Abstract

[FeFe]-hydrogenase enzymes employ a unique organometallic cofactor for efficient and reversible hydrogen conversion. This so-called H-cluster consists of a [4Fe–4S] cubane cysteine linked to a diiron complex coordinated by carbon monoxide and cyanide ligands and an azadithiolate ligand (adt = NH(CH₂S)₂)·[FeFe]-hydrogenase apo-protein binding only the [4Fe–4S] sub-complex can be fully activated in vitro by the addition of a synthetic diiron site precursor complex ([2Fe]^adt). Elucidation of the mechanism of cofactor assembly will aid in the design of improved hydrogen processing synthetic catalysts. We combined electron paramagnetic resonance, Fourier-transform infrared, and X-ray absorption spectroscopy to characterize intermediates of H-cluster assembly as initiated by mixing of the apo-protein (HydA1) from the green alga Chlamydomonas reinhardtii with [2Fe]^adt. The three methods consistently show rapid formation of a complete H-cluster in the oxidized, CO-inhibited state (Hox-CO) already within seconds after the mixing. Moreover, FTIR spectroscopy support a model in which Hox-CO formation is preceded by a short-lived Hred′-CO-like intermediate. Accumulation of Hox-CO was followed by CO release resulting in the slower conversion to the catalytically active state (Hox) as well as formation of reduced states of the H-cluster.

Graphic abstract

中文翻译：

---

[FeFe]-氢化酶成熟：通过电子顺磁共振、红外和 X 射线吸收光谱追踪 H 簇组装中间体。

摘要

[FeFe]-氢化酶采用独特的有机金属辅因子来实现高效且可逆的氢转化。这个所谓的 H 簇由一个 [4Fe–4S] 立方烷半胱氨酸组成，该半胱氨酸与二铁配合物相连，并由一氧化碳和氰化物配体以及氮杂二硫醇配体 (adt = NH(CH ₂ S) ₂ )·[FeFe]-氢化酶通过添加合成的二铁位点前体复合物 ([2Fe] ^adt )，只有 [4Fe–4S] 亚复合物才能在体外完全激活脱辅基蛋白结合。阐明辅因子组装的机制将有助于设计改进的氢加工合成催化剂。我们结合电子顺磁共振、傅里叶变换红外和 X 射线吸收光谱来表征 H 簇组装的中间体，该中间体是通过将来自绿藻莱茵衣藻的脱辅基蛋白 (HydA1) 与 [ 2Fe ] ^adt混合而引发的。这三种方法一致表明，在混合后的几秒钟内，就已经在氧化、CO 抑制状态 (Hox-CO) 下快速​​形成了完整的 H 簇。此外，FTIR 光谱支持一种模型，其中 Hox-CO 形成之前是一种短寿命的 Hred'-CO 类中间体。Hox-CO 的积累随后是 CO 的释放，导致向催化活性态 (Hox) 的转化速度变慢，并形成 H 簇的还原态。

图文摘要