Mechanistic Investigations of [FeFe] Hydrogenase H-Cluster Assembly

[FeFe]氢化酶 H 簇组装的机理研究

• 批准号: 9058117
• 负责人: Daniel Leif Migdow Suess
• 金额: $ 5.61万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9058117
• 关键词: Active Sites; Address; Affect; Amines; Anabolism; Anions; Attention; Carbon Dioxide; Catalysis; Cell Nucleus; Chemicals; Chemistry; Complex; Consumption; Conversion disorder; Data Reporting; Electron Nuclear Double Resonance; Electron Spin Resonance Spectroscopy; Electrons; Enzymes; Foundations; Frequencies; Goals; Health; Human; Hydrogen; Hydrogenase; Investigation; Isotopes; Label; Lead; Learning; Ligands; Light; Magnetism; Methods; Modeling; Molecular; Oxidation-Reduction; Positioning Attribute; Process; Production; Property; Proteins; Reaction; Research Training; Role; Site; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Staging; Structural Models; Structure; Testing; Thermodynamics; Tyrosine; Work; X-Ray Crystallography; atmospheric carbon dioxide; base; catalyst; design; electronic structure; fascinate; geometric structure; interest; member; migration; oxidation; research study

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.

描述（由适用提供）：大气二氧化碳水平的迅速上升对人类健康产生了重大的长期威胁。（1）当前二氧化碳水平的大部分增加可能归因于使用不可再生燃料的使用，因此，在研究h+减少和H2氧化等能量转换反应中，人们对能量转换反应产生了极大的兴趣。 -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) H-Cluster具有一个生物学上空前的结构，其2FE核心由三个CO和两个CN含量结扎，每个核心通常在其自由状态下都是有毒的。尽管众所周知，CO和cnligands源自酪氨酸，但（10-13）它们形成和组装到H群中的机械细节很少。此外，FE中心还被五个原子的丁香桥桥接。中央原子的身份被认为是n-has是很多争论的主题，很重要，因为它被认为是吊坠基础，可以在动力学上促进H+迁移。（9，14-20）由于H-Certuster的不寻常结构，这是由于H-Certist的不寻常结构及其在影响H2 Chem Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is Is的吊坠。（9，14-20）中。在这项拟议的工作中，我旨在阐明成熟酶Hyde，Hydf和Hydg的机制的几个方面，促进了H群集的形成。我将重点介绍三个问题：在集群成熟的早期阶段，含有中间体的几何和电子结构是什么？ 通过EPR光谱？为了解决这些问题，我将对H-Cluster合成和成熟的H群集中两个中间体的特定标记的同位素进行高级EPR实验。这些同位素标签将通过使用标记的底物（最常见的酪氨酸）引入，从而允许通过中间体从分子前体来追溯特定的同位素标签，最后进入成熟的H簇。对于每个中间，取向选择性的EPR实验，例如Hyscore和Endor，都可以确定相对于电子自旋（建模为点偶极子）的标记核的距离和方向，从而提供详细的几何和电子结构信息。这些EPR实验将与免费停止流量FTIR和M¿SSBAUER光谱镜一起进行。总体而言，这项工作将通过HYDG（在此过程中含有Fe的中间体的结构），HYDG反应的最终产物的身份以及对二硫代桥的分子前体的身份来解决酪氨酸程度对CO和CN-的机械细节。

项目成果

The Role of the Secondary Coordination Sphere in a Fungal Polysaccharide Monooxygenase.

• DOI: 10.1021/acschembio.7b00016
• 发表时间: 2017-04-21
• 期刊: ACS chemical biology
• 影响因子: 4
• 作者: Span EA;Suess DLM;Deller MC;Britt RD;Marletta MA
• 通讯作者: Marletta MA

Manganese-Cobalt Oxido Cubanes Relevant to Manganese-Doped Water Oxidation Catalysts.

• DOI: 10.1021/jacs.7b01792
• 发表时间: 2017-04-19
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: Nguyen AI;Suess DLM;Darago LE;Oyala PH;Levine DS;Ziegler MS;Britt RD;Tilley TD
• 通讯作者: Tilley TD