EPR Spectroscopic Studies of the Fe-S Clusters in the O2-Tolerant [NiFe]-Hydrogenase Hyd-1 from Escherichia coli and Characterization of the Unique [4Fe-3S] Cluster by HYSCORE
EPR Spectroscopic Studies of the Fe-S Clusters in the O2-Tolerant [NiFe]-Hydrogenase Hyd-1 from Escherichia coli and Characterization of the Unique [4Fe-3S] Cluster by HYSCORE
复制标题
DOI：
10.1021/ja307117y
复制
发表时间：
2012-09-19
影响因子：
15
通讯作者：
Armstrong, Fraser A.
中科院分区：
文献类型：
--
作者：
Roessler, Maxie M.;Evans, Rhiannon M.;Armstrong, Fraser A.
靠近活性位点的不寻常的[4Fe-3S]簇在允许一类[NiFe]-氢化酶在O-2存在下发挥关键作用，通过其独特的能力进行两次快速，连续的单电子转移。这种特性有助于中和活性氧。机制细节和中间和远端集群的作用仍未解决。为了探测铁硫接力，对大肠杆菌（Hyd-1）的o -2耐受性氢化酶（Hyd-1）和三个在近端和/或中部集群点突变的变体进行了连续波和脉冲电子顺磁共振（EPR）研究。电位测定法测定近端（[4Fe-3S](5+/4+/3+)）和内侧（[3Fe-4S](+/0)）簇的还原电位。内侧[3Fe-4S]（+/0）还原电位异常高，暗示在o -2耐受性中起机制作用。大量实验表明，远端簇在所有三种变体中都具有基态S > 1/2，并且表明天然的Hyd-1也是如此。与Hyd-1晶体结构同时，“超氧化”P242C变体的EPR数据显示，中间团簇是“磁沉默”的，揭示了近端[4Fe-3S]（5+）团簇的两个构象，x波段HYSCORE光谱显示两个N-14超精细耦合属于一个构象。最大的A(N-14) = [11.5,11.5,16.0] +/- 1.5 MHz，表征了一个Fe （Fe-4）与半胱氨酸-20的主酰胺- n之间不寻常的键。第二个，A(N-14) = [2.8,4.6,3.5] +/- 0.3 MHz，分配给N-C19。N-14的超细耦合是铁-4是超氧化态的价位化Fe3+的确凿证据，它的形成允许在O-2攻击时将额外的电子迅速转移回活性位点。
The unusual [4Fe-3S] cluster proximal to the active site plays a crucial role in allowing a class of [NiFe]-hydrogenases to function in the presence of O-2 through its unique ability to undergo two rapid, consecutive one-electron transfers. This property helps to neutralize reactive oxygen species. Mechanistic details and the role of the medial and distal clusters remain unresolved. To probe the Fe S relay, continuous wave and pulse electron paramagnetic resonance (EPR) studies were conducted on the O-2-tolerant hydrogenase from Escherichia colt (Hyd-1) and three variants with point mutations at the proximal and/or medial clusters. Reduction potentials of the proximal ([4Fe-3S](5+/4+/3+)) and medial ([3Fe-4S](+/0)) clusters were determined by potentiometry. The medial [3Fe-4S](+/0) reduction potential is exceptionally high, implicating a mechanistic role in O-2-tolerance. Numerous experiments establish that the distal cluster has a ground state S > 1/2 in all three variants and indicate that this is also the case for native Hyd-1. Concurrent with the Hyd-1 crystal structure, EPR data for the 'superoxidized' P242C variant, in which the medial cluster is 'magnetically silenced', reveal two conformations of the proximal [4Fe-3S](5+) cluster, and X-band HYSCORE spectroscopy shows two N-14 hyperfine couplings attributed to one conformer. The largest, A(N-14) = [11.5,11.5,16.0] +/- 1.5 MHz, characterizes the unusual bond between one Fe (Fe-4) and the backbone amide-N of cysteine-20. The second, A(N-14) = [2.8,4.6,3.5] +/- 0.3 MHz, is assigned to N-C19. The N-14 hyperfine couplings are conclusive evidence that Fe-4 is a valence-localized Fe3+ in the superoxidized state, whose formation permits an additional electron to be transferred rapidly back to the active site during O-2 attack.