Loss of Specific Active-Site Iron Atoms in Oxygen-Exposed [FeFe]-Hydrogenase Determined by Detailed X-ray Structure Analyses
Loss of Specific Active-Site Iron Atoms in Oxygen-Exposed [FeFe]-Hydrogenase Determined by Detailed X-ray Structure Analyses
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DOI：
10.1021/jacs.9b07808
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发表时间：
2019-11-06
影响因子：
15
通讯作者：
Happe, Thomas
中科院分区：
文献类型：
--
作者：
Esselborn, Julian;Kertess, Leonie;Happe, Thomas
[FeFe]-氢化酶在低过电位下以无与伦比的速度催化氢的吸收和释放。然而，氧诱导的活性位点内的独特的[6 Fe-6S]辅因子的降解，称为H-簇。我们使用X射线结构分析来确定不可逆氧驱动失活的可能模式。为此，我们将来自巴氏梭菌的[FeFe]-氢化酶CpI晶体暴露于氧气中，并使用多个数据集定量研究了在几个时间点对H-簇结构的影响，同时将其与酶活性的降低相关联。我们的研究结果揭示了H-团簇的二铁（2Fe（H））和[4Fe-4S]亚团簇（4Fe（H））中特定Fe原子的丢失。在2Fe（H）中，更远离4Fe（H）的Fe原子比更接近的Fe原子受到的影响更大。4Fe（H）在活性CpIADT的部分群体中相互转化为[2Fe-2S]簇，但在最初缺乏2Fe（H）的非活性apoCpI的晶体中不相互转化。因此，我们提出了两个平行的过程：远端Fe原子的解离和4Fe（H）相互转化。这两种途径似乎发挥重要作用，在氧化损伤的[FeFe]-氢化酶的电子供体剥夺条件下，我们的实验装置探测。
The [FeFe]-hydrogenases catalyze the uptake and evolution of hydrogen with unmatched speed at low over-potential. However, oxygen induces the degradation of the unique [6Fe-6S] cofactor within the active site, termed the H-cluster. We used X-ray structural analyses to determine possible modes of irreversible oxygen-driven inactivation. To this end, we exposed crystals of the [FeFe]-hydrogenase CpI from Clostridium pasteurianum to oxygen and quantitatively investigated the effects on the H-cluster structure over several time points using multiple data sets, while correlating it to decreases in enzyme activity. Our results reveal the loss of specific Fe atoms from both the diiron (2Fe(H)) and the [4Fe-4S] subcluster (4Fe(H)) of the H-cluster. Within the 2Fe(H), the Fe atom more distal to the 4Fe(H) is strikingly more affected than the more proximal Fe atom. The 4Fe(H) interconverts to a [2Fe-2S] cluster in parts of the population of active CpIADT, but not in crystals of the inactive apoCpI initially lacking the 2Fe(H). We thus propose two parallel processes: dissociation of the distal Fe atom and 4Fe(H) interconversion. Both pathways appear to play major roles in the oxidative damage of [FeFe]-hydrogenases under electron-donor deprived conditions probed by our experimental setup.