Correlation of three-dimensional and electronic structure of [FeFe] hydrogenases

该项目旨在结合结构、光谱、功能和理论对自然界中最活跃的氢转化酶[FeFe]氢化酶的活性位点h簇进行研究。h -簇是由一个典型的[4Fe-4S]亚簇偶联到一个独特的[2Fe]亚簇构成的，该亚簇含有一个独特的桥接氮丙烷二硫酯以及羰基和氰化物配体。多年来，h -簇已经进行了大量的光谱和功能研究，但迄今为止，关于催化循环如何运作以及如何通过光谱识别中间体可以在结构上合理化，还没有明确的共识。因此，我们将从硫酸盐还原菌Desulfovibrio脱硫菌（DdHydAB）中结晶出最活跃的双向[FeFe]氢化酶之一，并在广泛的催化状态下求解其结构。结构数据将得到电子顺磁共振和傅里叶变换红外光谱研究的支持。最后，将对合适的h簇模型进行分子理论计算，以解释与还原、质子化或配体结合相关的结构差异是如何导致光谱差异的。最终，这些研究将提供对[FeFe]氢化酶催化循环更深刻的理解，并揭示活性位点柔韧性如何影响催化性能。我们的研究结果将对其他铁硫酶以及非贵金属合成分子催化剂的开发具有广泛的指导意义。

The proposed project represents a dedicated effort toward combining structural, spectroscopic, functional and theoretical insight into the active site H-cluster of the [FeFe] hydrogenases, the most active hydrogen-converting enzymes in nature. The H-cluster is constructed from a canonical [4Fe-4S] subcluster coupled to a unique [2Fe] sub-cluster containing a unique bridging azapropane dithiolate as well as carbonyl and cyanide ligands. The H-cluster has been subjected to numerous spectroscopic and functional studies over the years, but as yet there is no clear consensus concerning how the catalytic cycle operates and how spectroscopically identified intermediates can be structurally rationalized. Hence, we will crystallize one of the most active and bidirectional [FeFe] hydrogenases from the sulfate-reducing bacterium Desulfovibrio desulfuricans (DdHydAB) and solve its structure in a wide range of catalytic states. The structural data will be supported by electron paramagnetic resonance, and Fourier-transform infrared spectroscopic studies on the same crystals. Finally, molecular theory calculations of suitable models of the H-cluster will be carried out in order to rationalize how structural differences related to reduction, protonation or ligand binding lead to spectroscopic differences. Ultimately, these studies will provide a more profound understanding of the [FeFe] hydrogenase catalytic cycle, and reveal how active site flexibility influences catalytic performance. Our results will have wide-reaching implications for other iron-sulfur enzymes, as well as for the development of synthetic molecular catalysts composed of non-precious metals.