Multifrequency (9, 34 and 94 GHz) EPR investigations of paramagnetic intermediates in heterodisulfide reductase and related enzymes

异二硫还原酶和相关酶中顺磁性中间体的多频（9、34 和 94 GHz）EPR 研究

• 批准号: 129926648
• 负责人: Professorin Dr. Marina Bennati
• 金额: --
• 依托单位: Max-Planck-Institut für Multidisziplinäre Naturwissenschaften
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2009
• 资助国家: 德国
• 起止时间: 2008-12-31 至 2013-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/129926648?language=en
• 关键词: Multifrequency; 34; 94; GHz; EPR

In this project we will employ state-of-the-art methods of electron paramagnetic resonance spectroscopy (EPR) at 9-, 34-, and 94 GHz frequencies to investigate the reaction mechanism of heterodisulfide reductase, a key enzyme in the energy metabolism of methane-producing archaea. The enzyme catalyses the reversible reduction of the heterodisulfide (CoM-S-S-CoB) to the thiol coenzymes, coenzyme M (CoM-SH) and coenzyme B (CoB-SH) in the final step of methanogenesis and employs an unusual 4Fe4S cluster to carry out substrate chemistry. Multifrequency ENDOR spectroscopy combined with selective isotopic labeling will permit determination of a detailed structure of the essential 4Fe4S-cluster and its interaction with the substrate. Freeze-quench EPR experiments on the millisecond time scale will be employed to identify further intermediates. In a major collaborative effort with other members of this consortium, including external collaborations, on Mössbauer spectroscopy, X-ray crystallography and quantum chemical calculations, the work will potentially emerge into a consistent mechanistic picture for this enzyme at the molecular level. The work will provide a basis to further investigate the hypothetical role of this enzyme in the energy conservation mechanism of other non-methanogenic organisms.

在这个项目中，我们将采用最先进的方法，电子顺磁共振光谱（EPR）在9-，34-和94 GHz的频率，调查的反应机制的杂二硫还原酶，在能量代谢的甲烷产生古菌的关键酶。该酶在产甲烷的最后步骤中催化杂二硫化物（CoM-S-S-Co B）可逆还原为硫醇辅酶、辅酶M（CoM-SH）和辅酶B（CoB-SH），并采用不寻常的4Fe 4S簇进行底物化学。多频ENDOR光谱结合选择性同位素标记将允许确定的基本4Fe 4S-簇的详细结构及其与基板的相互作用。冷冻淬火EPR实验的毫秒时间尺度将被用来确定进一步的中间体。在与该联盟其他成员的重大合作努力中，包括外部合作，在穆斯堡尔光谱学，X射线晶体学和量子化学计算方面，这项工作将有可能在分子水平上为这种酶提供一致的机制图。该研究为进一步探讨该酶在其他非产甲烷生物能量守恒机制中的假设作用提供了基础。