Department of Chemistry, Oxford University, South Parks Road, OX1 3QR Oxford, United Kingdom.
J Am Chem Soc. 2012 Sep 19;134(37):15581-94. doi: 10.1021/ja307117y. Epub 2012 Sep 4.
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 coli (Hyd-1) and three variants with point mutations at the proximal and/or medial clusters. Reduction potentials of the proximal (4Fe-3S) and medial (3Fe-4S) clusters were determined by potentiometry. The medial 3Fe-4S 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 cluster, and X-band HYSCORE spectroscopy shows two (14)N hyperfine couplings attributed to one conformer. The largest, A((14)N) = [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((14)N) = [2.8,4.6,3.5] ± 0.3 MHz, is assigned to N(C19). The (14)N hyperfine couplings are conclusive evidence that Fe(4) is a valence-localized Fe(3+) in the superoxidized state, whose formation permits an additional electron to be transferred rapidly back to the active site during O(2) attack.
活性位点附近不寻常的 [4Fe-3S] 簇在允许一类 [NiFe]-氢化酶在 O2 存在下发挥作用方面发挥着至关重要的作用，这要归功于其独特的能力，可以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物质。但其中的机制细节以及中、远端簇的作用仍未得到解决。为了探究铁硫中继，对来自大肠杆菌（Hyd-1）的耐氧氢化酶及其三个在近端和/或中簇处具有点突变的变体进行了连续波和脉冲电子顺磁共振（EPR）研究。通过电势测定法确定了近端 4Fe-3S和中 3Fe-4S簇的还原电位。中 3Fe-4S的还原电位异常高，暗示其在耐氧性方面具有机械作用。大量实验表明，在所有三种变体中，远端簇的基态 S > 1/2，并表明天然 Hyd-1 也是如此。与 Hyd-1 晶体结构同时，对“超氧化”P242C 变体的 EPR 数据进行研究，其中中簇“磁沉默”，揭示了近端 4Fe-3S簇的两种构象，并且 X 波段 HYSCORE 光谱显示了两个（14）N 超精细耦合归因于一个构象。最大的 A((14)N) = [11.5,11.5,16.0] ± 1.5 MHz，表征了一个 Fe（Fe(4)）与半胱氨酸-20 的酰胺-N 之间的不寻常键。第二个 A((14)N) = [2.8,4.6,3.5] ± 0.3 MHz，分配给 N(C19)。（14）N 超精细耦合是 Fe(4)在超氧化态下为价局部化 Fe(3+)的确凿证据，其形成允许在 O2 攻击期间将额外的电子快速转移回活性位点。
