO, agreement between experimental and calculated infrared frequencies improved by up to one order of magnitude for an apical CN- at the distal iron ion of the cofactor as opposed to an apical CO. For H-ox, two equally probable isomers with partially rotated ligands were suggested. Interconversion between these structures implies dynamic ligand reorientation at the H-cluster. Our experimental protocol for site-selective (CO)-C-13 isotope editing combined with computational species assignment opens new perspectives for characterization of functional intermediates in the catalytic cycle.