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

拟议的项目代表了一个致力于结合结构，光谱，功能和理论的洞察力的[FeFe]氢化酶，在自然界中最活跃的氢转化酶的活性位点H-簇。H-簇是由典型的[4Fe-4S]亚簇耦合到一个独特的[2Fe]亚簇，含有一个独特的桥接氮杂丙烷二硫纶以及羰基和氰化物配体。多年来，H-簇已经进行了许多光谱和功能研究，但迄今为止，关于催化循环如何运作以及光谱鉴定的中间体如何在结构上合理化，还没有明确的共识。因此，我们将从硫酸盐还原菌脱硫脱硫弧菌（DdHydAB）中结晶出最具活性和双向[FeFe]氢化酶之一，并在广泛的催化状态下解决其结构。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相同的晶体。最后，分子理论计算的H-集群的合适的模型将进行，以合理化如何与还原，质子化或配体结合的结构差异导致光谱差异。最终，这些研究将提供一个更深刻的理解[FeFe]氢化酶催化循环，并揭示活性位点的灵活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由非贵金属组成的合成分子催化剂的开发产生广泛的影响。

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.