High field EPR and ENDOR of [Fe]-Hydrogenase

[Fe]-氢化酶的高场 EPR 和 ENDOR

• 批准号: 5382067
• 负责人: Dr. Edward Reijerse
• 金额: --
• 依托单位: Max-Planck-Institut für Chemische Energiekonversion (CEC)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2002
• 资助国家: 德国
• 起止时间: 2001-12-31 至 2005-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5382067?language=en
• 关键词: field; EPR; ENDOR; Fe; Hydrogenase

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.

氢化酶是在微生物中发现的催化简单氧化还原反应的酶：H2>2H+ + 2e。已经确定了两类主要的氢化酶：铁-镍基团和纯铁基团。在过去的几年里，这两种酶的代表已经结晶，它们的x射线结构已经确定。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）中心和非蛋白质配体（CN， CO）。目前对这些酶的催化机制还知之甚少。电子顺磁共振（EPR）和电子核双共振（ENDOR）已被证明是研究氢化酶顺磁态的优良技术。我们打算使用最先进的EPR技术来研究[Fe]-氢化酶，即使用高磁场和毫米波频率（100-200 GHz）。主要优点是：a)非常高的绝对灵敏度（~108自旋/高斯）。2)使小蛋白质单晶的研究成为可能；2)在ENDOR实验中，非常高的泽曼结果导致了类似单晶的取向选择。我们项目的主要目标是揭示导致活性位点氢异解分裂的事件序列。这将通过使用光不稳定的CO抑制形式的[Fe]-氢化酶来识别质子接受碱来完成。高场EPR和ENDOR研究将提供活性位点在各种中间状态下的电子和几何结构的完整图像。由实验得到的磁相互作用校准的DFT计算将进一步完善我们对催化机理的理解。