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

在这个项目中，我们将使用9 GHz、34 GHz和94 GHz频率下最先进的电子顺磁共振波谱(EPR)方法来研究异二硫化物还原酶的反应机理，异二硫化物还原酶是产甲烷古细菌能量代谢的关键酶。该酶催化杂二硫化物(COM-S-S-COB)在产甲烷的最后一步可逆还原为硫醇辅酶M(COM-SH)和辅酶B(COB-SH)，并利用一个不寻常的4Fe4S簇进行底物化学。多频Endor光谱与选择性同位素标记相结合，将能够确定必要的4Fe4S-簇的详细结构及其与底物的相互作用。将使用毫秒时间尺度上的冷冻-淬火EPR实验来识别更多的中间体。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射线结晶学和量子化学计算方面，这项工作可能会在分子水平上形成这种酶的一致机制图景。这项工作将为进一步研究该酶在其他非产甲烷生物的能量节约机制中的假设作用提供基础。

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.