Esselborn J, Muraki N, Klein K, Engelbrecht V, Metzler-Nolte N, Apfel U-P, Hofmann E, Kurisu G, Happe T
AG Photobiotechnologie , Fakultät für Biologie und Biotechnologie , Ruhr-Universität Bochum , Universitätsstraße 150 , 44801 Bochum , Germany . Email:
Laboratory of Protein Crystallography , Institute for Protein Research , Osaka University , Suita , Osaka 565-0871 , Japan . Email:
Chem Sci. 2016 Feb 1;7(2):959-968. doi: 10.1039/c5sc03397g. Epub 2015 Oct 26.
[FeFe]-hydrogenases are nature's fastest catalysts for the evolution or oxidation of hydrogen. Numerous synthetic model complexes for the [2Fe] subcluster (2Fe) of their active site are known, but so far none of these could compete with the enzymes. The complex Feμ-(SCH)X(CO) with X = NH was shown to integrate into the apo-form of [FeFe]-hydrogenases to yield a fully active enzyme. Here we report the first crystal structures of the apo-form of the bacterial [FeFe]-hydrogenase CpI from at 1.60 Å and the active semisynthetic enzyme, CpI, at 1.63 Å. The structures illustrate the significant changes in ligand coordination upon integration and activation of the [2Fe] complex. These changes are induced by a rigid 2Fe cavity as revealed by the structure of apoCpI, which is remarkably similar to CpI. Additionally we present the high resolution crystal structures of the semisynthetic bacterial [FeFe]-hydrogenases CpI (X = CH), CpI (X = O) and CpI (X = S) with changes in the headgroup of the dithiolate bridge in the 2Fe cofactor. The structures of these inactive enzymes demonstrate that the 2Fe-subcluster and its protein environment remain largely unchanged when compared to the active enzyme CpI. As the active site shows an open coordination site in all structures, the absence of catalytic activity is probably not caused by steric obstruction. This demonstrates that the chemical properties of the dithiolate bridge are essential for enzyme activity.
[铁铁]氢化酶是自然界中催化氢气生成或氧化的最快催化剂。已知有许多针对其活性位点[2Fe]亚簇（2Fe）的合成模型配合物，但到目前为止，这些配合物都无法与酶相媲美。已证明配合物Feμ-(SCH)X(CO)（X = NH）可整合到[铁铁]氢化酶的脱辅基形式中，从而产生完全活性的酶。在此，我们报告了来自嗜热栖热菌的细菌[铁铁]氢化酶CpI脱辅基形式在1.60 Å分辨率下的首个晶体结构，以及活性半合成酶CpI在1.63 Å分辨率下的晶体结构。这些结构说明了[2Fe]配合物整合和激活后配体配位的显著变化。这些变化是由脱辅基CpI的结构所揭示的刚性2Fe腔诱导的，其与CpI非常相似。此外，我们还展示了半合成细菌[铁铁]氢化酶CpI（X = CH）、CpI（X = O）和CpI（X = S）的高分辨率晶体结构，这些酶的2Fe辅因子中二硫醇盐桥的头基发生了变化。这些无活性酶的结构表明，与活性酶CpI相比，2Fe亚簇及其蛋白质环境基本保持不变。由于在所有结构中活性位点都显示出一个开放的配位位点，催化活性的缺失可能不是由空间位阻引起的。这表明二硫醇盐桥的化学性质对酶活性至关重要。
