Mechanistic Investigations of [FeFe] Hydrogenase H-Cluster Assembly

描述（由申请人提供）：大气中二氧化碳水平的迅速上升对人类健康构成了重大的长期威胁。(1)目前CO2水平的上升大部分可归因于不可再生燃料的使用，因此，人们对研究能量转化反应如H+还原和H2氧化产生了极大的兴趣。(2)一些最好的H2生产和 - 消耗催化剂是[FeFe]氢化酶，其能够在反应的热力学势附近以高转换频率操作。(3-6)因为理解其高效率的化学基础可能有助于设计用于H2生产和消耗的新的合成催化剂，（7）已经对酶的活性位点（“H-簇”）的结构给予了很多关注。（8，9）H-簇具有生物学上前所未有的结构，具有由三个CO和两个CN-配体连接的2Fe核心，其中每一个在其游离状态下通常是有毒的。虽然已知CO和CN-配体衍生自酪氨酸，（10-13）它们形成和组装成H-簇的机理细节很少。此外，Fe中心是由一个五原子的二硫桥桥接;中心原子的身份-目前被认为是N-一直是争论的主题，是很重要的，因为它被认为是作为一个悬垂基地，可以动力学促进H+迁移。（9，14-20）由于H-簇的不寻常结构及其在影响特征H2化学的速率和氧化还原电位方面的核心重要性，其生物合成的机制受到高度关注。在这项拟议的工作中，我的目标是阐明几个方面的机制，其中一套成熟酶HydE，HydF，和HydG促进H-簇的形成。本文主要讨论三个问题：在团簇成熟的早期阶段，含铁中间体的几何结构和电子结构是什么？形成二硫键的分子前体是什么？ 电子顺磁共振波谱为了解决这些问题，我将进行先进的EPR实验，特别是标记的同位素的中间体在H-簇合成以及成熟的H-簇。这些同位素标记将通过使用标记的底物（最常见的是酪氨酸）引入，从而允许从分子前体通过中间体并最终进入成熟的H簇中追踪特定的同位素标记。对于每个中间体，取向选择性EPR实验，如HYSCORE和ENDOR将能够确定标记的核相对于电子自旋的距离和取向（建模为点偶极），从而提供详细的几何和电子结构信息。这些EPR实验将与补充的停流FTIR和穆斯堡尔谱一起进行。总体而言，这项工作将解决酪氨酸降解成CO和CN-的HydG，在这个过程中，含铁的中间体的结构，最终产品的身份（S）的HydG反应，和分子前体的二硫代硫醇桥的机械细节。

DESCRIPTION (provided by applicant): The rapid rise in atmospheric CO2 levels presents a significant long-term threat to human health.(1) Much of the current rise in CO2 levels may be ascribed to the use of non-renewable fuels and, as a result, there has been great interest in studying energy conversion reactions such as H+ reduction and H2 oxidation.(2) Some of the best H2-producing and -consuming catalysts are [FeFe] hydrogenase enzymes which are able to operate near the thermodynamic potential of the reaction at high turnover frequencies.(3-6) Because understanding the chemical basis for their high efficiency may aid in the design of new synthetic catalysts for H2 production and consumption,(7) much attention has been given to the structure of the active site of the enzyme (the "H- cluster").(8, 9) The H-cluster has a biologically unprecedented structure with a 2Fe core ligated by three CO and two CN- ligands, each of which are typically toxic in their free states. Although it is known that the CO and CN- ligands derive from tyrosine,(10-13) the mechanistic details of their formation and assembly into the H- cluster are scant. In addition, the Fe centers are bridged by a five-atom dithiolate bridge; the identity of the central atom-currently thought to be N-has been the subject of much debate, and is important because it is thought to serve as a pendant base that can kinetically facilitate H+ migration.(9, 14-20) Owing to the unusual structure of the H-cluster and its central importance in affecting the rate and redox potential of the featured H2 chemistry, the mechanism of its biosynthesis is of high interest. In this proposed work, I aim to elucidate several aspects of the mechanism by which the set of maturase enzymes HydE, HydF, and HydG promote H- cluster formation. I will focus on three questions: what are the geometric and electronic structures of Fe- containing intermediates in the early stages of cluster maturation, what are the molecular precursors that give rise to the dithiolate bridge, and can the central atom be identified directly by EPR spectroscopy? To address these questions I will perform advanced EPR experiments on specifically labeled isotopologs of both intermediates in H-cluster synthesis as well as the mature H-cluster. These isotopic labels will be introduced through the use of labeled substrates (most often tyrosine) thereby allowing for a specific isotopic label to be traced from the molecular precursor through intermediates and finally into the mature H-cluster. For each intermediate, orientation-selective EPR experiments such as HYSCORE and ENDOR will enable determination of the distances and orientations of the labeled nuclei with respect to the electron spin (modeled as a point- dipole), thereby providing detailed geometric and electronic structure information. These EPR experiments will be performed in conjunction with complimentary stopped-flow FTIR and M¿ssbauer spectroscopies. Overall, this work will address the mechanistic details of tyrosine degredation into CO and CN- by HydG, the structures of Fe-containing intermediates in this process, the identities of the final product(s) o the HydG reaction, and the molecular precursors to the dithiolate bridge.