Iron-sulfur cofactors involved in metal center assembly and catalysis of hydrogenase

铁硫辅助因子参与金属中心组装和氢化酶催化

• 批准号: 311062227
• 负责人: Dr. Oliver Lenz
• 金额: --
• 依托单位: Arbeitsgruppe Physikalische Chemie/ Biophysikalische Chemie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/311062227?language=en
• 关键词: Iron; sulfur; cofactors; involved; metal

In the second funding period of the Priority Program (PP) 1927, our joint project will focus, first, on the elucidation of the unusual active site properties of an O2-tolerant, thermostable NAD+-reducing [NiFe]-hydrogenase and their impact on the catalytic properties of this complex FeS cluster enzyme. The molecular and mechanistic details will be investigated in a collaborative approach, which will involve electron paramagnetic resonance (EPR), infrared (IR), and resonance Raman (RR) spectroscopy to identify and characterize catalytically relevant redox states both in vitro and in vivo. Comparative studies will be performed on a related F420-reducing [NiFe]-hydrogenase. In this context, we will apply a recently established experimental setup, which allows (cryogenic) IR and RR measurements on the same protein crystal under controlled gas atmospheres. In combination with X-ray crystallography and theoretical methods, these experiments will provide detailed structural and electronic information on individual catalytic intermediates. This interdisciplinary approach will be extended to analyze [Fe] hydrogenase and nitrogenase. In the second part of our joint project, we seek to improve the understanding of the multistep biosynthesis process of the NiFe(CN-)2(CO) cofactor of [NiFe] hydrogenase. We will investigate the reaction mechanism by which HypX converts formyl-tetrahydrofolate via formyl-CoA into the active site CO ligand of [NiFe] hydrogenases under oxic conditions. Experiments are planned to unveil the composition of the FeS cluster-containing HypCD maturation complex competent in receiving the (HypX-generated) CO molecule to eventually form the Fe(CN-)2(CO) unit. Based on a recently developed strategy, we are now able to purify isolated large subunits of [NiFe]-hydrogenases in different maturation stages of the catalytic center. This unique situation allows us to unravel the sequence of events of NiFe(CN-)2(CO) cofactor assembly. Vibrational spectroscopic techniques will be used to investigate the role and interplay of FeS centers and other metals in the maturation and catalysis of hydrogenase, nitrogenase(-like) enzymes and Complex I. In close collaboration with members of the PP consortium and external partners, we will exploit our results to gain a detailed picture of the overarching principles of FeS-based maturation and catalysis.

在1927年优先级计划（PP）的第二个融资期内，我们的联合项目将首先集中在阐明O2耐受耐热的NAD+NAD+降低[Nife]氢化酶及其对这种复合物群群蛋白酶蛋白酶催化特性的异常活性位点特性。分子和机械细节将以协作方法进行研究，该方法将涉及电子顺磁共振（EPR），红外（IR）和共振拉曼（RR）光谱，以识别和表征催化相关的氧化还原的氧化还原状态。比较研究将对相关的F420还原[Nife]氢化酶进行。在这种情况下，我们将采用最近建立的实验设置，该设置允许在受控气体大气中的相同蛋白质晶体上（低温）IR和RR测量。结合X射线晶体学和理论方法，这些实验将提供有关单个催化中间体的详细结构和电子信息。这种跨学科的方法将扩展以分析[Fe]氢化酶和氮气。在联合项目的第二部分中，我们试图提高对[Nife]氢化酶的Nife（CN-）2（CO）辅助因子的多步生物合成过程的理解。我们将研究HYPX通过甲基-COA将甲基四氢叶酸转化为[Nife]氢化酶的活性位点配体的反应机制。计划实验揭示含FES簇的HypCD成熟复合物的组成，该复合体有能力接收（Hypx生成的）CO分子，最终形成Fe（CN-）2（CO）单位。基于最近开发的策略，我们现在能够在催化中心的不同成熟阶段净化[Nife]氢化酶的分离大亚基。这种独特的情况使我们能够揭示Nife（CN-）2（CO）辅助因子组件的事件序列。振动光谱技术将用于研究FES中心和其他金属在氢化酶，氮酶（类似）酶的成熟和催化中的作用和相互作用。