High field EPR and ENDOR of [Fe]-Hydrogenase

氢酶是在微生物中发现的一种酶，它催化简单的氧化还原反应：H2&gt；2H++2E。氢酶主要有两类：铁镍基团和纯铁基团。在过去的几年里，代表这两类的酶已经结晶，并确定了它们的X射线结构。这两类化合物的活性中心都含有一个独特的双金属(Fe-Ni或Fe-Fe)中心，并带有非蛋白质配体(CN、CO)。目前对这些酶的催化机理仍知之甚少。电子顺磁共振(EPR)和电子核双共振(EndOR)已被证明是研究氢酶顺磁状态的优秀技术。我们打算使用现有的最先进的EPR技术来研究[Fe]-氢酶，即使用高磁场和毫米波频率(100-200 GHz)。它的主要优点是：a)非常高的绝对灵敏度(~108自旋/高斯.秒)，能够研究小的蛋白质单晶；b)在Endor实验中，非常高的塞曼结果导致了单晶的取向选择。我们项目的主要目标是揭开导致活性中心中氢的异质裂解的一系列事件。这将通过使用光不稳定的CO抑制形式的[Fe]-氢酶来鉴定质子接受碱来实现。高场EPR和Endor研究将提供活性中心在各种中间态的电子结构和几何结构的完整图景。由实验获得的磁相互作用校准的密度泛函计算将进一步完善我们对催化机理的理解。

Hydrogenases are enzymes found in microorganisms which catalyze the simple redox reaction: H2>2H+ + 2e. Two main classes of hydrogenases have been identified: the Iron-Nickel group and the Iron-only group. In the last few years enzymes representative of both groups have been crystallized and their X-ray structures have been determined. The active centre of both classes contains a unique bi-metallic (either Fe-Ni or Fe-Fe) centre with non-protein ligands (CN, CO). The catalytic mechanism of these enzymes is still poorly understood at present. Electron Paramagnetic Resonance (EPR) as well a Electron Nuclear Double Resonance (ENDOR) have proven to be excellent techniques for studying the paramagnetic states of hydrogenases. We intend to study [Fe]-hydrogenase using the most advanced EPR techniques available, i.e. using high magnetic fields and mm-wave frequencies (100-200 GHz). The main advantages will be: a) the very high absolute sensitivity (~108 spins/Gauss.sec) enabling the study of small protein single crystals, and b) the very high Zeeman-resulution leading to single crystal like orientation selection in ENDOR experiments. The main goal of our project is to unravel the sequence of events leading to the heterolytic splitting of hydrogen in the active site. This will be accomplished by identifying the proton-accepting base using the photo labile CO inhibited form of [Fe]-hydrogenase. High field EPR and ENDOR studies will provide a complete picture of the electronic and geometrical structure of the active site in various intermediate states. DFT calculations, calibrated by the experimentally obtained magnetic interactions will further refine our understanding of the catalytic mechanism.