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

拟议的项目代表着一项致力于将结构、光谱、功能和理论结合在一起的努力，以深入了解[FeFe]氢酶的活性部位H-簇，[FeFe]氢酶是自然界中最活跃的氢转换酶。H-簇是由一个典型的[4Fe-4S]亚簇连接到一个独特的[2Fe]亚簇构成的，该[2Fe]亚簇包含一个独特的桥联氮丙烷二硫酸盐以及羰基和氰基配体。多年来，H-簇已经进行了大量的光谱和功能研究，但对于催化循环是如何运作的，以及光谱确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细菌脱硫弧菌(DdHydAB)中结晶出最具活性和双向的[FeFe]氢酶之一，并解决其在广泛催化状态下的结构。结构数据将由电子顺磁共振和傅立叶变换红外光谱对同一晶体的研究支持。最后，将对合适的氢团簇模型进行分子理论计算，以合理地解释与还原、质子化或配体结合相关的结构差异如何导致光谱差异。最终，这些研究将提供对[FeFe]氢酶催化循环的更深刻的理解，并揭示活性中心灵活性如何影响催化性能。我们的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.