Mechanistic Investigations of [FeFe] Hydrogenase H-Cluster Assembly

描述(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)目前二氧化碳水平的上升在很大程度上可能归因于不可再生燃料的使用，因此，人们对H+还原和氢氧化等能量转换反应产生了极大的兴趣。(2)一些最好的氢气产生和 消耗的催化剂是[FeFe]氢酶，它们能够在反应的热力学潜力附近以较高的周转频率运行。(3-6)由于了解其高效率的化学基础可能有助于设计用于氢气生产和消费的新的合成催化剂，(7)人们非常关注酶的活性位置的结构(“H-簇”)。(8，9)H-簇具有生物史无前例的结构，其2FE核心由三个CO和两个CN-配体连接，每一个在自由状态下通常是有毒的。虽然已知CO和CN-配体来源于酪氨酸，但(10-13)它们形成和组装成H-簇的机理细节很少。此外，铁中心由五个原子的二硫代桥连接；中心原子的身份-目前被认为是N-一直是许多争论的主题，而且很重要，因为它被认为是一个悬基，可以在动力学上促进H+的迁移。(9，14-20)由于H-簇的不寻常结构及其在影响特征氢化学的速率和氧化还原电位中的中心重要性，其生物合成的机制引起了人们的极大兴趣。在这项拟议的工作中，我旨在阐明一组成熟酶Hyde、HydF和HydG促进H-簇形成的几个方面的机制。我将集中讨论三个问题：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，产生二硫代桥的分子前体是什么，中心原子能否被直接确定 用电子顺磁共振波谱？为了解决这些问题，我将对H-簇合成中的两个中间体以及成熟的H-簇的特定标记的同位素进行高级EPR实验。这些同位素标记物将通过使用标记底物(通常是酪氨酸)引入，从而允许从分子前体通过中间体追踪特定的同位素标记物，最终进入成熟的H-簇。对于每个中间体，选择性取向的EPR实验，如HYSCORE和Endor，将能够确定标记核相对于电子自旋的距离和取向(模拟为点-偶极)，从而提供详细的几何和电子结构信息。这些EPR实验将与免费的停流FTIR和穆斯堡尔光谱仪一起进行。总体而言，这项工作将解决酪氨酸脱氢为CO和CN-的机理细节，这一过程中含铁中间体的结构，HydG反应的最终产物(S)的身份，以及二硫代桥的分子前体。

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.